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Sample records for bluetongue virus isolates

  1. Genome Sequence of Bluetongue virus Serotype 17 Isolated in Brazil in 2014.

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    Matos, Ana Carolina Diniz; Rosa, Júlio César Câmara; Nomikou, Kyriaki; Guimarães, Lorena Lima Barbosa; Costa, Érica Azevedo; Guedes, Maria Isabel Maldonado Coelho; Driemeier, David; Lobato, Zélia Inês Portela; Mertens, Peter Paul Clement

    2016-10-27

    The complete genome sequence of Bluetongue virus (BTV) serotype 17 strain 17/BRA/2014/73, isolated from a sheep in Brazil in 2014, is reported here. All segments clustered with western topotype strains and indicated reassortment events with other BTV from the Americas. The strain 17/BRA/2014/73 represents a novel reference strain for BTV-17 from South America.

  2. Segment 2 based characterization of a novel Indian Bluetongue virus isolate

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    Gaya Prasad

    2013-10-01

    Full Text Available Aim: The study was conducted to characterize and serotype the novel isolate of bluetongue virus (BTV isolated from India. Materials and Methods: The BTV isolate was propagated in BHK-21 cell line. Nucleic acid (dsRNA was extracted using Trizol method and cDNA was prepared using a process called reverse transcription. The cDNA was subjected to group specific PCR using ns1 gene specific primer to confirm the isolate as BTV. The type specific PCR was conducted to confirm the serotype of the virus using vp2 gene specific primers for all the BTV serotype including BTV10. The vp2 gene specific PCR amplicon was sequenced and in-silico restriction enzyme analysis and phylogenetic analysis was conducted. Results: Group specific PCR using ns1 gene specific primers showed a single 274bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The type specific PCR using BTV10 vp2 gene specific primer showed a single amplicon of 647bp. Remaining BTV serotype specific primers didn't show any amplification. The vp2 gene PCR amplicon was sequenced. The in-silico restriction enzyme analysis of vp2 gene of Indian BTV10 isolate along with other isolates from GenBank database using HindIII, XhoII and ApoI showed a common pattern between Indian and USA isolates. Similarly, phylogenetic analyses using vp2 gene nucleotide as well as deduced amino acid sequence of Indian BTV10 isolate and global isolates showed that Indian and most of the USA isolates placed in a single clad. Conclusion: A novel BTV isolate was isolated and confirmed as BTV serotype 10. Upon molecular analysis Indian BTV10 isolate was found closer to that of USA isolates than other global isolates. [Vet World 2013; 6(5.000: 244-248

  3. Whole genome sequence analysis of circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates.

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    Gaudreault, Natasha N; Jasperson, Dane C; Dubovi, Edward J; Johnson, Donna J; Ostlund, Eileen N; Wilson, William C

    2015-07-01

    Bluetongue virus (BTV) is a vector-transmitted pathogen that typically infects and causes disease in domestic and wild ruminants. BTV is also known to infect domestic canines as discovered when dogs were vaccinated with a BTV-contaminated vaccine. Canine BTV infections have been documented through serological surveys, and natural infection by the Culicoides vector has been suggested. The report of isolation of BTV serotype 11 (BTV-11) from 2 separate domestic canine abortion cases in the states of Texas in 2011 and Kansas in 2012, were apparently unrelated to BTV-contaminated vaccination or consumption of BTV-contaminated raw meat as had been previously speculated. To elucidate the origin and relationship of these 2 domestic canine BTV-11 isolates, whole genome sequencing was performed. Six additional BTV-11 field isolates from Texas, Florida, and Washington, submitted for diagnostic investigation during 2011 and 2013, were also fully sequenced and analyzed. The phylogenetic analysis indicates that the BTV-11 domestic canine isolates are virtually identical, and both share high identity with 2 BTV-11 isolates identified from white-tailed deer in Texas in 2011. The results of the current study further support the hypothesis that a BTV-11 strain circulating in the Midwestern states could have been transmitted to the dogs by the infected Culicoides vector. Our study also expands the short list of available BTV-11 sequences, which may aid BTV surveillance and epidemiology.

  4. Isolation of bluetongue virus serotype 1 from Culicoides vector captured in livestock farms and sequence analysis of the viral genome segment-2.

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    Dadawala, A I; Biswas, S K; Rehman, W; Chand, K; De, A; Mathapati, B S; Kumar, P; Chauhan, H C; Chandel, B S; Mondal, B

    2012-08-01

    Bluetongue virus serotype-1 (BTV-1) was isolated from Culicoides oxystoma vectors captured on livestock farms in two places of Gujarat, India. The viruses were isolated on BHK-21 cells, which produced characteristic BTV-related cytopathic effects between 24 and 48 h post-infection. Virus antigen was demonstrated in infected cells at different passage by a BTV-specific sandwich ELISA. Further, polyacrylamide gel electrophoresis and silver staining of viral genomic RNA revealed ten double-stranded RNA segments characteristic of BTV. Serotype of the isolates was identified by virus neutralization and PCR coupled with sequencing. The isolates were designated as SKN-7 and SKN-8 and their genome segment-2 (VP2) were sequenced. Phylogenetic analyses revealed very close relationship between them although they are not identical. SKN-8 showed closer relationship with a recently isolated BTV-1 from goat. Bluetongue virus was earlier isolated from Culicoides in adjacent state more than 20 years ago, although the serotype of the virus was not determined.

  5. Studies of the Antigenic relationships between Bluetongue virus serotypes 2, 9 AND 15 isolated in Andhra Pradesh, India

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    Sreenivasulu Daggupati

    Full Text Available The presence of multiple serotypes of the midge-borne bluetongue virus and lack of effective vaccine are the major impediments in controlling bluetongue in sheep. Attempts are being made to develop a vaccine employing the available serotypes to control the disease in the state. Hence, it is essential to identify the antigenic relationships among the serotypes to identify the candidate strains to be incorporated in the preparation of vaccine. To understand the antigenic relationships between Bluetongue virus -2, 9 and 15 serotypes, the viruses were propagated in BHK21 cell lines, purified using PEG precipitation method and purified virus used to raise hyper immune serum in rabbits. Neutralizing antibodies for the BTV serotypes were detected by day 21 PI. Reciprocal cross neutralization test was employed to determine the R% values between BTV-2, 9 and 15 which indicated the extent of antigenic relationships among the serotypes. R% value between BTV-2 and BTV-9 was recorded as 2.8. R% value of 3.53 and 2.8 were observed between BTV-2 & 15 and BTV-9 & 15 respectively. The R% values recorded in the present study revealed a weak antigenic relationship between the BTV serotypes,indicating that the serotypes are highly divergent. [Vet. World 2011; 4(10.000: 444-448

  6. Whole genome sequencing and phylogenetic analysis of Bluetongue virus serotype 2 strains isolated in the Americas including a novel strain from the western United States.

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    Gaudreault, Natasha N; Mayo, Christie E; Jasperson, Dane C; Crossley, Beate M; Breitmeyer, Richard E; Johnson, Donna J; Ostlund, Eileen N; MacLachlan, N James; Wilson, William C

    2014-07-01

    Bluetongue is a potentially fatal arboviral disease of domestic and wild ruminants that is characterized by widespread edema and tissue necrosis. Bluetongue virus (BTV) serotypes 10, 11, 13, and 17 occur throughout much of the United States, whereas serotype 2 (BTV-2) was previously only detected in the southeastern United States. Since 1998, 10 other BTV serotypes have also been isolated from ruminants in the southeastern United States. In 2010, BTV-2 was identified in California for the first time, and preliminary sequence analysis indicated that the virus isolate was closely related to BTV strains circulating in the southeastern United States. In the current study, the whole genome sequence of the California strain of BTV-2 was compared with those of other BTV-2 strains in the Americas. The results of the analysis suggest co-circulation of genetically distinct viruses in the southeastern United States, and further suggest that the 2010 western isolate is closely related to southeastern strains of BTV. Although it remains uncertain as to how this novel virus was translocated to California, the findings of the current study underscore the need for ongoing surveillance of this economically important livestock disease.

  7. Whole genome sequencing and phylogenetic analysis of Bluetongue virus serotype 2 strains isolated in the Americas including a novel strain from the western United States

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    Bluetongue is caused by an arbovirus which produces widespread edema and tissue necrosis in domestic and wild ruminants that can be fatal. Bluetongue virus serotypes 10, 11, 13, and 17 are typically found throughout the United States (US), while serotype 2 was previously only detected in the southea...

  8. The molecular biology of Bluetongue virus replication.

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    Patel, Avnish; Roy, Polly

    2014-03-01

    The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention.

  9. Subclinical bluetongue virus infection in domestic ruminants in Taiwan.

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    Lee, Fan; Ting, Lu-Jen; Jong, Ming-Hwa; Chang, Wei-Ming; Wang, Fun-In

    2010-05-19

    Bluetongue is an arthropod-borne viral disease affecting domestic and wild ruminants. Taiwan, with the Tropic of Cancer crossing through it, was considered free of bluetongue virus (BTV) before 2001. The goals of this study are to identify the serotype and phylogeny of Taiwan BTV isolates and to understand the serological status and chronology of BTV infection. Analysis of the S10 gene segment revealed that Taiwan BTV isolates are closely related to Chinese strains. Seropositive results were found in 32.7% of the cattle and 8.2% of the goats by head, and 90.7% of the cattle herds and 28.9% of the goat flocks. Anti-BTV antibodies have existed in goat sera since 1989 and in bovine sera since 1993, and over the years, the seropositive rates in rapidly urbanized districts have decreased, most likely due to the loss of vector habitats. Seropositive rates for sheep were variable, due to a small sample size and a small sheep population. Thus far, all natural BTV infections have been subclinical, consistent with experimental sheep inoculation, revealing that the Taiwan isolate is of low virulence.

  10. Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.

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    Shad, G; Wilson, W C; Mecham, J O; Evermann, J F

    1997-04-01

    A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

  11. Potential role of ticks as vectors of bluetongue virus

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    Bouwknegt, C.; Rijn, van P.A.; Schipper, J.M.J.; Holzel, D.R.; Boonstra, J.; Nijhof, A.; Rooij, van E.M.A.; Jongejan, F.

    2010-01-01

    When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not

  12. Virus and host factors affecting the clinical outcome of Bluetongue Virus infection

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    Caporale, M.; Gialleonorado, L.; Janowicz, A.; Wilkie, G.; Shaw, A.; Savini, G.; Rijn, van P.A.; Mertens, P.; Ventura, M.; Palmarini, M.

    2014-01-01

    Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species

  13. 云南省师宗县蓝舌病病毒的分离及鉴定%ISOLATION AND IDENTIFICATION OF BLUETONGUE VIRUS IN 2012 IN SHIZONG COUNTY OF YUNNAN PROVINCE

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    肖雷; 孟锦昕; 李楠; 高林; 何于雯; 杨恒; 胡骑; 李华春; 朱建波

    2014-01-01

    The sentinel herd of 10 cattle was established in 2012 to monitor epidemiology of Bluetongue virus in Shizong county of Yunnan Province. Blood samples were taken weekly from May to October and then monthly from November to December 2014 and tested for serological response in C-ELISA and for virus isolation. The sero-conversion was revealed in August in some animals and then in November in whole sentinel herd. The erythrocyte preparations from blood samples were inoculated into chicken embryo veins. Chicken liver tissues were harvested, homogenized in PBS and centrifuged. The supernatants were inoculated into C6/36 cells and passaged in BHK-21 three times. The cytopathic effect (CPE) was observed in cell monolayers. The resulting 86 virus isolates were characterized in RT-PCR and virus neutralization (VN). The VP7 gene of Bluetongue virus was amplified in conventional RT-PCR using two pairs of primers designed according to the sequences in GenBank. A 1156 bp fragment of VP7 gene was amplified in RT-PCR from 67 out of 86 isolates. The virus neutralization was performed using 24 reference bluetongue viruses and 24 positive sera. Again, the same 67 isolates were confirmed as Bluetongue virus. The VP2 gene of two isolates was sequenced. One isolate was determined to be BTV-1 as it shared 92%identity with the reference strain Y863 (KC879616) and another isolate was BTV-16 as it shared 99%identity with the reference strain AB686221. The VN results also had an agreement with VP2 sequencing. In conclusion, 67 isolates isolated from the sentinel herd belonged to Bluetongue virus, indicating the virus transmission among bovine herds.%为了解近年来云南省师宗县蓝舌病病毒流行情况,2012年在师宗县五龙乡建立了10头蓝舌病血清学阴性黄牛的监控动物群。从2012年5~10月,每周采血1次,11~12月,每月采血1次,采用C-ELISA进行血清学监测。8月开始动物血清学检测结果转阳性,至11月,监控动物全

  14. Short communication. Bluetongue virus serotype-1 in goats in the Pithoragarh area of Uttarakahand, India

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    Bimalendu Mondal

    2013-12-01

    Full Text Available This short communication reports the results of a bluetongue sero-surveillance conducted in the Pithoragarh hills of Uttarakhand in India during the autumn of 2011. Unclotted blood and serum samples were collected from 51 goats for detection of bluetongue virus (BTV antigen and antibodies. Of the 51 collected samples, 18 (35% were positive to an indirect ELISA and 33 (64% resulted positive to a BTV ELISA antigen. From a strong antigen-positive blood sample, a BTV was isolated (named as PTG-13 on cell culture and was subsequently confirmed as BTV‑1 by RT‑PCR and partial sequencing of genome segment-2. The goat serum samples were found to contain high titer of neutralising antibodies against BTV-23, nonetheless the virus could not be isolated. Interestingly, no neutralizing antibodies were detected against PTG-13 or other BTV-1 isolate, which suggests that sampling was probably done before the development of neutralizing antibodies against PTG-13 virus in the host. Isolation of BTV-1 (PTG-13 and presence of BTV-23 neutralizing antibodies in serum samples indicate that goats were probably infected with BTV-1 and 23 in different periods.

  15. Molecular evolution of American field strains of bluetongue and epizootic haemorrhagic disease viruses.

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    Wilson, William C; Gaudreault, Natasha N; Jasperson, Dane C; Johnson, Donna J; Ostlund, Eileen N; Chase, Christopher L; Ruder, Mark G; Stallknecht, David E

    2015-01-01

    Recent Orbivirus occurrences in the Americas have been investigated using whole genome amplification and sequencing followed by phylogenetic analysis. The bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) whole genomes were amplified without prior sequence knowledge and deep sequenced. This technology was applied to evaluate BTV‑3 isolates spanning 4 decades from Florida, Arkansas, Mississippi, South Dakota, Central America, and the Caribbean basin. The results of the dataset analysis are consistent with the hypothesis that these viruses were introduced into the United States from Central America and the Caribbean basin. A similar analysis has been performed on a recent BTV‑2 isolate from California. It indicates that the BTV‑2 strain was likely introduced into Florida and then moved South to the Caribbean and West to California. A historical (1955‑2012) molecular characterisation of EHDV strains was also completed, and subsequently used as reference sequence for comparison of genomes from recent 2012 cattle isolates associated with clinical disease. Finally, this analysis was performed on BTV‑11 isolated from 2 canine cases and demonstrated that the genome sequences of the virus isolates from these cases were almost identical. These studies indicate the value of this technology in understanding virus epidemiology and ecology.

  16. Bluetongue virus serotype 6 in Europe in 2008 - Emergence and disappearance of an unexpected non-virulent BTV

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    Rijn, van P.A.; Geurts, Y.; Spek, van der A.N.; Veldman, D.; Gennip, van H.G.P.

    2012-01-01

    Bluetongue viruses (BTVs) could invade N-W Europe similar to BTV serotype 8 (BTV8/net06), since the source and route of introduction of this virus has not been solved. Therefore, the Dutch survey for Bluetongue by PCR testing was extended by further analysis of PCR positives to identify the involved

  17. Production and Characterization of Monoclonal Antibodies to Bluetongue Virus

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    Veerakyathappa Bhanuprakash; Madhusudhan Hosamani; Vinayagamurthy Balamurugan; Pradeep Narayan Gandhale; Gnanavel Venkatesan; Raj Kumar Singh

    2011-01-01

    In the present study, a total of 24 Mabs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein, titres, isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones, a majority of them (n = 18)belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA, the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However, this clone showed a variable percent of inhibition ranging from 16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones, only 4A 10 was found to have a possible diagnostic application.

  18. Structural constraints in the packaging of bluetongue virus genomic segments.

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    Burkhardt, Christiane; Sung, Po-Yu; Celma, Cristina C; Roy, Polly

    2014-10-01

    The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5' and 3' ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was packaged successfully, confirming that the motif identified directs the correct packaging of the segment.

  19. Genetic modification of Bluetongue virus by uptake of "synthetic" genome segments

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    Gennip, van H.G.P.; Veldman, D.; Water, van de S.G.P.; Rijn, van P.A.

    2010-01-01

    Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was repor

  20. The first survey for antibody against Bluetongue virus in sheep flocks in Southeast of Iran

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    Ali Asghar Mozaffari; Mohammad Khalili

    2012-01-01

    Objective: Bluetongue virus is an arthropod-borne Orbivirus in the family Reoviridae which infects both domestic and wild ruminants. Bluetongue disease is a "List A" disease of the Office of International Epizootics. To the best of our knowledge, no report has been published on bluetongue disease of sheep flocks of Southeast of Iran. The objective of this study was to describe the seroprevalence rates of BTV in sheep flocks in southeast of Iran. Methods: The blood samples were collected randomly from herds of Southeast of Iran. A total of 188 sera samples (94 male, 94 female) collected between 2009 and 2010, were available. Antibodies to BTV in sera were detected by using a commercial competitive ELISA (Institute Pourquier, Montpellier, France) according to manufacturer’s instructions. Results: The seroprevalence rates were 6.57 %for sheep herds. Within a herd, prevalence of BTV seropositive animals ranged from 0% to 42.85%. 33.3% sheep flocks were positive to BTV antibodies. Sex didn't affect the rate of seropositivity, but the rate of seropositivity was significantly changed in different age groups. Conclusion: This study describes the seroprevalence rates of Bluetongue virus (BTV) in sheep flocks in southeast of Iran for the first time.

  1. Genetic diversity of the S10 RNA segment of field and vaccine strains of bluetongue virus from the P. R. China.

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    Zhang, Yifang; Du, Xiaogang; Li, Wengui; Li, Jinyao; Liu, Jianping; Zhu, Jianbo; Zhang, Nianzu

    2010-02-01

    Bluetongue virus (BTV) infection of ruminants is endemic throughout tropical and subtropical regions of the world. However, the molecular epidemiology of BTV infection in China has not yet been reported. In this study, the S10 gene segments from 30 BTV isolates, one attenuated BTV strain, one vaccine BTV strain, and one South Africa BTV prototype strain, were sequenced. Phylogenetic analysis of the S10 genes showed that Chinese BTV isolates could be classified into two phyletic subgroups, and the clustering of Chinese BTV viruses was dependent on their geographical origin and the number of generations for which they had been propagated, rather than their host species or year of isolation.

  2. Evidence for transmission of bluetongue virus serotype 26 through direct contact.

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    Carrie Batten

    Full Text Available The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26 in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.

  3. Widespread Reassortment Shapes the Evolution and Epidemiology of Bluetongue Virus following European Invasion.

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    Kyriaki Nomikou

    2015-08-01

    Full Text Available Genetic exchange by a process of genome-segment 'reassortment' represents an important mechanism for evolutionary change in all viruses with segmented genomes, yet in many cases a detailed understanding of its frequency and biological consequences is lacking. We provide a comprehensive assessment of reassortment in bluetongue virus (BTV, a globally important insect-borne pathogen of livestock, during recent outbreaks in Europe. Full-genome sequences were generated and analysed for over 150 isolates belonging to the different BTV serotypes that have emerged in the region over the last 5 decades. Based on this novel dataset we confirm that reassortment is a frequent process that plays an important and on-going role in evolution of the virus. We found evidence for reassortment in all ten segments without a significant bias towards any particular segment. However, we observed biases in the relative frequency at which particular segments were associated with each other during reassortment. This points to selective constraints possibly caused by functional relationships between individual proteins or genome segments and genome-wide epistatic interactions. Sites under positive selection were more likely to undergo amino acid changes in newly reassorted viruses, providing additional evidence for adaptive dynamics as a consequence of reassortment. We show that the live attenuated vaccines recently used in Europe have repeatedly reassorted with field strains, contributing to their genotypic, and potentially phenotypic, variability. The high degree of plasticity seen in the BTV genome in terms of segment origin suggests that current classification schemes that are based primarily on serotype, which is determined by only a single genome segment, are inadequate. Our work highlights the need for a better understanding of the mechanisms and epidemiological consequences of reassortment in BTV, as well as other segmented RNA viruses.

  4. Evaluation of cross-protection of bluetongue virus serotype 4 with other serotypes in sheep

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    Gcwalisile B. Zulu

    2014-02-01

    Full Text Available Bluetongue (BT is a non-contagious disease of sheep and other domestic and wild ruminants caused by the bluetongue virus (BTV. Currently 26 serotypes of the virus have been identified. In South Africa, 22 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze-dried live attenuated polyvalent BTV vaccine. The vaccine is constituted of 15 BTV serotypes divided into three separate bottles and the aim is to develop a vaccine using fewer serotypes without compromising the immunity against the disease. This study is based on previously reported cross-neutralisation of specific BTV serotypes in in vitro studies. Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control, 1 (unrelated serotype, 9, 10 and 11 in sheep using the serum neutralisation test. The purpose of the study was to determine possible cross-protection of different serotypes in sheep. Of those vaccinated with BTV-4 and challenged with BTV-1, which is not directly related to BTV-4, 20% were completely protected and 80% showed clinical signs, but the reaction was not as severe as amongst the unvaccinated animals. In the group challenged with BTV-10, some showed good protection and some became very sick. Those challenged with BTV-9 and BTV-11 had good protection. The results showed that BTV-4 does not only elicit a specific immune response but can also protect against other serotypes.

  5. Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

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    Feenstra, Femke; Drolet, B.S.; Boonstra, Jan; Rijn, Van P.A.

    2015-01-01

    Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. Howe

  6. Full-Genome Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India.

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    Sushila Maan

    Full Text Available Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV. Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp. and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV 'topotype'. However, genome-segment 5 (Seg-5 encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03 are closely related (>99% identity to a South African BTV-2 vaccine-strain (western topotype. Similarly BTV-10 isolates (IND2003/06; IND2005/04 show >99% identity in all genome segments, to the prototype BTV-10 (CA-8 strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of 'live' South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

  7. Presence of bluetongue virus in the marginal zone of the spleen in acute infected sheep.

    Science.gov (United States)

    Lee, Fan; Chen, Jiun-Liang; Lin, Chun-Ming; Wang, Fun-In

    2011-08-26

    Bluetongue virus, a member of the genus Orbivirus of the family Reoviridae, is the causative agent of bluetongue, which is a non-contagious Culicoides mediated blood-borne disease. The present study characterizes the pathogenicity of a Taiwan prototype BTV2/KM/2003 in Corriedale sheep inoculated subcutaneously into the ear pinna. Histologically, multifocal petechiated hemorrhage, with mild to moderate inflammation and edema, were present in the contralateral ear pinna, tongue, and facial skin, without remarkable lesions in lymphoid organs. By days post-infection (DPI) 7, viral VP7 antigen, detected by immunohistochemistry, presented in the spleen, chiefly located in the outer rim of macrophages bordering the marginal zone and red pulp, and T lymphocytes of the red pulp. By DPI 11, viral signals shifted from the marginal zone to macrophages and small lymphocytes within follicles of the spleen. In situ hybridization with VP7 gene probe detected strong signals in the spleen, chiefly spanning the whole width of 5-10 cell thickness of the marginal zone, including the marginal zone macrophages and marginal zone B cells, as well as macrophages of sheathed capillaries in the red pulp. This study demonstrates molecular as well as morphologic evidence of the presence of bluetongue virus in the marginal zone of the spleen, most likely associated with viremia in acute infection, as previously demonstrated by the authors.

  8. Turnover rate of NS3 proteins modulates bluetongue virus replication kinetics in a host-specific manner

    NARCIS (Netherlands)

    Ftaich, Najate; Ciancia, Claire; Viarouge, Cyril; Barry, Gerald; Ratinier, Maxime; Rijn, van P.A.; Breard, Emmanuel; Vitour, Damien; Zientara, Stephan; Palmarini, Massimo; Terzian, Christophe; Arnaud, Frédérick

    2015-01-01

    Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they b

  9. Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress

    Directory of Open Access Journals (Sweden)

    Roy Polly

    2007-01-01

    Full Text Available Abstract Background The VP2 outer capsid protein Bluetongue Virus (BTV is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown. Results In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of aminio acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells. Conclusion The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii Deletion of amino acids 65–114 or mutation of amino acids 70–72 to DVD abrogates vimentin association. iii Finally

  10. Validation of a commercial ELISA for the detection of bluetongue virus (BTV) specific antibodies in individual milk samples of Dutch dairy cows

    NARCIS (Netherlands)

    Kramps, J.A.; Maanen, van K.; Mars, M.H.; Popma, J.K.; Rijn, van P.A.

    2008-01-01

    recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum a

  11. A Rapid Field-Deployable Reverse Transcription-Insulated Isothermal Polymerase Chain Reaction Assay for Sensitive and Specific Detection of Bluetongue Virus.

    Science.gov (United States)

    Ambagala, A; Pahari, S; Fisher, M; Lee, P-Y A; Pasick, J; Ostlund, E N; Johnson, D J; Lung, O

    2017-04-01

    Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus (BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan-BTV reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '-' without the need for data interpretation. The RT-iiPCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-iiPCR assay was as sensitive as real-time RT-PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory.

  12. Circulation of bluetongue virus in goats in the Karamoja region of Uganda

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    Elijah N. Mulabbi

    2013-02-01

    Full Text Available The presence of bluetongue virus (BTV in indigenous goats from the Karamoja region of northern Uganda was investigated. A total of 300 goats were sampled (serum and whole blood from five districts within the Karamoja region. The samples were analysed for the presence of bluetongue (BT antibodies using a commercial Enzyme-linked immunosorbent assay (ELISA and for the presence of BTV viral RNA by real-time Reverse transcription polymerase chain reaction (RT-PCR, because BTV is an RNA virus. Of the 300 goats tested, 269 (90% were positive for BTV antibodies, indicating high levels of BTV circulation within the region. Out of the 150 whole blood samples tested for the presence of the virus by real-time RT-PCR, 84 (56% were positive for BTV RNA. This study, which is the first of its kind in Uganda, showed a high seroprevalence of BT antibodies and active circulation of BTV in a high proportion of goats in the Karamoja region.

  13. Circulation of bluetongue virus in goats in the Karamoja region of Uganda

    Directory of Open Access Journals (Sweden)

    Carrie A. Batten

    2013-04-01

    Full Text Available The presence of bluetongue virus (BTV in indigenous goats from the Karamoja region of northern Uganda was investigated. A total of 300 goats were sampled (serum and whole blood from five districts within the Karamoja region. The samples were analysed for the presence of bluetongue (BT antibodies using a commercial Enzyme-linked immunosorbent assay (ELISA and for the presence of BTV viral RNA by real-time Reverse transcription polymerase chain reaction (RT-PCR, because BTV is an RNA virus. Of the 300 goats tested, 269 (90% were positive for BTV antibodies, indicating high levels of BTV circulation within the region. Out of the 150 whole blood samples tested for the presence of the virus by real-time RT-PCR, 84 (56% were positive for BTV RNA. This study, which is the first of its kind in Uganda, showed a high seroprevalence of BT antibodies and active circulation of BTV in a high proportion of goats in the Karamoja region.

  14. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines

    Science.gov (United States)

    Celma, Cristina C.; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A.; Beer, Martin

    2016-01-01

    ABSTRACT Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new

  15. Pathological Characterization Of IFNAR(-/-) Mice Infected With Bluetongue Virus Serotype 4

    Science.gov (United States)

    Marín-López, Alejandro; Bermúdez, Roberto; Calvo-Pinilla, Eva; Moreno, Sandra; Brun, Alejandro; Ortego, Javier

    2016-01-01

    Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1β mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses. PMID:27994510

  16. Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

    NARCIS (Netherlands)

    Backx, A.; Heutink, C.G.; Rooij, van E.M.A.; Rijn, van P.A.

    2009-01-01

    Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven ou

  17. Mapping the basic reproduction number (Ro) for vector-borne diseases: A case study on bluetongue virus.

    NARCIS (Netherlands)

    Hartemink, N.; Purse, B.V.; Meiswinkel, R.; Brown, H.E.; Koeijer, de A.A.; Elbers, A.R.W.; Boender, G.J.; Rogers, D.J.; Heesterbeek, J.A.P.

    2009-01-01

    Geographical maps indicating the value of the basic reproduction number, R0, can be used to identify areas of higher risk for an outbreak after an introduction. We develop a methodology to create R0 maps for vector-borne diseases, using bluetongue virus as a case study. This method provides a tool f

  18. 9 CFR 113.303 - Bluetongue Vaccine.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bluetongue Vaccine. 113.303 Section... Virus Vaccines § 113.303 Bluetongue Vaccine. Bluetongue Vaccine shall be prepared from virus-bearing... be used for preparing the seeds for vaccine production. All serials of vaccine shall be prepared...

  19. Epidemiology of Bluetongue in India.

    Science.gov (United States)

    Rao, P P; Hegde, N R; Reddy, Y N; Krishnajyothi, Y; Reddy, Y V; Susmitha, B; Gollapalli, S R; Putty, K; Reddy, G H

    2016-04-01

    Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine

  20. Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

    Science.gov (United States)

    Top, Sokunthea; Foucras, Gilles; Deplanche, Martine; Rives, Germain; Calvalido, Jérôme; Comtet, Loic; Bertagnoli, Stéphane; Meyer, Gilles

    2012-02-21

    Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

  1. Quantitative assessment of the probability of bluetongue virus overwintering by horizontal transmission: application to Germany

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    Napp Sebastian

    2011-01-01

    Full Text Available Abstract Even though bluetongue virus (BTV transmission is apparently interrupted during winter, bluetongue outbreaks often reappear in the next season (overwintering. Several mechanisms for BTV overwintering have been proposed, but to date, their relative importance remain unclear. In order to assess the probability of BTV overwintering by persistence in adult vectors, ruminants (through prolonged viraemia or a combination of both, a quantitative risk assessment model was developed. Furthermore, the model allowed the role played by the residual number of vectors present during winter to be examined, and the effect of a proportion of Culicoides living inside buildings (endophilic behaviour to be explored. The model was then applied to a real scenario: overwintering in Germany between 2006 and 2007. The results showed that the limited number of vectors active during winter seemed to allow the transmission of BTV during this period, and that while transmission was favoured by the endophilic behaviour of some Culicoides, its effect was limited. Even though transmission was possible, the likelihood of BTV overwintering by the mechanisms studied seemed too low to explain the observed re-emergence of the disease. Therefore, other overwintering mechanisms not considered in the model are likely to have played a significant role in BTV overwintering in Germany between 2006 and 2007.

  2. Cross-sectional serosurvey and associated factors of bluetongue virus antibodies presence in small ruminants of Nepal

    OpenAIRE

    Gaire, Tara Nath; Karki, Surendra; DHAKAL, Ishwari Prasad; Khanal, Doj Raj; Joshi, Nanda Prakash; Sharma, Bishwas; Richard A Bowen

    2014-01-01

    Background Bluetongue (BT) is an infectious, insect-borne viral disease primarily affecting sheep and occasionally cattle and goats. In Nepal, BT is an emerging disease of economic importance. The objective of this study was to estimate the seroprevalence of BT virus (BTV) in small ruminants of two eco-zones of Nepal, Hills and Terai, and to identify the factors associated with virus exposure. We conducted a cross-sectional serosurvey from March 2012 through February 2013 by sampling 318 smal...

  3. Evaluation of Metarhizium anisopliae for the control of Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae), the principal vector of bluetongue virus in Australia.

    Science.gov (United States)

    Nicholas, A H; McCorkell, B

    2014-06-01

    Four isolates of the entomopathogenic fungus Metarhizium anisopliae were tested for their potential to control the biting midge Culicoides brevitarsis, the principal vector of bluetongue virus in Australia. Adult C. brevitarsis died three to eight days after walking on paper substrate treated with 0.7 g/m(2) conidia of any of the isolates, indicating that M. anisopliae has potential as a surface treatment or topical application control strategy. Incorporation of the fungus into freshly excreted cattle dung at rates of between 0.25 and 1 g conidia/kg reduced the emergence of adult midges by up to 98.5% compared to untreated dung indicating that M. anisopliae has the potential to control C. brevitarsis larvae in cattle dung. Three of the isolates produced similar mortality rates on adult and immature C. brevitarsis while the fourth isolate produced lower, but still significant, mortality rates on adult and immature stages.

  4. Involvement of the skin during bluetongue virus infection and replication in the ruminant host.

    Science.gov (United States)

    Darpel, Karin E; Monaghan, Paul; Simpson, Jennifer; Anthony, Simon J; Veronesi, Eva; Brooks, Harriet W; Elliott, Heather; Brownlie, Joe; Takamatsu, Haru-Hisa; Mellor, Philip S; Mertens, Peter Pc

    2012-04-30

    Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence".BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.

  5. An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

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    Estelle H. Venter

    2011-02-01

    Full Text Available Bluetongue (BT, a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV, can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

  6. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

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    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  7. Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue Virus Serotypes 1 and 8 in a Subclinical Experimental Infection

    Science.gov (United States)

    Lorca-Oró, Cristina; Pujols, Joan; García-Bocanegra, Ignacio; Mentaberre, Gregorio; Granados, José Enrique; Solanes, David; Fandos, Paulino; Galindo, Iván; Domingo, Mariano; Lavín, Santiago; López-Olvera, Jorge Ramón

    2012-01-01

    Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication. PMID:22666321

  8. Anticorpos contra o vírus da língua azul em bovinos do sertão da Paraíba Antibodies to bluetongue virus in bovines of Paraíba State, Brazil

    OpenAIRE

    C.B. Melo; Oliveira, A. M. de; Azevedo,E.O.; Z.I.P. Lobato; R.C. Leite

    2000-01-01

    In June of 1997 the prevalence of antibodies to bluetongue virus was between 3.94 and 4.82% in 137 bovine serum samples from 12 herds in Paraiba State, Brazil. This is the first report of antibodies to bluetongue virus in Paraiba State herds.

  9. Viral emergence and consequences for reproductive performance in ruminants: two recent examples (bluetongue and Schmallenberg viruses).

    Science.gov (United States)

    Zientara, Stéphan; Ponsart, Claire

    2014-12-01

    Viruses can emerge unexpectedly in different regions of the world and may have negative effects on reproductive performance. This paper describes the consequences for reproductive performance that have been reported after the introduction to Europe of two emerging viruses, namely the bluetongue (BTV) and Schmallenberg (SBV) viruses. Following the extensive spread of BTV in northern Europe, large numbers of pregnant cows were infected with BTV serotype 8 (BTV-8) during the breeding season of 2007. Initial reports of some cases of abortion and hydranencephaly in cattle in late 2007 were followed by quite exhaustive investigations in the field that showed that 10%-35% of healthy calves were infected with BTV-8 before birth. Transplacental transmission and fetal abnormalities in cattle and sheep had been previously observed only with strains of the virus that were propagated in embryonated eggs and/or cell culture, such as vaccine strains or vaccine candidate strains. After the unexpected emergence of BTV-8 in northern Europe in 2006, another arbovirus, namely SBV, emerged in Europe in 2011, causing a new economically important disease in ruminants. This new virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the UK, France, Italy, Luxembourg, Spain, Denmark and Switzerland. Adult animals show no or only mild clinical symptoms, whereas infection during a critical period of gestation can lead to abortion, stillbirth or the birth of severely malformed offspring. The impact of the disease is usually greater in sheep than in cattle. The consequences of SBV infection in domestic ruminants and more precisely the secondary effects on off-springs will be described.

  10. Autophagy Activated by Bluetongue Virus Infection Plays a Positive Role in Its Replication

    Directory of Open Access Journals (Sweden)

    Shuang Lv

    2015-08-01

    Full Text Available Bluetongue virus (BTV is an important pathogen of wild and domestic ruminants. Despite extensive study in recent decades, the interplay between BTV and host cells is not clearly understood. Autophagy as a cellular adaptive response plays a part in many viral infections. In our study, we found that BTV1 infection triggers the complete autophagic process in host cells, as demonstrated by the appearance of obvious double-membrane autophagosome-like vesicles, GFP-LC3 dots accumulation, the conversion of LC3-I to LC3-II and increased levels of autophagic flux in BSR cells (baby hamster kidney cell clones and primary lamb lingual epithelial cells upon BTV1 infection. Moreover, the results of a UV-inactivated BTV1 infection assay suggested that the induction of autophagy was dependent on BTV1 replication. Therefore, we investigated the role of autophagy in BTV1 replication. The inhibition of autophagy by pharmacological inhibitors (3-MA, CQ and RNA interference (siBeclin1 significantly decreased viral protein synthesis and virus yields. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted viral protein expression and the production of infectious BTV1. These findings lead us to conclude that autophagy is activated by BTV1 and contributes to its replication, and provide novel insights into BTV-host interactions.

  11. Autophagy Activated by Bluetongue Virus Infection Plays a Positive Role in Its Replication.

    Science.gov (United States)

    Lv, Shuang; Xu, Qingyuan; Sun, Encheng; Yang, Tao; Li, Junping; Feng, Yufei; Zhang, Qin; Wang, Haixiu; Zhang, Jikai; Wu, Donglai

    2015-08-01

    Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. Despite extensive study in recent decades, the interplay between BTV and host cells is not clearly understood. Autophagy as a cellular adaptive response plays a part in many viral infections. In our study, we found that BTV1 infection triggers the complete autophagic process in host cells, as demonstrated by the appearance of obvious double-membrane autophagosome-like vesicles, GFP-LC3 dots accumulation, the conversion of LC3-I to LC3-II and increased levels of autophagic flux in BSR cells (baby hamster kidney cell clones) and primary lamb lingual epithelial cells upon BTV1 infection. Moreover, the results of a UV-inactivated BTV1 infection assay suggested that the induction of autophagy was dependent on BTV1 replication. Therefore, we investigated the role of autophagy in BTV1 replication. The inhibition of autophagy by pharmacological inhibitors (3-MA, CQ) and RNA interference (siBeclin1) significantly decreased viral protein synthesis and virus yields. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted viral protein expression and the production of infectious BTV1. These findings lead us to conclude that autophagy is activated by BTV1 and contributes to its replication, and provide novel insights into BTV-host interactions.

  12. Activation of TLR3/interferon signaling pathway by bluetongue virus results in HIV inhibition in macrophages.

    Science.gov (United States)

    Dai, Ming; Wang, Xu; Li, Jie-Liang; Zhou, Yu; Sang, Ming; Liu, Jin-Biao; Wu, Jian-Guo; Ho, Wen-Zhe

    2015-12-01

    Bluetongue virus (BTV), a nonenveloped double-stranded RNA virus, is a potent inducer of type Ι interferons in multiple cell systems. In this study, we report that BTV16 treatment of primary human macrophages induced both type I and III IFN expression, resulting in the production of multiple antiviral factors, including myxovirus resistance protein A, 2',5'-oligoadenylate synthetase, and the IFN-stimulated gene 56. Additionally, BTV-treated macrophages expressed increased HIV restriction factors (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3 G/F/H) and CC chemokines (macrophage inflammatory protein 1-α, macrophage inflammatory protein 1-β, regulated on activation of normal T cell expressed and secreted), the ligands for HIV entry coreceptor CC chemokine receptor type 5. BTV16 also induced the expression of tetherin, which restricts HIV release from infected cells. Furthermore, TLR3 signaling of macrophages by BTV16 resulted in the induction of several anti-HIV microRNAs (miRNA-28, -29a, -125b, -150, -223, and -382). More importantly, the induction of antiviral responses by BTV resulted in significant suppression of HIV in macrophages. These findings demonstrate the potential of BTV-mediated TLR3 activation in macrophage innate immunity against HIV.

  13. Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus.

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    Eva Calvo-Pinilla

    Full Text Available Bluetongue virus (BTV belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR((-/- mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8(+ T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV.

  14. Development of a novel protein chip for the detection of bluetongue virus in China.

    Science.gov (United States)

    Xu, Q Y; Sun, E C; Feng, Y F; Li, J P; Lv, S; Zhang, Q; Wang, H X; Zhang, J K; Wu, D L

    2016-08-01

    Bluetongue (BT), which is caused by the BT virus (BTV), is an important disease in ruminants that leads to significant economic losses in the husbandry industry. To detect BTV-specific antibodies in serum, a protein chip detection method based on a novel solid supporting material known as polymer-coated initiator-integrated poly (dimethyl siloxane) (iPDMS) was developed. With a threshold of 25% (signal-to-noise percentage), the sensitivity and specificity of the protein chip were 98.6% and 94.8%, respectively. Furthermore, spot serum samples obtained from six provinces of China were tested with the protein chip and a commercially available BTV enzyme-linked immunosorbent assay (ELISA) kit (IDEXX). Of 615 samples, BTV-specific antibodies were detected in 200 (32.52%) by the protein chip and in 176 (28.62%) by the IDEXX BTV ELISA kit. Comparison of the protein chip with the commercial IDEXX BTV ELISA kit yielded the following spot serum detection results: a total coincidence, a negative coincidence and a positive coincidence of 95.12%, 99.28% and 86.5%, respectively. With the protein chip, the BTV-specific serum antibody was detected in samples from all six provinces, and the positive rates ranged from 4.12 to 74.4%. These results indicate that this protein chip detection method based on iPDMS is useful for the serological diagnosis of BTV infection and for epidemiological investigation.

  15. Prevalence of Bluetongue virus serotype 4 in cattle in the State of Sao Paulo, Brazil.

    Science.gov (United States)

    Hellmeister de Campos Nogueira, Adriana; De Stefano, Eliana; de Souza Nunes Martins, Maira; Okuda, Liria Hiromi; Dos Santos Lima, Michele; da Silva Garcia, Thais; Heinz Hellwig, Otto; Alves de Lima, José Eduardo; Savini, Giovanni; Pituco, Edviges Maristela

    2016-09-30

    Bluetongue (BT) is considered endemic in several regions of Brazil. The State of Sao Paulo was divided into 7 cattle production regions (circuits) according the different systems of breeding, operational and logistical capacity of the state veterinary service. At least 1 animal from each property (a total of 1,716 farms) was tested by competitive ELISA for the presence of antibodies against BTV. Sero‑positive sera were subsequently also tested by virus neutralization tests (VNT) using serial dilutions from 1:10 (cutoff) up to 1:640 (in MEM). BTV‑4 neutralizing antibodies were detected in 86% (1,483/1,716) of the animals tested. These results show that BTV‑4 is endemic and widespread in the State of San Paulo and indirectly confirm that in the State there are favourable conditions for the multiplication of competent vectors. However, as no clinical signs have ever been reported in cattle in the region, BTV‑4 infection is likely to occur silently in the State of Sao Paulo.

  16. Establishment of a bluetongue virus infection model in mice that are deficient in the alpha/beta interferon receptor.

    Directory of Open Access Journals (Sweden)

    Eva Calvo-Pinilla

    Full Text Available Bluetongue (BT is a noncontagious, insect-transmitted disease of ruminants caused by the bluetongue virus (BTV. A laboratory animal model would greatly facilitate the studies of pathogenesis, immune response and vaccination against BTV. Herein, we show that adult mice deficient in type I IFN receptor (IFNAR((-/- are highly susceptible to BTV-4 and BTV-8 infection when the virus is administered intravenously. Disease was characterized by ocular discharges and apathy, starting at 48 hours post-infection and quickly leading to animal death within 60 hours of inoculation. Infectious virus was recovered from the spleen, lung, thymus, and lymph nodes indicating a systemic infection. In addition, a lymphoid depletion in spleen, and severe pneumonia were observed in the infected mice. Furthermore, IFNAR((-/- adult mice immunized with a BTV-4 inactivated vaccine showed the induction of neutralizing antibodies against BTV-4 and complete protection against challenge with a lethal dose of this virus. The data indicate that this mouse model may facilitate the study of BTV pathogenesis, and the development of new effective vaccines for BTV.

  17. European bluetongue serotype 8

    NARCIS (Netherlands)

    Drolet, Barbara S.; Reister-Hendricks, Lindsey M.; Podell, Brendan K.; Breitenbach, Jonathan E.; Mcvey, D.S.; Rijn, van Piet A.; Bowen, Richard A.

    2016-01-01

    Bluetongue virus (BTV) is an orbivirus transmitted by biting midges (Culicoides spp.) that can result in moderate to high morbidity and mortality primarily in sheep and white-tailed deer. Although only 5 serotypes of BTV are considered endemic to the United States, as many as 11 incursive serotyp

  18. A Serological Investigation of Pestivirus, Bluetongue Virus and Peste Des Petits Ruminants Virus in Small Ruminants in Samsun Province of Turkey

    OpenAIRE

    Albayrak, Harun; ÖZAN, Emre; BEYHAN, Yunus Emre; KURT, Mitat; KILIÇOĞLU, Yunus

    2013-01-01

    Abstract: Bluetongue virus (BTV) is a vector-borne disease of ruminants disseminated in the tropic and sub-tropic zone of the world. All pestiviruses are important veterinary pathogens causing economic losses in cattle, sheep and pigs. Peste des petits ruminants (PPR) is a contagious viral disease of the domestic and wild ruminants. In this study, blood samples randomly collected from 144 sheep and 50 goats were analysed for the presence of antibodies to pestiviruses, BTV and PPRV using an en...

  19. Seroprevalence and S7 gene characterization of bluetongue virus in the West of Iran

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    Mohammad Khezri

    Full Text Available Aim: The objective of this study was conducted to determine the seroprevalence and S7 gene characterization of BTV of sheep in the West of Iran, during 2007-2008. Materials and Methods: A total 372 sheep blood samples were collected from known seropositive regions in the West of Iran. Anti-BTV antibodies were detected in the serum samples by group specific, c-ELISA. Extractions of the dsRNA from whole blood samples were carried out. The One-step RT-PCR kit was used for the detection of S7 BTV gene in the blood samples. PCR products of the first amplification (RT-PCR were used; template in the nested PCR. Products were separated by 1.2% Agarose gel electrophoresis. Nested PCR products of S7 segment from positive samples and the reference strain; BTV1 (RSA vvvv/01 were prepared for sequencing. All sequences were subjected to multiple sequence alignments and phylogenetic analysis. Results: The results showed widespread presence of the anti-BTV antibodies in the province's sheep population, where 46.77% of the tested sera were positive on ELISA. Bluetongue viruses were diagnosed in some animals by RT-PCR and nested PCR, by targeting S7 segment. This genome segment was sequenced and analyzed in four samples as a conserved gene in BTV serogroup. This group was very similar to the West BTV strains from US, Africa and Europe. This clustered was categorized with BTV4 from Turkey. Conclusion: Increases in epidemic disease may constitute a serious problem for Iran's rural economy in future, and the situation is likely to worsen in the next few years as the proportion of unvaccinated livestock increases. [Vet World 2012; 5(9.000: 549-555

  20. Prediction of Bluetongue virus seropositivity on pastoral properties in northern Australia using remotely sensed bioclimatic variables.

    Science.gov (United States)

    Klingseisen, Bernhard; Stevenson, Mark; Corner, Robert

    2013-06-01

    To monitor Bluetongue virus (BTV) activity in northern and eastern Australia the National Arbovirus Monitoring Program (NAMP) collects data from a network of sentinel herds. Groups of young cattle, previously unexposed to infection, are regularly tested to detect evidence of seroconversion. While this approach has been successful in fulfilling international surveillance requirements, it is labour and cost intensive and operationally challenging in the remote area of the northern Australian rangelands. The aim of this study was to assess the suitability of remotely sensed data as a means for predicting the distribution of BTV seroprevalence. For the period 2000-2009, bioclimatic variables were derived from the Moderate Resolution Imaging Spectroradiometer (MODIS) and Tropical Rainfall Measuring Mission (TRMM) data products for the entire Northern Territory. A generalised linear model, based on the seasonal Normalised Difference Vegetation Index (NDVI) and minimum land surface temperature, was developed to predict BTV seropositivity. The odds of seropositivity in locations with NDVI estimates >0.45 was 3.90 (95% CI 1.11 to 13.7) times that of locations where NDVI estimates were between 0 and 0.45. Unit increases in minimum night land surface temperature in the previous winter increased the odds of seropositivity by a factor of 1.40 (95% CI 1.02 to 1.91). The area under a Receiver Operator Characteristic curve generated on the basis of the model predictions was 0.8. Uncertainty in the model's predictions was attributed to the spatio-temporal inconsistency in the precision of the available serosurveillance data. The discriminatory ability of models of this type could be improved by ensuring that exact location details and date of NAMP BTV test events are consistently recorded.

  1. Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

    DEFF Research Database (Denmark)

    Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J

    2010-01-01

    A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward mu...

  2. Epidemiologic characteristics of bluetongue virus serotype 8 laboratory-confirmed outbreaks in The Netherlands in 2007 and a comparison with the situation in 2006

    NARCIS (Netherlands)

    Elbers, A.R.W.; Spek, van der A.N.; Rijn, van P.A.

    2009-01-01

    A major epidemic of bluetongue virus serotype 8 (BTV-8) occurred in Western Europe in 2006. During 2007 it became evident that BTV-8 had survived the winter and a re-emerging epidemic quickly developed. The objective of this study was to describe the severity and clinical impact of the BTV-8 epidemi

  3. DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1.

    Science.gov (United States)

    Li, Junping; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Feng, Yufei; Lv, Shuang; Zhang, Qin; Wang, Haixiu; Wu, Donglai

    2015-10-01

    Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

  4. Simulating spread of Bluetongue Virus by flying vectors between hosts on pasture

    DEFF Research Database (Denmark)

    Græsbøll, Kaare; Bødker, Rene; Enøe, Claes;

    2012-01-01

    Bluetongue is a disease of ruminants which reached Denmark in 2007. We present a process-based stochastic simulation model of vector-borne diseases, where host animals are not confined to a central geographic farm coordinate, but can be distributed onto pasture areas. Furthermore vectors fly freely...

  5. How does increasing immunity change spread kernel parameters in subsequent outbreaks? – A simulation study on Bluetongue Virus

    DEFF Research Database (Denmark)

    Græsbøll, Kaare; Bødker, Rene; Enøe, Claes;

    Modelling the spatial spread of vector borne diseases, one may choose methods ranging from statistic to process oriented. One often used statistic tool is the empirical spread kernel. An empiric spread kernel fitted to outbreak data provides hints on the spread mechanisms, and may provide a good...... of such changes are: vaccinations, acquired immunity, vector density and control, meteorological variations, wind pattern, and so on. Including more and more variables leads to a more process oriented model. A full process oriented approach simulates the movement of virus between vectors and host, describing...... detailed simulation spread model. And by using empirical spread kernels from past outbreaks we have fitted some of the more uncertain parameters for this case study. A stochastic simulation model was developed for the spread of bluetongue virus. In the model hosts (cattle) and vectors (Culicoides...

  6. The role of wildlife in bluetongue virus maintenance in Europe: lessons learned after the natural infection in Spain.

    Science.gov (United States)

    Ruiz-Fons, Francisco; Sánchez-Matamoros, Almudena; Gortázar, Christian; Sánchez-Vizcaíno, José Manuel

    2014-03-01

    Bluetongue (BT) is a re-emergent vector-borne viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), a member of the genus Orbivirus. A complex multi-host, multi-vector and multi-pathogen (26 serotypes) transmission and maintenance network has recently emerged in Europe, and wild ruminants are regarded as an important node in this network. This review analyses the reservoir role of wild ruminants in Europe, identifying gaps in knowledge and proposing actions. Wild ruminant species are indicators of BTV circulation. Excepting the mouflon (Ovis aries musimon), European wild ungulates do not develop clinical disease. Diagnostic techniques used in wildlife do not differ from those used in domestic ruminants provided they are validated. Demographic, behavioural and physiological traits of wild hosts modulate their relationship with BTV vectors and with the virus itself. While BTV has been eradicated from central and northern Europe, it is still circulating in the Mediterranean Basin. We propose that currently two BTV cycles coexist in certain regions of the Mediterranean Basin, a wild one largely driven by deer of the subfamily Cervinae and a domestic one. These are probably linked through shared Culicoides vectors of several species. We suggest that wildlife might be contributing to this situation through vector maintenance and virus maintenance. Additionally, differences in temperature and other environmental factors add complexity to the Mediterranean habitats as compared to central and northern European ones. Intervention options in wildlife populations are limited. There is a need to know the role of wildlife in maintaining Culicoides populations, and to know which Culicoides species mediate the wildlife-livestock-BTV transmission events. There is also a clear need to study more in depth the links between Cervinae deer densities, environmental factors and BTV maintenance. Regarding disease control, we suggest that research efforts should be

  7. Does the Bluetongue virus circulates in cattle population of Mat district, Albania?

    Directory of Open Access Journals (Sweden)

    KLODIAN DEDOLLI

    2014-06-01

    Full Text Available Bluetongue is a viral, infectious, non-contiguous, vector transmitted disease of ruminants animals, caused by an Orbivurus. Despite the disease is not zoonoses, it is with high economic importance and as other OIE listed disease, significantly interfere with animal health and trade. Clinically, most affected species are sheep, however cattle serve as reservoir of infection and play major role on epidemiology of disease. Presence of Blue tongue disease proved only when it is based on laboratory tests.

  8. PCR identification of culicoid biting midges (Diptera, Ceratopogonidae of the Obsoletus complex including putative vectors of bluetongue and Schmallenberg viruses

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    Lehmann Kathrin

    2012-09-01

    Full Text Available Abstract Background Biting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV in northern and central Europe during the 2006 outbreak of bluetongue disease (BT. Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus plus the isomorphic species C. dewulfi. Methods Biting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer. Results The newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic

  9. Host-seeking activity of bluetongue virus vectors: endo/exophagy and circadian rhythm of Culicoides in Western Europe.

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    Elvina Viennet

    Full Text Available Feeding success of free-living hematophagous insects depends on their ability to be active when hosts are available and to reach places where hosts are accessible. When the hematophagous insect is a vector of pathogens, determining the components of host-seeking behavior is of primary interest for the assessment of transmission risk. Our aim was to describe endo/exophagy and circadian host-seeking activity of Palaearctic Culicoides species, which are major biting pests and arbovirus vectors, using drop traps and suction traps baited with four sheep, as bluetongue virus hosts. Collections were carried out in the field, a largely-open stable and an enclosed stable during six collection periods of 24 hours in April/May, in late June and in September/October 2010 in western France. A total of 986 Culicoides belonging to 13 species, mainly C. brunnicans and C. obsoletus, was collected on animal baits. Culicoides brunnicans was clearly exophagic, whereas C. obsoletus was able to enter stables. Culicoides brunnicans exhibited a bimodal pattern of host-seeking activity with peaks just after sunrise and sunset. Culicoides obsoletus was active before sunset in spring and autumn and after sunset in summer, thus illustrating influence of other parameters than light, especially temperature. Description of host-seeking behaviors allowed us to discuss control strategies for transmission of Culicoides-borne pathogens, such as bluetongue virus. However, practical vector-control recommendations are difficult to provide because of the variation in the degree of endophagy and time of host-seeking activity.

  10. Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

    Energy Technology Data Exchange (ETDEWEB)

    Matsuo, Eiko [Microbiology and Immunology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe-City 657-8501 (Japan); Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom); Leon, Esther; Matthews, Steve J. [Division of Molecular Biosciences, Centre for Structural Biology, Imperial College London, South Kensington, London SW7 2AZ (United Kingdom); Roy, Polly, E-mail: polly.roy@lshtm.ac.uk [Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT (United Kingdom)

    2014-09-05

    Highlights: • NMR analysis on BTV VP6 reveals two large loop regions. • The loss of a loop (aa 34–130) does not affect the overall fold of the protein. • A region of VP6 (aa 34–92) is not required for BTV replication. • A region of VP6 (aa 93–130) plays an essential role in the virus replication. - Abstract: Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.

  11. Longitudinal study of the detection of Bluetongue virus in bull semen and comparison of real-time polymerase chain reaction assays.

    Science.gov (United States)

    Gu, Xingnian; Davis, Rodney J; Walsh, Susan J; Melville, Lorna F; Kirkland, Peter D

    2014-01-01

    Infection with Bluetongue virus (BTV) is a significant impediment to the global movement of bovine semen. Repeat testing of blood from donor animals is specified in the World Organization for Animal Health (OIE) Manual for the export of semen from regions where BTV may be present. Screening of blood or semen samples has usually been carried out by virus isolation (VI) either by inoculation of chicken embryos followed by passage onto insect and mammalian cell cultures or in vivo inoculation of sheep followed by serology to detect seroconversion. Direct testing of semen for BTV would enable earlier release of semen samples and avoid repeat testing of the donor, as well as provide an option for releasing batches of semen that were collected without certification of the donor. Quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) assays overcome most of the limitations of other methods and have the potential to provide higher sensitivity. The present study compared 5 qRT-PCR assays, including 2 commercially available kits, for the detection of BTV in semen serially collected from 8 bulls over a period of 90 days after experimental infection. The results of the study show that at least one of the qRT-PCR assays is extremely reproducible and has both very high sensitivity and specificity to reliably detect all available serotypes. The preferred qRT-PCR gave consistently superior results to VI, sheep inoculation, and conventional RT-PCR. Therefore, the assay can be recommended for the screening of bovine semen for freedom from BTV.

  12. Why did bluetongue spread the way it did? Environmental factors influencing the velocity of bluetongue virus serotype 8 epizootic wave in France.

    Science.gov (United States)

    Pioz, Maryline; Guis, Hélène; Crespin, Laurent; Gay, Emilie; Calavas, Didier; Durand, Benoît; Abrial, David; Ducrot, Christian

    2012-01-01

    Understanding where and how fast an infectious disease will spread during an epidemic is critical for its control. However, the task is a challenging one as numerous factors may interact and drive the spread of a disease, specifically when vector-borne diseases are involved. We advocate the use of simultaneous autoregressive models to identify environmental features that significantly impact the velocity of disease spread. We illustrate this approach by exploring several environmental factors influencing the velocity of bluetongue (BT) spread in France during the 2007-2008 epizootic wave to determine which ones were the most important drivers. We used velocities of BT spread estimated in 4,495 municipalities and tested sixteen covariates defining five thematic groups of related variables: elevation, meteorological-related variables, landscape-related variables, host availability, and vaccination. We found that ecological factors associated with vector abundance and activity (elevation and meteorological-related variables), as well as with host availability, were important drivers of the spread of the disease. Specifically, the disease spread more slowly in areas with high elevation and when heavy rainfall associated with extreme temperature events occurred one or two months prior to the first clinical case. Moreover, the density of dairy cattle was correlated negatively with the velocity of BT spread. These findings add substantially to our understanding of BT spread in a temperate climate. Finally, the approach presented in this paper can be used with other infectious diseases, and provides a powerful tool to identify environmental features driving the velocity of disease spread.

  13. Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

    Science.gov (United States)

    Kochinger, Stefanie; Renevey, Nathalie; Hofmann, Martin A; Zimmer, Gert

    2014-06-13

    Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

  14. Why did bluetongue spread the way it did? Environmental factors influencing the velocity of bluetongue virus serotype 8 epizootic wave in France.

    Directory of Open Access Journals (Sweden)

    Maryline Pioz

    Full Text Available Understanding where and how fast an infectious disease will spread during an epidemic is critical for its control. However, the task is a challenging one as numerous factors may interact and drive the spread of a disease, specifically when vector-borne diseases are involved. We advocate the use of simultaneous autoregressive models to identify environmental features that significantly impact the velocity of disease spread. We illustrate this approach by exploring several environmental factors influencing the velocity of bluetongue (BT spread in France during the 2007-2008 epizootic wave to determine which ones were the most important drivers. We used velocities of BT spread estimated in 4,495 municipalities and tested sixteen covariates defining five thematic groups of related variables: elevation, meteorological-related variables, landscape-related variables, host availability, and vaccination. We found that ecological factors associated with vector abundance and activity (elevation and meteorological-related variables, as well as with host availability, were important drivers of the spread of the disease. Specifically, the disease spread more slowly in areas with high elevation and when heavy rainfall associated with extreme temperature events occurred one or two months prior to the first clinical case. Moreover, the density of dairy cattle was correlated negatively with the velocity of BT spread. These findings add substantially to our understanding of BT spread in a temperate climate. Finally, the approach presented in this paper can be used with other infectious diseases, and provides a powerful tool to identify environmental features driving the velocity of disease spread.

  15. Why Did Bluetongue Spread the Way It Did? Environmental Factors Influencing the Velocity of Bluetongue Virus Serotype 8 Epizootic Wave in France

    Science.gov (United States)

    Pioz, Maryline; Guis, Hélène; Crespin, Laurent; Gay, Emilie; Calavas, Didier; Durand, Benoît; Abrial, David; Ducrot, Christian

    2012-01-01

    Understanding where and how fast an infectious disease will spread during an epidemic is critical for its control. However, the task is a challenging one as numerous factors may interact and drive the spread of a disease, specifically when vector-borne diseases are involved. We advocate the use of simultaneous autoregressive models to identify environmental features that significantly impact the velocity of disease spread. We illustrate this approach by exploring several environmental factors influencing the velocity of bluetongue (BT) spread in France during the 2007–2008 epizootic wave to determine which ones were the most important drivers. We used velocities of BT spread estimated in 4,495 municipalities and tested sixteen covariates defining five thematic groups of related variables: elevation, meteorological-related variables, landscape-related variables, host availability, and vaccination. We found that ecological factors associated with vector abundance and activity (elevation and meteorological-related variables), as well as with host availability, were important drivers of the spread of the disease. Specifically, the disease spread more slowly in areas with high elevation and when heavy rainfall associated with extreme temperature events occurred one or two months prior to the first clinical case. Moreover, the density of dairy cattle was correlated negatively with the velocity of BT spread. These findings add substantially to our understanding of BT spread in a temperate climate. Finally, the approach presented in this paper can be used with other infectious diseases, and provides a powerful tool to identify environmental features driving the velocity of disease spread. PMID:22916249

  16. Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

    Science.gov (United States)

    Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí

    2015-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062

  17. Occurrence of Bluetongue in ruminants in Tamil Nadu, South India.

    Science.gov (United States)

    Reddy, Y Krishnamohan; Brindha, K; Ganesan, P I; Srinivas, K; Reddy, G S; Minakshi, P

    2016-09-30

    Tamil Nadu is located in the South-Eastern part of Indian peninsula, between 8.087° and 13.09°N and 76.50° and 80.27°E. Bluetongue (BT) was first reported in this region in sheep during 1982 with regular occurrence thereafter. In 1989-1990, 1997-1998 and 2005-2006, there was wide spread occurrence of BT resulting in huge mortality of sheep. The present study had the goal of isolating the BTV from outbreaks in sheep occurred in Tamil Naadu between 2003-2011 and comparing the VP2 gene sequences of the BTV isolates involved in such outbreaks. Serotypes 1, 2, 16, and 23 of the Bluetongue virus (BTV) have been isolated from sheep during BT outbreaks. BTV-16 has also been isolated in goats and cattle in the region; BTV-2 isolated in Tamil Nadu has homology with BTV-2 isolated in Africa; whereas the BTV-23 isolated in this area has homology with BTV-23 from South East Asia, indicating that both Eastern and Western topotypes of BTV are circulating in ruminant population in Tamil Nadu.

  18. Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows.

    Science.gov (United States)

    Kramps, Johannes A; van Maanen, Kees; Mars, Maria H; Popma, Johan K; van Rijn, Piet A

    2008-07-27

    A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.

  19. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    Science.gov (United States)

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

  20. Viruses isolated from Panamanian sloths.

    Science.gov (United States)

    Seymour, C; Peralta, P H; Montgomery, G G

    1983-11-01

    Seven virus strains were isolated in Vero cells from whole blood samples from 80 wild-caught sloths, Bradypus variegatus and Choloepus hoffmanni, from Central Panamá. Four strains of at least two different serotypes are related to Changuinola virus; two of these were associated with prolonged or recrudescent viremias. One strain is an antigenic subtype of Punta Toro virus, and another, described here as Bradypus-4 virus, is a new, antigenically ungrouped virus. A second new virus from sloths, Utive virus, forms an antigenic complex within the Simbu serogroup with Utinga and Pintupo viruses. Tests on sequential plasma samples from radio-marked free-ranging sloths and from recently captured animals maintained in captivity showed that both species develop neutralizing antibodies following naturally acquired virus infections. Antibodies against the Changuinola and Simbu serogroup viruses are widespread in both sloth species and are especially prevalent in Choloepus, but are virtually absent in all other wild vertebrate species tested.

  1. Bluetongue virus detection by real-time RT-PCR in Culicoides captured during the 2006 epizootic in Belgium and development of an internal control.

    Science.gov (United States)

    Vanbinst, T; Vandenbussche, F; Vandemeulebroucke, E; De Leeuw, I; Deblauwe, I; De Deken, G; Madder, M; Haubruge, E; Losson, B; De Clercq, K

    2009-06-01

    After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

  2. Purification, stability, and immunogenicity analyses of five bluetongue virus proteins for use in development of a subunit vaccine that allows differentiation of infected from vaccinated animals.

    Science.gov (United States)

    Anderson, Jenna; Bréard, Emmanuel; Lövgren Bengtsson, Karin; Grönvik, Kjell-Olov; Zientara, Stéphan; Valarcher, Jean-Francois; Hägglund, Sara

    2014-03-01

    Bluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus or Escherichia coli expression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or -80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n = 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.

  3. Application of syndromic surveillance on routinely collected cattle reproduction and milk production data for the early detection of outbreaks of Bluetongue and Schmallenberg viruses.

    Science.gov (United States)

    Veldhuis, Anouk; Brouwer-Middelesch, Henriëtte; Marceau, Alexis; Madouasse, Aurélien; Van der Stede, Yves; Fourichon, Christine; Welby, Sarah; Wever, Paul; van Schaik, Gerdien

    2016-02-01

    This study aimed to evaluate the use of routinely collected reproductive and milk production data for the early detection of emerging vector-borne diseases in cattle in the Netherlands and the Flanders region of Belgium (i.e., the northern part of Belgium). Prospective space-time cluster analyses on residuals from a model on milk production were carried out to detect clusters of reduced milk yield. A CUSUM algorithm was used to detect temporal aberrations in model residuals of reproductive performance models on two indicators of gestation length. The Bluetongue serotype-8 (BTV-8) epidemics of 2006 and 2007 and the Schmallenberg virus (SBV) epidemic of 2011 were used as case studies to evaluate the sensitivity and timeliness of these methods. The methods investigated in this study did not result in a more timely detection of BTV-8 and SBV in the Netherlands and BTV-8 in Belgium given the surveillance systems in place when these viruses emerged. This could be due to (i) the large geographical units used in the analyses (country, region and province level), and (ii) the high level of sensitivity of the surveillance systems in place when these viruses emerged. Nevertheless, it might be worthwhile to use a syndromic surveillance system based on non-specific animal health data in real-time alongside regular surveillance, to increase the sense of urgency and to provide valuable quantitative information for decision makers in the initial phase of an emerging disease outbreak.

  4. Seroepidemiology of bluetongue disease in small ruminants of north-east of Iran

    Directory of Open Access Journals (Sweden)

    Vahid Najarnezhad

    2013-06-01

    Conclusions: The results showed that the majority of animals in the north-east of Iran are infected with bluetongue virus. High correlation between abortion history and seroposivity emphasize the economical importance of bluetongue virus in the sheep herds of the region.

  5. Inter-laboratory evaluation of the performance parameters of a Lateral Flow Test device for the detection of Bluetongue virus-specific antibodies.

    Science.gov (United States)

    Hanon, Jean-Baptiste; Vandenberge, Valerie; Deruelle, Matthias; De Leeuw, Ilse; De Clercq, Kris; Van Borm, Steven; Koenen, Frank; Liu, Lihong; Hoffmann, Bernd; Batten, Carrie Anne; Zientara, Stéphan; Breard, Emmanuel; Van der Stede, Yves

    2016-02-01

    Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities.

  6. Bluetongue: control, surveillance and safe movement of animals

    DEFF Research Database (Denmark)

    Nielsen, Søren Saxmose

    2017-01-01

    The performance of different bluetongue control measures related to both vaccination and protection from bluetongue virus (BTV) vectors was assessed. By means of a mathematical model, it was concluded that when vaccination is applied on 95% of animals even for 3 years, bluetongue cannot...... it was highlighted that under the current surveillance policy bluetongue circulation might occur undetected. For the safe movement of animals, newborn ruminants from vaccinated mothers with neutralising antibodies can be considered protected against infection, although a protective titre threshold cannot...... be identified. The presence of colostral antibodies interferes with the vaccine immunisation in the newborn for more than 3 months after birth, whereas the minimum time after vaccination of animal to be considered immune can be up to 48 days. The knowledge about vectors ecology, mechanisms of over...

  7. Questionnaire survey about the motives of commercial livestock farmers and hobby holders to vaccinate their animals against Bluetongue virus serotype 8 in 2008-2009 in the Netherlands.

    Science.gov (United States)

    Elbers, A R W; de Koeijer, A A; Scolamacchia, F; van Rijn, P A

    2010-03-16

    After a massive epidemic of Bluetongue virus serotype 8 (BTV-8) among ruminants in 2006-2007 in the European Union (EU), the Netherlands started a voluntary emergency vaccination campaign in May 2008, subsidized by the EU. At the start of a new campaign in 2009, without subsidized vaccination, we investigated by mail survey the motives of farmers and hobby holders to vaccinate against BTV-8 in 2008 and 2009. Mean vaccine uptake in 2008 was: 73% in sheep, 71% in cattle, 43% in goat farms and 67% in hobby holdings. Top-5 motives pro-vaccination were: prevention of production loss; subsidized vaccination; recommendation by practitioner; welfare reasons; contribution to the eradication campaign. Top-5 motives against vaccination were: vaccination costs; absence of clinical BT-problems; presumed low infection risk; balance between vaccination costs and loss without vaccination; bad experience with earlier vaccination campaigns. Willingness to vaccinate was significantly lower in 2009: 42% in sheep, 58% in cattle, 19% in goat farms and 49% in hobby holdings. Measures to stimulate vaccination among those that did not want to vaccinate in 2009 were: subsidized vaccination; possibility to vaccinate their own animals; more information on efficacy/safety of vaccine and why animals had to be vaccinated again; availability of a BT vaccine combined with vaccine(s) against other diseases.

  8. Comparative efficacy of standard AGID, CCIE and competitive ELISA for detecting bluetongue virus antibodies in indigenous breeds of sheep and goats in Rajasthan, India.

    Science.gov (United States)

    Shringi, Smriti; Shringi, B N

    2005-03-01

    The sero-prevalence of antibodies against blue tongue virus (BTV) in 408 local breeds of sheep in Rajasthan state in India was investigated using standard agar gel immunodiffusion (AGID) test. Maximum seropositivities of 11.3% (13/115), 10.7% (13/121), 7.1% (11/155) and 5.9% (1/17) were recorded in the Chokla, Magra, Nali and Pugal breeds, respectively. Out of 107 goat serum samples, 6 (5.6%) were AGID positive. The performance of the standard AGID, counter current immuno-electrophoresis (CCIE) and the competitive enzyme-linked immunosorbent assay (cELISA) for the detection of serum antibody against BTV in indigenous breeds of sheep were compared. Out of 178 sheep serum samples tested, 17 (9.5%), 22 (12.3%) and 54 (30.3%) were positive for group-specific bluetongue antibodies by AGID, CCIE and cELISA, respectively. There was appreciable difference in the seroprevalence detected by AGID, CCIE and cELISA in clinically healthy and diseased sheep with regard to relative sensitivities and specificities of the tests with cELISA being highly sensitive and specific followed by CCIE and AGID test. It was concluded that these indigenous breeds of sheep may be a potential reservoir of BTV infection and cELISA should be routinely used for the detection of antibodies against BTV in these local breeds of sheep.

  9. Coinfections of Sudanese dairy cattle with bovine herpes virus 1, bovine viral diarrhea virus, bluetongue virus and bovine herpes virus 4 and their relation to reproductive disorders

    Directory of Open Access Journals (Sweden)

    Amira M. Elhassan

    2016-12-01

    Reults: The meta-analysis of the data indicated high seroprevalence of coinfections with various combinations of these agents; only few animals were singly infected. An infection with BHV-1 was observed to be higher than the prevalence of associations between BHV-1 and the other three viral agents. Prevalence of seropositivities to coinfection with BHV-1/BTV; BHV-1/BVD; BHV-1/BTV/BVD were the highest while seropositivities prevalences that involved BHV-4 were much lower. The highest abortion rates were encountered in coinfections with BHV-1/BVD/BTV (31% and BHV-1/BVD/BTV/BHV-4 (30% while most infertility cases were noticed in coinfection with BHV-1/BVD/BTV (44% and BHV-1/BVD/BTV/BHV-4 (21%, and coinfections with the four viruses were encountered in most of the death after birth cases (25%. Overall mixed infections with BHV-1/BVD/BTV (34% and BHV-1/BVD/BTV/BHV-4 (22.5% were involved in the majority of reproductive problems studied. Conclusion: Mixed infections constitutes the vast majority of cases and are involved in the majority of reproductive disorders investigated. The high prevalence of seropositivity to all of the four viruses should call for an intervention strategy to reduce the impact of these viruses. [J Adv Vet Anim Res 2016; 3(4.000: 332-337

  10. Bluetongue virus serotype 1 outbreak in the Basque Country (Northern Spain 2007-2008. Data support a primary vector windborne transport.

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    Rodrigo García-Lastra

    Full Text Available BACKGROUND: Bluetongue (BT is a vector-borne disease of ruminants that has expanded its traditional global distribution in the last decade. Recently, BTV-1 emerged in Southern Spain and caused several outbreaks in livestock reaching the north of the country. The aim of this paper was to review the emergence of BTV-1 in the Basque Country (Northern Spain during 2007 and 2008 analyzing the possibility that infected Culicoides were introduced into Basque Country by winds from the infected areas of Southern Spain. METHODOLOGY/PRINCIPAL FINDINGS: We use a complex HYSPLIT (Hybrid Single-Particle Lagrangian Integrated Trajectory model to draw wind roses and backward wind trajectories. The analysis of winds showed September 28 to October 2 as the only period for the introduction of infected midges in the Basque Country. These wind trajectories crossed through the areas affected by serotype 1 on those dates in the South of the Iberian Peninsula. Additionally meteorological data, including wind speed and humidity, and altitude along the trajectories showed suitable conditions for Culicoides survival and dispersion. CONCLUSIONS/SIGNIFICANCE: An active infection in medium-long distance regions, wind with suitable speed, altitude and trajectory, and appropriate weather can lead to outbreaks of BTV-1 by transport of Culicoides imicola, not only over the sea (as reported previously but also over the land. This shows that an additional factor has to be taken into account for the control of the disease which is currently essentially based on the assumption that midges will only spread the virus in a series of short hops. Moreover, the epidemiological and serological data cannot rule out the involvement of other Culicoides species in the spread of the infection, especially at a local level.

  11. Expression of VP7, a Bluetongue virus group specific antigen by viral vectors: analysis of the induced immune responses and evaluation of protective potential in sheep.

    Directory of Open Access Journals (Sweden)

    Coraline Bouet-Cararo

    Full Text Available Bluetongue virus (BTV is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0 or a leporipoxvirus (SG33-VP7, to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

  12. Expression of VP7, a Bluetongue Virus Group Specific Antigen by Viral Vectors: Analysis of the Induced Immune Responses and Evaluation of Protective Potential in Sheep

    Science.gov (United States)

    Bouet-Cararo, Coraline; Contreras, Vanessa; Caruso, Agathe; Top, Sokunthea; Szelechowski, Marion; Bergeron, Corinne; Viarouge, Cyril; Desprat, Alexandra; Relmy, Anthony; Guibert, Jean-Michel; Dubois, Eric; Thiery, Richard; Bréard, Emmanuel; Bertagnoli, Stephane; Richardson, Jennifer; Foucras, Gilles; Meyer, Gilles; Schwartz-Cornil, Isabelle; Zientara, Stephan; Klonjkowski, Bernard

    2014-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity. PMID:25364822

  13. Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, J.L.; Langeveld, J.P.M.; Venteo, A.;

    1999-01-01

    African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological...... immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule...

  14. Possible routes of introduction of bluetongue serotype 8 virus into the epicentre of the 2006 epidemic in north-western Europe

    NARCIS (Netherlands)

    Mintiens, K.; Meroc, E.; Mellor, P.S.; Staubach, C.; Gerbier, G.; Elbers, A.R.W.; Hendrickx, G.; Clercq, K.

    2008-01-01

    In August 2006, bluetongue (BT) was notified in The Netherlands on several animal holdings. This was the onset of a rapidly spreading BT-epidemic in north-western Europe (latitude >51°N) that affected cattle and sheep holdings in The Netherlands, Belgium, Germany, France and Luxembourg. The outbr

  15. Did vaccination slow the spread of bluetongue in France?

    Directory of Open Access Journals (Sweden)

    Maryline Pioz

    Full Text Available Vaccination is one of the most efficient ways to control the spread of infectious diseases. Simulations are now widely used to assess how vaccination can limit disease spread as well as mitigate morbidity or mortality in susceptible populations. However, field studies investigating how much vaccines decrease the velocity of epizootic wave-fronts during outbreaks are rare. This study aimed at investigating the effect of vaccination on the propagation of bluetongue, a vector-borne disease of ruminants. We used data from the 2008 bluetongue virus serotype 1 (BTV-1 epizootic of southwest France. As the virus was newly introduced in this area, natural immunity of livestock was absent. This allowed determination of the role of vaccination in changing the velocity of bluetongue spread while accounting for environmental factors that possibly influenced it. The average estimated velocity across the country despite restriction on animal movements was 5.4 km/day, which is very similar to the velocity of spread of the bluetongue virus serotype 8 epizootic in France also estimated in a context of restrictions on animal movements. Vaccination significantly reduced the propagation velocity of BTV-1. In comparison to municipalities with no vaccine coverage, the velocity of BTV-1 spread decreased by 1.7 km/day in municipalities with immunized animals. For the first time, the effect of vaccination has been quantified using data from a real epizootic whilst accounting for environmental factors known to modify the velocity of bluetongue spread. Our findings emphasize the importance of vaccination in limiting disease spread across natural landscape. Finally, environmental factors, specifically those related to vector abundance and activity, were found to be good predictors of the velocity of BTV-1 spread, indicating that these variables need to be adequately accounted for when evaluating the role of vaccination on bluetongue spread.

  16. Did vaccination slow the spread of bluetongue in France?

    Science.gov (United States)

    Pioz, Maryline; Guis, Hélène; Pleydell, David; Gay, Emilie; Calavas, Didier; Durand, Benoît; Ducrot, Christian; Lancelot, Renaud

    2014-01-01

    Vaccination is one of the most efficient ways to control the spread of infectious diseases. Simulations are now widely used to assess how vaccination can limit disease spread as well as mitigate morbidity or mortality in susceptible populations. However, field studies investigating how much vaccines decrease the velocity of epizootic wave-fronts during outbreaks are rare. This study aimed at investigating the effect of vaccination on the propagation of bluetongue, a vector-borne disease of ruminants. We used data from the 2008 bluetongue virus serotype 1 (BTV-1) epizootic of southwest France. As the virus was newly introduced in this area, natural immunity of livestock was absent. This allowed determination of the role of vaccination in changing the velocity of bluetongue spread while accounting for environmental factors that possibly influenced it. The average estimated velocity across the country despite restriction on animal movements was 5.4 km/day, which is very similar to the velocity of spread of the bluetongue virus serotype 8 epizootic in France also estimated in a context of restrictions on animal movements. Vaccination significantly reduced the propagation velocity of BTV-1. In comparison to municipalities with no vaccine coverage, the velocity of BTV-1 spread decreased by 1.7 km/day in municipalities with immunized animals. For the first time, the effect of vaccination has been quantified using data from a real epizootic whilst accounting for environmental factors known to modify the velocity of bluetongue spread. Our findings emphasize the importance of vaccination in limiting disease spread across natural landscape. Finally, environmental factors, specifically those related to vector abundance and activity, were found to be good predictors of the velocity of BTV-1 spread, indicating that these variables need to be adequately accounted for when evaluating the role of vaccination on bluetongue spread.

  17. Decode Fish Lymphocystis Virus Isolated from China

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    @@ Aresearch group headed by Prof. Zhang Qiya from the CAS Institute of Hydrobiology (IHB) has succeeded in sequencing the complete genome of lymphocystis disease virus isolated from China (LCDV-C), a virus isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. Their work has been published in the recent issue of Journal of Virology (2004, 78 (13): 6982- 6994).

  18. Seroepidemiology of bluetongue disease in small ruminants of north-east of Iran

    Institute of Scientific and Technical Information of China (English)

    Vahid Najarnezhad; Mahin Rajae

    2013-01-01

    To estimate the prevalence and distribution of bluetongue virus antibody in sheep and goats in 25 townships of Khorasan Razavi. Bluetongue is an infectious, non-contagious, arthropod born viral disease of ruminants and has been reported from most of the tropical and subtropical regions of the world. Methods: A total number of 1 034 serum samples from sheep and goats were collected and transmitted to Serological Laboratory of Veterinary Council of Khorasan Razavi. Serums were screened for the presence of group-specific bluetongue virus antibody using competitive Enzyme Linked Immuno Sorbent Assay (c-ELISA). Results: The seropositivity of sheep and goats for bluetongue was found to be 89.2%. The highest prevalence rate was seen in Taybad, Khalil-abad and Torbat-jam (100%) and the least prevalence rate was seen in Jovein (55%). Conclusions: The results showed that the majority of animals in the north-east of Iran are infected with bluetongue virus. High correlation between abortion history and seroposivity emphasize the economical importance of bluetongue virus in the sheep herds of the region.

  19. Seroepidemiology of bluetongue disease in small ruminants of north-east of Iran

    OpenAIRE

    Vahid Najarnezhad; Mahin Rajae

    2013-01-01

    Objective: To estimate the prevalence and distribution of bluetongue virus antibody in sheep and goats in 25 townships of Khorasan Razavi. Bluetongue is an infectious, non-contagious, arthropod born viral disease of ruminants and has been reported from most of the tropical and subtropical regions of the world. Methods: A total number of 1 034 serum samples from sheep and goats were collected and transmitted to Serological Laboratory of Veterinary Council of Khorasan Razavi. Serums were scr...

  20. A Multicomponent Animal Virus Isolated from Mosquitoes.

    Science.gov (United States)

    Ladner, Jason T; Wiley, Michael R; Beitzel, Brett; Auguste, Albert J; Dupuis, Alan P; Lindquist, Michael E; Sibley, Samuel D; Kota, Krishna P; Fetterer, David; Eastwood, Gillian; Kimmel, David; Prieto, Karla; Guzman, Hilda; Aliota, Matthew T; Reyes, Daniel; Brueggemann, Ernst E; St John, Lena; Hyeroba, David; Lauck, Michael; Friedrich, Thomas C; O'Connor, David H; Gestole, Marie C; Cazares, Lisa H; Popov, Vsevolod L; Castro-Llanos, Fanny; Kochel, Tadeusz J; Kenny, Tara; White, Bailey; Ward, Michael D; Loaiza, Jose R; Goldberg, Tony L; Weaver, Scott C; Kramer, Laura D; Tesh, Robert B; Palacios, Gustavo

    2016-09-14

    RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

  1. [Isolation and purification of virus damaging sunflower].

    Science.gov (United States)

    Zakusilo, A O; Didenko, L F; Kniazieva, N A; Boĭko, A L

    1994-01-01

    A procedure has been developed for purifying intact virus's isolate particles evoking yellow spot mosaic disease in sunflower. Purification of pathogen in 0.1 M sodium phosphate buffer, pH 8.0 containing 0.05 M Na3SO3 and 0.2% 2-mercaptoethanol is used. After first clarification extract was exposed to two cycles of high-speed centrifugation and fractionated in linear 10-40% (wt vol-1) sucrose density gradient. Virus was recovered from appropriate fractions after dialysis against 0.01 M Na2SO3.

  2. EFSA Panel on Animal Health and Welfare (AHAW); Scientific Opinion on bluetongue serotype 8

    DEFF Research Database (Denmark)

    Bøtner, Anette; Oura, Chris; Saegerman, Claude

    To answer a question from the European Commission on the potential special characteristics of bluetongue virus (BTV) serotype 8 (BTV-8) compared to other serotypes and their possible impact on the epidemiology of the disease, a systematic literature review was carried out by a working group...

  3. Seroepidemiology of bluetongue in South Bengal

    Directory of Open Access Journals (Sweden)

    Arkendu Halder

    2016-01-01

    Full Text Available Aim: With the aim of revealing the epidemiological intricacies of bluetongue (BT in the southern part of West Bengal state, the present study was undertaken to assess seroprevalence of BT along with identification of the vector of the disease, i.e., Culicoides midges available in the region in their breeding season with conducive environmental factors, if any. Materials and Methods: A total of 1509 (sheep-504, goat-1005 samples were collected from three different agroclimatic zones of South Bengal viz. new alluvial, red laterite and coastal saline. To detect anti-BT antibodies in the collected serum samples, indirect-enzyme-linked immunosorbent assay (i-ELISA was performed. Culicoides midges were collected from those agro-climatic zones of South Bengal for species identification. The meteorological parameters, viz. temperature (maximum and minimum, rainfall and relative humidity of three agro-climatic zones of South Bengal were analyzed for the months of July to December during 2010-2013. Results: The overall seropositivity was 33.13% and 30.24% in sheep and goat, respectively as assessed by i-ELISA. In South Bengal, the predominant species of Culicoides found were Culicoides schultzei, Culicoides palpifer and Culicoides definitus. Conclusion: Since virus transmitting species of Culicoides midges could be detected in South Bengal, besides high seropositivity in ruminants, the possibility of circulating BT virus in South Bengal is quite imminent.

  4. Seroprevalence of bluetongue disease in sheep in west and northwest provinces of Iran

    Directory of Open Access Journals (Sweden)

    Mohammad Khezri

    2013-09-01

    Full Text Available The objective of this study was to describe the seroprevalence rates of bluetongue virus (BTV in sheep in west and northwest provinces of Iran. Bluetongue virus, an economically important orbivirus of the Reoviridae family, causes a hemorrhagic disease mainly in sheep and occasionally in cattle and some species of deer. Bluetongue virus is transmitted between its mammalian hosts by certain species of biting midges (Culicoides spp. and it can infect all ruminant species. Overall, 26 serotypes have been reported around the world. Due to its economic impact, bluetongue (BT is an Office of International des Epizooties (OIE-listed disease. A total of 756 sera samples collected during 2007-2008, were available. Sera were tested with competitive enzyme-linked immunosorbent assay (C-ELISA. The seroprevalence rate in sheep was 40.87%. The rate of positivity in sheep in west and northwest was 46.10% and 33.75%, respectively. The highest prevalence of antibodies in serum was in West Azerbaijan (64.86%, and lower was in Ardabil (23.77%.

  5. Mayaro virus isolated from a Trinidadian mosquito, Mansonia venezuelensis.

    Science.gov (United States)

    AITKEN, T H; DOWNS, W G; ANDERSON, C R; SPENCE, L; CASALS, J

    1960-04-01

    A strain of Mayaro virus has been isolated in Trinidad from the mosquito Mansonia venezuelensis. This is the first record of isolation of this agent from naturally infected mosquitoes, caught in the wild.

  6. Seroprevalence of bluetongue in ruminants of Jharkhand

    Directory of Open Access Journals (Sweden)

    Pinky Tigga

    2015-03-01

    Full Text Available Aim: This study was carried out to assess the presence of anti-bluetongue (BT antibodies in sheep, goat and cattle of different agro-climatic zones of Jharkhand. Materials and Methods: Serum samples were collected from apparently healthy as well as suspected sheep, goat and cattle from different districts of Jharkhand covering different agro-climatic zones. Serum samples were screened by indirect enzyme linked immunosorbent assay (iELISA for detecting anti-BT antibodies. Results: Out of a total of 480 animal serum samples (sheep-190, goats-210 and cattle-80 screened, 83 (43.68% of sheep, 91 (43.33% of goat and 46 (57.50% of cattle sera were found positive. The % positivity ranged between 41% and 51% in different agro-climatic zones. The results showed slight higher seroprevalence, although not significantly, in cattle than sheep and goats in different agro-climatic zones of Jharkhand. Conclusions: The above data indicate widespread prevalence of BT virus antibodies in studied areas. The incidence of BT is not detected officially, so far. The present seroprevalence status of BT in Jharkhand indicates presence of BT infection in the state for the first time.

  7. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

    Directory of Open Access Journals (Sweden)

    Jonathan S Towner

    2009-07-01

    Full Text Available In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus based on detection of Marburg virus RNA in 31/611 (5.1% bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

  8. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

    Science.gov (United States)

    Towner, Jonathan S; Amman, Brian R; Sealy, Tara K; Carroll, Serena A Reeder; Comer, James A; Kemp, Alan; Swanepoel, Robert; Paddock, Christopher D; Balinandi, Stephen; Khristova, Marina L; Formenty, Pierre B H; Albarino, Cesar G; Miller, David M; Reed, Zachary D; Kayiwa, John T; Mills, James N; Cannon, Deborah L; Greer, Patricia W; Byaruhanga, Emmanuel; Farnon, Eileen C; Atimnedi, Patrick; Okware, Samuel; Katongole-Mbidde, Edward; Downing, Robert; Tappero, Jordan W; Zaki, Sherif R; Ksiazek, Thomas G; Nichol, Stuart T; Rollin, Pierre E

    2009-07-01

    In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

  9. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

    Science.gov (United States)

    Fenyö, E M; Morfeldt-Månson, L; Chiodi, F; Lind, B; von Gegerfelt, A; Albert, J; Olausson, E; Asjö, B

    1988-01-01

    According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only. Images PMID:2459416

  10. Isolation of surface tubules of fowlpox virus.

    Science.gov (United States)

    Carter, J K; Cheville, N F

    1981-01-01

    Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

  11. Tobacco streak virus isolated from lettuce.

    Science.gov (United States)

    Abtahi, F S; Khodai Motlagh, M

    2009-05-01

    Tobacco streak virus (TSV) is an ilarvirus with a worldwide distribution. This virus infects many plants and causes significant yield losses. In this study, 300 samples of lettuce were collected from lettuce fields in Tehran Province. Infected plants show symptoms such as: mosaic, vein clearing, vein necrosis, yellowing and leaf distortion. DAS-ELISA (Double Antibody Sandwich-ELISA) was used with a polyclonal antiserum against TSV. Five isolates (T1, T2, T3, T4 and T5), which are collected, respectively from Mohammad Abad (Karaj), Malek Abad (Karaj), Hashtgerd (Karaj), Tarand Balla (Varamin) and Deh mah sin (Pishva) were inoculated on 29 species of Cucurbitaceae, Amaranthaceae, Solanacea, Compositae, Leguminosae and Chenopodiacea. Chenopodium quinoa 6 days after inoculation showed necrotic local lesions. Gomphrena globosa 10 days after inoculation developed chlorotic local lesions. Systemic symptoms were produced in Datura stramonium. Phaseolus vulgaris cv. Red Kidney 5 days after inoculation developed necrotic local lesions. Nicotiana tabacum 7 days after inoculation showed necrotic and chlorotic local lesions. Nicotiana clevelandii 15 days after inoculation developed leaf distortion and vein necrosis. Lactuca sativa 10-15 days after inoculation developed leaf istortion and mosaic. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using one primer pairs designed by DSMZ. An approximately 710 bp fragment was amplified with a specific primer.

  12. An Improved Culture System for Virus Isolation and Detection

    Institute of Scientific and Technical Information of China (English)

    Yu-chen XIA; Zhi-hong HU; Zhi-juan QIU; Zhong-bin MA; Hua-lin WANG; Fei DENG

    2008-01-01

    Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.

  13. High sequence conservation among cucumber mosaic virus isolates from lily.

    Science.gov (United States)

    Chen, Y K; Derks, A F; Langeveld, S; Goldbach, R; Prins, M

    2001-08-01

    For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV isolates of Alstroemeria and crocus were classified as subgroup II isolates, whereas 8 other isolates, from lily, gladiolus, amaranthus, larkspur, and lisianthus, were identified as subgroup I members. In general, nucleotide sequence comparisons correlated well with geographic distribution, with one notable exception: the analyzed nucleotide sequences of 5 lily isolates showed remarkably high homology despite different origins.

  14. Bluetongue disease and seroprevalence in South American camelids from the northwestern region of the United States.

    Science.gov (United States)

    Allen, Andrew J; Stanton, James B; Evermann, James F; Fry, Lindsay M; Ackerman, Melissa G; Barrington, George M

    2015-03-01

    In late summer/early fall of 2013, 2 South American camelids from central Washington were diagnosed with fatal bluetongue viral disease, an event which is rarely reported. A 9-year-old intact male llama (Lama glama), with a 1-day history of anorexia, recumbency, and dyspnea before death. Abundant foam discharged from the mouth and nostrils, and the lungs were severely edematous on postmortem examination. Histologically, there was abundant intra-alveolar edema with fibrin. Hemorrhage and edema disrupted several other organs. Bluetongue viral RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and serotype 11 was identified by sequencing a segment of the VP2 outer capsid gene. Approximately 1 month later, at a site 150 miles north of the index case, a 2-year-old female alpaca with similar, acutely progressive clinical signs was reported. A postmortem examination was performed, and histologic lesions from the alpaca were similar to those of the llama, and again serotype 11 was detected by PCR. The occurrence of bluetongue viral infection and disease is described in the context of seasonal Bluetongue virus activity within the northwestern United States and southwestern Canada.

  15. Complete Genome Sequences of Five Zika Virus Isolates.

    Science.gov (United States)

    Ladner, Jason T; Wiley, Michael R; Prieto, Karla; Yasuda, Chadwick Y; Nagle, Elyse; Kasper, Matthew R; Reyes, Daniel; Vasilakis, Nikolaos; Heang, Vireak; Weaver, Scott C; Haddow, Andrew; Tesh, Robert B; Sovann, Ly; Palacios, Gustavo

    2016-05-12

    Zika virus is an emerging human pathogen of great concern due to putative links to microcephaly and Guillain-Barre syndrome. Here, we report the complete genomes, including the 5' and 3' untranslated regions, of five Zika virus isolates, one from the Asian lineage and four from the African lineage.

  16. High sequence conservation among cucumber mosaic virus isolates from Lily

    NARCIS (Netherlands)

    Chen, Y.K.; Derks, A.F.L.M.; Langeveld, S.; Goldbach, R.; Prins, M.

    2001-01-01

    For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV i

  17. Characterization of a Zika Virus Isolate from Colombia

    Science.gov (United States)

    Lahon, Anismrita; Arya, Ravi P.; Kneubehl, Alexander R.; Vogt, Megan B.; Dailey Garnes, Natalie J. M.; Rico-Hesse, Rebecca

    2016-01-01

    Background Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments. Methodology/Clinical findings We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient’s clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3’ un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains. Conclusions/Significance We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas. PMID:27654889

  18. Isolation and characterization of cidofovir resistant vaccinia viruses

    Directory of Open Access Journals (Sweden)

    Prichard Mark N

    2008-05-01

    Full Text Available Abstract Background The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase. Results We have isolated several CDV resistant (CDVR vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3–7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated. Conclusion Resistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents.

  19. Persistent parainfluenza virus shedding during isolation at the South Pole.

    Science.gov (United States)

    Muchmore, H G; Parkinson, A J; Humphries, J E; Scott, E N; McIntosh, D A; Scott, L V; Cooney, M K; Miles, J A

    1981-01-15

    Persistent parainfluenza virus shedding in healthy young adults occurred throughout the 8 1/2-month winter isolation period at Amundsen-Scott South Pole Station during 1978. Two episodes of respiratory illness were observed after 10 and 29 weeks of complete social isolation. Throat swabs collected both routinely, and during each outbreak of respiratory illness, were directly inoculated into cell cultures. Parainfluenza virus types 1 and 3 were recovered from both symptomatic and asymptomatic subjects throughout the winter. No other viruses were obtained by these efforts. The presence of parainfluenza virus in these subjects long after the accepted incubation period for viral upper respiratory illness, and when the introduction of new virus to this community was impossible, suggests its persistence in man.

  20. Chikungunya virus isolation using simplified cell culture technique in Mauritius.

    Science.gov (United States)

    Pyndiah, M N; Pursem, V; Meetoo, G; Daby, S; Ramuth, V; Bhinkah, P; Chuttoo, R; Paratian, U

    2012-03-01

    During the chikungunya outbreak of 2005 - 2006, the only laboratory facilities available in Mauritius were virus isolation in cell culture tubes and serology. The laboratory was submerged with large numbers of blood samples. Comparative isolation was made in human embryonic lung (HEL) and VERO cells grown in 96-well plate. Culture on HEL cells was found to be more sensitive and presence of cytopathic effect (CPE) was observed earlier than in VERO cells. Out of the 18 300 blood samples inoculated on HEL, 11 165 were positive. This virus isolation method was of great help for the surveillance and control of the vectors. In cases of an outbreak a cheap, rapid and simple method of isolating chikungunya virus is described.

  1. Bluetongue: a historical and epidemiological perspective with the emphasis on South Africa

    Directory of Open Access Journals (Sweden)

    Coetzee Peter

    2012-09-01

    Full Text Available Abstract Bluetongue (BT is a non-contagious, infectious, arthropod transmitted viral disease of domestic and wild ruminants that is caused by the bluetongue virus (BTV, the prototype member of the Orbivirus genus in the family Reoviridae. Bluetongue was first described in South Africa, where it has probably been endemic in wild ruminants since antiquity. Since its discovery BT has had a major impact on sheep breeders in the country and has therefore been a key focus of research at the Onderstepoort Veterinary Research Institute in Pretoria, South Africa. Several key discoveries were made at this Institute, including the demonstration that the aetiological agent of BT was a dsRNA virus that is transmitted by Culicoides midges and that multiple BTV serotypes circulate in nature. It is currently recognized that BT is endemic throughout most of South Africa and 22 of the 26 known serotypes have been detected in the region. Multiple serotypes circulate each vector season with the occurrence of different serotypes depending largely on herd-immunity. Indigenous sheep breeds, cattle and wild ruminants are frequently infected but rarely demonstrate clinical signs, whereas improved European sheep breeds are most susceptible. The immunization of susceptible sheep remains the most effective and practical control measure against BT. In order to protect sheep against multiple circulating serotypes, three pentavalent attenuated vaccines have been developed. Despite the proven efficacy of these vaccines in protecting sheep against the disease, several disadvantages are associated with their use in the field.

  2. Isolation of influenza viruses in MDCK 33016PF cells and clearance of contaminating respiratory viruses.

    Science.gov (United States)

    Roth, Bernhard; Mohr, Hannah; Enders, Martin; Garten, Wolfgang; Gregersen, Jens-Peter

    2012-01-11

    This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼10(4)), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses.

  3. Gene detection, virus isolation, and sequence analysis of avian leukosis viruses in Taiwan country chickens.

    Science.gov (United States)

    Chang, Shu-Wei; Hsu, Meng-Fang; Wang, Ching-Ho

    2013-06-01

    Avian leukosis virus (ALV) infection in Taiwan Country chickens (TCCs) was investigated by using gene detection, virus isolation, and sequence analysis. The blood samples of 61 TCC flocks at market ages from a slaughter house were screened for exogenous ALVs using polymerase chain reaction to investigate the ALV infection status. The buffy coats from three breeder and four commercial chicken flocks were cocultured with DF-1 cells to isolate the virus. The full proviral DNA genomes of two ALV isolates were sequenced, analyzed, and compared with reference ALV strains. The gene detection results showed that 60 and 43 of the 61 flocks were infected with subgroup A of ALV (ALV-A) and subgroup J of ALV (ALV-J), respectively. Virus isolation results showed that five ALV-As and two ALV-Js were isolated from those seven TCC flocks. The full sequences of the isolates showed that isolate TW-3577 possessed a myeloblastosis-associated virus 1 gp85 coding region and an ALV-J 3'-untranslated region (3'UTR) and was similar to ordinary ALV-A. However, TW-3593 was unique. The 3'UTR of this isolate displayed high identity to endogenous counterpart sequence and its gp85 was different from all subgroups. This unique ALV is common in Taiwan.

  4. Colostral antibody protection and interference with immunity in lambs born from sheep vaccinated with an inactivated Bluetongue serotype 8 vaccine.

    Science.gov (United States)

    Oura, C A L; Wood, J L N; Floyd, T; Sanders, A J; Bin-Tarif, A; Henstock, M; Edwards, L; Simmons, H; Batten, C A

    2010-03-24

    Widespread vaccination programmes against Bluetongue virus serotype 8 (BTV-8), using inactivated vaccines, are being carried out across many countries in northern, western and southern Europe. This study investigates the extent and length of colostral antibody protection, as well as the degree of colostral antibody induced interference of the immune response to BTV-8, in sheep. Significantly lower titres of neutralising antibodies were transferred in colostrum to lambs born from sheep vaccinated once as opposed those vaccinated twice (single vaccine in the first year and a booster vaccine in the second year). On BTV-8 challenge, lambs born from sheep vaccinated on two occasions, with the second booster vaccine given approximately 1 month prior to lambing, were protected from clinical disease for up to 14 weeks. BTV-8 was isolated from 5 of the 22 challenged lambs, although only one of these lambs showed a transient rise in body temperature with no other clinical signs. Lambs born from ewes given a second booster vaccine 1 month prior to lambing, are likely to be protected from clinical disease for at least 14 weeks, whereas lambs born from ewes vaccinated once are likely to be protected for a shorter time. Colostral antibodies present in the 13-14-week-old lambs appeared to interfere with the humoral response to challenge virus. These results suggest that colostral antibodies may interfere with vaccination in lambs up to at least 14 weeks of age.

  5. Differences among isolates of simian hemorrhagic fever (SHF) virus.

    Science.gov (United States)

    Gravell, M; London, W T; Leon, M E; Palmer, A E; Hamilton, R S

    1986-01-01

    Simian hemorrhagic fever (SHF) virus is a member of the Togaviridae family which currently is unclassified to genus. We have studied the relatedness of four different SHF virus isolates obtained from infected macaque or patas monkeys. Differences were found among isolates in type and severity of disease produced in patas monkeys, cell sensitivity to infection, viral antigens, and levels of specific antibody induced in patas monkeys. Based on these criteria, the four isolates have been grouped in two categories: those producing acute infections in patas monkeys (LVR, P-180) and those producing persistent infections (P-248, P-741). The P-180 isolate induced the most severe disease in experimentally infected patas monkeys, but only occasionally were their infections fatal. Persistently infected patas monkeys were viremic over a period of years, but showed no signs or symptoms of infection. All four isolates were found to be antigenically related by use of enzyme-linked immunosorbent assay (ELISA); the P-248 isolate showing the weakest antigenic relationship. However, none of the four isolates induced cross-neutralizing antibodies in infected patas monkeys. High titers of specific IgG antibody (up to 31,250 as determined by ELISA) were induced in acutely infected patas monkeys (LVR, P-180), but antibody was barely detectable (less than or equal to 50) in persistently infected patas monkeys (P-248, P-741). LVR lytically infected USU-104 cells, patas monkey peritoneal macrophages (PMAC), and rhesus monkey PMAC. The P-180 isolate lytically infected both patas monkey PMAC and rhesus monkey PMAC, but not USU-104 cells. The isolates producing persistent infections (P-248, P-741) lytically infected only rhesus monkey PMAC. These results show that marked differences exist among isolates of SHF virus from naturally infected animals. These differences should be useful in categorizing new isolates.

  6. BLUETONGUE VIRUS ANTIBODIES DETECTIONS IN SHEEP FROM ARAÇATUBA REGION –SAO PAULO, BRAZIL DETECÇÃO DE ANTICORPOS CONTRA O VÍRUS DA LÍNGUA AZUL EM OVINOS NA REGIÃO DE ARAÇATUBA – SÃO PAULO, BRASIL

    Directory of Open Access Journals (Sweden)

    Adriana Hellmeister de Campos Nogueira

    2009-12-01

    Full Text Available

    Bluetongue (BT is an infectious, insect-born viral disease of ruminants. The causative agent of BT is bluetongue virus (BTV that belongs to the family Reoviridae genus Orbivirus. Insect vectors in the genus Culicoides transmit this virus. BT affects domestic and wild ruminants, however small ruminants are considered the most affected specie. The aim of the study was to detect antibodies against BTV in commercial sheep farms, of the Northeastern region of Sao Paulo State, Brazil. A total of 1002 sera samples collected from adult sheep (above 1 year-old, comprising a total of 31 farms, were screened for the presence of BTV antibodies, by agar gel immunodiffusion test (AGID and ELISA-CFS (Enzyme Linked Immunosorbent Assay – competitive solid phase, both produced by Pan American Center of FMDV. From a total of 1002 samples, 651 (65% were positive by AGID and 742 (74.1%, were positive by ELISA-CFS. These results suggest that the BTV is widespread among farms, probably causing subclinical infections.

    KEY WORDS: AGID, bluetongue virus, ELISA-CFS, seroepidemiological survey.

    A língua azul é uma doença viral, cujo agente etiológico pertence à família Reoviridae, gênero Orbivirus, transmitida por um vetor (artrópode hematófago, do gênero Culicoides. Os animais acometidos são ruminantes domésticos e selvagens, porém os pequenos ruminantes são os mais afetados. O estudo teve como objetivo detectar a presença de anticorpos para língua azul em ovinos da região de Araçatuba, por possuir um rebanho expressivo e condições climáticas favoráveis à multiplicação de insetos. Foram analisadas 1.002 amostras de soros ovinos, provenientes de 31 cabanhas, pelas provas de imunodifusão dupla em gel de ágar (AGID e ELISA (Enzyme Linked immunosorbent Assay de competição da fase sólida (ELISA CFS, provenientes do Centro Panamericano de Febre Aftosa. Desses soros, 651 (65% foram

  7. Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

    Science.gov (United States)

    Lowery, D Thomas; Vickers, Patricia M; Bittner, Lori A; Stobbs, Lorne W; Foottit, Robert G

    2015-10-01

    Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs.

  8. Complete Genome Sequence of Zika Virus Isolated from Semen

    Science.gov (United States)

    Graham, Victoria; Lewandowski, Kuiama; Dowall, Stuart D.; Pullan, Steven T.; Hewson, Roger

    2016-01-01

    Zika virus (ZIKV) is an emerging pathogenic flavivirus currently circulating in numerous countries in South America, the Caribbean, and the Western Pacific Region. Using an unbiased metagenomic sequencing approach, we report here the first complete genome sequence of ZIKV isolated from a clinical semen sample. PMID:27738033

  9. Increased virulence of Marek's disease virus field isolates.

    Science.gov (United States)

    Witter, R L

    1997-01-01

    The continuation of an apparent evolutionary trend of Marek's disease virus (MDV) towards greater virulence may explain recent increased losses from Marek's disease (MD) in vaccinated flocks. To address this question, the virulence of 31 isolates of serotype 1 MDV obtained from layer or broiler flocks between 1987 and 1995 were characterized. Each isolate was cultured in duck embryo fibroblasts for four to six passages, and ascertained to be free from contamination with avian retroviruses, chicken anemia virus, and MDVs of other serotypes. The viruses, along with prototype viruses JM/102W and Md5, were tested for virulence by inoculation at 6 days of age into laboratory strain 15I5 x 7(1) chickens of three types: nonvaccinated, vaccinated with turkey herpesvirus (HVT) and bivalent (HVT + SB-1)-vaccinated. The results showed that three isolates did not differ from JM/102W and were classified in the virulent (vMDV) pathotype. Twenty-one isolates produced significantly higher levels of MD in HVT-vaccinated chickens than did the JM/102W control and were classified in the very virulent (vvMDV) pathotype. Seven isolates, five of which were isolated in 1994 or 1995, produced significantly higher levels of MD in bivalent-vaccinated chickens than did the Md5 (vvMDV) control. These isolates, provisionally designated as the vv+MDV pathotype, appeared to be at the high end of a virulence continuum. Several MD response parameters, including lymphoma mortality, early mortality with bursal/thymic atrophy, and frequency of visceral lymphomas or ocular lesions in nonvaccinated chickens were positively correlated with virulence. These findings support the continued evolution of MDV towards greater virulence.

  10. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  11. An evolutionary insight into Newcastle disease viruses isolated in Antarctica.

    Science.gov (United States)

    Soñora, Martin; Moreno, Pilar; Echeverría, Natalia; Fischer, Sabrina; Comas, Victoria; Fajardo, Alvaro; Cristina, Juan

    2015-08-01

    The disease caused by Newcastle disease virus (NDV) is a severe threat to the poultry industry worldwide. Recently, NDV has been isolated in the Antarctic region. Detailed studies on the mode of evolution of NDV strains isolated worldwide are relevant for our understanding of the evolutionary history of NDV. For this reason, we have performed Bayesian coalescent analysis of NDV strains isolated in Antarctica to study evolutionary rates, population dynamics, and patterns of evolution. Analysis of F protein cleavage-site sequences of NDV isolates from Antarctica suggested that these strains are lentogenic. Strains isolated in Antarctica and genotype I reference strain Ulster/67 diverged from ancestors that existed around 1958. The time of the most recent common ancestor (MRCA) was established to be around 1883 for all class II viruses. A mean rate of evolution of 1.78 × 10(-3) substitutions per site per year (s/s/y) was obtained for the F gene sequences of NDV strains examined in this study. A Bayesian skyline plot indicated a decline in NDV population size in the last 25 years. The results are discussed in terms of the possible role of Antarctica in emerging or re-emerging viruses and the evolution of NDV populations worldwide.

  12. Mayaro virus fever in French Guiana: isolation, identification, and seroprevalence.

    Science.gov (United States)

    Talarmin, A; Chandler, L J; Kazanji, M; de Thoisy, B; Debon, P; Lelarge, J; Labeau, B; Bourreau, E; Vié, J C; Shope, R E; Sarthou, J L

    1998-09-01

    This paper reports the first isolation of Mayaro (MAY) virus from a patient infected in French Guiana. The identification was initially performed using immunofluorescent antibody testing with specific mouse antibody, and confirmed by plaque-reduction neutralization testing and reverse transcription-polymerase chain reaction. To determine if MAY virus infection is widespread in French Guiana, a serosurvey was performed to determine the prevalence of antibody to this virus in various ethnic groups and areas of French Guiana. Human sera (n = 1,962) were screened using the hemagglutination inhibition (HI) test. To determine whether MAY virus circulates in the rain forest, a serosurvey in monkey populations was performed. Monkey sera (n = 150) were also screened for antibody to MAY virus using HI testing. Of the human sera tested, 6.3% were positive for anti-MAY virus antibodies. Significant differences in MAY virus seroprevalence between different age groups were observed. Seroprevalence rates increased with age, with a large increase in people 10-19 years of age in comparison with those less than 10 years of age. After adjustment for age, significant differences were also found between places of residence. The prevalence of anti-MAY virus antibody was higher in people living in contact with the forest, especially in the Haut Oyapock area (odds ratio [OR] = 97.7, 95% confidence interval [CI] = 48.2-197.9) and along the Maroni River (OR = 39.7, 95% CI = 20.6-76.6). The ethnic differences observed in this study were probably due to differences in residence. Among monkeys, higher seroprevalence rates were found in Alouatta seniculus (66.0%) than in Saguinus midas (18.2%). Among Alouatta, the seroprevalence increased significantly with weight (and therefore with age). This study indicates that MAY virus is present in French Guiana, and human infections occur in areas where people live near the tropical rain forest.

  13. Molecular characterization of Duck Plague virus isolated from Bangladesh

    Directory of Open Access Journals (Sweden)

    Md. Mostakin Ahamed

    2015-09-01

    Full Text Available Duck plague (DP is the most feared duck disease in the world. For isolation, identification, molecular detection and characterization of DP virus (DPV, a total of 94 samples were collected from commercial farms (n=6 and households (n=13 from Rajshahi (n=37, Netrokona (n=35 and Mymensingh (n=22 districts of Bangladesh. The samples were processed and inoculated into 11-13 days old embryonated duck eggs for virus propagation. Virus was identified using agar gel immunodiffusion test (AGIT and passive hemagglutination (PHA test, and was confirmed by polymerase chain reaction (PCR targeting DNA polymerase and gC genes, followed by sequencing. Pathogenicity tests were performed using duck embryos, ducklings and ducks. Among the 94 samples, 17 isolates were confirmed as DPV by PCR amplification of partial DNA polymerase (446-bp and gC genes (78-bp, respectively. One of the isolates (Anatid herpes 1 BAU DMH was sequenced and found to be closely related with a Chinese variant of DPV (GenBank: JQ647509.1. Thus, we assume that both Bangladeshi and Chinese isolates of DPV may have a common ancestor. [J Adv Vet Anim Res 2015; 2(3.000: 296-303

  14. Genetic diversity of Hungarian Maize dwarf mosaic virus isolates.

    Science.gov (United States)

    Gell, Gyöngyvér; Balázs, Ervin; Petrik, Kathrin

    2010-04-01

    The genetic diversity of the coat-protein (CP) region and the untranslated C-terminal region (3'UTR) of Maize dwarf mosaic virus (MDMV) was analyzed to evaluate the variability between isolates (inter-isolate sequence diversity). The results of inter-isolate sequence diversity analysis showed that the diversity of the MDMV CP gene is fairly high (p-distance: up to 0.136). During sequence analysis, a 13 amino-acid residue insertion and an 8 amino-acid residue deletion were found within the N-terminal region of the CP gene. The phylogenetic analysis showed that-unlike other potyvirus species in this subgroup-the MDMV isolates could not be distinguished on the basis of their host plants or geographic origins.

  15. Detection of dengue virus in platelets isolated from dengue patients.

    Science.gov (United States)

    Noisakran, Sansanee; Gibbons, Robert V; Songprakhon, Pucharee; Jairungsri, Aroonroong; Ajariyakhajorn, Chuanpis; Nisalak, Ananda; Jarman, Richard G; Malasit, Prida; Chokephaibulkit, Kulkanya; Perng, Guey Chuen

    2009-03-01

    Though thrombocytopenia or dysfunction of platelets is common in dengue virus infection, the role of platelets has not been established. We enrolled 33 hospitalized children with serologically confirmed dengue virus infection. Blood specimens were collected during hospitalization. Platelets and plasma were isolated from the whole blood. Detection of dengue virus in plasma and platelets was carried out by RT-PCR with primers that can differentiate different dengue serotypes simultaneously, and by electron transmission microscopy (EM). Dengue viral RNA was detected in the platelets and plasma by conventional RT-PCR. A significantly higher percentage of dengue viral RNA was detected in platelets than in plasma (p = 0.03). Platelets isolated 5 days after onset of fever were most likely positive for viral RNA. Concurrent infection or co-circulation with multiple dengue serotypes was observed in 12% of patients. Infrequently, negative-stranded dengue viral RNA was detected in platelets and in plasma. Importantly, EM confirmed the presence of dengue viral-like particles inside platelets prepared from dengue patients. Our findings suggest the presence of dengue virus in platelets may be associated with the dysfunction of platelets observed in dengue patients.

  16. Identification of a strain of maize dwarf mosaic virus, related to sugarcane mosaic virus isolated from maize in Burundi

    Directory of Open Access Journals (Sweden)

    Verhoyen, M.

    1983-01-01

    Full Text Available A strain of maize dwarf mosaic virus related to sugarcane mosaic virus has been isolated from maize in Burundi. The properties (including electron microscopy and serology of the virus are described, and elements for a control strategy are reviewed.

  17. Genome sequencing and characterization analysis of a Beijing isolate of chicken corona virus infectious bronchitis virus

    Institute of Scientific and Technical Information of China (English)

    JIN Weiwu; YU Jialin; LI Ning; GONG Yuanshi; SUN Qixin; CHEN Zhangliang; CHEN Chen; ZHANG Ying; ZHAO Yiqiang; FENG Jidong; CHEN Fuyong; WU Qingming; YANG Hanchun; WANG Ming

    2004-01-01

    Avian infectious bronchitis virus (AIBV) is lassified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases,which has a 5′ cap structure and 3′ polyadenylation tract.The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames: 5′-orfla-orflab-s-3a-3b-e-m- 6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2%similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Beijing isolate is lone virus in group Ⅲ and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.

  18. Antigenic and genetic characterization of rabies virus isolates from Uruguay.

    Science.gov (United States)

    Guarino, Helena; Castilho, Juliana Galera; Souto, Juanita; Oliveira, Rafael de Novaes; Carrieri, Maria Luiza; Kotait, Ivanete

    2013-05-01

    After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp.) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149-1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to T. brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp. specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report of rabies

  19. Characterization of RD-114 Virus Isolated from a Commercial Canine Vaccine Manufactured Using CRFK Cells ▿

    Science.gov (United States)

    Yoshikawa, Rokusuke; Sato, Eiji; Igarashi, Tatsuhiko; Miyazawa, Takayuki

    2010-01-01

    Recently, we found that several commercial pet vaccines were contaminated with an infectious endogenous retrovirus, RD-114-related virus. Here, we determined the entire nucleotide sequences of RD-114-related viruses isolated from CRFK cells and a vaccine manufactured using CRFK cells. These RD-114-related viruses were nearly identical to the authentic RD-114 virus. PMID:20631117

  20. Cedar virus: a novel Henipavirus isolated from Australian bats.

    Directory of Open Access Journals (Sweden)

    Glenn A Marsh

    Full Text Available The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV and Nipah virus (NiV for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV, which shares significant features with the known henipaviruses. The genome size (18,162 nt and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin-B2 for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.

  1. Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

    Science.gov (United States)

    Donis, Ruben O; Davis, C Todd; Foust, Angie; Hossain, M Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, Odewijk; Neumeier, Elisabeth; Ziegler, Thedi

    2014-11-12

    Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine

  2. Molecular analysis of Korean isolate of barley yellow mosaic virus.

    Science.gov (United States)

    Lee, Kui Jae; Choi, Min Kyung; Lee, Wang Hyu; Rajkumar, Mani

    2006-04-01

    The complete sequences of both RNAs of an isolate of barley yellow mosaic virus (BaYMV) from Haenam, Korea, were determined. RNA1 is 7639 nucleotides long [excluding the 3'-poly(A)], and codes for a 270 kDa polyprotein of 2411 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNA2 is 3582 nucleotides long and codes for a polyprotein of 890 amino acids, which contains a 28 kDa putative proteinase (P1) and a 73 kDa polypeptide (P2). The whole sequences of Korean isolate (BaYMV-K) closely resembled those of an isolate from Japan (BaYMV-J) (99.6 identical nucleotides for RNA1; 99.4 for RNA2) and china (BaYMV-C) (96.7 and 96.2%, respectively) than from Germany (BaYMV-G) (93.6 and 90.4%, respectively). The greatest differences between the BaYMV-K and BaYMV-J isolates were in the 3'-NCRs of RNA1 and 5' NCRs of RNA2 and there were also some other regions of difference in Nib Pro (RNA1) and P1 (RNA2). Further, the phylogenetic analysis of CP region showed that Asian and European isolates formed distinct clusters. However, molecular variations between isolates could not be linked to earlier results showing differences in cultivar response.

  3. Dengue-1 virus isolation during first dengue fever outbreak on Easter Island, Chile.

    Science.gov (United States)

    Perret, Cecilia; Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-11-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription-polymerase chain reaction as being dengue type 1.

  4. Dengue-1 Virus Isolation during First Dengue Fever Outbreak on Easter Island, Chile

    OpenAIRE

    Perret, Cecilia; Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-01-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1.

  5. Characterization of viruses infecting potato plants from a single location in Shetland, an isolated scottish archipelago

    DEFF Research Database (Denmark)

    Mortensen, R.J.; Shen, Xinyi; Reid, Alex

    2010-01-01

    , as were 29 Scottish mainland isolates of the same four potato virus species, and these 58 isolates were compared to previously published sequence data. This has allowed the characterization of viruses from a relatively isolated location, where there is little production of ware potatoes and no seed potato...

  6. Isolation, Genetic Characterization, and Seroprevalence of Adana Virus, a Novel Phlebovirus Belonging to the Salehabad Virus Complex, in Turkey

    Science.gov (United States)

    Alkan, Cigdem; Alwassouf, Sulaf; Piorkowski, Géraldine; Bichaud, Laurence; Tezcan, Seda; Dincer, Ender; Ergunay, Koray; Ozbel, Yusuf; Alten, Bulent; de Lamballerie, Xavier

    2015-01-01

    ABSTRACT A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies. IMPORTANCE Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or

  7. Hepatitis B virus infection in isolated Afro-Brazilian communities.

    Science.gov (United States)

    Motta-Castro, Ana R C; Martins, Regina M B; Yoshida, Clara F T; Teles, Sheila A; Paniago, Anamaria M; Lima, Kátia M B; Gomes, Selma A

    2005-10-01

    The prevalence and genotypes of hepatitis B virus (HBV) have distinct geographical distribution. In Brazil, some African-descendants have been maintained as small isolated communities since the slavery period. In this study, HBV infection among these communities of African origin was examined. Individuals (1,058) living in 12 communities were interviewed and serum samples screened for the presence of HBV markers. HBsAg-positive sera were tested for HBV DNA by PCR and positive samples were genotyped by restriction fragment length polymorphism (RFLP). The overall prevalence of HBV infection was 19.8% (95% CI: 17.5-22.3), ranging from 5.5% to 42.4%, depending on the communities studied. Multivariate analysis of risk factors showed that increasing age, family history of hepatitis, and sexual activity were associated significantly with this infection. HBsAg was detected in 23/1,058 (2.2%) individuals. HBV DNA was present in 2/2 of HBeAg-positive serum samples and in 18/21 (85.7%) anti-HBe-positive samples. All HBV isolates belonged to genotype A, subtype Aa. Three RFLP patterns were identified: AI (17 isolates), AIV (1 isolate), and AVI (2 isolates). These findings suggest a common introduction of HBV during the slave trade from Africa to Brazil.

  8. First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

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    Mondher Boughalmi

    2013-11-01

    Full Text Available Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3 and France (2. The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection.

  9. First isolation of West Nile virus from a dromedary camel.

    Science.gov (United States)

    Joseph, Sunitha; Wernery, Ulrich; Teng, Jade Ll; Wernery, Renate; Huang, Yi; Patteril, Nissy Ag; Chan, Kwok-Hung; Elizabeth, Shyna K; Fan, Rachel Yy; Lau, Susanna Kp; Kinne, Jörg; Woo, Patrick Cy

    2016-06-08

    Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154-156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection.

  10. First isolation of West Nile virus from a dromedary camel

    Science.gov (United States)

    Joseph, Sunitha; Wernery, Ulrich; Teng, Jade LL; Wernery, Renate; Huang, Yi; Patteril, Nissy AG; Chan, Kwok-Hung; Elizabeth, Shyna K; Fan, Rachel YY; Lau, Susanna KP; Kinne, Jörg; Woo, Patrick CY

    2016-01-01

    Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154–156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection. PMID:27273223

  11. Isolation of saint louis encephalitis virus from a horse with neurological disease in Brazil.

    Directory of Open Access Journals (Sweden)

    Roberta Rosa

    2013-11-01

    Full Text Available St. Louis encephalitis virus (SLEV is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil.

  12. Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures.

    Science.gov (United States)

    Sotomayor, E A; Josephson, S L

    1999-07-01

    Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation. Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture. We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures. Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus. To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture. This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines.

  13. Isolation & molecular characterization of human parainfluenza virus in Chennai, India

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    C P Indumathi

    2015-01-01

    Full Text Available Background & objectives: Human parainfluenza virus (HPIV accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI. Methods: Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2 cell line. Results: Of the 232 samples, 26(11.2% were positive by mRT-PCR and nine (34.6% showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297. Interpretation & conclusions: The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.

  14. Isolation of Zika Virus Imported from Tonga into Australia

    Science.gov (United States)

    Pyke, Alyssa T.; Moore, Peter R.; Hall-Mendelin, Sonja; McMahon, Jamie L.; Harrower, Bruce J; Constantino, Tanya R; van den Hurk, Andrew F

    2016-01-01

    Introduction: The globally emergent Zika virus (ZIKV) is a threat to Australia, given the number of imported cases from epidemic regions and the presence of competent mosquito vectors. We report the isolation of ZIKV from a female traveler who recently returned from Tonga to Brisbane, Queensland, Australia in 2016. Methods: A specific TaqMan real-time reverse transcriptase polymerase chain reaction assay (RT-PCR) assay was used to detect ZIKV in serum and urine samples. Conventional cell culture techniques and suckling mice were employed in an attempt to isolate ZIKV from serum and urine. Results: A ZIKV isolate (TS17-2016) was recovered from the serum sample after one passage in suckling mouse brains and harvested 11 days post inoculation. Phylogenetic analysis of complete envelope (E) gene sequences demonstrated TS17-2016 shared 99.9% nucleotide identity with other contemporary sequences from Tonga 2016, Brazil 2015 and French Polynesia 2013 within the Asian lineage. Discussion: This is the first known report of successful isolation of ZIKV from a human clinical sample in Australia and the first from a traveler from Tonga. This study highlights the potential difficulties in isolating ZIKV from acute clinical samples using conventional cell culture techniques, particularly in non-endemic countries like Australia where access to samples of sufficient viral load is limited. The successful isolation of TS17-2016 will be essential for continued investigations of ZIKV transmission and pathogenicity and will enable the advancement of new preventative control measures extremely relevant to the Australian and Pacific region. PMID:27679739

  15. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis

    NARCIS (Netherlands)

    Wahyuningsih, Retno; SahBandar, Ivo N.; Theelen, Bart; Hagen, Ferry; Poot, Ge; Meis, Jacques F.; Rozalyani, Anna; Sjam, Ridhawati; Widodo, Djoko; Djauzi, Samsuridjal; Boekhout, Teun

    2008-01-01

    Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavueonazole and wa

  16. West Nile virus in Tunisia, 2014: First isolation from mosquitoes.

    Science.gov (United States)

    Wasfi, F; Dachraoui, K; Cherni, S; Bosworth, A; Barhoumi, W; Dowall, S; Chelbi, I; Derbali, M; Zoghlami, Z; Beier, J C; Zhioua, E

    2016-07-01

    Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection.

  17. Virus isolations from mosquitoes collected during the 1982 Japanese encephalitis epidemic in northern Thailand.

    Science.gov (United States)

    Leake, C J; Ussery, M A; Nisalak, A; Hoke, C H; Andre, R G; Burke, D S

    1986-01-01

    From 16 June to 15 August, 1982 CDC light traps were used to collect mosquitoes in the province of Kamphaengphet, N. Thailand. 353,042 mosquitoes comprising 59 species were collected and identified, and 345,173 were placed in pools for attempted virus isolation by inoculation of C6/36 Aedes albopictus mosquito cell cultures. Viruses were isolated from 63 mosquito pools. These comprised 56 flaviviruses, identified as 35 isolates of Japanese encephalitis (JE) virus strains, 18 strains of Tembusu (TEM) virus and three untyped flaviviruses (FLA); three alphaviruses, identified as the first isolates of Getah (GET) virus to have been made in Thailand; and four viruses which are still unidentified. Most virus isolates were from Culex tritaeniorhynchus mosquitoes collected in carbon dioxide baited light traps. JE virus was isolated only over a ten-day period and the last isolate was obtained one week before the peak of admission of human encephalitis cases at Kamphaengphet Provincial Hospital. Rapid screening of isolates grown on Ae. pseudoscutellaris (LSTM-AP-61) mosquito cells by indirect immunofluorescence using flavivirus group-specific and JE-specific monoclonal antibodies showed a high degree of correlation with plaque reduction neutralization tests. An antigen capture enzyme immunoassay (EIA) test successfully identified about 50% of the JE virus positive pools, but the method saved considerable processing time.

  18. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene.

    Science.gov (United States)

    Kim, W-S; Oh, M-J; Nishizawa, T; Park, J-W; Kurath, G; Yoshimizu, M

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan.

  19. Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses.

    Science.gov (United States)

    Hause, Ben M; Ducatez, Mariette; Collin, Emily A; Ran, Zhiguang; Liu, Runxia; Sheng, Zizhang; Armien, Anibal; Kaplan, Bryan; Chakravarty, Suvobrata; Hoppe, Adam D; Webby, Richard J; Simonson, Randy R; Li, Feng

    2013-02-01

    Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

  20. Effect of Culicoides sonorensis salivary proteins on clinical disease outcome in experimental Bleutongue virus serotype 8 infection of Dorset sheep

    NARCIS (Netherlands)

    Drolet, B.S.; Reister, L.M.; Lehiy, C.J.; Rijn, van P.A.; Bowen, R.A.

    2015-01-01

    The severity of Bluetongue clinical disease in ruminants varies greatly depending on the outbreak serotype/strain, animal species/breed, and immune status of the herd. To predict disease risk from any of the 26 Bluetongue virus (BTV) serotypes identified to date, experimental animal susceptibility s

  1. Lethal Effect of Bluetongue Virus Strain HbC3 on Mouse Prostata Cancer RM-1 Cells%蓝舌病毒湖北株对小鼠前列腺癌RM-1细胞的杀伤效应

    Institute of Scientific and Technical Information of China (English)

    王肖; 张杰; 杜贤进; 周晓光

    2011-01-01

    Objective: To investigate the characteristics and the mechanism of bluetongue virus strain HbC3(HCMV) infecting mouse prostate cancer RM-1 cells in vitro.Methods: BTV-HbC3 was used to infect RM-1 cells, the the cytopathic effect (CPE) was observed, and the inhibition activity of RM-1 cell infected with BTV-HbC3 was determined by MTT.Transmission electron microscope (TEM) was adopted to study the changes of cell ultrastructure.DNA Ladder was taken to detect the apoptosis of RM-1 cells induced by BTV-HbC3.The apoptosis was detected by flow cytometry (FCM).Results: RM-1 cells were sensitive to BTV-HbC3 infection, CPE was found in BTV-HbC3 infected RM-1 cells, and lots of virus particles were found in cytoplasm by TEM.Apoptotic cells were detected by FCM.Conclusion: BTV-HbC3 could infect RM-1 cells and replicate efficiently, and induce apoptosis in tumor cells.%目的:体外研究蓝舌病毒湖北株3(BTV-HbC3)对小鼠前列腺癌细胞RM-1的感染性并探讨BTV-HbC3靶向性溶瘤的机制.方法:观察RM-1细胞感染BTV-HbC3的细胞病变效应;MTT法研究病毒致细胞病变率的特征;透射电镜观察感染病毒后细胞超微结构的变化;DNA Ladder分析病毒诱导细胞凋亡的情况;流式细胞仪测定病毒对RM-1细胞凋亡的影响.结果:BTV-HbC3感染RM-1细胞后有明显的细胞病变效应;DNA Ladder分析为阶梯状条带;透射电镜发现胞质内有大量病毒颗粒和典型细胞凋亡形态变化;流式细胞仪可见明显的细胞凋亡.结论:BTV-HbCs在体外能有效的感染RM-1细胞,并能诱导RM-1细胞凋亡.

  2. Molecular-genetic analysis of field isolates of Avian Leucosis Viruses in the Russian Federation

    Science.gov (United States)

    Commercial poultry farms in 14 regions of Russian Federation were monitored for avian leukosis virus (ALV) infection using virus isolation tests and serology. Results indicated the presence of two subgroups of ALV in farms located in 11 of 14 regions. Analysis of the genomes of 12 field isolates of...

  3. Complete Genome Sequence of an African Swine Fever Virus Isolate from Sardinia, Italy

    Science.gov (United States)

    Torresi, Claudia; Oggiano, Annalisa; Malmberg, Maja; Iscaro, Carmen; De Mia, Gian Mario; Belák, Sándor

    2016-01-01

    Previous genetic characterization of African swine fever virus isolates from the Italian island of Sardinia, where the virus has been present since 1978, has largely been limited to a few selected genomic regions. Here, we report the complete genome sequence of the isolate 47/Ss/08 collected during an outbreak in 2008. PMID:27856577

  4. First Complete Genome Sequence of a Chikungunya Virus Strain Isolated from a Patient Diagnosed with Dengue Virus Infection in Malaysia

    Science.gov (United States)

    Gan, Han Ming; Rohani, Ahmad

    2016-01-01

    Here, we report the complete genome sequence of a chikungunya virus coinfection strain isolated from a dengue virus serotype 2-infected patient in Malaysia. This coinfection strain was determined to be of the Asian genotype and contains a novel insertion in the nsP3 gene. PMID:27563048

  5. GENOTYPING OF ISOLATED VIRUSES FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS) IN CROATIA

    OpenAIRE

    Irena Vardić; Damir Kapetanović; Damir Valić; Božidar Kurtović; Zlatica Teskeredžić; Emin Teskeredžić

    2007-01-01

    Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout aquaculture. Detection of these viruses in Croatia initiated investigation of their genetic relatedness to the worldwide IHNV and IPNV isolates. For this purpose, determination of nucleotide sequences of G and NV genes for IHNV and VP2/NS region for IPNV was performed. Phylogenetic analyses revealed that Croatian IHNV isolate was clustering within European cl...

  6. Isolation and Identification of Virus dsRNA from Strawberry Plants

    Institute of Scientific and Technical Information of China (English)

    LI He; DAI Hong-yan; ZHANG Zhi-hong; GAO Xiu-yan; DU Guo-dong; ZHANG Xin-yu

    2007-01-01

    The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid (dsRNA) and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction (RT-PCR). The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease Ⅰ (DNase Ⅰ ), but evidently, it became resistant to ribonuclease A (RNase A) only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase Ⅰ /RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.

  7. The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses

    NARCIS (Netherlands)

    Souza Richards, R.; Adams, I.P.; Kreuze, J.F.; Souza, de J.; Cuellar, W.; Dullemans, A.M.; Vlugt, van der R.A.A.; Glover, R.; Hany, U.; Dickinson, M.; Boonham, N.

    2014-01-01

    The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), whi

  8. Isolation of recombinant field strains of Marek's disease virus integrated with reticuloendotheliosis virus genome fragments

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Zhi; CUI; Zhizhong

    2005-01-01

    Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.

  9. Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

    Directory of Open Access Journals (Sweden)

    Botton S.A.

    1998-01-01

    Full Text Available Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs (Corapi WV, Donis RO and Dubovi EJ (1990 American Journal of Veterinary Research, 55: 1388-1394. Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3% reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R was calculated, 49 of 66 comparisons (74.24% between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

  10. New Brazilian giant viruses isolation from environmental samples using a panel of protozoa.

    Directory of Open Access Journals (Sweden)

    Fábio Pio Dornas

    2015-10-01

    Full Text Available The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini and Vermamoeba vermiformis as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in Acanthamoeba polyphaga (46.38% and the lowest in Vermamoeba vermiformis (0%. Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation.

  11. Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa

    Science.gov (United States)

    Dornas, Fábio P.; Khalil, Jacques Y. B.; Pagnier, Isabelle; Raoult, Didier; Abrahão, Jônatas; La Scola, Bernard

    2015-01-01

    The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation. PMID:26500630

  12. Separation and isolation of BTV dsRNA segments and viral proteins.

    Science.gov (United States)

    Li, Joseph K-K; Huang, I-Jen; Hayama, Emiko

    2012-05-01

    Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.

  13. Isolation of a new herpes virus from human CD4 sup + T cells

    Energy Technology Data Exchange (ETDEWEB)

    Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.; Katsafanas, G.; Roffman, E.; Danovich, R.M. (National Institutes of Health, Rockville, MD (USA)); June, C.H. (Naval Medical Research Institute, Bethesda, MD (USA))

    1990-01-01

    A new human herpes virus has been isolated from CD4{sup +} T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpes virus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpes virus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hydridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. The authors conclude that the RK virus is distinct from previously characterized human herpesviruses. The authors propose to designate it as the prototype of a new herpes virus, the seventh human herpes virus identified to date.

  14. Adaptive evolution of simian immunodeficiency viruses isolated from two conventional progressor macaques with neuroaids

    Energy Technology Data Exchange (ETDEWEB)

    Foley, Brian T [Los Alamos National Laboratory; Korber, Bette T [Los Alamos National Laboratory

    2008-01-01

    Simian immunodeficiency virus infection of macaques may result in neuroAIDS, a feature more commonly observed in macaques with rapid progressive disease than in those with conventional disease. This is the first report of two conventional progressors (H631 and H636) with encephalitis in rhesus macaques inoculated with a derivative of SIVsmES43-3. Phylogenetic analyses of viruses isolated from the cerebral spinal fluid (CSF) and plasma from both animals demonstrated tissue compartmentalization. Additionally, virus from the central nervous system (CNS) was able to infect primary macaque monocyte-derived macrophages more efficiently than virus from plasma. Conversely, virus isolated from plasma was able to replicate better in peripheral blood mononuclear cells than virus from CNS. We speculate that these viruses were under different selective pressures in their separate compartments. Furthermore, these viruses appear to have undergone adaptive evolution to preferentially replicate in their respective cell targets. Analysis of the number of potential N-linked glycosylation sites (PNGS) in gp160 showed that there was a statistically significant loss of PNGS in viruses isolated from CNS in both macaques compared to SIVsmE543-3. Moreover, virus isolated from the brain in H631, had statistically significant loss of PNGS compared to virus isolated from CSF and plasma of the same animal. It is possible that the brain isolate may have adapted to decrease the number of PNGS given that humoral immune selection pressure is less likely to be encountered in the brain. These viruses provide a relevant model to study the adaptations required for SIV to induce encephalitis.

  15. Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

    Science.gov (United States)

    Turell, M J; O'Guinn, M L; Jones, J W; Sardelis, M R; Dohm, D J; Watts, D M; Fernandez, R; Travassos da Rosa, A; Guzman, H; Tesh, R; Rossi, C A; Ludwig, V; Mangiafico, J A; Kondig, J; Wasieloski, L P; Pecor, J; Zyzak, M; Schoeler, G; Mores, C N; Calampa, C; Lee, J S; Klein, T A

    2005-09-01

    As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

  16. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    OpenAIRE

    Kim, W. -S.; Oh, M. -J.; Nishizawa, T; Park, J. -W.; Kurath, G; Yoshimizu, M

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogen...

  17. Phylogenetic analysis of Newcastle disease viruses isolated from commercial poultry in Mozambique (2011-2016).

    Science.gov (United States)

    Mapaco, Lourenço P; Monjane, Iolanda V A; Nhamusso, Antonieta E; Viljoen, Gerrit J; Dundon, William G; Achá, Sara J

    2016-10-01

    The complete sequence of the fusion (F) protein gene from 11 Newcastle disease viruses (NDVs) isolated from commercial poultry in Mozambique between 2011 and 2016 has been generated. The F gene cleavage site motif for all 11 isolates was (112)RRRKRF(117) indicating that the viruses are virulent. A phylogenetic analysis using the full F gene sequence revealed that the viruses clustered within genotype VIIh and showed a higher similarity to NDVs from South Africa, China and Southeast Asia than to viruses previously described in Mozambique in 1994, 1995 and 2005. The identification of these new NDVs has important implications for Newcastle disease management and control in Mozambique.

  18. Emergence of a new arbovirus disease in Brazil. I. Isolation and characterization of the etiologic agent, Rocio virus.

    Science.gov (United States)

    de Souza Lopes, O; Coimbra, T L; de Abreu Sacchetta, L; Calisher, C H

    1978-05-01

    In April, 1975, an epidemic of human encephalitis was detected in several counties in the State of São Paulo, Brazil; the epidemic continued into 1976. A virus was isolated from central nervous system (CNS) tissues of a 39-year-old male who died on December 8, 1975; the virus was found to be a new flavivirus for which the name Rocio virus is proposed. Nine further isolations of Rocio virus were obtained from CNS tissues of 17 patients who died with clinical symptoms of encephalitis. Isolations of virus and serologic evidence of Rocio virus infection in a significant proportion of the encephalitis patients suggested that Rocio virus was the etiologic agent of the epidemic. Rocio virus was isolated only from patients who died within 5 days of onset of illness. The virus was isolated from two sentinel mice exposed in the epidemic zone and from a rufous collared sparrow (Zonotrichia capensis) collected in the area.

  19. [Subtypes of dengue virus serotypes 2, 3 and 4 isolated in Santander District, Colombia].

    Science.gov (United States)

    Cortés, Fabián M; Gómez, Sergio Y; Ocazionez, Raquel E

    2007-01-01

    Virus serotypes 2, 3 and 4 that had circulated in Santander District, Colombia in the period 1998-2004 were analyzed. Identifying the subtype of a dengue virus serotype is a useful tool for surveillance of severe risk factors because the strain potential to cause hemorrhagic dengue makes the difference among them. Simultaneous sequence amplification technique known as restriction site specific-polymerase chain reaction (RSS-PCR) was used to determine the subtype by comparing the electrophoretic pattern of the local isolate to the reference virus. Virus serotype 2 corresponded to subtype A similar to the one isolated in Thailand (1996) and to the other isolated in Porto Rico (1986); virus serotypes 3 were of subtype C like the virus found in Sri Lanka (1990), Honduras (1995) and Porto Rico (2000); virus serotypes 4 were a variant of subtype B similar to a virus from Porto Rico (1987) and to another virus from Tahiti (1985). The study confirmed the presence in Colombia of dengue virus subtypes circulating now in the Americas.

  20. Isolation of Tacaribe virus, a Caribbean arenavirus, from host-seeking Amblyomma americanum ticks in Florida.

    Directory of Open Access Journals (Sweden)

    Katherine A Sayler

    Full Text Available Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573, with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2% of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

  1. Isolation of Tacaribe virus, a Caribbean arenavirus, from host-seeking Amblyomma americanum ticks in Florida.

    Science.gov (United States)

    Sayler, Katherine A; Barbet, Anthony F; Chamberlain, Casey; Clapp, William L; Alleman, Rick; Loeb, Julia C; Lednicky, John A

    2014-01-01

    Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

  2. Animal viral diseases and global change: Bluetongue and West Nile fever as paradigms

    Directory of Open Access Journals (Sweden)

    Miguel Angel eJimenez-Clavero

    2012-06-01

    Full Text Available Environmental changes have an undoubted influence on the appearance, distribution and evolution of infectious diseases, and notably on those transmitted by vectors. Global change refers to environmental changes arising from human activities affecting the fundamental mechanisms operating in the biosphere. This paper discusses the changes observed in recent times with regard to some important arboviral (arthropod-borne viral diseases of animals, and the role global change could have played in these variations. Two of the most important arboviral diseases of animals, bluetongue and West Nile fever/encephalitis, have been selected as models. In both cases, in the last 15 years an important leap forward has been observed, which has lead to considering them emerging diseases in different parts of the world. Bluetongue, affecting domestic ruminants, has recently afflicted livestock in Europe in an unprecedented epizootic, causing enormous economic losses. West Nile fever/encephalitis affects wildlife (birds, domestic animals (equines and humans, thus, beyond the economic consequences of its occurrence, as a zoonotic disease, it poses an important public health threat. West Nile virus has expanded in the last 12 years worldwide, and particularly in the Americas, where it first occurred in 1999, extending throughout the Americas relentlessly since then, causing a severe epidemic of disastrous consequences for public health, wildlife and livestock. In Europe, West Nile virus is known long time ago, but it is since the last years of the XXth century that its incidence has risen substantially. Circumstances such as global warming, changes in land use and water management, increase in travel, trade of animals, and others, can have an important influence in the observed changes in both diseases. The following question is raised: What is the contribution of global changes to the current increase of these diseases in the world?

  3. Dengue virus serotype 2 from a sylvatic lineage isolated from a patient with dengue hemorrhagic fever.

    Directory of Open Access Journals (Sweden)

    Jane Cardosa

    Full Text Available Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF with thrombocytopenia (12,000/ul and a raised hematocrit (29.5% above baseline in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975.

  4. Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

    Directory of Open Access Journals (Sweden)

    J. A. Swanstrom

    2016-07-01

    Full Text Available Zika virus (ZIKV is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73% failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], 1:100 serum dilution; 9% levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.

  5. Virus excretion and antibody dynamics in goats inoculated with a field isolate of peste des petits ruminants virus.

    Science.gov (United States)

    Liu, W; Wu, X; Wang, Z; Bao, J; Li, L; Zhao, Y; Li, J

    2013-11-01

    A field isolate of peste des petits ruminants virus (PPRV) from an outbreak in Tibet, China, was inoculated into goats to investigate the dynamics of virus excretion and antibody production. Further, animals received PPRV vaccine strain Nigeria 75/1. Ocular, nasal and oral samples were tested for the presence of virus antigen by one-step real-time qualitative RT-PCR (qRT-PCR); competitive ELISA (c-ELISA) was used for the measurement of specific antibodies against PPRV. Virus particles could be detected as early as day 3 post-inoculation (pi) and virus excretion lasted for up to day 26 pi. All four goats inoculated with the PPRV field isolate were seropositive as early as day 10 pi. In animals inoculated with the vaccine strain, antibody was detected at day 14 pi, and levels of neutralizing antibodies remained above the protection threshold level (1 : 8) for 8 months. Both virus particles and neutralizing antibodies were detected earlier in goats challenged with the field isolate than in those receiving the vaccine strain.

  6. First Complete Genome Sequence of a Simian Foamy Virus Isolate from a Cynomolgus Macaque

    Science.gov (United States)

    Sakai, Koji; Ami, Yasushi; Suzaki, Yuriko

    2016-01-01

    We report here the first complete proviral genome sequence (DDBJ/ENA/GenBank accession no. LC094267) of a simian foamy virus, SFVmfa/Cy5061, isolated from a cynomolgus macaque (Macaca fascicularis). This proviral genome consists of 12,965 nucleotides and has five open reading frames, gag, pol, env, tas, and bet, as with other foamy viruses. PMID:27908992

  7. Genome Sequence of Lassa Virus Isolated from the First Domestically Acquired Case in Germany

    Science.gov (United States)

    Wolff, Svenja; Schultze, Tilman; Fehling, Sarah Katharina; Mengel, Jan Philipp; Kann, Gerrit; Wolf, Timo; Eickmann, Markus; Becker, Stephan

    2016-01-01

    Lassa virus (LASV) is a zoonotic, hemorrhagic fever-causing virus endemic in West Africa, for which no approved vaccines or specific treatment options exist. Here, we report the genome sequence of LASV isolated from the first case of acquired Lassa fever disease outside of Africa. PMID:27660771

  8. Dengue-1 Virus Isolation during First Dengue Fever Outbreak on Easter Island, Chile

    Science.gov (United States)

    Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-01-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1. PMID:14718094

  9. Genetic analysis of influenza B viruses isolated in Uganda during the 2009–2010 seasons

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    Byarugaba Denis K

    2013-01-01

    Full Text Available Abstract Background Influenza B viruses can cause morbidity and mortality in humans but due to the lack of an animal reservoir are not associated with pandemics. Because of this, there is relatively limited genetic sequences available for influenza B viruses, especially from developing countries. Complete genome analysis of one influenza B virus and several gene segments of other influenza B viruses isolated from Uganda from May 2009 through December 2010 was therefore undertaken in this study. Methods Samples were collected from patients showing influenza like illness and screened for influenza A and B by PCR. Influenza B viruses were isolated on Madin-Darby Canine Kidney cells and selected isolates were subsequently sequenced and analyzed phylogenetically. Findings Of the 2,089 samples collected during the period, 292 were positive by PCR for influenza A or B; 12.3% of the PCR positives were influenza B. Thirty influenza B viruses were recovered and of these 25 that grew well consistently on subculture were subjected to further analysis. All the isolates belonged to the B/Victoria-lineage as identified by hemagglutination inhibition assay and genetic analysis except one isolate that grouped with the B-Yamagata-lineage. The Ugandan B/Victoria-lineage isolates grouped in clade 1 which was defined by the N75K, N165K and S172P substitutions in hemagglutinin (HA protein clustered together with the B/Brisbane/60/2008 vaccine strain. The Yamagata-like Ugandan strain, B/Uganda/MUWRP-053/2009, clustered with clade 3 Yamagata viruses such as B/Bangladesh/3333/2007 which is characterized by S150I and N166Y substitutions in HA. Conclusion In general there was limited variation among the Ugandan isolates but they were interestingly closer to viruses from West and North Africa than from neighboring Kenya. Our isolates closely matched the World Health Organization recommended vaccines for the seasons.

  10. Two biologically distinct isolates of Zucchini yellow mosaic virus lack seed transmissibility in cucumber.

    Science.gov (United States)

    Glasa, M; Kollerova, E

    2007-01-01

    The seed transmission of the Zucchini yellow mosaic virus (ZYMV) was studied in cucumber using two isolates unrelated in their biological characteristics. Although the virus could be readily detected in mature seeds harvested from infected cucumbers, the seedlings obtained from infected germinated seeds tested negative for ZYMV using both ELISA and RT-PCR assays. No evidence was obtained for transmission of two ZYMV isolates through seeds.

  11. Isolation of Madre de Dios Virus (Orthobunyavirus; Bunyaviridae), an Oropouche Virus Species Reassortant, from a Monkey in Venezuela.

    Science.gov (United States)

    Navarro, Juan-Carlos; Giambalvo, Dileyvic; Hernandez, Rosa; Auguste, Albert J; Tesh, Robert B; Weaver, Scott C; Montañez, Humberto; Liria, Jonathan; Lima, Anderson; Travassos da Rosa, Jorge Fernando Soares; da Silva, Sandro P; Vasconcelos, Janaina M; Oliveira, Rodrigo; Vianez, João L S G; Nunes, Marcio R T

    2016-08-03

    Oropouche virus (OROV), genus Orthobunyavirus, family Bunyaviridae, is an important cause of human illness in tropical South America. Herein, we report the isolation, complete genome sequence, genetic characterization, and phylogenetic analysis of an OROV species reassortant, Madre de Dios virus (MDDV), obtained from a sick monkey (Cebus olivaceus Schomburgk) collected in a forest near Atapirire, a small rural village located in Anzoategui State, Venezuela. MDDV is one of a growing number of naturally occurring OROV species reassortants isolated in South America and was known previously only from southern Peru.

  12. Genetic characterization and evolutionary analysis of Newcastle disease virus isolated from domestic duck in South Korea.

    Science.gov (United States)

    Gaikwad, Satish; Kim, Ji-Ye; Lee, Hyun-Jeong; Jung, Suk Chan; Choi, Kang-Seuk

    2016-03-15

    Domestic ducks are considered a potential reservoir of Newcastle disease virus. In the study, a Newcastle disease virus (NDV) isolated from a domestic duck during surveillance in South Korea was characterized. The complete genome of the NDV isolate was sequenced, and the phylogenetic relationship to reference strains was studied. Phylogenetic analysis revealed that the strain clustered in genotype I of Class II ND viruses, has highly phylogenetic similarity to NDV strains isolated from waterfowl in China, but was distant from the viruses isolated in chickens and vaccine strains used in South Korea. Pathogenicity experiment in chickens revealed it to be a lentogenic virus. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the avirulent motif (112)GKQGRL(117) at the cleavage site and caused no apparent disease in chickens and ducks. With phylogeographic analysis based on fusion gene, we estimate the origin of an ancestral virus of the isolate and its sister strain located in China around 1998. It highlights the need of continuous surveillance to enhance current understanding of the molecular epidemiology and evolution of the pathogenic strains.

  13. Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

    Science.gov (United States)

    Kuwata, Ryusei; Satho, Tomomitsu; Isawa, Haruhiko; Yen, Nguyen Thi; Phong, Tran Vu; Nga, Phan Thi; Kurashige, Tomokazu; Hiramatsu, Yukihiro; Fukumitsu, Yuki; Hoshino, Keita; Sasaki, Toshinori; Kobayashi, Mutsuo; Mizutani, Tetsuya; Sawabe, Kyoko

    2013-11-01

    In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

  14. Sequence analysis of a soil-borne wheat mosaic virus isolate from Italy shows that it is the same virus as European wheat mosaic virus and Soil-borne rye mosaic virus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The complete sequence of the two RNAs of a furovirus isolate fromdurum wheat in Italy was determined. Sequence comparisons and phylogenetic analysis were done to compare the Italian virus with Soil-borne wheat mosaic virus (SBWMV) from the USA and with furovirus sequences recently published as European wheat mosaic virus (EWMV), from wheat in France, and Soil-borne rye mosaic virus (SBRMV), from rye and wheat in Germany. Over the entire genome, the Italian isolate RNA1 and RNA2 had respectively 97.5% and 98.6% nucleotide identity with EWMV, 95.5% and 85.8% with SBRMV-G and 70.6% and 64.5% with SBWMV. The Italian isolate was therefore clearly distinct from SBWMV. The European isolates all appear to belong to the same virus and the name Soil-borne cereal mosaic virus may resolve earlier ambiguities.

  15. Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

    Directory of Open Access Journals (Sweden)

    Abdel-Moneim Ahmed S

    2012-08-01

    Full Text Available Abstract Background Avian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness. Methods Recently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated. Results Phylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens. Conclusion The study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.

  16. CPm gene diversity in field isolates of Citrus tristeza virus from Colombia.

    Science.gov (United States)

    Oliveros-Garay, Oscar Arturo; Martinez-Salazar, Natalhie; Torres-Ruiz, Yanneth; Acosta, Orlando

    2009-01-01

    The nucleotide sequence diversity of the CPm gene from 28 field isolates of Citrus tristeza virus (CTV) was assessed by SSCP and sequence analyses. These isolates showed two major shared haplotypes, which differed in distribution: A1 was the major haplotype in 23 isolates from different geographic regions, whereas R1 was found in isolates from a discrete region. Phylogenetic reconstruction clustered A1 within an independent group, while R1 was grouped with mild isolates T30 from Florida and T385 from Spain. Some isolates contained several minor haplotypes, which were very similar to, and associated with, the major haplotype.

  17. Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido, Japan.

    Science.gov (United States)

    Abe, Yuri; Tamura, Tomokazu; Torii, Shiho; Wakamori, Shiho; Nagai, Makoto; Mitsuhashi, Kazuya; Mine, Junki; Fujimoto, Yuri; Nagashima, Naofumi; Yoshino, Fumi; Sugita, Yukihiko; Nomura, Takushi; Okamatsu, Masatoshi; Kida, Hiroshi; Sakoda, Yoshihiro

    2016-01-01

    In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5'-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.

  18. Isolation and life-cycle characterization of lytic viruses infecting heterotrophic bacteria and cyanobacteria

    DEFF Research Database (Denmark)

    Middelboe, Mathias; Chan, Amy; Bertelsen, Sif Koldborg

    2010-01-01

    Basic knowledge on viruses infecting heterotrophic bacteria and cyanobacteria is key to future progress in understanding the role of viruses in aquatic systems and the influence of virus–host interactions on microbial mortality, biogeochemical cycles, and genetic exchange. Such studies require......, and discusses the applications and limitations of different isolation procedures. Most work on phage isolation has been carried out with aerobic heterotrophic bacteria and cyanobacteria, culturable both on agar plates and in enriched liquid cultures. The procedures presented here are limited to lytic viruses...

  19. The antigenic property of the H5N1 avian influenza viruses isolated in central China

    Directory of Open Access Journals (Sweden)

    Zou Wei

    2012-08-01

    Full Text Available Abstract Background Three influenza pandemics outbroke in the last century accompanied the viral antigen shift and drift, resulting in the change of antigenic property and the low cross protective ability of the existed antibody to the newly emerged pandemic virus, and eventually the death of millions of people. The antigenic characterizations of the viruses isolated in central China in 2004 and 2006–2007 were investigated in the present study. Results Hemagglutinin inhibition assay and neutralization assay displayed differential antigenic characteristics of the viruses isolated in central China in two periods (2004 and 2006–2007. HA genes of the viruses mainly located in two branches in phylogeny analysis. 53 mutations of the deduced amino acids of the HA genes were divided into 4 patterns. Mutations in pattern 2 and 3 showed the main difference between viruses isolated in 2004 and 2006–2007. Meanwhile, most amino acids in pattern 2 and 3 located in the globular head of the HA protein, and some of the mutations evenly distributed at the epitope sites. Conclusions The study demonstrated that a major antigenic drift had occurred in the viruses isolated in central China. And monitoring the antigenic property should be the priority in preventing the potential pandemic of H5N1 avian influenza virus.

  20. Genetic characterization of a noncytopathic bovine viral diarrhea virus 2b isolated from cattle in China.

    Science.gov (United States)

    Wang, Wei; Shi, Xinchuan; Chen, Chaoyang; Wu, Hua

    2014-10-01

    In January 2013, several clinical signs of cattle with diarrhea, cough, nasal discharge, and fever were reported in Jilin province, China. One virus named SD1301 was isolated and identified. Complete genome of the virus is 12258nt in length and contains a 5'UTR, one open reading frame encoding a polyprotein of 3,897 amino acids and a 3'UTR. Phylogenetic analysis of 5'UTR, N(pro), E1 and E2 gene demonstrated the virus belonged to BVDV 2b, and genetically related to the BVDV strain Hokudai-Lab/09 from Japan in 2010. This bovine viral diarrhea virus displays a unique genetic signature with 27-nucleotide deletion in the 5'UTR, which is similar to the bovine viral diarrhea virus C413 (AF002227). This was the first confirmed isolation of ncp BVDV2b circulating in bovine herd of China.

  1. [Genome sequencing and analysis of a peste des petits ruminants virus isolate, China/Tib/07].

    Science.gov (United States)

    Liu, Wen-Hua; Bao, Jing-Yue; Wu, Xiao-Dong; Wang, Zhi-Liang

    2010-07-01

    Peste des petits ruminants virus is a member of Morbillivirus Paramyxoviridae. The complete genome of a Peste des petits ruminants virus (PPRV) isolate, China/Tib/07 was sequenced and molecular characteristics was analyzed. The internal sequences of the virus genome were amplified by RT-PCR with primers designed according to the published data in GenBank, while the sequences of the 3' and 5' ends of the genome were determined by RACE. Amplification products were directly sequenced,assembled and analyzed with DNAStar4.0. Results showed that China/Tib/07 genome consisted of 15 948 nucleotides in length, encoding six structural proteins and two non-structural proteins just like other known PPRV genomes. Phylogenetically, the virus genome shared homology of 91.6%-98.1% with Southwest Asian isolates among PPRV strains and the highest homology of 64.3% with rinderpest virus among morbillivirus members.

  2. Genomic 3' terminal sequence comparison of three isolates of rabbit haemorrhagic disease virus.

    Science.gov (United States)

    Milton, I D; Vlasak, R; Nowotny, N; Rodak, L; Carter, M J

    1992-05-15

    Comparison of sequence data is necessary in older to investigate virus origins, identify features common to virulent strains, and characterize genomic organization within virus families. A virulent caliciviral disease of rabbits recently emerged in China. We have sequenced 1100 bases from the 3' ends of two independent European isolates of this virus, and compared these with previously determined calicivirus sequences. Rabbit caliciviruses were closely related, despite the different countries in which isolation was made. This supports the rapid spread of a new virus across Europe. The capsid protein sequences of these rabbit viruses differ markedly from those determined for feline calicivirus, but a hypothetical 3' open reading frame is relatively well conserved between the caliciviruses of these two different hosts and argues for a functional role.

  3. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses.

    Science.gov (United States)

    Kuno, G; Gubler, D J; Vélez, M; Oliver, A

    1985-01-01

    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability.

  4. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses*

    Science.gov (United States)

    Kuno, G.; Gubler, D. J.; Vélez, M.; Oliver, A.

    1985-01-01

    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability. PMID:2861916

  5. Phylogenetic analysis of recent isolates of classical swine fever virus from Colombia.

    Science.gov (United States)

    Sabogal, Zonia Yubyll; Mogollón, José Darío; Rincón, Maria Antonia; Clavijo, Alfonso

    2006-01-01

    The ability to discriminate between different classical Swine fever virus (CSFV) isolates is a prerequisite for identifying the possible origin of an outbreak. To determine the relatedness between Colombian isolates from different geographical regions, genetic sequences of the glycoprotein E2 and the 5'UTR of CSFV were amplified by PCR, sequenced and compared with reference strains of different genetic grouping. The viruses originated from classical swine fever (CSF) outbreaks in Colombia during 1998-2002. All viruses characterized belonged to genogroup 1 and were members of the subgroup 1.1. The results indicate that the outbreaks from the year 2002 are caused by a strain related to the virus CSF/Santander, isolated in 1980, suggesting that the current CSF outbreaks are the consequence of a single strain that continues to circulate in the field. For the first time, an association between isolates from outbreaks in Colombia in the 1990s was established with a virus isolate from Brazil, indicating a possible origin of the virus causing the outbreak.

  6. Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil.

    Science.gov (United States)

    Pauvolid-Corrêa, Alex; Solberg, Owen; Couto-Lima, Dinair; Kenney, Joan; Serra-Freire, Nicolau; Brault, Aaron; Nogueira, Rita; Langevin, Stanley; Komar, Nicholas

    2015-01-01

    We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses.

  7. Genetic detection and isolation of crimean-congo hemorrhagic fever virus, Kosovo, Yugoslavia.

    Science.gov (United States)

    Papa, Anna; Bozovi, Bojana; Pavlidou, Vassiliki; Papadimitriou, Evangelia; Pelemis, Mijomir; Antoniadis, Aantonis

    2002-08-01

    Crimean-Congo hemorrhagic fever virus (C-CHFV) strains were isolated from a fatal case and the attending physician in Kosovo, Yugoslavia. Early, rapid diagnosis of the disease was achieved by reverse transcription-polymerase chain reaction. The physician was successfully treated with oral ribavirin. These cases yielded the first genetically studied C-CHFV human isolates in the Balkans.

  8. Biological characterization and complete nucleotide sequence of a Tunisian isolate of Moroccan watermelon mosaic virus.

    Science.gov (United States)

    Yakoubi, S; Desbiez, C; Fakhfakh, H; Wipf-Scheibel, C; Marrakchi, M; Lecoq, H

    2008-01-01

    During a survey conducted in October 2005, cucurbit leaf samples showing virus-like symptoms were collected from the major cucurbit-growing areas in Tunisia. DAS-ELISA showed the presence of Moroccan watermelon mosaic virus (MWMV, Potyvirus), detected for the first time in Tunisia, in samples from the region of Cap Bon (Northern Tunisia). MWMV isolate TN05-76 (MWMV-Tn) was characterized biologically and its full-length genome sequence was established. MWMV-Tn was found to have biological properties similar to those reported for the MWMV type strain from Morocco. Phylogenetic analysis including the comparison of complete amino-acid sequences of 42 potyviruses confirmed that MWMV-Tn is related (65% amino-acid sequence identity) to Papaya ringspot virus (PRSV) isolates but is a member of a distinct virus species. Sequence analysis on parts of the CP gene of MWMV isolates from different geographical origins revealed some geographic structure of MWMV variability, with three different clusters: one cluster including isolates from the Mediterranean region, a second including isolates from western and central Africa, and a third one including isolates from the southern part of Africa. A significant correlation was observed between geographic and genetic distances between isolates. Isolates from countries in the Mediterranean region where MWMV has recently emerged (France, Spain, Portugal) have highly conserved sequences, suggesting that they may have a common and recent origin. MWMV from Sudan, a highly divergent variant, may be considered an evolutionary intermediate between MWMV and PRSV.

  9. Complete Genome Sequence of a Tomato Isolate of Parietaria Mottle Virus from Italy.

    Science.gov (United States)

    Martínez, Carolina; Aramburu, José; Rubio, Luis; Galipienso, Luis

    2015-12-17

    We report here the complete genome sequence of isolate T32 of parietaria mottle virus (PMoV) infecting tomato plants in Turin, Italy, obtained by Sanger sequencing. T32 shares 90.48 to 96.69% nucleotide identity with other two PoMV isolates, CR8 and Pe1, respectively, whose complete genome sequences are available.

  10. Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal

    Science.gov (United States)

    Karlsson, Erik A.; Ip, Hon S.; Hall, Jeffrey S.; Yoon, Sun W.; Johnson, Jordan; Beck, Melinda A.; Webby, Richard J.; Schultz-Cherry, Stacey

    2014-01-01

    The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

  11. Estimating front-wave velocity of infectious diseases: a simple, efficient method applied to bluetongue

    Directory of Open Access Journals (Sweden)

    Pioz Maryline

    2011-04-01

    Full Text Available Abstract Understanding the spatial dynamics of an infectious disease is critical when attempting to predict where and how fast the disease will spread. We illustrate an approach using a trend-surface analysis (TSA model combined with a spatial error simultaneous autoregressive model (SARerr model to estimate the speed of diffusion of bluetongue (BT, an infectious disease of ruminants caused by bluetongue virus (BTV and transmitted by Culicoides. In a first step to gain further insight into the spatial transmission characteristics of BTV serotype 8, we used 2007-2008 clinical case reports in France and TSA modelling to identify the major directions and speed of disease diffusion. We accounted for spatial autocorrelation by combining TSA with a SARerr model, which led to a trend SARerr model. Overall, BT spread from north-eastern to south-western France. The average trend SARerr-estimated velocity across the country was 5.6 km/day. However, velocities differed between areas and time periods, varying between 2.1 and 9.3 km/day. For more than 83% of the contaminated municipalities, the trend SARerr-estimated velocity was less than 7 km/day. Our study was a first step in describing the diffusion process for BT in France. To our knowledge, it is the first to show that BT spread in France was primarily local and consistent with the active flight of Culicoides and local movements of farm animals. Models such as the trend SARerr models are powerful tools to provide information on direction and speed of disease diffusion when the only data available are date and location of cases.

  12. Estimating front-wave velocity of infectious diseases: a simple, efficient method applied to bluetongue.

    Science.gov (United States)

    Pioz, Maryline; Guis, Hélène; Calavas, Didier; Durand, Benoît; Abrial, David; Ducrot, Christian

    2011-04-20

    Understanding the spatial dynamics of an infectious disease is critical when attempting to predict where and how fast the disease will spread. We illustrate an approach using a trend-surface analysis (TSA) model combined with a spatial error simultaneous autoregressive model (SAR(err) model) to estimate the speed of diffusion of bluetongue (BT), an infectious disease of ruminants caused by bluetongue virus (BTV) and transmitted by Culicoides. In a first step to gain further insight into the spatial transmission characteristics of BTV serotype 8, we used 2007-2008 clinical case reports in France and TSA modelling to identify the major directions and speed of disease diffusion. We accounted for spatial autocorrelation by combining TSA with a SAR(err) model, which led to a trend SAR(err) model. Overall, BT spread from north-eastern to south-western France. The average trend SAR(err)-estimated velocity across the country was 5.6 km/day. However, velocities differed between areas and time periods, varying between 2.1 and 9.3 km/day. For more than 83% of the contaminated municipalities, the trend SAR(err)-estimated velocity was less than 7 km/day. Our study was a first step in describing the diffusion process for BT in France. To our knowledge, it is the first to show that BT spread in France was primarily local and consistent with the active flight of Culicoides and local movements of farm animals. Models such as the trend SAR(err) models are powerful tools to provide information on direction and speed of disease diffusion when the only data available are date and location of cases.

  13. Economic comparison of the monitoring programmes for bluetongue vectors in Austria and Switzerland.

    Science.gov (United States)

    Pinior, B; Brugger, K; Köfer, J; Schwermer, H; Stockreiter, S; Loitsch, A; Rubel, F

    2015-05-02

    With the bluetongue virus serotype 8 (BTV-8) outbreak in 2006, vector monitoring programmes (according to EU regulation 1266/2007) were implemented by European countries to obtain information on the spatial distribution of vectors and the vector-free period. This study investigates the vector monitoring programmes in Austria and Switzerland by performing a retrospective cost analysis for the period 2006-2010. Two types of costs were distinguished: costs financed directly via the national bluetongue programmes and costs contributed in-kind by the responsible institutions and agricultural holdings. The total net costs of the monitoring programme in Austria amounted to €1,415,000, whereby in Switzerland the costs were valued at €94,000. Both countries followed the legislation complying with requirements, but differed in regard to sampling frequency, number of trap sites and sampling strategy. Furthermore, the surface area of Austria is twice the area of Switzerland although the number of ruminants is almost the same in both countries. Thus, for comparison, the costs were normalised with regard to the sampling frequency and the number of trap sites. Resulting costs per trap sample comprised €164 for Austria and €48 for Switzerland. In both countries, around 50 per cent of the total costs can be attributed to payments in-kind. The benefit of this study is twofold: first, veterinary authorities may use the results to improve the economic efficiency of future vector monitoring programmes. Second, the analysis of the payment in-kind contribution is of great importance to public authorities as it makes the available resources visible and demonstrates how they have been used.

  14. Complete nucleotide sequence analysis of a Dengue-1 virus isolated on Easter Island, Chile.

    Science.gov (United States)

    Cáceres, C; Yung, V; Araya, P; Tognarelli, J; Villagra, E; Vera, L; Fernández, J

    2008-01-01

    Dengue-1 viruses responsible for the dengue fever outbreak in Easter Island in 2002 were isolated from acute-phase sera of dengue fever patients. In order to analyze the complete genome sequence, we designed primers to amplify contiguous segments across the entire sequence of the viral genome. RT-PCR products obtained were cloned, and complete nucleotide and deduced amino acid sequences were determined. This report constitutes the first complete genetic characterization of a DENV-1 isolate from Chile. Phylogenetic analysis shows that an Easter Island isolate is most closely related to Pacific DENV-1 genotype IV viruses.

  15. Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005.

    Science.gov (United States)

    Fringe, Raul; Bosman, Anna-Mari; Ebersohn, Karen; Bisschop, Shahn; Abolnik, Celia; Venter, Estelle

    2012-08-31

    Newcastle disease (ND) is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV) virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F) protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28), as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in Mozambique.

  16. Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005

    Directory of Open Access Journals (Sweden)

    Raul Fringe

    2012-02-01

    Full Text Available Newcastle disease (ND is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28, as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in

  17. Construction and identification of reverse genetics system of Dengue type 2 virus isolated in China

    Institute of Scientific and Technical Information of China (English)

    ZHU Wuyang; CHEN Shuiping; QIN Chenggeng; YU Man; JIANG Tao; DENG Yongqiang; QIN Ede

    2006-01-01

    To construct infectious full-length cDNA clone of dengue virus type 2 isolated in China (DEN2-43), according to the published nucleotide sequence of the virus strain, the approximately 11 kb full-length cDNAs of DEN2-43 were amplified by long RT-PCR and fusion PCR. Full-length cDNA clones were constructed by inserting the full-length cDNA into a low copy vector pWSK29, from which rescued virus D212 was acquired by transcription in vitro and electroporation. The full-length cDNA clone pD212 was infectious, and rescued virus acquired in C6/36 cells was indistinguishable from DEN2-43 virus in biological properties including suckling mice neurovirulence. The reverse genetics system helps elucidate the mechanism of pathogenesis of dengue virus and develop novel vaccine against dengue.

  18. GENOTYPING OF ISOLATED VIRUSES FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS IN CROATIA

    Directory of Open Access Journals (Sweden)

    Irena Vardić

    2007-07-01

    Full Text Available Infectious hematopoietic necrosis virus (IHNV and infectious pancreatic necrosis virus (IPNV are important pathogens in rainbow trout aquaculture. Detection of these viruses in Croatia initiated investigation of their genetic relatedness to the worldwide IHNV and IPNV isolates. For this purpose, determination of nucleotide sequences of G and NV genes for IHNV and VP2/NS region for IPNV was performed. Phylogenetic analyses revealed that Croatian IHNV isolate was clustering within European clade most closely related to the North American M genogroup. Croatian IPNV isolate appeared in the cluster of genogroup III, together with French, English, Danish and Norwegian isolates. These results are important for further epidemiological studies of IHNV and IPNV outbreaks in Croatia.

  19. Characterization of West Nile viruses isolated from captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

    Science.gov (United States)

    Osorio, Jorge E; Ciuoderis, Karl A; Lopera, Juan G; Piedrahita, Leidy D; Murphy, Darby; Levasseur, James; Carrillo, Lina; Ocampo, Martha C; Hofmeister, Erik

    2012-09-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

  20. Characterization of West Nile viruses isolated form captive American flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

    Science.gov (United States)

    Osorio, Jorge E.; Ciuoderis, Karl A.; Lopera, Juan G.; Piedrahita, Leidy D.; Murphy, Darby; LeVasseur, James; Carrillo, Lina; Ocampo, Martha C.; Hofmeister, Erik

    2012-01-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

  1. Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    OpenAIRE

    Nishizawa, T; Kinoshita, S; Kim, W. -S.; Higashi, S.; Yoshimizu, M

    2006-01-01

    Infectious hematopoietic necrosis virus (IHNV), a member of the genus Novirhabdovirus, causes a highly lethal disease of salmonid fish. In the present study, G gene nucleotide sequences of 9 Japanese IHNV isolates obtained from 1971 to 1996 were analyzed to evaluate the genetic diversity and compared with IHNV isolates from North America and Europe. A radial phylogenetic tree revealed 5 major clusters including 3 genogroups (U, M and L) for North American isolates and 1 genogroup for European...

  2. Molecular characterization of Belgian pseudorabies virus isolates from domestic swine and wild boar.

    Science.gov (United States)

    Verpoest, Sara; Cay, Ann Brigitte; De Regge, Nick

    2014-08-06

    Aujeszky's disease is an economically important disease in domestic swine caused by suid herpesvirus 1, also called pseudorabies virus (PRV). In several European countries, including Belgium, the virus has successfully been eradicated from the domestic swine population. The presence of PRV in the wild boar population however poses a risk for possible reintroduction of the virus into the domestic pig population. It is therefore important to assess the genetic relatedness between circulating strains and possible epidemiological links. In this study, nine historical Belgian domestic swine isolates that circulated before 1990 and five recent wild boar isolates obtained since 2006 from Belgium and the Grand Duchy of Luxembourg were genetically characterized by restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis. While all wild boar isolates were characterized as type I RFLP genotypes, the RFLP patterns of the domestic swine isolates suggest that a shift from genotype I to genotype II might have occurred in the 1980s in the domestic population. By phylogenetic analysis, Belgian wild boar isolates belonging to both clade A and B were observed, while all domestic swine isolates clustered within clade A. The joint phylogenetic analysis of both wild boar and domestic swine strains showed that some isolates with identical sequences were present within both populations, raising the question whether these strains represent an increased risk for reintroduction of the virus into the domestic population.

  3. Characterization of influenza A viruses isolated from wild waterfowl in Zambia.

    Science.gov (United States)

    Simulundu, Edgar; Ishii, Akihiro; Igarashi, Manabu; Mweene, Aaron S; Suzuki, Yuka; Hang'ombe, Bernard M; Namangala, Boniface; Moonga, Ladslav; Manzoor, Rashid; Ito, Kimihito; Nakamura, Ichiro; Sawa, Hirofumi; Sugimoto, Chihiro; Kida, Hiroshi; Simukonda, Chuma; Chansa, Wilbroad; Chulu, Jack; Takada, Ayato

    2011-06-01

    Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.

  4. Genetic characterization and pathogenicity assessment of Newcastle disease virus isolated from wild peacock.

    Science.gov (United States)

    Khulape, Sagar A; Gaikwad, Satish S; Chellappa, Madhan Mohan; Mishra, Bishnu Prasad; Dey, Sohini

    2014-12-01

    The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 3'-N-P-M-F-HN-L-5', which was limited by 55-nts leader region at the 3' end and a 114-nts trailer sequence at 5' end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site (112)R-R-Q-K-R-F(117), a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population.

  5. Characterization of influenza A (H7N9 viruses isolated from human cases imported into Taiwan.

    Directory of Open Access Journals (Sweden)

    Ji-Rong Yang

    Full Text Available A novel avian influenza A (H7N9 virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave. The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.

  6. Molecular Characteristics of S1 Gene of Infectious Bronchitis Virus Isolated from Chicken Proventriculus

    Institute of Scientific and Technical Information of China (English)

    CHENG Li-qin; ZHOU Ji-yong; John Dikki; SHEN Xing-yan; CHEN Ji-gang; ZHANG De-yong

    2003-01-01

    Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. Thesuspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation andidentification. All the virus isolates were able to agglutinate chicken erythrocytes after treatment with trypsin,and interfer with the reproduction of Newcastle disease virus in chicken embryos, and have low antigenic relat-edness values with reference positive IBV. The isolates 2/97, 3/97, 1/98 RNAs extracted from the allantoicfluid of inoculated embryonated eggs were converted to cDNA by reverse transcription with 3'-primer of S1gene of (IBV). Polymerase chain reaction (PCR) was performed with two primers which span the S1 gene.Amplified product of 1.93 kb was subjected to EcoR Ⅰ and BamH Ⅰ digestion and the fragments obtainedwere the same as expected size. The PCR product was ligated to pBlueScript-SK (+) vector, and its nucleotidesequence was determined by the dideoxy-mediated chain termination method. Nucleotide sequence analysisshowed 73.6 - 99.7 % homology between the isolated IBV and the IBV strains in GenBank. The homology ofamino acid was 71.4 - 99.4 %.

  7. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

    Science.gov (United States)

    2016-01-01

    Avian leukosis virus (ALV) belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate. PMID:27597865

  8. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

    Directory of Open Access Journals (Sweden)

    Faruku Bande

    2016-01-01

    Full Text Available Avian leukosis virus (ALV belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate.

  9. Genetic diversity of Japanese encephalitis virus isolates obtained from the Indonesian archipelago between 1974 and 1987.

    Science.gov (United States)

    Schuh, Amy J; Guzman, Hilda; Tesh, Robert B; Barrett, Alan D T

    2013-07-01

    Five genotypes (GI-V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI-III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate

  10. Nucleotide sequencing and serologic analysis of Cache Valley virus isolates from the Yucatan Peninsula of Mexico.

    Science.gov (United States)

    Blitvich, Bradley J; Loroño-Pino, Maria A; Garcia-Rejon, Julian E; Farfan-Ale, Jose A; Dorman, Karin S

    2012-08-01

    Nucleotide sequencing was performed on part of the medium and large genome segments of 17 Cache Valley virus (CVV) isolates from the Yucatan Peninsula of Mexico. Alignment of these sequences to all other sequences in the Genbank database revealed that they have greatest nucleotide identity (97-98 %) with the equivalent regions of Tlacotalpan virus (TLAV), which is considered to be a variety of CVV. Next, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice that had been inoculated with a representative isolate from the Yucatan Peninsula (CVV-478) or the prototype TLAV isolate (61-D-240). The PRNT titers exhibited a twofold difference in one direction and no difference in the other direction suggesting that CVV-478 and 61-D-240 belong to the same CVV subtype. In conclusion, we demonstrate that the CVV isolates from the Yucatan Peninsula of Mexico are genetically and antigenically similar to the prototype TLAV isolate.

  11. IDENTIFICATION AND EFFECTS OF MIXED INFECTION OF Potyvirus ISOLATES WITH Cucumber mosaic virus IN CUCURBITS

    Directory of Open Access Journals (Sweden)

    GRAZIELA DA SILVA BARBOSA

    2016-01-01

    Full Text Available Mixed infections in cucurbits are frequently observed in natural conditions between viruses from the Potyvirus genus and Cucumber mosaic virus (CMV, which significantly decreases productivity. The objectives of the present study was to compare the host range of PRSV - W, WMV, and ZYMV isolates and evaluate the effects of mixed infections with CMV in zucchini plants ( Cucurbita pepo L.. Host range studies comprising 23 plant species confirmed some similarities and biological differences among the isolates of PRSV - W, ZYMV, and WMV. RT - PCR confirmed the amplification of DNA fragments of the PRSV - W, WMV, and ZYMV coat protein gene ( cp and cytoplasm inclusion gene ( ci . The virus interaction studies in zucchini Caserta plants indicated synergistic interactions, particularly among species from the Potyvirus genus, and some CMV interference with some virus combinations.

  12. Molecular characterization of the complete genome of a street rabies virus WH11 isolated from donkey in China.

    Science.gov (United States)

    Xie, Tingbo; Yu, Hua; Wu, Jie; Ming, Pinggang; Huang, Sijia; Shen, Zhijun; Xu, Gelin; Yan, Jiaxin; Yu, Bin; Zhou, Dunjin

    2012-12-01

    The complete genomic sequence of a rabies virus isolate WH11, isolated from brain tissue of a rabid donkey in China, was determined and compared with other rabies viruses. This is the first Chinese street strain which was isolated from donkey and the entire length and organization of the virus was similar to that of other rabies viruses. Multiple alignments of amino acid sequences of the nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and large protein of WH11 with those of other rabies viruses were undertaken to examine the conservative degree of functional regions. Phylogenetic analysis using the complete genomic sequence of WH11 determined that this isolate is most closely related with rabies viruses previously isolated in China and the attenuated Chinese vaccine strain CTN181.

  13. Existence of variant strains Fowlpox virus integrated with Reticuloendotheliosis virus in its genome in field isolates in Tanzania.

    Science.gov (United States)

    Mzula, Alexanda; Masola, Selemani N; Kasanga, Christopher J; Wambura, Philemon N

    2014-06-01

    Fowlpox virus (FPV) is one example of poultry viruses which undergoes recombination with Reticuloendotheliosis virus (REV). Trepidation had been raised, and it was well established on augmented pathogenicity of the FPV upon integration of the full intact REV. In this study, we therefore intended at assessing the integration of REV into FPV genome of the field isolates obtained in samples collected from different regions of Tanzania. DNA extraction of 85 samples (scabs) was performed, and FPV-specific PCR was done by the amplification of the highly conserved P4b gene. Evaluation of FPV-REV recombination was done to FPV-specific PCR positively identified samples by amplifying the env gene and REV long terminal repeats (5' LTR). A 578-bp PCR product was amplified from 43 samples. We are reporting for the first time in Tanzania the existence of variant stains of FPV integrated with REV in its genome as 65 % of FPV identified isolates were having full intact REV integration, 21 % had partial FPV-REV env gene integration and 5 % had partial 5' LTR integration. Despite of the fact that FPV-REV integrated stains prevailed, FPV-REV-free isolates (9 %) also existed. In view of the fact that full intact REV integration is connected with increased pathogenicity of FPV, its existence in the FPV genome of most field isolates could have played a role in increased endemic, sporadic and recurring outbreaks in selected areas in Tanzania.

  14. Isolation of a virulent Newcastle disease virus from confiscated LaSota vaccine.

    Science.gov (United States)

    Pedersen, Janice C; Hines, Nichole L; Killian, Mary Lea; Predgen, Ann S; Schmitt, Beverly J

    2013-06-01

    Vials of Newcastle disease vaccine labeled as LaSota were confiscated by the Arizona Division of Customs and Border Protection officials. Two different avian type 1 paramyxoviruses were isolated from all three vials of vaccine submitted to the National Veterinary Services Laboratories. The LaSota strain of avian paramyxovirus type 1 virus was isolated from all three vials and analyzed by nucleotide sequence analysis. A virulent Newcastle disease virus was also present in all three vials, but in low concentration. The virulence of the Newcastle disease virus was characterized by the intracerebral chicken pathogenicity index chicken inoculation assay but could not be determined by nucleotide sequence analysis from the virus isolated from embryonating chicken eggs. The intracerebral chicken pathogenicity index value for the isolated Newcastle disease virus was 1.55. Strains of Newcastle disease virus with intracerebral pathogenicity indexes significantly above 1.0 have been found to selectively kill many types of cancer cells while not affecting normal nonneoplastic cells and are considered to be a viable option for cancer treatment in humans by alternative medical researchers; however, the treatment is not approved for use in the United States by the Food and Drug Administration. Customs and Border Protection officials have been notified of an increased risk of Newcastle disease virus entering the United States for use as a nonapproved cancer treatment. Illegal importation of Newcastle disease vaccine for vaccination of backyard poultry is also a threat. This case report emphasizes the importance of conducting chicken inoculation for complete virus pathotyping and demonstrates the need for stringent security procedures at U.S. borders to detect known livestock pathogens that may be smuggled in for use in animal agriculture and reasons unrelated to animal agriculture.

  15. Heterogeneity in pepper isolates of cucumber mosaic virus

    Science.gov (United States)

    Rodriguez-Alvarado, G.; Kurath, G.; Dodds, J.A.

    1995-01-01

    Twenty-four cucumber mosaic cucumovirus (CMV) field isolates from pepper crops in Cali-fornia were characterized and compared by nucleic acid hybridization subgrouping, virion electrophoresis, and biological effects in several hosts. Isolates, belonging to subgroup I or subgroup II, were found that induced severe symptoms in mechanically inoculated bell pep-pers. Only two isolates, both from subgroup II, were mild. A group of 19 isolates collected from a single field were all in subgroup II and appeared identical by virion electrophoresis, but they exhibited varying degrees of symptom severity in peppers. As a more detailed indicator of heterogeneity, these 19 isolates were examined by RNase protection assays to delect sequence variation in the coat protein gene region of their genomes. The patterns of bands observed were complex and a high degree of genomic heterogeneity was detected between isolates, with no apparent correlation to symptomatology in bell pepper.

  16. The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

    Institute of Scientific and Technical Information of China (English)

    程晔; 陈炯; 陈剑平

    2002-01-01

    The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3′-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and CI proteins.

  17. Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

    Science.gov (United States)

    Dornas, Fábio P; Silva, Lorena C F; de Almeida, Gabriel M; Campos, Rafael K; Boratto, Paulo V M; Franco-Luiz, Ana P M; La Scola, Bernard; Ferreira, Paulo C P; Kroon, Erna G; Abrahão, Jônatas S

    2014-01-01

    Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

  18. Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

    Directory of Open Access Journals (Sweden)

    Fábio P Dornas

    Full Text Available Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water and hospital (ventilator plastic device tube substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

  19. Sequence analysis and genetic diversity of five new Indian isolates of cucumber mosaic virus.

    Science.gov (United States)

    Kumar, S; Gautam, K K; Raj, S K

    2015-12-01

    Cucumber mosaic virus (CMV) is an important virus since it causes severe losses to many economically important crops worldwide. Five new isolates of CMV were isolated from naturally infected Hippeastrum hybridum, Dahlia pinnata, Hemerocallis fulva, Acorus calamus and Typhonium trilobatum plants, all exhibiting severe leaf mosaic symptoms. For molecular identification and sequence analyses, the complete coat protein (CP) gene of these isolates was amplified by RT-PCR. The resulting amplicons were cloned and sequenced and isolates were designated as HH (KP698590), DP (JF682239), HF (KP698589), AC (KP698588) and TT (JX570732). For study of genetic diversity among these isolates, the sequence data were analysed by BLASTn, multiple alignment and generating phylogenetic trees along with the respective sequences of other CMV isolates available in GenBank Database were done. The isolates under study showed 82-99% sequence diversity among them at nucleotide and amino acid levels; however they showed close relationships with CMV isolates of subgroup IB. In alignment analysis of amino acid sequences of HH and AC isolates, we have found fifteen and twelve unique substitutions, compared to HF, DP and TT isolates, suggesting the cause of high genetic diversity.

  20. Molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from Saudi Arabia.

    Science.gov (United States)

    Almajhdi, Fahad N; Alshaman, Mohamed S; Amer, Haitham M

    2012-08-01

    Human parainfluenza virus 3 (HPIV-3) is a leading cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most variable gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this report, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two successive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in comparison to 20 strains isolated by others from different countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis confirmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution.

  1. Phylogenetic analysis of surface proteins of novel H1N1 virus isolated from 2009 pandemic.

    Science.gov (United States)

    Danishuddin, Mohd; Khan, Shahper N; Khan, Asad U

    2009-09-30

    Swine Influenza Virus (H1N1) is a known causative agent of swine flu. Transmission of Swine Influenza Virus form pig to human is not a common event and may not always cause human influenza. The 2009 outbreak by subtype H1N1 in humans is due to transfer of Swine Influenza Virus from pig to human. Thus to analyze the origin of this novel virus we compared two surface proteins (HA and NA) with influenza viruses of swine, avian and humans isolates recovered from 1918 to 2008 outbreaks. Phylogenetic analyses of hemagglutinin gene from 2009 pandemic found to be clustered with swine influenza virus (H1N2) circulated in U.S.A during the 1999-2004 outbreaks. Whereas, neuraminidase gene was clustered with H1N1 strains isolated from Europe and Asia during 1992-2007 outbreaks. This study concludes that the new H1N1 strain appeared in 2009 outbreak with high pathogenicity to human was originated as result of re-assortment (exchange of gene). Moreover, our data also suggest that the virus will remain sensitive to the pre-existing therapeutic strategies.

  2. Receptor binding properties of human and animal H1 influenza virus isolates.

    Science.gov (United States)

    Rogers, G N; D'Souza, B L

    1989-11-01

    It has been previously reported that several human H1 influenza viruses isolated prior to 1956, in contrast to human H3 isolates which are quite specific for SA alpha 2,6Gal sequences, apparently recognize both SA alpha 2,3Gal and SA alpha 2,6Gal sequences (Rogers, G.N., and Paulson, J.C., Virology 127, 361-373, 1983). In this report human H1 isolates representative of two epidemic periods, from 1934 to 1957 and from 1977 to 1986, and H1 influenza isolated from pigs, ducks, and turkeys were compared for their ability to utilize sialyloligosaccharide structures containing terminal SA alpha 2,3Gal or SA alpha 2,6Gal sequences as receptor determinants. Five of the eight human isolates from the first epidemic period recognize both SA alpha 2,3Gal and SA alpha 2,6Gal linkages, in agreement with our previous results. Of the remaining three strains, all isolated towards the end of the first epidemic, two appear to prefer SA alpha 2,6Gal sequences while the third preferentially binds SA alpha 2,3Gal sequences. In contrast to the early isolates, 11 of 13 human strains isolated during the second epidemic period preferentially bind SA alpha 2,6Gal containing oligosaccharides. On the basis of changes in receptor binding associated with continued passage in the laboratory for some of these later strains, it seems likely that human H1 isolates preferentially bind SA alpha 2,6Gal sequences in nature, and that acquisition of SA alpha 2,3Gal-binding is associated with laboratory passage. Influenza H1 viruses isolated from pigs were predominantly SA alpha 2,6Gal-specific while those isolated from ducks were primarily SA alpha 2,3Gal-specific. Thus, as has been previously reported for H3 influenza isolates, receptor specificity for influenza H1 viruses appears to be influenced by the species from which they were isolated, human isolates binding preferentially to SA alpha 2,6Gal-containing oligosaccharides while those isolated from ducks prefer SA alpha 2,3Gal

  3. Fish viruses: isolation of an icosahedral cytoplasmic deoxyribovirus from sheatfish (Silurus glanis).

    Science.gov (United States)

    Ahne, W; Schlotfeldt, H J; Thomsen, I

    1989-07-01

    An icosahedral cytoplasmic deoxyvirus has been isolated from moribund sheatfish (Silurus glanis) fry of a commercial warm water recirculation aquaculture unit with cumulative mortalities of up to 100%. The agent replicated in BF-2 and in FHM cells at 20-30 degrees C producing cytoplasmatic inclusion bodies followed by lysis of the cells. The DNA containing virus proved to be labile to chloroform. Infected BF-2 cells revealed hexagonal particles in the cytoplasm measuring about 125-135 nm in diameter. The virus consisted of a central electron-dense core and a electron-translucent zone. The isolate shares characteristics with the Iridoviridae.

  4. Prevalence of HIV infection in seronegative high-risk individuals examined by virus isolation and PCR

    DEFF Research Database (Denmark)

    Nielsen, C; Teglbjærg, Lars Stubbe; Pedersen, C;

    1991-01-01

    HIV seronegative individuals with high-risk behavior were tested for HIV infection by sensitive virus isolation techniques using T4 lymphocytes and monocyte/macrophages, and by detection of proviral DNA using PCR with three different sets of nested primers. No evidence of HIV infection was found...... among the 31 seronegative high-risk subjects, either by virus isolation of by PCR (97.5% confidence limits, 0-11). Our results indicate that ongoing HIV infection in seronegative persons at high risk of infection is a rare event....

  5. [Genetic characterisation of Powassan virus (POWV) isolated from Haemophysalis longicornis ticks in Primorye and two strains of Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus): Alma-Arasan virus (AAV) isolated from Ixodes persulcatus ticks in Kazakhstan and Malyshevo virus isolated from Aedes vexans nipponii mosquitoes in Khabarovsk kray].

    Science.gov (United States)

    L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Deriabin, P G; Gitel'man, A K; Botikov, A G; Aristova, V A

    2014-01-01

    The complete genomes of the three tick-borne flaviviruses (genus Flavivirus, fam. Bunyaviridae) were sequenced: Povassan virus (POWV, strain LEIV-3070Prm, isolated from Haemophysalis logicornis in Primorsky Krai, Russia in 1977), Alma-Arasan virus (AAV, strain LEIV-1380Kaz, isolated from Ixodes persulcatus ticks in Kazakhstan in 1977) and Malyshevo virus (isolated from a pool of Aedes vexans nipponii mosquitoes, in the Khabarovsk Krai, Russia in 1978). It is shown that AAV and Malyshevo virus are the strains of Tick-borne encephalitis virus (TBEV) and belong to Sibirian and Far-Eastern genotypes, respectively (GenBank ID: AAV KJ744033; strain Malyshevo KJ744034). Phylogenetically AAV is closest related (94,6% nt and 98,3% aa identity) to TBEV strains, isolated in Sibiria (Vasilchenko, Aino, Chita-653, Irkutsk-12). Malyshevo virus is closest related (96,4% nt and 98,3% nt identity) to strains of TBEV, isolated in Far Eastern part of Russia (1230, Spassk-72, Primorye-89). POWV LEIV-3070Prm has 99.7% identity with the prototype strain POWV LB, isolated in Canada and 99.5% of isolates with Far-Eastern strains of POWV (Spassk-9 and Nadezdinsk-1991).

  6. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presence...

  7. Molecular analysis of hepatitis B virus isolates in Mexico: Predominant circulation of hepatitis B virus genotype H

    Institute of Scientific and Technical Information of China (English)

    Cosme Alvarado-Esquivel; Erwin Sablon; Carlos Jesús Conde-González; Luis Juárez-Figueroa; Lilia Ruiz-Maya; Sergio Aguilar-Benavides

    2006-01-01

    AIM:To determine the genotypes in Mexican hepatitis B virus (HBV) isolates and characterize their precore and core promoter mutations.METHODS: Forty-nine HBV isolates of Mexico obtained from sera of 15 hepatitis patients, 6 hemodialysis patients, 20 men seeking HIV testing, and 8 AIDS patients were analyzed. HBV isolates were amplified by PCR,and genotyped by line probe assay (INNO-LiPA HBV Genotyping; INNOGENETICS N V, Ghent, Belgium).HBV genotype confirmation was performed by DNA sequencing part of the sAg region. Precore and core promoter mutation characterization was performed by line probe assay (INNO-LiPA HBV PreCore; INNOGENETICS N.V.; Ghent, Belgium).RESULTS: Overall, HBV genotype H was found in 37(75.5%) out of the 49 isolates studied. HBV genotypes G, A, and D were found in 5 (10.2%), 4 (8.2%), and 3(6.1%) isolates, respectively. HBV genotype H was predominant in isolates from hemodialysis patients (100%),hepatitis patients (80%), and men seeking HIV testing (75%), and accounted for half of infections in AIDS patients (50%). Six (12.2%) out of the 49 HBV isolates showed both wild type and mutant populations at precore codon 28. These mixed wild type and precore murant populations were observed in one HBV genotype A isolate and in all HBV genotype G isolates. A dual variant core promoter mutation was observed in 1 (2%) of the isolates, which was genotype H.CONCLUSION: HBV genotype H is highly predominant in HBV isolates of Mexico followed by genotypes G, A and D. A low frequency of precore and core promoter mutations is observed in HBV Mexican isolates.

  8. Molecular characterization of infectious bronchitis viruses isolated from broiler chicken farms in Iran, 2014-2015.

    Science.gov (United States)

    Najafi, Hamideh; Langeroudi, Arash Ghalyanchi; Hashemzadeh, Masoud; Karimi, Vahid; Madadgar, Omid; Ghafouri, Seyed Ali; Maghsoudlo, Hossein; Farahani, Reza Khaltabadi

    2016-01-01

    Infectious bronchitis (IB) is a viral avian disease with economic importance in the world, including Iran. S1 gene sequencing has been used for molecular epidemiological studies and genotypic characterization of infectious bronchitis virus (IBV). A total of 118 IBV isolates were obtained from tissue samples from chickens with clinically suspected IB from Iranian broiler farms (eight provinces, 200 samples). The isolates were confirmed by real-time polymerase chain reaction (PCR) and characterized by sequencing the spike glycoprotein gene. The isolates formed six distinct phylogenetic groups (IS/1494/06 [Var2] like, 4/91-like, IS/720-like, QX-like, IR-1 and Mass-like) that were related to variants isolated in the region. The most frequently detected viruses were of the Var2-like (IS/1494/06-like) genotype, with an overall prevalence of 34 %. Twenty-one percent of the isolates formed a cluster together with the 4/91 IBV type, 10 % were of the QX genotype, and 8 % were of the IS/720 genotype. In addition, 4 % and 3 % of the isolates belonged to the Massachusetts and IR-1 genotype, respectively. For the first time, we have isolated and characterized IBV variants from broiler farms in different provinces of Iran. This study demonstrates a constant evolution of IBV in Iran, demonstrating the need for continuous monitoring and development of new vaccines based on indigenous viruses.

  9. Diversity of Papaya ringspot virus isolates in Puerto Rico

    Science.gov (United States)

    Papaya ringspot virus (PRSV) devastates papaya production worldwide. In Puerto Rico, papaya fields can be completely infected with PRSV within a year of planting. Information about the diversity of the Puerto Rican PRSV population is relevant in order to establish a control strategy in the island. T...

  10. Complete Genome Sequence of Zika Virus Isolated in Mexico, 2016

    Science.gov (United States)

    Peña-Alonso, Rocío; Mendieta-Condado, Edgar; Garcés-Ayala, Fabiola; González-Durán, Elizabeth; Escobar-Escamilla, Noé; Vázquez-Pichardo, Mauricio; Torres-Rodríguez, María de la Luz; Núñez-León, Alma; Torres-Longoria, Belem; López-Martínez, Irma; Ruíz-Matus, Cuitláhuac; Kuri-Morales, Pablo

    2016-01-01

    Zika virus belongs to the genus Flavivirus, and its spread remains an international public health emergency. In this report, we describe the obtainment and molecular characterization of a complete viral genome through the direct metagenomic analysis from saliva from an autochthonous transmission case in Mexico. PMID:27491989

  11. Complete Genome Sequence of Zika Virus Isolated in Mexico, 2016.

    Science.gov (United States)

    Díaz-Quiñonez, José Alberto; Peña-Alonso, Rocío; Mendieta-Condado, Edgar; Garcés-Ayala, Fabiola; González-Durán, Elizabeth; Escobar-Escamilla, Noé; Vázquez-Pichardo, Mauricio; Torres-Rodríguez, María de la Luz; Núñez-León, Alma; Torres-Longoria, Belem; López-Martínez, Irma; Ruíz-Matus, Cuitláhuac; Kuri-Morales, Pablo; Ramírez-González, José Ernesto

    2016-08-04

    Zika virus belongs to the genus Flavivirus, and its spread remains an international public health emergency. In this report, we describe the obtainment and molecular characterization of a complete viral genome through the direct metagenomic analysis from saliva from an autochthonous transmission case in Mexico.

  12. Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates

    DEFF Research Database (Denmark)

    Johansson, Tove; Einer-Jensen, Katja; Batts, William;

    2009-01-01

    /98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than......Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13...... the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay...

  13. Genomic analysis of highly virulent Georgia 2007/1 isolate of African swine fever virus.

    Science.gov (United States)

    Chapman, David A G; Darby, Alistair C; Da Silva, Melissa; Upton, Chris; Radford, Alan D; Dixon, Linda K

    2011-04-01

    African swine fever is widespread in Africa but has occasionally been introduced into other continents. In June 2007, African swine fever was isolated in the Caucasus Region of the Republic of Georgia and subsequently in neighboring countries (Armenia, Azerbaijan, and 9 states of the Russian Federation). Previous data for sequencing of 3 genes indicated that the Georgia 2007/1 isolate is closely related to isolates of genotype II, which has been identified in Mozambique, Madagascar, and Zambia. We report the complete genomic coding sequence of the Georgia 2007/1 isolate and comparison with other isolates. A genome sequence of 189,344 bp encoding 166 open reading frames (ORFs) was obtained. Phylogeny based on concatenated sequences of 125 conserved ORFs showed that this isolate clustered most closely with the Mkuzi 1979 isolate. Some ORFs clustered differently, suggesting that recombination may have occurred. Results provide a baseline for monitoring genomic changes in this virus.

  14. Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China.

    Science.gov (United States)

    Wang, Jingjing; Lv, Yan; Zhang, Yi; Zheng, Dongxia; Zhao, Yunling; Castellan, David; Liu, Hualei; Wang, Zhiliang

    2016-01-01

    One class I Newcastle disease virus (NDV), designated as duck/Guangxi/1261/2015 (GX1261), was isolated from asymptomatic ducks in live bird markets (LBM) from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China.

  15. Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China.

    Directory of Open Access Journals (Sweden)

    Jingjing Wang

    Full Text Available One class I Newcastle disease virus (NDV, designated as duck/Guangxi/1261/2015 (GX1261, was isolated from asymptomatic ducks in live bird markets (LBM from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China.

  16. Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M.

    Science.gov (United States)

    Enzmann, P J; Kurath, G; Fichtner, D; Bergmann, S M

    2005-09-23

    Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

  17. An avian leukosis virus subgroup J isolate with a Rous sarcoma virus-like 5'-LTR shows enhanced replication capability.

    Science.gov (United States)

    Gao, Yanni; Guan, Xiaolu; Liu, Yongzhen; Li, Xiaofei; Yun, Bingling; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Wang, Xiaomei; Gao, Yulong

    2015-01-01

    Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.

  18. Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Nielsen, Jens; Bille-Hansen, Vivi

    1994-01-01

    The first case of porcine reproductive and respiratory syndrome (PRRS) in Denmark was diagnosed in March 1992 by the detection of specific antibodies against PRRS virus in serum samples originating from sows in a herd located on the island of Als. Subsequently, PRRS virus was isolated from a 200......-sow farrow-to-finish herd with clinical signs consistent with PRRS. The virus was isolated by inoculation of pleural fluid from a stillborn piglet onto porcine pulmonary alveolar macrophages. The isolate was identified as PRRS virus by staining with a specific antiserum. By electron microscopy...... infection was demonstrated by the re-isolation of PRRS virus from approximately 45% of the piglets from the experimentally infected gilts. However, the experimental infection produced no significant reproductive disorders or other clinical signs. At autopsy, histopathological examination revealed slight...

  19. Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene.

    Science.gov (United States)

    Nishizawa, T; Kinoshita, S; Kim, W S; Higashi, S; Yoshimizu, M

    2006-08-30

    Infectious hematopoietic necrosis virus (IHNV), a member of the genus Novirhabdovirus, causes a highly lethal disease of salmonid fish. In the present study, G gene nucleotide sequences of 9 Japanese IHNV isolates obtained from 1971 to 1996 were analyzed to evaluate the genetic diversity and compared with IHNV isolates from North America and Europe. A radial phylogenetic tree revealed 5 major clusters including 3 genogroups (U, M and L) for North American isolates and 1 genogroup for European isolates. Five Japanese isolates from 1971 to 1982 appeared in the cluster for genogroup U, while the remaining Japanese isolates from 1980 to 1996 formed a new genogroup, JRt (Japanese rainbow trout). Maximum nucleotide diversity among the Japanese isolates was 4.5%, which was greater than that within the North American isolates (3.6%), and the degree of nucleotide diversity within Japanese isolates was increased by inclusion of the genogroup JRt isolates. It was concluded that Japanese isolates shared a common source with the genogroup U of the North American isolates and that there were large divergences between Japanese isolates before and after the 1980s.

  20. Isolation and characterization of bovine parainfluenza virus type 3 from water buffaloes (Bubalus bubalis in Argentina

    Directory of Open Access Journals (Sweden)

    Maidana Silvina S

    2012-06-01

    Full Text Available Abstract Background Parainfluenza virus type 3 (PIV3 was isolated from dairy buffaloes (Bubalus bubalis naturally affected with respiratory and reproductive clinical conditions. Results Examination of nasal and vaginal swabs collected from 12 diseased buffaloes led to the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological and biological characteristics of these three isolates were essentially similar to those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3. Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied. Buffalo isolates were similar to genotype B (BPIV3b while the six BPIV3 isolates were similar to genotypes A (BPIV3a and C (BPIV3c. Conclusions This is the first characterization of BPIV3 in water buffalo. According to the samples analyzed, in Argentina, the genotype B was found in buffalo and the genotypes A and C were found in cattle.

  1. Complete Genome Sequences of Zika Virus Strains Isolated from the Blood of Patients in Thailand (2014) and Philippines (2012)

    Science.gov (United States)

    2016-03-09

    Complete genome sequences of Zika Virus strains isolated from the blood of patients in 1 Thailand (2014) and Philippines (2012). 2 Ellison,D.W.1...Institute, Seoul, Republic of Korea. 20 21 Running Head: Zika Virus Genomes 22 23 ABSTRACT 24 ZIKV is an arbovirus and member of the family...genome sequences of two Zika Virus (ZIKV) strains, Zika virus/H.sapiens-27 tc/THA/2014/SV0127-14 and Zika virus/H.sapiens-tc/PHL/2012/CPC-0740, isolated

  2. Characterization of H3N6 avian influenza virus isolated from a wild white pelican in Zambia.

    Science.gov (United States)

    Simulundu, Edgar; Mweene, Aaron S; Tomabechi, Daisuke; Hang'ombe, Bernard M; Ishii, Akihiro; Suzuki, Yuka; Nakamura, Ichiro; Sawa, Hirofumi; Sugimoto, Chihiro; Ito, Kimihito; Kida, Hiroshi; Saiwana, Lewis; Takada, Ayato

    2009-01-01

    We characterized an influenza virus isolated from a great white pelican in Zambia. Phylogenetic analysis showed that all of its gene segments belonged to the Eurasian lineage and that they appear to have evolved in distinct geographical regions in Europe, Asia, and Africa, suggesting reassortment of virus genes maintained in wild aquatic birds whose flyways overlap across these continents. It is notable that this virus might possess some genes of the same origin as those of highly pathogenic H7 and H5 viruses isolated in Eurasia. The present study underscores the need for continued monitoring of avian influenza viruses in Eurasia and Africa.

  3. Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine phytoplankton.

    Science.gov (United States)

    Suttle, C A; Chan, A M; Cottrell, M T

    1991-03-01

    Viruses may be major structuring elements of phytoplankton communities and hence important regulators of nutrient and energy fluxes in aquatic environments. In order to ascertain whether viruses are potentially important in dictating phytoplankton community structure, it is essential to determine the extent to which representative phytoplankton taxa are susceptible to viral infection. We used a spiral ultrafiltration cartridge (30,000-molecular-weight cutoff) to concentrate viruses from seawater at efficiencies approaching 100%. Natural virus communities were concentrated from stations in the Gulf of Mexico, a barrier island pass, and a hypersaline lagoon (Laguna Madre) and added to cultures of potential phytoplankton hosts. By following changes in in vivo fluorescence over time, it was possible to isolate several viruses that were pathogens to a variety of marine phytoplankton, including a prasinophyte (Micromonas pusilla), a pennate diatom (likely a Navicula sp.), a centric diatom (of unknown taxa), and a chroococcoid cyanobacterium (a Synechococcus sp.). As well, we observed changes in fluorescence in cultures of a cryptophyte (a Rhodomonas sp.) and a chlorophyte (Nannochloropsis oculata) which were consistent with the presence of viral pathogens. Although pathogens were isolated from all stations, all the pathogens were not isolated from every station. Filterability studies on the viruses infecting M. pusilla and the Navicula sp. showed that the viruses were consistently infective after filtration through polycarbonate and glass-fiber filters but were affected by most other filter types. Establishment of phytoplankton-pathogen systems will be important in elucidating the effect that viruses have on primary producers in aquatic systems.

  4. Sequence diversity and virulence in Zea mays of Maize streak virus isolates.

    Science.gov (United States)

    Martin, D P; Willment, J A; Billharz, R; Velders, R; Odhiambo, B; Njuguna, J; James, D; Rybicki, E P

    2001-09-30

    Full genomic sequences were determined for 12 Maize streak virus (MSV) isolates obtained from Zea mays and wild grass species. These and 10 other publicly available full-length sequences were used to classify a total of 66 additional MSV isolates that had been characterized by PCR-restriction fragment length polymorphism and/or partial nucleotide sequence analysis. A description is given of the host and geographical distribution of the MSV strain and subtype groupings identified. The relationship between the genotypes of 21 fully sequenced virus isolates and their virulence in differentially MSV-resistant Z. mays genotypes was examined. Within the only MSV strain grouping that produced severe symptoms in maize, highly virulent and widely distributed genotypes were identified that are likely to pose the most serious threat to maize production in Africa. Evidence is presented that certain of the isolates investigated may be the products of either intra- or interspecific recombination.

  5. Characterization of a New Tomato Spotted Wilt Virus Isolates Found in Hippeastrum hybridum (Hort. Plants in Poland

    Directory of Open Access Journals (Sweden)

    Berniak Hanna

    2016-06-01

    Full Text Available Two Tomato spotted wilt virus (TSWV isolates H1 and H2 found in Hippeastrum hybridum plants were characterized based on biological, serological, and molecular properties. Virus isolates showed differences in symptom expression – H1 isolate displayed severe necrotic spots and patterns, whereas mild mosaic symptoms were observed on H2-infected H. hybridum plants. Both TSWV isolates showed comparable reactivity with TSWV-specific antibodies and they induced similar symptoms on herbaceous indicator plants, but some differences between these isolates were detected at the nucleotide sequence level of genomic S and M ssRNAs segment fragments. The nucleotide sequences encoding nucleocapsid (N and nonstructural (NSs and NSm proteins showed 98.2%, 97.5%, and 96.5% identity, respectively. Phylogenetic analysis of N and NSs sequences conducted for tested isolates and 31 TSWV isolates included for comparison revealed that H1 and H2 isolates fell into the same cluster and they were grouped together with isolates found previously in different vegetables, ornamentals, and weeds. When NSm ORF was analyzed, the tested isolates formed a separate cluster: H1 isolate showed the highest affinity with TSWV isolates infecting chrysanthemum and pepper plants, whereas H2 isolate was most closely related to other virus isolates found in sweet pepper and tomatoes. These results indicate that both isolates were reassortants between different virus isolates, and represented two novel genetic patterns of TSWV.

  6. Characterisation of bovine viral diarrhoea virus (BVDV) isolates from an outbreak with haemorrhagic enteritis and severe pneumonia.

    Science.gov (United States)

    Yeşilbağ, Kadir; Förster, Christine; Ozyiğit, M Ozgür; Alpay, Gizem; Tuncer, Pelin; Thiel, Heinz-Jürgen; König, Matthias

    2014-02-21

    During 2007 a disease outbreak occurred in cattle in the Marmara region of western Turkey characterised by severe pneumonia and haemorrhagic enteritis in calves. Cases from three farms at different locations were examined and bovine viral diarrhoea virus (BVDV) isolated in all cases. Phylogenetic characterisation of the virus isolates allocated them in a new cluster tentatively named as BVDV-1r.

  7. Status of sheep sera to bluetongue, peste des petits ruminants and sheep pox in a few northern states of India

    Directory of Open Access Journals (Sweden)

    Vinayagamurthy Balamurugan

    2008-09-01

    Full Text Available Bluetongue (BT, peste des petits ruminants (PPR and sheep pox are the most economically important viral diseases of sheep in India. Serum samples obtained from sheep in five northern states of the country were screened for antibody against these agents to explore the extent of spread of these infections. A total of 516 serum samples were screened for the presence of antibodies against BT and PPR viruses. Of these, 155 samples were also tested for antibodies against sheep pox virus. BT antibodies were found in 293 (56.8% animals, PPR virus antibodies in 215 (41.7% and sheep pox virus antibodies in 106 (68.3%. Of the serum samples tested, 25.2% were positive for antibodies against all three viruses. These findings clearly demonstrated not only the enzootic nature of disease, but also the co-existence of antibodies to more than one of these viruses which would indicate that concurrent infections were common. Therefore, control measures should focus in combating all three diseases simultaneously by exploring the possibility of a trivalent vaccine or the use of multiple genes expressing vectored vaccine.

  8. Analysis of Arbovirus Isolates from Australia Identifies Novel Bunyaviruses Including a Mapputta Group Virus from Western Australia That Links Gan Gan and Maprik Viruses.

    Science.gov (United States)

    Briese, Thomas; Williams, David T; Kapoor, Vishal; Diviney, Sinead M; Certoma, Andrea; Wang, Jianning; Johansen, Cheryl A; Chowdhary, Rashmi; Mackenzie, John S; Lipkin, W Ian

    2016-01-01

    The Mapputta group comprises antigenically related viruses indigenous to Australia and Papua New Guinea that are included in the family Bunyaviridae but not currently assigned to a specific genus. We determined and analyzed the genome sequences of five Australian viruses isolated from mosquitoes collected during routine arbovirus surveillance in Western Australia (K10441, SW27571, K13190, and K42904) and New South Wales (12005). Based on matching sequences of all three genome segments to prototype MRM3630 of Trubanaman virus (TRUV), NB6057 of Gan Gan virus (GGV), and MK7532 of Maprik virus (MPKV), isolates K13190 and SW27571 were identified as TRUV, 12005 as GGV, and K42904 as a Mapputta group virus from Western Australia linking GGV and MPKV. The results confirmed serum neutralization data that had linked SW27571 to TRUV. The fifth virus, K10441 from Willare, was most closely related to Batai orthobunyavirus, presumably representing an Australian variant of the virus. Phylogenetic analysis also confirmed the close relationship of our TRUV and GGV isolates to two other recently described Australian viruses, Murrumbidgee virus and Salt Ash virus, respectively. Our findings indicate that TRUV has a wide circulation throughout the Australian continent, demonstrating for the first time its presence in Western Australia. Similarly, the presence of a virus related to GGV, which had been linked to human disease and previously known only from the Australian southeast, was demonstrated in Western Australia. Finally, a Batai virus isolate was identified in Western Australia. The expanding availability of genomic sequence for novel Australian bunyavirus variants supports the identification of suitably conserved or diverse primer-binding target regions to establish group-wide as well as virus-specific nucleic acid tests in support of specific diagnostic and surveillance efforts throughout Australasia.

  9. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

    OpenAIRE

    Wang Nan; Sun Changjiang; Wang Quankai; Du Rui; Wang Shijie; Gao Yugang; Zhang Pengju; Zhang Lianxue

    2011-01-01

    Abstract Background Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical...

  10. Genomic Characterization of Recent Chicken Anemia Virus Isolates in China

    Science.gov (United States)

    Chicken infectious anemiavirus (CIAV) causes diseases in young chickens, which include increased pathogenicity of secondary infectious agents, generalized lymphoid depletion, and immune-repression. In the present study, we have identified 22 CIAV strains isolated from several commercial chicken farm...

  11. Genomic characterization of influenza A (H7N9) viruses isolated in Shenzhen, Southern China, during the second epidemic wave.

    Science.gov (United States)

    Fang, Shisong; Wang, Xin; Dong, Fangyuan; Jin, Tao; Liu, Guang; Lu, Xing; Peng, Bo; Wu, Weihua; Liu, Hui; Kong, Dongfeng; Tang, Xiujuan; Qin, Yanmin; Mei, Shujiang; Xie, Xu; He, Jianfan; Ma, Hanwu; Zhang, Renli; Cheng, Jinquan

    2016-08-01

    There were three epidemic waves of human infection with avian influenza A (H7N9) virus in 2013-2014. While many analyses of the genomic origin, evolution, and molecular characteristics of the influenza A (H7N9) virus have been performed using sequences from the first epidemic wave, genomic characterization of the virus from the second epidemic wave has been comparatively less reported. In this study, an in-depth analysis was performed with respect to the genomic characteristics of 11 H7N9 virus strains isolated from confirmed cases and four H7N9 virus strains isolated from environmental samples in Shenzhen during the second epidemic wave. Phylogenetic analysis demonstrated that six internal segments of the influenza A (H7N9) virus isolated from confirmed cases and environmental samples in Shenzhen were clustered into two different clades and that the origin of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen was different from that of viruses isolated during the first wave. In addition, H9N2 viruses, which were prevalent in southern China, played an important role in the reassortment of the influenza A (H7N9) virus isolated in Shenzhen. HA-R47K and -T122A, PB2-V139I, PB1-I397M, and NS1-T216P were the signature amino acids of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen. We found that the HA, NA, M, and PA genes of the A(H7N9) viruses underwent positive selection in the human population. Therefore, enhanced surveillance should be carried out to determine the origin and mode of transmission of the novel influenza A (H7N9) virus and to facilitate the formulation of effective policies for prevention and containment of a human infection epidemics.

  12. Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry.

    Science.gov (United States)

    Nagarajan, S; Rajukumar, K; Tosh, C; Ramaswamy, V; Purohit, K; Saxena, G; Behera, P; Pattnaik, B; Pradhan, H K; Dubey, S C

    2009-01-01

    A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.

  13. Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism.

    Science.gov (United States)

    Renz, Katrin G; Cooke, Julie; Clarke, Nadeene; Cheetham, Brian F; Hussain, Zahid; Fakhrul Islam, A F M; Tannock, Gregory A; Walkden-Brown, Stephen W

    2012-01-01

    We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.

  14. Ilheus virus isolation in the Pantanal, west-central Brazil.

    Science.gov (United States)

    Pauvolid-Corrêa, Alex; Kenney, Joan L; Couto-Lima, Dinair; Campos, Zilca M S; Schatzmayr, Hermann G; Nogueira, Rita M R; Brault, Aaron C; Komar, Nicholas

    2013-01-01

    The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.

  15. Isolation of Japanese encephalitis virus from clinical specimens using a continuous mosquito cell line.

    Science.gov (United States)

    Leake, C J; Burke, D S; Nisalak, A; Hoke, C H

    1986-09-01

    During the 1983 Japanese encephalitis (JE) epidemic in northern Thailand, we systematically attempted to isolate JE virus (JEV) from clinical specimens collected from 49 consecutive JE patients at 1 provincial hospital. Fresh acute plasma and cerebrospinal fluid (CSF) samples and postmortem brain samples were immediately inoculated onto cultured monolayers of Aedes pseudoscutellaris (LSTM-AP-61) cells which had been shipped to the epidemic site. None of 49 plasma samples yielded virus. None of 30 fresh CSF samples from nonfatal cases yielded virus, but 5 of 15 (33%) CSF samples from fatal cases did. Inoculation of fresh brain specimens obtained at autopsy yielded virus in every case attempted (7 of 7), whereas postmortem needle biopsy specimens of brain yielded virus in only 1 of 4 cases. Isolates were most frequently successful using thalamic tissue (6 of 7 cases), but isolates were also commonly obtained from frontal cortex (4/7), occipital cortex (4/7), cerebellum (4/7), medulla (4/7) and pons (2/7).

  16. Avian H11 influenza virus isolated from domestic poultry in a Colombian live animal market

    Science.gov (United States)

    Jiménez-Bluhm, Pedro; Karlsson, Erik A; Ciuoderis, Karl A; Cortez, Valerie; Marvin, Shauna A; Hamilton-West, Christopher; Schultz-Cherry, Stacey; Osorio, Jorge E

    2016-01-01

    Live animal markets (LAMs) are an essential source of food and trade in Latin American countries; however, they can also serve as ‘hotbeds' for the emergence and potential spillover of avian influenza viruses (AIV). Despite extensive knowledge of AIV in Asian LAMs, little is known about the prevalence South American LAMs. To fill this gap in knowledge, active surveillance was carried out at the major LAM in Medellin, Colombia between February and September 2015. During this period, overall prevalence in the market was 2.67% and a North American origin H11N2 AIV most similar to a virus isolated from Chilean shorebirds asymptomatically spread through multiple bird species in the market resulting in 17.0% positivity at peak of infection. Phenotypically, the H11 viruses displayed no known molecular markers associated with increased virulence in birds or mammals, had α2,3-sialic acid binding preference, and caused minimal replication in vitro and little morbidity in vivo. However, the Colombian H11N2 virus replicated and transmitted effectively in chickens explaining the spread throughout the market. Genetic similarity to H11 viruses isolated from North and South American shorebirds suggest that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover event. The ability to spread in domestic poultry as well as potential for human infection by H11 viruses highlight the need for enhanced AIV surveillance in South America in both avian species and humans. PMID:27924808

  17. Avian H11 influenza virus isolated from domestic poultry in a Colombian live animal market.

    Science.gov (United States)

    Jiménez-Bluhm, Pedro; Karlsson, Erik A; Ciuoderis, Karl A; Cortez, Valerie; Marvin, Shauna A; Hamilton-West, Christopher; Schultz-Cherry, Stacey; Osorio, Jorge E

    2016-12-07

    Live animal markets (LAMs) are an essential source of food and trade in Latin American countries; however, they can also serve as 'hotbeds' for the emergence and potential spillover of avian influenza viruses (AIV). Despite extensive knowledge of AIV in Asian LAMs, little is known about the prevalence South American LAMs. To fill this gap in knowledge, active surveillance was carried out at the major LAM in Medellin, Colombia between February and September 2015. During this period, overall prevalence in the market was 2.67% and a North American origin H11N2 AIV most similar to a virus isolated from Chilean shorebirds asymptomatically spread through multiple bird species in the market resulting in 17.0% positivity at peak of infection. Phenotypically, the H11 viruses displayed no known molecular markers associated with increased virulence in birds or mammals, had α2,3-sialic acid binding preference, and caused minimal replication in vitro and little morbidity in vivo. However, the Colombian H11N2 virus replicated and transmitted effectively in chickens explaining the spread throughout the market. Genetic similarity to H11 viruses isolated from North and South American shorebirds suggest that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover event. The ability to spread in domestic poultry as well as potential for human infection by H11 viruses highlight the need for enhanced AIV surveillance in South America in both avian species and humans.

  18. The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses.

    Science.gov (United States)

    Richards, R Souza; Adams, I P; Kreuze, J F; De Souza, J; Cuellar, W; Dullemans, A M; Van Der Vlugt, R A A; Glover, R; Hany, U; Dickinson, M; Boonham, N

    2014-04-01

    The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.

  19. Molecular characterization of banana bunchy top virus isolate from Sri Lanka and its genetic relationship with other isolates.

    Science.gov (United States)

    Wickramaarachchi, W A R T; Shankarappa, K S; Rangaswamy, K T; Maruthi, M N; Rajapakse, R G A S; Ghosh, Saptarshi

    2016-06-01

    Bunchy top disease of banana caused by Banana bunchy top virus (BBTV, genus Babuvirus family Nanoviridae) is one of the most important constraints in production of banana in the different parts of the world. Six genomic DNA components of BBTV isolate from Kandy, Sri Lanka (BBTV-K) were amplified by polymerase chain reaction (PCR) with specific primers using total DNA extracted from banana tissues showing typical symptoms of bunchy top disease. The amplicons were of expected size of 1.0-1.1 kb, which were cloned and sequenced. Analysis of sequence data revealed the presence of six DNA components; DNA-R, DNA-U3, DNA-S, DNA-N, DNA-M and DNA-C for Sri Lanka isolate. Comparisons of sequence data of DNA components followed by the phylogenetic analysis, grouped Sri Lanka-(Kandy) isolate in the Pacific Indian Oceans (PIO) group. Sri Lanka-(Kandy) isolate of BBTV is classified a new member of PIO group based on analysis of six components of the virus.

  20. Identification and isolation of Genotype-I Japanese Encephalitis virus from encephalitis patients

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    Gao Xiaoyan

    2010-11-01

    Full Text Available Abstract Historically, Japanese Encephalitis virus (JEV genotype III (GIII has been responsible for human diseases. In recent years, JEV genotype I (GI has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.

  1. Emergence of a new arbovirus disease in Brazil. III. Isolation of Rocio virus from Psorophora Ferox (Humboldt, 1819).

    Science.gov (United States)

    de Souza Lopes, O; de Abreu Sacchetta, L; Francy, D B; Jakob, W L; Calisher, C H

    1981-02-01

    A newly described flavivirus was responsible for a large encephalitis epidemic in São Paulo State, Brazil. The etiologic agent, Rocio virus, was isolated from human patients and sentinel mice. The natural history of the virus is unknown although presumed to be arthropod-borne. Rocio virus was isolated from a single pool containing 19 Psorophora ferox of 47 pools (283 specimens) of this species tested. The positive pool contained 16 deplete, 2 gravid, and 2 engorged mosquitoes. No isolations were made from 2183 pools of other species. The positive pool was collected during the year of the epidemic at the same approximate time and place where vertebrate isolations were made.

  2. Molecular characterization and phylogenetic study of Newcastle disease virus isolates from recent outbreaks in eastern Uganda

    DEFF Research Database (Denmark)

    Otim, Maxwell O.; Christensen, Henrik; Jørgensen, Poul Henrik;

    2004-01-01

    Newcastle disease virus isolates from chickens in eastern Uganda in 2001 were found to be velogenic by fusion protein cleavage site sequence analysis and biological characterization; the intracerebral pathogenicity index was 1.8. Analysis of their hemagglutinin-neuraminidase protein gene sequences...

  3. First complete genome sequence of an emerging cucumber green mottle mosaic virus isolate in North America

    Science.gov (United States)

    The complete genome sequence (6,423 nt) of an emerging Cucumber green mottle mosaic virus (CGMMV) isolate on cucumber in North America was determined through deep sequencing of sRNA and rapid amplification of cDNA ends. It shares 99% nucleotide sequence identity to the Asian genotype, but only 90% t...

  4. Genome sequences of nine vesicular stomatitis virus isolates from South America

    Science.gov (United States)

    We report nine full-genome sequences of vesicular stomatitis virus obtrained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species, clade III, while two...

  5. Alstroemeria-infecting cucumber mosaic virus isolates contain additional sequences in the RNA 3 segment.

    NARCIS (Netherlands)

    Chen, Y.K.; Prins, M.W.; Derks, A.F.L.M.; Langeveld, S.A.; Goldbach, R.W.

    2002-01-01

    The coat protein (CP) genes and flanking regions of three alstroemeria-infecting cucumber mosaic virus isolates (CMV-ALS), denoted ALS-LBO, ALS-IPO, and ALS-NAK, were cloned and their nucleotide sequence determined and compared at both nucleic acid and deduced protein level with the published sequen

  6. Complete Genome Sequence of a South Korean Isolate of Habenaria mosaic virus.

    Science.gov (United States)

    Igori, Davaajargal; Lim, Seungmo; Zhao, Fumei; Baek, Dasom; Moon, Jae Sun

    2016-09-08

    Habenaria mosaic virus (HaMV), a member of the genus Potyvirus in the family Potyviridae, was first discovered from Habenaria radiata in Japan. The complete genomic sequence of a South Korean isolate (PA1) of HaMV infecting Plantago asiatica L. was determined with high-throughput RNA sequencing.

  7. Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus

    NARCIS (Netherlands)

    Komatsu, Ken; Yamashita, Kazuo; Sugawara, Kota; Verbeek, Martin; Fujita, Naoko; Hanada, Kaoru; Uehara-Ichiki, Tamaki; Fuji, Shin Ichi

    2017-01-01

    Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and t

  8. Complete Genome Sequence of a Newcastle Disease Virus Isolate from an Outbreak in Central India

    Science.gov (United States)

    Gogoi, Polakshee; Morla, Sudhir; Kaore, Megha; Kurkure, Nitin Vasantrao

    2015-01-01

    The complete genome sequence of a Newcastle disease virus (NDV) strain NDV/Chicken/Nagpur/01/12 was isolated from vaccinated chicken farms in India during outbreaks in 2012. The genome is 15,192 nucleotides in length and is classified as genotype VII in class II. PMID:25593257

  9. In vitro and in vivo isolation and characterization of Duvenhage virus

    NARCIS (Netherlands)

    P. Koraka (Penelope); B.E.E. Martina (Byron); J.M. Roose (Jouke M.); P.P.A.M. van Thiel (Pieter); G. van Amerongen (Geert); T. Kuiken (Thijs); A.D.M.E. Osterhaus (Albert)

    2012-01-01

    textabstractA fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization o

  10. First Complete Genome Sequences of Zika Virus Isolated from Febrile Patient Sera in Ecuador

    Science.gov (United States)

    Márquez, S.; Carrera, J.; Pullan, S. T.; Lewandowski, K.; Paz, V.; Loman, N.; Quick, J.; Bonsall, D.; Powell, R.; Thézé, J.; Pybus, O. G.; Klenerman, P.; Eisenberg, J.; Coloma, J.; Carroll, M. W.; Trueba, G.

    2017-01-01

    ABSTRACT Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador. PMID:28232448

  11. Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

    Directory of Open Access Journals (Sweden)

    M.P. Miagostovich

    2002-08-01

    Full Text Available The genetic characterization of dengue virus type 3 (DEN-3 strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550 of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

  12. Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009.

    Directory of Open Access Journals (Sweden)

    Jiu-Song Zhang

    Full Text Available BACKGROUND: Several studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China. METHODS AND RESULTS: In 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: Ser(E-123-Asn in the two Yunnan isolates and Lys(E-166-Arg in the Shanxi isolate. CONCLUSIONS: Our findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

  13. R5 to X4 coreceptor switch of human immunodeficiency virus type 1 B' and B'/C recombinant subtype isolates in China

    Institute of Scientific and Technical Information of China (English)

    GUO Yan-fang; ZHANG Xiao-yan; RUAN Yu-hua; ZHANG Yao-xin; SHAO Yi-ming; MA Li-ying; YUAN Lin; WANG Shu-hua; SUN Jian-ping; XU Wei-si; Xu Jian-qing; XING Hui; HONG Kun-xue

    2007-01-01

    @@ The chemokine receptors CCR5 and CXCR4 play an important role as coreceptors for human immunodeficiency virus type 1 (HIV-1) entring into cells.HIV-1 isolates can be distinguished by the chemokine coreceptors. Nonsyncytium inducing (NSI), macrophage tropic viruses utilizing CCR5, are called R5 viruses;syncytium inducing (SI) isolates use CXCR4 and known as X4 viruses.

  14. Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru.

    Science.gov (United States)

    Vasilakis, Nikos; Castro-Llanos, Fanny; Widen, Steven G; Aguilar, Patricia V; Guzman, Hilda; Guevara, Carolina; Fernandez, Roberto; Auguste, Albert J; Wood, Thomas G; Popov, Vsevolod; Mundal, Kirk; Ghedin, Elodie; Kochel, Tadeusz J; Holmes, Edward C; Walker, Peter J; Tesh, Robert B

    2014-04-01

    Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.

  15. Biological and Molecular Characterization of Cucumber mosaic virus Subgroup II Isolate Causing Severe Mosaic in Cucumber.

    Science.gov (United States)

    Kumari, Reenu; Bhardwaj, Pooja; Singh, Lakhmir; Zaidi, Aijaz A; Hallan, Vipin

    2013-06-01

    Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricultural and ornamental crops. Earlier reports showed the prevalence of CMV subgroup I isolates in India. However, some recent reports point towards increasing incidence of subgroup II isolates in the country. The complete genome of a CMV isolate causing severe mosaic in cucumber was characterized and its phylogenetic analysis with other 21 CMV isolates reported worldwide clustered it with subgroup II strains. The genome comprised of RNA 1 (3,379 nucleotides), RNA 2 (3,038 nucleotides) and RNA 3 (2,206 nucleotides). The isolate showed highest homology with subgroup II isolates: 95.1-98.7, 87.7-98.0, and 85.4-97.1 % within RNA1, RNA2, and RNA3, respectively. RNA1 and RNA2 were closely related to the Japanese isolate while RNA3 clustered with an American isolate. Host range studies revealed that isolate showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The isolate induced leaf deformation and mild filiform type symptoms in tomato. To best of our knowledge this is the first report of complete genome of CMV subgroup II isolate from India.

  16. Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

    Directory of Open Access Journals (Sweden)

    Simon Delgado

    2008-04-01

    Full Text Available A small focus of hemorrhagic fever (HF cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá. RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

  17. Genotypic characteristics of bovine viral diarrhea virus 2 strains isolated in northern Italy.

    Science.gov (United States)

    Giangaspero, M; Harasawa, R; Zecconi, A; Luzzago, C

    2001-09-01

    Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.

  18. Complete genome sequence of a J subgroup avian leukosis virus isolated from local commercial broilers.

    Science.gov (United States)

    Li, Hongxin; Xue, Chunyi; Ji, Jun; Chang, Shuang; Shang, Huiqin; Zhang, Lingjun; Ma, Jingyun; Bi, Yingzuo; Xie, Qingmei

    2012-11-01

    Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5'-GGCACCTCC-3') for c-myb was identified in the GDKP1202 genome. These findings will provide additional insights into the molecular characteristics in the genomes and pathogenicity of ALV-J.

  19. Comparison of the pathogenicity of Nipah virus isolates from Bangladesh and Malaysia in the Syrian hamster.

    Directory of Open Access Journals (Sweden)

    Blair L DeBuysscher

    Full Text Available Nipah virus is a zoonotic pathogen that causes severe disease in humans. The mechanisms of pathogenesis are not well described. The first Nipah virus outbreak occurred in Malaysia, where human disease had a strong neurological component. Subsequent outbreaks have occurred in Bangladesh and India and transmission and disease processes in these outbreaks appear to be different from those of the Malaysian outbreak. Until this point, virtually all Nipah virus studies in vitro and in vivo, including vaccine and pathogenesis studies, have utilized a virus isolate from the original Malaysian outbreak (NiV-M. To investigate potential differences between NiV-M and a Nipah virus isolate from Bangladesh (NiV-B, we compared NiV-M and NiV-B infection in vitro and in vivo. In hamster kidney cells, NiV-M-infection resulted in extensive syncytia formation and cytopathic effects, whereas NiV-B-infection resulted in little to no morphological changes. In vivo, NiV-M-infected Syrian hamsters had accelerated virus replication, pathology and death when compared to NiV-B-infected animals. NiV-M infection also resulted in the activation of host immune response genes at an earlier time point. Pathogenicity was not only a result of direct effects of virus replication, but likely also had an immunopathogenic component. The differences observed between NiV-M and NiV-B pathogeneis in hamsters may relate to differences observed in human cases. Characterization of the hamster model for NiV-B infection allows for further research of the strain of Nipah virus responsible for the more recent outbreaks in humans. This model can be used to study NiV-B pathogenesis, transmission, and countermeasures that could be used to control outbreaks.

  20. Genomic characterization of three bovine viral diarrhea virus isolates from cattle.

    Science.gov (United States)

    Cai, Dongjie; Song, Quanjiang; Wang, Jiufeng; Zhu, Yaohong

    2016-12-01

    Three strains of the bovine viral diarrhea virus (BVDV) were isolated from cattle in Beijing, China. To investigate their genomic features, we sequenced and characterized the complete genome of each of the isolates. Each of the three virus genomes is about 12,220 bp in length, containing a 5' untranslated region (UTR), one open reading frame (ORF) encoding a 3897-amino-acid polypeptide, and a 3' UTR. The nucleotide sequence of the three isolates were 99.0 % identical to each and other shared nucleotide sequence identities of 73.4 % to 98.3 % with other BVDV-1 strains, about 70.0 % with BVDV-2 strains, about 67.0 % with BVDV-3, and less than 67.0 % with other pestiviruses. Phylogenetic analysis of the full-length genome, 3' UTR, and the N(pro) gene demonstrated that the three viruses were BVDV-1 isolates. This is the first report of complete genome sequences of BVDV 1d isolates from China and might have implications for vaccine development.

  1. Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The potyvirus Papaya ringspot virus (PRSV) is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein (CP) genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting (PRSV-P). The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.

  2. Genotypic and pathotypic characterization of Newcastle disease virus isolated from racing pigeons in China.

    Science.gov (United States)

    Liu, Mengda; Qu, Yajin; Wang, Fangkun; Liu, Sidang; Sun, Honglei

    2015-07-01

    A Newcastle disease virus (NDV) isolated from an outbreak in racing pigeons in China was characterized in this study. Complete gene of the NDV isolate was sequenced and phylogenetic analysis. Pathogenicity experiment was carried out in pigeons, chickens, and ducks. Phylogenetic analysis revealed that the strain clustered with the Class II viruses, has highly phylogenetically similar to NDV strains isolated from pigeons in China, but was distant from the viruses prevalence in chickens and vaccine strains used in China. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the virulent motif (112)RRQKRF(117) at the cleavage site, but it caused no appearance disease in chickens and ducks. However, the isolate had virulence in pigeons, resulting in severe nervous signs and highly mortality. Pigeons were considered as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the NDV infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.

  3. First Report of Cucumber mosaic virus Isolated from Wild Vigna angularis var. nipponensis in Korea

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    Mi-Kyeong Kim

    2014-06-01

    Full Text Available A viral disease causing severe mosaic, necrotic, and yellow symptoms on Vigna angularis var. nipponensis was prevalent around Suwon area in Korea. The causal virus was characterized as Cucumber mosaic virus (CMV on the basis of biological and nucleotide sequence properties of RNAs 1, 2 and 3 and named as CMV-wVa. CMV-wVa isolate caused mosaic symptoms on indicator plants, Nicotiana tabacum cv. Xanthi-nc, Petunia hybrida, and Cucumis sativus. Strikingly, CMV-wVa induced severe mosaic and malformation on Cucurbita pepo, and Solanum lycopersicum. Moreover, it caused necrotic or mosaic symptoms on V. angularis and V. radiate of Fabaceae. Symptoms of necrotic local or pin point were observed on inoculated leaves of V. unguiculata, Vicia fava, Pisum sativum and Phaseolus vulgaris. However, CMV-wVa isolate failed to infect in Glycine max cvs. ‘Sorok’, ‘Sodam’ and ‘Somyeong’. To assess genetic variation between CMV-wVa and the other known CMV isolates, phylogenetic analysis using 16 complete nucleotide sequences of CMV RNA1, RNA2, and RNA3 including CMV-wVa was performed. CMV-wVa was more closely related to CMV isolates belonging to CMV subgroup I showing about 85.1–100% nucleotide sequences identity to those of subgroup I isolates. This is the first report of CMV as the causal virus infecting wild Vigna angularis var. nipponensis in Korea.

  4. First Report of Cucumber mosaic virus Isolated from Wild Vigna angularis var. nipponensis in Korea.

    Science.gov (United States)

    Kim, Mi-Kyeong; Jeong, Rae-Dong; Kwak, Hae-Ryun; Lee, Su-Heon; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeongjin; Choi, Hong-Soo

    2014-06-01

    A viral disease causing severe mosaic, necrotic, and yellow symptoms on Vigna angularis var. nipponensis was prevalent around Suwon area in Korea. The causal virus was characterized as Cucumber mosaic virus (CMV) on the basis of biological and nucleotide sequence properties of RNAs 1, 2 and 3 and named as CMV-wVa. CMV-wVa isolate caused mosaic symptoms on indicator plants, Nicotiana tabacum cv. Xanthi-nc, Petunia hybrida, and Cucumis sativus. Strikingly, CMV-wVa induced severe mosaic and malformation on Cucurbita pepo, and Solanum lycopersicum. Moreover, it caused necrotic or mosaic symptoms on V. angularis and V. radiate of Fabaceae. Symptoms of necrotic local or pin point were observed on inoculated leaves of V. unguiculata, Vicia fava, Pisum sativum and Phaseolus vulgaris. However, CMV-wVa isolate failed to infect in Glycine max cvs. 'Sorok', 'Sodam' and 'Somyeong'. To assess genetic variation between CMV-wVa and the other known CMV isolates, phylogenetic analysis using 16 complete nucleotide sequences of CMV RNA1, RNA2, and RNA3 including CMV-wVa was performed. CMV-wVa was more closely related to CMV isolates belonging to CMV subgroup I showing about 85.1-100% nucleotide sequences identity to those of subgroup I isolates. This is the first report of CMV as the causal virus infecting wild Vigna angularis var. nipponensis in Korea.

  5. Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

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    FAVORETTO Silvana Regina

    2002-01-01

    Full Text Available Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog, 3 (Desmodus rotundus, 4 (Tadarida brasiliensis, 5 (vampire bat from Venezuela, 6 (Lasiurus cinereus and Lab (reacted to all used antibodies. Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus. These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.

  6. Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells

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    Coyle Peter V

    2011-01-01

    Full Text Available Abstract Background Human respiratory syncytial virus (RSV causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs. Methods To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. Results RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. Conclusions The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

  7. Phylogenetic analysis of Chinese sheeppox and goatpox virus isolates

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    Zhou Tao

    2012-01-01

    Full Text Available Abstract Background Sheeppox virus (SPPV and goatpox virus (GTPV, members of the Capripoxvirus genus of the Poxviridae family are causative agents of sheep pox and goat pox respectively, which are important contagious diseases and endemic in central and northern Africa, the Middle and Far East, and the Indian sub-continent. Both sheep pox and goat pox can cause wool and hide damage, and reduce the production of mutton and milk, which may result in significant economic losses and threaten the stockbreeding. In this study, three SPPVs and two GTPVs were collected from China in 2009 and 2011. We described the sequence features and phylogenetic analysis of the P32 gene, GPCR gene and RPO30 gene of the SPPVs and GTPVs to reveal their genetic relatedness. Results Sequence and phylogenetic analysis showed that there was a close relationship among SPPV/GanS/2/2011/China, SPPV/GanS/1/2011/China and SPPV/NingX/2009/China. They were clustered on the same SPPV clade. GTPV/HuB/2009/China and GS-V1 belonged to the GTPV lineage. GS-V1 was closely related to other GTPV vaccine strains. GTPV/HuB/2009/China and GS-V1 were clustered with GTPVs from China and some southern Asian countries. Conclusion This study may expand the datum for spread trend research of Chinese SPPVs and GTPVs, meanwhile provide theoretical references to improve the preventive and control strategy.

  8. Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India.

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    Morla, Sudhir; Deka, Pankaj; Kumar, Sachin

    2016-04-01

    Infectious bursal disease virus (IBDV) is a highly infectious disease of young chicken that predominantly affects the immune system. In the present study, we are reporting first comprehensive study of IBDV outbreaks from the Northeastern part of India. Northeast India shares a porous border with four different countries; and as a rule any outbreak in the neighboring countries substantially affects the poultry population in the adjoining states. Nucleotide sequence analysis of the VP2 gene of the IBDV isolates from the Northeastern part of India suggested the extreme virulent nature of the virus. The virulent marker amino acids (A222, I242, Q253, I256 and S299) in the hypervariable region of the Northeastern isolates were found identical with the reported very virulent strains of IBDV. A unique insertion of I/L294V was recorded in all the isolates of the Northeastern India. The study will be useful in understanding the circulating pathotypes of IBDV in India.

  9. Nucleotide sequences of two Korean isolates of Cucumber green mottle mosaic virus.

    Science.gov (United States)

    Kim, Sang-Min; Lee, Jung-Myung; Yim, Kyu-Ock; Oh, Man-Ho; Park, Jin-Woo; Kim, Kook-Hyung

    2003-12-31

    The nucleotide sequences of the genomic RNAs of Cucumber green mottle mosaic virus Korean watermelon isolate (CGMMV-KW) and Korean oriental melon isolate (CGMMV-KOM) were determined and compared to the sequences of other tobamoviruses including CGMMV strains W and SH. Each CGMMV isolate had a genome of 6,424 nucleotides. Each also had 60 and 176 nucleotides of 5' and 3' untranslated regions (UTRs), respectively, and four open reading frames (ORF1-4). ORFs 1 to 4 encode proteins of 129, 186, 29, and 17.4 kDa, respectively. The nucleotide and deduced amino acid sequences of CGMMV-KOM and CGMMV-KW were more than 98.3% identical. When compared to other CGMMV strains in a phylogenetic analysis they were found to form a distinct virus clade, and were more distantly related to other tobamoviruses (23.5-56.7% identity).

  10. Molecular characterization of Dasheen mosaic virus isolates infecting edible aroids in India.

    Science.gov (United States)

    Babu, B; Hegde, V

    2014-01-01

    Dasheen mosaic virus (DsMV) infecting three major edible aroids namely Amorphophallus paeoniifolius, Colocasia esculenta, and Xanthosoma sagittifolium cultivated in India was characterized. Infected plants showing typical DsMV symptoms were subjected to reverse transcription-polymerase chain reaction, and an amplification of a 963 bp fragment which encoded the coat protein (CP) gene was obtained. BLAST analysis of the cloned DNA amplicon revealed the identity of the virus to be that of DsMV. Sequence identity matrix of the nucleotide sequences among the three isolates showed that the DsMV isolate infecting A. paeoniifolius and C. esculenta shared an identity as high as 93%, while the DsMV isolate from X. sagittifolium shared an identity of only 73% and 76% with the DsMV isolates from A. paeoniifolius and C. esculenta, respectively. Comparative analysis of the coat protein of the three DsMV isolates showed the presence of DVG motif (A. paeoniifolius and C. esculenta) and DTG motif in X. sagittifolium and several varying potential threonine and asparagine rich N-glycosylation motifs. Single amino acid substitution of the several conserved motifs occurs in all the three DsMV isolates. This is the first characterization of DsMV isolates infecting A. paeoniifolius, C. esculenta, and X. sagittifolium plants in India.

  11. Lassa Virus Isolates from Mali and the Ivory Coast Represent an Emerging Fifth Lineage

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    John Tyler Manning

    2015-10-01

    Full Text Available Previous imported cases of Lassa fever into the United Kingdom from the Ivory Coast and Mali, as well as the detection of Lassa virus among the Mastomys natalensis population within Mali has led to the suggestion that the endemic area for Lassa fever is expanding. Initial phylogenetic analyses arrange isolates from Mali and the Ivory Coast separately from the classical lineage IV isolates taken from Sierra Leone, Guinea, and Liberia. The availability of full genome sequences continues to increase, allowing for a more complete phylogenetic comparison of the isolates from Mali and the Ivory Coast to the other existing isolates. In this study, we utilized a Bayesian approach to infer the demographic histories of each Lassa virus isolate for which the full sequence was available. Our results indicate that the isolates from Mali and the Ivory Coast group separately from the isolates of lineage IV, comprising a distinct fifth lineage. The split between lineages IV and V is estimated to have occurred around 200-300 years ago, which coincides with the colonial period of West Africa.

  12. Partial sequencing and phylogenetic analysis of Soybean mosaic virus isolated in Ukraine

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    Polischuk V. P.

    2011-12-01

    Full Text Available The aim of the present study is to compare the biological and molecular properties of Ukrainian soybean mosaic virus (SMV isolates with those of known strains or isolates from other countries, and to trace their possible origin. The methods of mechanical inoculation, reverse transcription polymerase chain reaction, DNA sequencing and phylogenetic analysis have been used. Results. Five SMV isolates have been collected and biologically purified from breeding plots in Vinnitsa region of Ukraine. It has been found that all these isolates show the same reaction patterns when infecting 11 differential soybean cultivars. Phylogenetic analysis of sequences of the coat protein coding region and P1 coding region revealed strong genetic relationships between representative Ukrainian (UA1Gr and SMV-VA2 isolates which together were sorted in one clade with G2 strain. The investigation of sequence identity showed that different genomic regions of SMV were under different evolutionary constraints. Conclusions. SMV, isolated in Ukraine for the first time, belongs to the G2 strain group that is widespread in North America. The SMV isolates obtained in this work may be employed in the Ukrainian national breeding programs to create soybean with durable virus resistance.

  13. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

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    Wang Nan

    2011-02-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. Results we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE, indirect immunoperoxidase test (IPX and electron microscopy(EM. The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV and 3 strains of border disease virus(BDV in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. Conclusion To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

  14. Molecular characterization of cucumber mosaic virus isolates infecting tomato in Hamedan and Tehran provinces of Iran.

    Science.gov (United States)

    Safaeizadeh, M; Saidi, A; Palukaitis, P

    2015-06-01

    Here we identified four isolates, MS, 3H, 50A, and 2K of cucumber mosaic virus (CMV) infecting tomato, on the basis of their non-coding intergenic region and a part of the coat protein (CP) sequence in the CMV genomic RNA3. The sequences from the four isolates were compared with other previously characterized isolates of CMV isolated from different plant species across the globe. Sequence comparisons revealed that the two CMV isolates from Hamedan province (MS and 3H) had the highest sequence identity with CMV-G10 (98%), which was previously reported as a severe Hellenic tomato isolate of CMV, while the CMV isolates from Tehran province, including CMV-2K (isolated from Karaj region) and CMV-50A (isolated from Varamin region), had the highest sequence identity with that of CMV-ALF (99%). Phylogenetic analysis of the nucleotide sequences showed that CMV-MS and CMV-3H belong to group IB, while CMV-2K and CMV-50A belong to group IA. This is the first report on the molecular characterization of novel isolates of CMV infecting tomato plants in Iran.

  15. Comparison of multiple genes of spring viremia of carp viruses isolated in the United States.

    Science.gov (United States)

    Warg, Janet V; Dikkeboom, Audrey L; Goodwin, Andrew E; Snekvik, Kevin; Whitney, John

    2007-08-01

    Five spring viremia of carp viruses (SVCV), Rhabdovirus carpio, were isolated in the United States (US) between 2002 and 2004. Single tube reverse transcription-polymerase chain reaction (RT-PCR) was used to generate overlapping cDNA fragments from the US isolates of SVCV. Multiple pairs of specific primers were designed to amplify a portion of the phosphoprotein gene, the matrix gene, and the glycoprotein gene of SVCV genogroup Id (corresponding to nucleotides 2174-4942 of GenBank accession NC_002803). Sequences were proofread and aligned to generate a consensus sequence for each isolate. Phylogenetic analysis of the 2705 nucleotide consensus sequence revealed that all five US isolates belong to SVCV genogroup Ia, Asian origin isolates, and a PCR primer binding site unique to SVCV genogroup Ia was identified.

  16. Diagnosis and sequence analysis of avian leukosis virus subgroup J isolated from Chinese Partridge Shank chickens.

    Science.gov (United States)

    Dong, Xuan; Zhao, Peng; Li, Weihua; Chang, Shuang; Li, Jianliang; Li, Yang; Ju, Sidi; Sun, Peng; Meng, Fanfeng; Liu, Juan; Cui, Zhizhong

    2015-04-01

    The diagnosis of avian leukosis virus subgroup J (ALV-J) infection in Chinese Partridge Shank chickens was confirmed by necropsy, histopathological examinations, antibody tests, viral isolation, immunofluorescence assays, and sequence analysis. Myelocytoma, myeloma, and fibrosarcoma were simultaneously found in Partridge Shank flock with ALV-J infection. Sequence analysis of the env genes of ALV-J demonstrated that both gp85 and gp37 were highly homologous among the three strains from local chickens of those among ALV-J strains isolated from white meat-type chickens. The phylogenetic trees indicated that the three strains isolated in this study were closely related to reference strains isolated in so-called Chinese yellow chickens and some strains isolated from white meat-type chickens, both from the USA and China. The observed ALV-J infection was the first report on Partridge Shank chickens, and myelocytoma, myeloma, and fibrosarcoma were found at the same time in this batch of local chickens.

  17. Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

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    Abdel-Ghany Ahmad E

    2010-04-01

    Full Text Available Abstract Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE. Reverse transcriptase polymerase chain reaction (RT-PCR and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas.

  18. Hepatitis delta virus:Making the point from virus isolation up to 2014

    Institute of Scientific and Technical Information of China (English)

    Raffaella; Romeo; Riccardo; Perbellini

    2015-01-01

    Chronic infection with hepatitis delta virus(HDV) has lately regained clinical importance because of the recent evidence of increasing prevalence in several European countries,due to immigration from highly endemic areas. HDV requires the mandatory presence ofhepatitis B virus(HBV) for propagation to hepatocytes. It is transmitted by the same routes of HBV and it can be acquired either by co-infection(simultaneous transmission of the two viruses) or super-infection(acquisition of HDV by an already chronic carrier of HBV). As a consequence,every HBV carrier is potentially at risk for HDV superinfection. Since the clinical course of superinfection can be severe,early diagnosis of HDV infection is necessary.

  19. Phylogenetic and pathogenic analyses of avian influenza A H5N1 viruses isolated from poultry in Vietnam.

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    Dongming Zhao

    Full Text Available Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature.

  20. Field Trials of CpGV Virus Isolates Overcoming Resistance to CpGV-M

    Institute of Scientific and Technical Information of China (English)

    M. Berling; J. -B. Rey; S. -J. Ondet; Y. Tallot; O. Soubabère; A. Bonhomme; B. Sauphanor; M. Lopez-Ferber

    2009-01-01

    The Cydia pomonella granulovirus (CpGV) has been used for many years as biological agent for codling moth control in apple orchards. Resistance to the Mexican strain of CpGV was detected in orchards in Germany, France and Italy. A laboratory insect colony was started from insects collected in a French resistant orchard. It was named RGV. Various virus isolates were identified as active against this resistant insect colony. Field tests were carried out in 2007 to test if the two virus isolates CpGV-I12 and NPP-R1 were effective in the field. Although these virus isolates were not able to reduce insect caused fruit damages, they significantly reduced the overwintering insect populations. NPP-R1 was subjected to eight passages on RGV larvae (NPP-R1.8) that improved its biological activity on RGV larvae. 2008 field trials were set up to test this improved virus strain, compared to CpGV-I12 and Madex plus active on RGV. These tests confirmed the ability to control both in susceptible and resistant insect populations.

  1. SPF rabbits infected with rabbit hepatitis E virus isolate experimentally showing the chronicity of hepatitis.

    Science.gov (United States)

    Han, Jian; Lei, Yaxin; Liu, Lin; Liu, Peng; Xia, Junke; Zhang, Yulin; Zeng, Hang; Wang, Lin; Wang, Ling; Zhuang, Hui

    2014-01-01

    This study focused on investigating the pathogenesis seen in specific-pathogen-free (SPF) rabbits following infection with a homologous rabbit HEV isolate (CHN-BJ-rb14) and comparing it to that seen following infection with a heterologous swine genotype 4 HEV isolate (CHN-XJ-SW13). Three of the four animals inoculated with the homologous rabbit HEV became infected, exhibiting an intermittent viremia, obvious fluctuations of liver function biomarkers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and persistent fecal virus shedding throughout the nine month study. In addition, liver histopathology showed both chronic inflammation and some degree of fibrosis. Both positive and negative-stranded HEV RNA and HEV antigen expression were detected in liver, brain, stomach, duodenum and kidney from the necropsied rabbits. Inflammation of extrahepatic tissue (duodenum and kidney) was also observed. Three of the four rabbits inoculated with the heterologous genotype 4 swine HEV also became infected, showing similar levels of anti-HEV antibody to that generated following infection with the homologous virus isolate. The duration of both viremia and fecal shedding of virus was however shorter following infection with the heterologous virus and there was no significant elevation of liver function biomarkers. These results suggest that rabbit HEV infection may cause more severe hepatitis and prolong the course of the disease, with a possible chronic trend of hepatitis in SPF rabbits.

  2. Isolation of avian influenza H5N1 virus from vaccinated commercial layer flock in Egypt

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    El-Zoghby Elham F

    2012-11-01

    Full Text Available Abstract Background Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade. Findings In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (Log2 3.2± 4.2. The hemagglutinin (HA and neuraminidase (NA genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed. Conclusions Despite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans.

  3. Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis

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    Budzanivska I. G.

    2014-03-01

    Full Text Available Aim. Identification of the widespread Ukrainian isolate(s of PVY (Potato virus Y in different potato cultivars and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY has been identified serologically in potato cultivars of Ukrainian selection. In this work we have optimized a method for total RNA extraction from potato samples and offered a sensitive and specific PCR-based test system of own design for diagnostics of the Ukrainian PVY isolates. Part of the CP gene of the Ukrainian PVY isolate has been sequenced and analyzed phylogenetically. It is demonstrated that the Ukrainian isolate of Potato virus Y (CP gene has a higher percentage of homology with the recombinant isolates (strains of this pathogen (approx. 98.8– 99.8 % of homology for both nucleotide and translated amino acid sequences of the CP gene. The Ukrainian isolate of PVY is positioned in the separate cluster together with the isolates found in Syria, Japan and Iran; these isolates possibly have common origin. The Ukrainian PVY isolate is confirmed to be recombinant. Conclusions. This work underlines the need and provides the means for accurate monitoring of Potato virus Y in the agroecosystems of Ukraine. Most importantly, the phylogenetic analysis demonstrated the recombinant nature of this PVY isolate which has been attributed to the strain group O, subclade N:O.

  4. Types of variation in DNA-A among isolates of East African cassava mosaic virus from Kenya, Malawi and Tanzania.

    Science.gov (United States)

    Zhou, X; Robinson, D J; Harrison, B D

    1998-11-01

    Complete nucleotide sequences of the DNA-A-like molecules of three East African cassava mosaic virus (EACMV) isolates from Kenya (-K, 2801 nt) and Malawi (-MH and -MK, both 2804 nt) were determined. These sequences were compared with that published for a Tanzanian isolate (-T, 2801 nt) and the partial sequence of a third Malawian isolate. Intergenic region sequences of all isolates, and deduced amino acid sequences of their AC1 (Rep) proteins, each formed a tightly related cluster that was distinct from the comparable components of other begomoviruses. Other complementary-sense genes (AC2, AC3, AC4) differed between EACMV isolates in a way consistent with the accumulation of point mutations. In contrast, virus-sense genes (CP, AV2) of isolates -MH and -MK differed (substantially for AV2) from those of other EACMV isolates but somewhat resembled those of tomato yellow leaf curl virus-Israel, suggesting they had been acquired by recombination with an unidentified begomovirus.

  5. Viruses as teratogens.

    Science.gov (United States)

    Oberst, R D

    1993-03-01

    The ability of certain viruses to affect prenatal development in domestic animals is well documented. However, differentiating a viral-induced malformation from those caused by genetic or other environmental causes is a diagnostic dilemma. Understanding how viruses interact with their embryo-fetal hosts and the potential consequences on prenatal development requires refining and dispelling some old concepts and injecting new insights into this diagnostic challenge. This article discusses several viral teratogens affecting domestic animals: Akabane, bluetongue, Cache Valley, Japanese B encephalitis, bovine viral diarrhea, Border disease, Chuzan, epizootic hemorrhagic disease, hog cholera, Rift Valley fever, and Wesselsbron disease viruses.

  6. Prevalence and genetic diversity of fig mosaic virus isolates infecting fig tree in Iran.

    Science.gov (United States)

    Danesh-Amuz, S; Rakhshandehroo, F; Rezaee, S

    2014-01-01

    Commercial and outdoor fig orchards in four Iranian provinces were surveyed for the incidence of fig mosaic virus (FMV), fig leaf mottle associated virus 2 (FLMaV-2) and fig mild mottle associated virus (FMMaV) from March 2011 to October 2012. A total of 350 asymptomatic and symptomatic fig samples were collected and tested by dot-immunobinding assay (DIBA) for the fig mosaic disease (FMD) using a polyclonal antiserum. According to DIBA results, FMD was present in 73% of the collected symptomatic samples from all visited regions. Samples with positive reactions in DIBA were then analyzed by RT-PCR using with specific primers. PCR results showed that about 14.8% of the FMD-positive samples from three inspected provinces are infected with at least one virus. FMV was the most widely spread virus (14%) followed by FLMaV-2 (1.5%), whereas FMMaV was not found. Phylogenetic analysis of the glycoprotein nucleotide and amino acid sequences of known FMV isolates showed two independent groups with high bootstrap values, with all Iranian isolates distinctly clustered in group I, subgroup IA beside those reported in Turkey. Nucleotide diversity was high within but low between different selected geographic regions and except for Europe, nucleotide distance within geographic regions was low. Statistical analyses indicated a correlation between the genetic structure of the FMV isolates and the geographical origin of isolation. Our analyses suggested that the FMV population is in a state of increase following a bottleneck or founder event in Iran.

  7. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    A. Sasikalaveni

    2015-05-01

    Full Text Available Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA cells, which enable quicker isolation of street rabies virus with minimum passages. Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

  8. Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

    Indian Academy of Sciences (India)

    A Srivastava; S K Raj

    2008-06-01

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

  9. Seroprevalence of Q fever, Brucellosis, and Bluetongue in Selected Provinces in Lao People's Democratic Republic

    Science.gov (United States)

    Douangngeun, Bounlom; Theppangna, Watthana; Soukvilay, Vilayvahn; Senaphanh, Chanthana; Phithacthep, Kamphok; Phomhaksa, Souk; Yingst, Samuel; Lombardini, Eric; Hansson, Eric; Selleck, Paul W.; Blacksell, Stuart D.

    2016-01-01

    This study has determined the proportional seropositivity of two zoonotic diseases, Q fever and brucellosis, and bluetongue virus (BTV) which is nonzoonotic, in five provinces of Lao People's Democratic Republic (PDR) (Loungphabang, Luangnumtha, Xayaboury, Xiengkhouang, and Champasak, and Vientiane Province and Vientiane capital). A total of 1,089 samples from buffalo, cattle, pigs, and goats were tested, with seropositivity of BTV (96.7%), Q fever (1.2%), and brucellosis (0.3%). The results of this survey indicated that Q fever seropositivity is not widely distributed in Lao PDR; however, Xayaboury Province had a cluster of seropositive cattle in seven villages in four districts (Botan, Kenthao, Paklaiy, and Phiang) that share a border with Thailand. Further studies are required to determine if Xayaboury Province is indeed an epidemiological hot spot of Q fever activity. There is an urgent need to determine the levels of economic loss and human health-related issues caused by Q fever, brucellosis, and BTV in Lao PDR. PMID:27430548

  10. Seroepidemiology of bluetongue disease and risk factors in small ruminants of Shiraz suburb, Fars province, Iran.

    Science.gov (United States)

    Mohammadi, A; Tanzifi, P; Nemati, Y

    2012-12-01

    Bluetongue virus (BTV) is a member of the genus Orbivirus in the family Reoviridae which is transmitted by insects and could cause considerable damages in sheep and goat flocks, such as mortality, decreased production and fertility, medical costs and commercial limits for flocks and their biologic production. As no study has been conducted on small ruminants about this disease in Fars province of Iran, the present study was conducted to determine the seroprevalence of the disease and some risk factors that might be related to it. A total of 200 serum samples were collected from 13 flocks including nomadic animals (70%) and resident flocks (30%) using a cluster random sampling method with an equal proportion of sheep and goats during the last three months of 2010. Some risk factors such as age, breed and abortion, exposure to other flocks, density and female replacement origin were reported in a question sheet. Totally, 73.5% of the samples were positive for presence of BTV antibody.The results showed that age and contact with other herds are influential risk factors on seroprevalence of the disease.

  11. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

    Directory of Open Access Journals (Sweden)

    Vanessa Danielle Muller

    Full Text Available The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

  12. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

    Science.gov (United States)

    Muller, Vanessa Danielle; Soares, Ricardo Oliveira; dos Santos, Nilton Nascimento; Trabuco, Amanda Cristina; Cintra, Adelia Cristina; Figueiredo, Luiz Tadeu; Caliri, Antonio; Sampaio, Suely Vilela; Aquino, Victor Hugo

    2014-01-01

    The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

  13. Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs.

    Science.gov (United States)

    Stevens, James; Chen, Li-Mei; Carney, Paul J; Garten, Rebecca; Foust, Angie; Le, Jianhua; Pokorny, Barbara A; Manojkumar, Ramanunninair; Silverman, Jeanmarie; Devis, Rene; Rhea, Karen; Xu, Xiyan; Bucher, Doris J; Paulson, James C; Paulson, James; Cox, Nancy J; Klimov, Alexander; Donis, Ruben O

    2010-08-01

    Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of alpha2-6 and alpha2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to alpha2-6- and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.

  14. Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil

    Science.gov (United States)

    Drumond, Betania Paiva; da Silva Fagundes, Luiz Gustavo; Rocha, Raissa Prado; Fumagalli, Marcilio Jorge; Araki, Carlos Shigueru; Colombo, Tatiana Elisa; Nogueira, Mauricio Lacerda; Castilho, Thiago Elias; da Silveira, Nelson José Freitas; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-01-01

    Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population. PMID:26887252

  15. Molecular analysis of bovine viral diarrhoea virus isolates from South Africa

    Directory of Open Access Journals (Sweden)

    N. Kabongo

    2003-11-01

    Full Text Available The presence of bovine viral diarrhoea virus in South Africa has been confirmed by several serological surveys. However, little is known about its biological properties. Twenty five isolates obtained by isolation in tissue culture and detected by means of the antigen capture ELISA from clinically sick cattle and from foetal calf serum in South Africa were characterized on the basis of analysis of the 5' non-translated (NTR region of the genome. A reverse-transcription polymerase chain reaction (RT-PCR was used to amplify specific sequences from the 5'NTR of the genome. The oligonucleotide primers corresponding to positions 105-125 and 399-378, respectively, in the sequence of BVDV strain NADL were used to generate the PCR products. Both strands were sequenced directly with these primers and fluorescence-labelled dideoxynucleotides in an automated nucleic acid sequencer. Reference strains of pestiviruses [(BVDV type I, BVDV type II, border disease virus (BDV and hog cholera virus (HCV] and isolates from a previous investigation on BVDV in southern Africa were included for comparative purposes. All the BVDV strains obtained during this study belong to subgroups of BVDV genotype I. No association could be demonstrated between the geographic origin of the isolates. A number of isolates formed another branch separate from the existing branches Ia, Ib and Ic. These findings suggest that extensive genetic diversity can be found within BVDV type I isolates from southern Africa. Isolates that group with the classical BVDV type I strains, particularly of American origin, coexist with variants that appear to represent a local genetic pool and or variants evolving from the classical strains.

  16. Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil.

    Science.gov (United States)

    Drumond, Betania Paiva; Fagundes, Luiz Gustavo da Silva; Rocha, Raissa Prado; Fumagalli, Marcilio Jorge; Araki, Carlos Shigueru; Colombo, Tatiana Elisa; Nogueira, Mauricio Lacerda; Castilho, Thiago Elias; da Silveira, Nelson José Freitas; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-01-01

    Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1-4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.

  17. Phylogenetic characterization of virulent Newcastle disease viruses isolated during outbreaks in northwestern Iran in 2010.

    Science.gov (United States)

    Ahmadi, Elham; Pourbakhsh, Seyed Ali; Ahmadi, Malahat; Mardani, Karim; Talebi, Alireza

    2016-11-01

    The northwest of Iran shares long borders with three neighboring countries; therefore, it is considered one of the main entry portals of Newcastle disease virus (NDV) into the country. Ten virulent NDVs were recovered from 19 poultry farms of various prefectures in northwestern Iran during Newcastle disease outbreaks in 2010. The isolates were genotypically analyzed using an F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) assay. The amplified F gene (nucleotides 189-1666) sequences of the NDV isolates were compared phylogenetically with those of previously published strains in GenBank. All of the NDV isolates belonged to genotype VIIb and were closely related to some isolates from Iran, Russia, and Sweden. Therefore, it can be postulated that these isolates evolved from previously reported strains. The velogenic viruses carried the motif (112)R-R-Q-K-R/F(117) at the F0 cleavage site and a unique substitution of (190)L→F which had never been reported in any NDV genotype VIIb isolate. They shared high sequence similarity with each other but were distinct from current NDV vaccines and NDV strains reported from other countries. This information is fundamental for improving the efficacy of controlling strategies and vaccine development for NDV.

  18. Phylogenetic characterization and virulence of two Newcastle disease viruses isolated from wild birds in China.

    Science.gov (United States)

    Liu, Haijin; Zhang, Peng; Wu, Pengpeng; Chen, Shengli; Mu, Guohui; Duan, Xuji; Hao, Huafang; Du, Enqi; Wang, Xinglong; Yang, Zengqi

    2013-12-01

    Wild birds are considered as a natural reservoir of Newcastle disease virus (NDV). However, there is no information about genotype IX NDV from wild birds, especially from Columbiformes. In this study, two genotype IX NDV viruses were isolated from wild birds. One was from Eurasian Blackbird, while the other was from Spotted-necked dove. After purification by plaque technique, complete genomes of both viruses were sequenced. Phylogenetic analysis of partial fusion (F) gene and complete genome indicated both strains belonged to genotype IX. Based on intracerebral pathogenicity index (ICPI), the virus from Eurasian Blackbird was velogenic virus, while the strain from Spotted-necked dove was lentogenic virus. However, both strains showed one of velogenic cleavage sites. In addition, the strain from Eurasian Blackbird showed greater replication ability and generated larger fusion foci in vitro than that of strain from Spotted-necked dove. Comparing all the corresponding protein sequences of both strains, there were only 9 different amino acid residues between them. Furthermore, after analysis of these differences, the information about lentogenic NDV with multi-basic cleavage site was presented.

  19. Isolation and characterization of influenza C virus inhibitor in rat serum.

    Science.gov (United States)

    Kitame, F; Nakamura, K; Saito, A; Sinohara, H; Homma, M

    1985-10-01

    Two hemagglutination inhibitors for influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the hemagglutination by influenza C virus strains but did not affect the hemagglutination by influenza A and B virus strains. Hemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with influenza C virus at 37 degrees C but not with the other types of influenza virus, indicating that the inhibitors are specific for influenza C virus. The inhibitory activity was also destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is cleavable by the former neuraminidase but not by the latter is essential for the hemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.

  20. Pinhal Virus, a New Arenavirus Isolated from Calomys tener in Brazil.

    Science.gov (United States)

    Bisordi, Ivani; Levis, Silvana; Maeda, Adriana Y; Suzuki, Akemi; Nagasse-Sugahara, Teresa K; de Souza, Renato P; Pereira, Luiz E; Garcia, Jorge B; Cerroni, Matheus de P; de A e Silva, Franko; dos Santos, Cecília L S; da Fonseca, Benedito A L

    2015-11-01

    Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease.

  1. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

    Science.gov (United States)

    Shin, Dongyoung; Richards, Stephanie L; Alto, Barry W; Bettinardi, David J; Smartt, Chelsea T

    2013-01-01

    Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

  2. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

    Directory of Open Access Journals (Sweden)

    Dongyoung Shin

    Full Text Available Dengue virus (DENV is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL, including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1 and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW, FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

  3. Characterization of pigeon paramyxoviruses (Newcastle disease virus isolated in South Africa from 2001 to 2006

    Directory of Open Access Journals (Sweden)

    C. Abolnik

    2008-08-01

    Full Text Available Pigeon paramyxovirus type 1 (PPMV-1, a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced in to South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbil(l Bucorvus leadbeateri which becamea cutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had lCPl and MDT values characteristic of PPMV-1s trains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

  4. Molecular and biological characterization of Potato mop-top virus (PMTV, Pomovirus) isolates from potato-growing regions in Colombia

    DEFF Research Database (Denmark)

    Gil, José; Adams, Ian; Boonham, Neil

    2016-01-01

    samples were taken from the main potato-producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons......Potato mop-top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil...

  5. Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates.

    Science.gov (United States)

    Johansson, Tove; Einer-Jensen, Katja; Batts, William; Ahrens, Peter; Björkblom, Carina; Kurath, Gael; Björklund, Harry; Lorenzen, Niels

    2009-11-09

    Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) against the N protein. MAbs 136-1 and 136-3 reacted equally at all concentrations with the isolates tested, indicating that these antibodies identify a common epitope. MAb 34D3 separated the M and L genogroup isolates from the U genogroup isolate. MAb 1DW14D divided the European isolates into 2 groups. MAb 1DW14D reacted more strongly with DE-DF 13/98 11621 and RU-FR1 than with IT-217A, FR-32/87, DE-DF 4/99-8/99 and AU-9695338. In the phylogenetic studies, the Italian, French, German and Austrian isolates clustered in the M genogroup, whereas in the serological studies using MAbs, the European M genogroup isolates could not be placed in the same specific group. These results indicate that genotypic and serotypic classification do not correlate.

  6. ISOLATION OF VIRUS IN PRODUCTS OF CONCEPTION IN SPONTANEOUS ABORTION

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    KAMYAB S.D. AZARI S.D. NATEGH R

    1980-08-01

    Full Text Available Two hundred pa t ient s o f t he l ow socioe conomic g roup wi th an aborti on o f the f i r s t t r i mest e r were stud 1ed . The s ter i l e produc ts o f concept ion were c ul t ured on Gr e e n Monke y Kidney ti s sue cul t ure f or v i r a l de t e c - tion. Four po s i t i ve Herpe s virus, two positive Rubella"nv~ru s and t hree pos i tive unidentifie d Entero v irus l ike Vlrus were obtained The sera of 192 of these patients were titrated for Rubella antibody. The sera of a control group of 150 normal pregnant women were titrated for Rubella antibody and 8.8 per cent of these patients had a negative titre and 21.3 per cent , 1 had a tltre over 40

  7. A survey of fish viruses isolated from wild marine fishes from the coastal waters of southern Korea.

    Science.gov (United States)

    Kim, Wi-Sik; Choi, Shin-Young; Kim, Do-Hyung; Oh, Myung-Joo

    2013-11-01

    A survey was conducted to investigate viral infection in 253 wild marine fishes harvested in the southern coastal area of Korea from 2010 to 2012. The fish that were captured by local anglers were randomly bought and sampled for virus examination. The samples were tested for presence of virus by virus isolation with FHM, FSP, and BF-2 cells and molecular methods (polymerase chain reaction and sequencing). Of the 253 fish sampled, 9 fish were infected with virus. Aquabirnaviruses (ABVs), Viral hemorrhagic septicemia virus (VHSV), and Red seabream iridovirus (RSIV) were detected in 7, 1, and 1 fish, respectively. Molecular phylogenies demonstrated the detected viruses (ABV, VHSV, and RSIV) were more closely related to viruses reported of the same type from Korea and Japan than from other countries, suggesting these viruses may be indigenous to Korean and Japanese coastal waters.

  8. High-throughput isolation of giant viruses in liquid medium using automated flow cytometry and fluorescence staining.

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    Jacques Yaacoub Bou Khalil

    2016-01-01

    Full Text Available The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than ten strains of previously known species of giant viruses and 7 new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

  9. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

    Science.gov (United States)

    Khalil, Jacques Y. B.; Robert, Stephane; Reteno, Dorine G.; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  10. Ecologic observations of Venezuelan encephalitis virus in vertebrates and isolations of Nepuyo and Patois viruses from sentinel hamsters at Pacific and Atlantic habitats in Guatemala, 1968-1980.

    Science.gov (United States)

    Scherer, W F; Dickerman, R W; Cupp, E W; Ordonez, J V

    1985-07-01

    La Avellana and Puerto Barrios, two enzootic foci of Venezuelan encephalitis (VE) virus on the Pacific and Caribbean lowlands (respectively) of Guatemala have been studied over a 13-year period. Data from sentinel hamsters and guinea pigs and wild and domestic vertebrates are reported. VE virus strains were isolated from hamsters each period they were exposed during the rainy seasons 1968-1980 and at the end of the dry season 1974. Rates of isolation of VE virus ranged from 0.2%-5.7% hamster/days/exposure. All strains tested were free of epizootic virions. Although virus was isolated from sentinel guinea pigs, their deaths were not attributable to infection with VE virus. Antibody titers in 26 of 28 terrestrial mammals bled at La Avellana in 1971 were higher to enzootic than to epizootic VE strains. Thirty-seven percent of 109 residents of Puerto Barrios had antibody to VE virus. In 13 of 20 tested, antibodies were engendered by the enzootic strain. Nepuyo and Patois viruses were isolated from sentinel hamsters at both La Avellana and Puerto Barrios.

  11. Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

    Science.gov (United States)

    Albariño, César G; Uebelhoer, Luke S; Vincent, Joel P; Khristova, Marina L; Chakrabarti, Ayan K; McElroy, Anita; Nichol, Stuart T; Towner, Jonathan S

    2013-11-01

    Recent investigations have shown the Egyptian fruit bat (Rousettus aegyptiacus) to be a natural reservoir for marburgviruses. To better understand the life cycle of these viruses in the natural host, a new reverse genetics system was developed for the reliable rescue of a Marburg virus (MARV) originally isolated directly from a R. aegyptiacus bat (371Bat). To develop this system, the exact terminal sequences were first determined by 5' and 3' RACE, followed by the cloning of viral proteins NP, VP35, VP30 and L into expression plasmids. Novel conditions were then developed to efficiently replicate virus mini-genomes followed by the construction of full-length genomic clones from which recombinant wild type and GFP-containing MARVs were rescued. Surprisingly, when these recombinant MARVs were propagated in primary human macrophages, a dramatic difference was found in their ability to grow and to elicit anti-viral cytokine responses.

  12. Complete genome sequences of two biologically distinct isolates of Asparagus virus 1.

    Science.gov (United States)

    Blockus, S; Lesker, T; Maiss, E

    2015-02-01

    The complete genome sequences of two asparagus virus 1 (AV-1) isolates differing in their ability to cause systemic infection in Nicotiana benthamiana were determined. Their genomes had 9,741 nucleotides excluding the 3'-terminal poly(A) tail, encoded a polyprotein of 3,112 amino acids, and shared 99.6 % nucleotide sequence identity. They differed at 37 nucleotide and 15 amino acid sequence positions (99.5 % identity) scattered over the polyprotein. The closest relatives of AV-1 in amino acid sequence identity were plum pox virus (54 %) and turnip mosaic virus (53 %), corroborating the classification of AV-1 as a member of a distinct species in the genus Potyvirus.

  13. Isolation and characterization of influenza C virus inhibitors in rat serum.

    Science.gov (United States)

    Kitame, F; Nakamura, K; Saito, A; Sinohara, H; Homma, M

    1985-09-01

    Two inhibitors against haemagglutination by influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 2, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the haemagglutination by influenza C virus strains but did not affect the haemagglutination by influenza A and B virus strains. Haemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is excised by the former neuraminidase but not by the latter is essential for the haemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.

  14. Cymbidium chlorotic mosaic virus, a new sobemovirus isolated from a spring orchid (Cymbidium goeringii) in Japan.

    Science.gov (United States)

    Kondo, Hideki; Takemoto, Shogo; Maruyama, Kazuyuki; Chiba, Sotaro; Andika, Ida Bagus; Suzuki, Nobuhiro

    2015-08-01

    Cymbidium chlorotic mosaic virus (CyCMV), isolated from a spring orchid (Cymbidium goeringii), was characterized molecularly. CyCMV isometric virions comprise a single, positive-strand RNA genome of 4,083 nucleotides and 30-kDa coat protein. The virus genome contains five overlapping open reading frames with a genomic organization similar to that of sobemoviruses. BLAST searches and phylogenetic analysis revealed that CyCMV is most closely related to papaya lethal yellowing virus, a proposed dicot-infecting sobemovirus (58.8 % nucleotide sequence identity), but has a relatively distant relationship to monocot-infecting sobemoviruses, with only modest sequence identities. This suggests that CyCMV is a new monocot-infecting member of the floating genus Sobemovirus.

  15. [Sequencing and analysis of the complete genome of a rabies virus isolate from Sika deer].

    Science.gov (United States)

    Zhao, Yun-Jiao; Guo, Li; Huang, Ying; Zhang, Li-Shi; Qian, Ai-Dong

    2008-05-01

    One DRV strain was isolated from Sika Deer brain and sequenced. Nine overlapped gene fragments were amplified by RT-PCR through 3'-RACE and 5'-RACE method, and the complete DRV genome sequence was assembled. The length of the complete genome is 11863bp. The DRV genome organization was similar to other rabies viruses which were composed of five genes and the initiation sites and termination sites were highly conservative. There were mutated amino acids in important antigen sites of nucleoprotein and glycoprotein. The nucleotide and amino acid homologies of gene N, P, M, G, L in strains with completed genomie sequencing were compared. Compared with N gene sequence of other typical rabies viruses, a phylogenetic tree was established . These results indicated that DRV belonged to gene type 1. The highest homology compared with Chinese vaccine strain 3aG was 94%, and the lowest was 71% compared with WCBV. These findings provided theoretical reference for further research in rabies virus.

  16. First genome analysis and molecular characterization of Chickpea chlorotic dwarf virus Egyptian isolate infecting squash.

    Science.gov (United States)

    Fahmy, Inas Farouk; Taha, Omnia; El-Ashry, Abdel Nasser

    2015-06-01

    This study aims to identifying and characterizing some molecular properties of geminiviruses co-infection in squash field crop cultivated in Egypt. Squash crops observed to be heavily infected with several insect vectors, also severe chlorosis and stunting was observed. Electron microscopic analysis has revealed geminate capsid particles which indicate the infection of Geminiviruses, especially SqLCV which represent an economic problem to squash filed crop in Egypt. We have investigated possible mixed infections with different plant viruses associated with chlorotic stunt diseases and or other genus groups of geminiviruses. The main objective of this study is to investigate the recombination events, possible recombinants and variants among these genera in the same family differing in vector transmission. This is the first report of the molecular characterization, phylogenetic analysis and putative recombination events of the full length genome of the Chickpea Chlorotic Dwarf Mastrevirus in Egypt. And the first report of co-infection with another begomovirus infecting squash plants. A full length clone of both viruses were isolated and characterized at the molecular level. The complete nucleotide sequence of DNA-A was determined (2,572 bp) and submitted to the genbank under accession no. KF692356. The isolate from Egypt has about 97.8 % homology with the Chickpea chlorotic dwarf virus (CpCDV) isolate from Syria DNA-A isolate FR687959, a 83.2 % homology with the Sudan isolate AM933134 and a 82.7 % homology with Pakistan isolate FR687960. To best of our knowledge this is the first report of complete genome of CpCDV that infect squash plants in Egypt and worldwide.

  17. Typing of feline calicivirus isolates from different clinical groups by virus neutralisation tests.

    Science.gov (United States)

    Dawson, S; McArdle, F; Bennett, M; Carter, M; Milton, I P; Turner, P; Meanger, J; Gaskell, R M

    1993-07-03

    One hundred and thirteen isolates of feline calicivirus originating from seven different clinical groups were typed by virus neutralisation tests using eight different cat antisera. The clinical groups comprised 'healthy' cats, cases of acute oral/respiratory disease, chronic stomatitis, acute febrile lameness syndrome, vaccine reactions (clinical disease seen within 21 days of vaccination) and vaccine breakdowns (clinical disease seen more than 21 days after but within one year of vaccination). Isolates from the vaccine reaction cases were grouped into those associated with acute oral/respiratory disease alone and those associated with the lameness syndrome, and the latter group was further subdivided according to the vaccine used. Two groups appeared significantly different from others with some of the antisera. Thus the lameness vaccine reaction isolates associated with vaccine B were significantly different from the isolates from all the other clinical groups, including other lameness isolates, with a number of the antisera. In addition, the chronic stomatitis isolates were significantly different from those from the 'healthy' and the acute oral/respiratory disease groups with one or two of the antisera. Eighty-five to 88 per cent of the isolates were neutralised by antisera raised against F9 or F9-like vaccine strains at a dilution of 1 in 2. Twenty antibody units of such antisera neutralised 42 to 80 per cent of the isolates. A bivalent antiserum raised against a vaccine F9 strain and field strain LS015 neutralised 96 per cent of the isolates at a dilution of 1 in 2, and 20 antibody units neutralised 68 per cent of isolates. Antisera to field strain F65 neutralised all the remaining isolates at a dilution of 1 in 2 and 44 per cent of the remaining isolates at a dilution of 20 antibody units. Therefore, strains LS015 and F65 may be of use in the production of a polyvalent feline calicivirus vaccine, together with the widely used strain F9.

  18. Cloning of full genome sequence of hepatitis E virus of Shanghai swine isolate using RACE method

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    Mou Jing

    2007-10-01

    Full Text Available Abstract Genotype 4 hepatitis E virus (HEV was reportedly transmitted freely between humans and swine in eastern China. The full-length genomic sequence of Shanghai swine isolate (SH-SW-zs1 recovered from feces sample of a pig which was infected with HEV RNA positive swine serum was determined using RT-PCR and RACE (Rapid Amplification of cDNA Ends methods. The full genome of the SH-SW-zs1 isolate was 7265 nucleotides in length and phylogenetic analysis indicated that this isolate belonged to genotype 4. Comparison of the 3' UTR sequence with the corresponding regions of other 38 HEV strains from different region revealed that the Shanghai swine isolate is 21–49 bp longer than the other stains.

  19. Emergence of simian virus 40 variants during serial passage of plaque isolates.

    Science.gov (United States)

    Norkin, L C; Tirrell, S M

    1982-05-01

    Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant.

  20. Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010.

    Science.gov (United States)

    Nougairede, Antoine; Bichaud, Laurence; Thiberville, Simon-Djamel; Ninove, Laetitia; Zandotti, Christine; de Lamballerie, Xavier; Brouqui, Philippe; Charrel, Remi N

    2013-09-01

    Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is an emerging arthropod-borne virus transmitted by phlebotomine sandflies. TOSV is a frequent cause of central nervous system infection during the warm season in several countries bordering the Mediterranean Sea. Here, we report a case of TOSV aseptic meningitis diagnosed in 2012 in Marseille, France. The virus strain was recovered in cell culture from the cerebrospinal fluid. New-generation sequencing based on Ion Torrent technology was used to determine its complete genome sequence. Phylogenetic analysis based on the partial L segment revealed that this isolate belongs to the lineage B together with other French, Spanish, and Moroccan strains. Although several cases of TOSV meningitis are reported in the literature, few of them are diagnosed by RT-PCR combined with virus isolation and further sequence characterization. This case report supports that virus isolation should be attempted whenever possible because this remains the gold standard technique for diagnosis of arthropod-borne viral infections.

  1. Vanilla mosaic virus isolates from French Polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively infect vanilla.

    Science.gov (United States)

    Farreyrol, K; Pearson, M N; Grisoni, M; Cohen, D; Beck, D

    2006-05-01

    Sequence was determined for the coat protein (CP) gene and 3' non-translated region (3'NTR) of two vanilla mosaic virus (VanMV) isolates from Vanilla tahitensis, respectively from the Cook Islands (VanMV-CI) and French Polynesia (VanMV-FP). Both viruses displayed distinctive features in the N-terminal region of their CPs; for VanMV-CI, a 16-amino-acid deletion including the aphid transmission-related DAG motif, and for VanMV-FP, a stretch of GTN repeats that putatively belongs to the class of natively unfolded proteins. VanMV-FP CP also has a novel DVG motif in place of the DAG motif, and an uncommon Q//V protease cleavage site. The sequences were compared to a range of Dasheen mosaic virus (DsMV) strains and to potyviruses infecting orchids. Identity was low to DsMV strains across the entire CP coding region and across the 3'NTR, but high across the CP core and the CI-6K2-NIa region. In accordance with current ICTV criteria for species demarcation within the family Potyviridae, VanMV-CI and VanMV-FP are strains of DsMV that exclusively infect vanilla.

  2. Short communication. Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV isolates in Kosovo

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    Izedin Goga

    2014-03-01

    Full Text Available Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of BVDV and represent the first documented data about Kosovo BVDV isolates.

  3. Fish viruses: transmission and pathogenicity of an icosahedral cytoplasmic deoxyribovirus isolated from sheatfish (Silurus glanis).

    Science.gov (United States)

    Ahne, W; Ogawa, M; Schlotfeldt, H J

    1990-05-01

    An iridovirus-like agent which was isolated from sheatfish fry showed to be of high virulence to those fish. 100% of sheatfish fry infected by water bath died within 8 days. The agent could be transmitted by infected fish causing 100% mortality in 11 days. Moribund fry showed spiralic swimming and exhibited hemorrhages in the skin. The infection studies demonstrate that the isolated virus was responsible for a sudden epizootic in 1988 with 100% mortality occurring in a warm water recirculating aquaculture unit in Northwest Germany, regularly controlled by the State Fish Epidemics Control Service of Lower Saxony and Fish Health Service.

  4. Occurrence and characterization of plum pox virus strain D isolates from European Russia and Crimea.

    Science.gov (United States)

    Chirkov, Sergei; Ivanov, Peter; Sheveleva, Anna; Kudryavtseva, Anna; Prikhodko, Yuri; Mitrofanova, Irina

    2016-02-01

    Numerous plum pox virus (PPV) strain D isolates have been found in geographically distant regions of European Russia and the Crimean peninsula on different stone fruit hosts. Phylogenetic analysis of their partial and complete genomes suggests multiple introductions of PPV-D into Russia. Distinct natural isolates from Prunus tomentosa were found to bear unique amino acid substitutions in the N-terminus of the coat protein (CP) that may contribute to the adaptation of PPV-D to this host. Serological analysis using the PPV-D-specific monoclonal antibody 4DG5 provided further evidence that mutations at positions 58 and 59 of the CP are crucial for antibody binding.

  5. Tissue distribution of virus replication in cats experimentally infected with distinct feline calicivirus isolates.

    Science.gov (United States)

    Truyen, U; Geissler, K; Hirschberger, J

    1999-09-01

    Four specific pathogen-free (SPF) cats were each inoculated with one of two genetically and antigenically well characterized feline caliciviruses originally isolated from cats with acute respiratory disease (FCV-KS100/2), or with chronic stomatitis (FCV-KS20). Two cats of each group were euthanized at day 10 post infection and two cats at day 28. No clear differences between the clinical disease induced by the two isolates could be observed, and no apparent differences in the tissue spectrum were seen between day 10 and 28. No persistent virus shedding was observed over the 4-week period of this experiment.

  6. Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea

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    Ju Yeon Yoon

    2014-06-01

    Full Text Available Apple stem pitting virus (ASPV, of the Foveavirus genus in the family Betaflexiviridae, is one of the most common viruses of apple and pear trees. To examine variability of the coat protein (CP gene from ASPV, eight isolates originating from 251 apple trees, which were collected from 22 apple orchards located in intensive apple growing areas of the North Gyeongsang and North Jeolla Provinces in Korea, were sequenced and compared. The nucleotide sequence identity of the CP gene of eight ASPV isolates ranged from 77.0 to 97.0%, while the amino acid sequence identity ranged from 87.7 to 98.5%. The N-terminal region of the viral CP gene was highly variable, whereas the C-terminal region was conserved. Genetic algorithm recombination detection (GARD and single breakpoint recombination (SBP analyses identified base substitutions between eight ASPV isolates at positions 54 and 57 and position 771, respectively. GABranch analysis was used to determine whether the eight isolates evolved due to positive selection. All values in the GABranch analysis showed a ratio of substitution rates at non-synonymous and synonymous sites (dNS/dS below 1, suggestive of strong negative selection forces during ASPV CP history. Although negative selection dominated CP evolution in the eight ASPV isolates, SLAC and FEL tests identified four possible positive selection sites at codons 10, 22, 102, and 158. This is the first study of the ASPV genome in Korea.

  7. Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea.

    Science.gov (United States)

    Yoon, Ju Yeon; Joa, Jae Ho; Choi, Kyung San; Do, Ki Seck; Lim, Han Cheol; Chung, Bong Nam

    2014-06-01

    Apple stem pitting virus (ASPV), of the Foveavirus genus in the family Betaflexiviridae, is one of the most common viruses of apple and pear trees. To examine variability of the coat protein (CP) gene from ASPV, eight isolates originating from 251 apple trees, which were collected from 22 apple orchards located in intensive apple growing areas of the North Gyeongsang and North Jeolla Provinces in Korea, were sequenced and compared. The nucleotide sequence identity of the CP gene of eight ASPV isolates ranged from 77.0 to 97.0%, while the amino acid sequence identity ranged from 87.7 to 98.5%. The N-terminal region of the viral CP gene was highly variable, whereas the C-terminal region was conserved. Genetic algorithm recombination detection (GARD) and single breakpoint recombination (SBP) analyses identified base substitutions between eight ASPV isolates at positions 54 and 57 and position 771, respectively. GABranch analysis was used to determine whether the eight isolates evolved due to positive selection. All values in the GABranch analysis showed a ratio of substitution rates at non-synonymous and synonymous sites (dNS/dS) below 1, suggestive of strong negative selection forces during ASPV CP history. Although negative selection dominated CP evolution in the eight ASPV isolates, SLAC and FEL tests identified four possible positive selection sites at codons 10, 22, 102, and 158. This is the first study of the ASPV genome in Korea.

  8. Detection and phylogenetic analysis of peste des petits ruminants virus isolated from outbreaks in Punjab, Pakistan.

    Science.gov (United States)

    Munir, M; Zohari, S; Saeed, A; Khan, Q M; Abubakar, M; LeBlanc, N; Berg, M

    2012-02-01

    Peste des Petits Ruminants (PPR) is an important viral disease of small ruminants and is endemic in Pakistan. In the following study, samples from two outbreaks of PPR in goats have been subjected to laboratory investigations. The Peste des Petits Ruminants virus (PPRV) genome was detected using both conventional and real-time PCR. Genetic characterization of the local PPRV field isolates was conducted by sequencing 322 bp of the fusion (F) gene and 255 bp of the nucleoprotein (N) gene. The phylogenetic tree based on the F gene clustered samples from both outbreaks into lineage 4 along with other Asian isolates, specifically into subcluster 1 along with isolates from Middle East. Analysis of N gene revealed a different pattern. In this case, the Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates, which probably represents the true geographical pattern of virus circulation. This is the first report presenting the phylogenetic tree based on N gene as well as performing a parallel comparison of the trees of F and N gene together from Pakistani isolates. The results of this study shed light on the PPRV population in Pakistan and emphasize the importance of using molecular methods to understand the epidemiology. Such understanding is essential in any efforts to control the number and impact of outbreaks that are occurring in endemic countries such as Pakistan, especially in the current scenario where OIE and FAO are eager to control and subsequently eradicate PPR from the globe, as has been achieved for Rinderpest.

  9. Screening, isolation and optimization of anti-white spot syndrome virus drug derived from terrestrial plants

    Institute of Scientific and Technical Information of China (English)

    Upasana Ghosh; Somnath Chakraborty; Thangavel Balasubramanian; Punyabrata Das

    2014-01-01

    Objective: To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model.Methods:Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.Results: Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.Conclusions:The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug.

  10. Isolation and diagnosis of Chikungunya virus causing outbreaks in Andhra Pradesh, India

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    C.V.M. Naresh Kumar

    2013-01-01

    Full Text Available Background: Chikungunya fever has recently re-emerged in India with a high morbidity. However, the prevalence of chikungunya fever in India has been underreported due to non-availability of specialized kits to confirm the disease in most of the laboratories. Methods: Nine hundred and fifty six serum samples were collected from subjects presenting with a short febrile illness from various places in Chittoor district, Andhra Pradesh, between January to October 2009 and were screened for Chikungunya Virus (CHIKV infection. Virus isolation, reverse transcriptase - polymerase chain reaction (RT-PCR and immunoglobulin M (IgM rapid strip method were employed for the identification of the causative agent. Results: Chikungunya Virus (CHIKV infection was confirmed in 520 (68.1% patients by RT-PCR. Seventy seven (40.1% patients showed the presence of anti-CHIKV IgM antibodies while 12 (6.3% patients showed the presence of both anti-CHIKV IgM and immunoglobulin G (IgG antibodies respectively. The isolation of CHIKV was successful from five patients. Conclusions: The re-emergence and persistence of CHIKV in Andhra Pradesh suggests the need for continuous monitoring and identification of the pathogen and thereby prevention of the spread of the virus to other parts of the country.

  11. Complete genome sequence analysis of two Citrus tatter leaf virus (CTLV) isolates from China

    Institute of Scientific and Technical Information of China (English)

    SONG Zhen; LI Zhong-an; LIU Ke-hong; ZHOU Chang-yong

    2015-01-01

    In order to understand molecular characterization of Citrus tatter leaf virus (CTLV) isolated from China, ful-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome ampliifcation by RT-PCR. The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6 497 nucleotides in length and shared 79.9–91.0%and 78.8–98.0%nucleotide sequence identity, respectively, with other Apple stem grooving virus (ASGV) or CTLV strains available in GenBank. Unexpectedly, CTLV-MTH showed the highest nucleo-tide sequence identity (91%) with an apple isolate of ASGV, fol owed by 86.5%with ASGV-HH and 85.7%with ASGV-CHN. Furthermore, CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree, suggesting it has a closer relationship to ASGV than to CTLV. Therefore, it can be concluded roughly that CTLV may be not a distinct strains of ASGV. We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.

  12. Laboratory diagnosis of contagious ecthyma: comparison of different PCR protocols with virus isolation in cell culture.

    Science.gov (United States)

    Kottaridi, Christine; Nomikou, Kiki; Lelli, Rossella; Markoulatos, Panayotis; Mangana, Olga

    2006-06-01

    A new polymerase chain reaction (PCR) assay for rapid diagnosis of contagious ecthyma was designed and applied to 21 clinical samples from Greece. This assay, which detects a highly conserved gene from the parapox genome, was evaluated for its sensitivity and specificity in order to be considered as a useful diagnostic tool. A comparative study with two published PCR protocols one using primers PPP1-PPP3, PPP1-PPP4 which targets putative virion envelope gene B2L and the other using VIR1-VIR2 primers which amplifies ORF virus interferon resistant (VIR) gene, as well as cell culture virus neutralization assay was carried out. All samples tested were amplified successfully with the PCR protocol established in the laboratory. The combination of primers PPP1-PPP3 and PPP1-PPP4 in a semi-nested PCR gave a positive result in 20 of 21 samples while primers VIR1-VIR2 failed to amplify successfully 7 of 21 samples. The diagnostic value of parapox viral DNA amplification was also compared with the results of virus isolation by cell culture and was positive in three samples that the virus isolation was obtained.

  13. Tobacco curly shoot virus iso-lated in Yunnan is a distinct species of Begomovirus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Y1 is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).

  14. Isolation and characterization of tick-borne encephalitis virus from Ixodes persulcatus in Mongolia in 2012.

    Science.gov (United States)

    Muto, Memi; Bazartseren, Boldbaatar; Tsevel, Bazartseren; Dashzevge, Erdenechimeg; Yoshii, Kentaro; Kariwa, Hiroaki

    2015-07-01

    Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus, in the family Flaviviridae. The virus, which is endemic in Europe and northern parts of Asia, causes severe encephalitis. Tick-borne encephalitis (TBE) has been reported in Mongolia since the 1980s, but details about the biological characteristics of the endemic virus are lacking. In this study, 680 ticks (Ixodes persulcatus) were collected in Selenge aimag, northern Mongolia, in 2012. Nine Mongolian TBEV strains were isolated from tick homogenates. A sequence analysis of the envelope protein gene revealed that all isolates belonged to the Siberian subtype of TBEV. Two strains showed similar growth properties in cultured cells, but their virulence in mice differed. Whole genome sequencing revealed only thirteen amino acid differences between these Mongolian TBEV strains. Our results suggest that these naturally occurring amino acid mutations affected the pathogenicity of Mongolian TBEV. Our results may be an important platform for monitoring TBEV to evaluate the epidemiological risk in TBE endemic areas of Mongolia.

  15. Isolation and pathogenic analysis of virulent Marek's disease virus field strain in China.

    Science.gov (United States)

    Cui, Ning; Su, Shuai; Sun, Peng; Zhang, Yankun; Han, Ni; Cui, Zhizhong

    2016-07-01

    Marek's disease (MD) has become increasingly common in China, resulting in considerable economic loss. The etiological agent is unclear. In this study, we isolated a field MD virus (MDV) strain, designated SX1301, from CVI988/Rispens-vaccinated chickens with tumors. Co-infection of avian leukosis virus, reticuloendotheliosis virus, and chicken infectious anemia virus was excluded by polymerase chain reaction, enzyme-linked immunosorbant assay, DNA blotting hybridization, and indirect immunofluorescence assay. As with most strains isolated in China, SX1301 had the same amino acid mutation of meq protein at positions 77(E), 80(Y), and 115(A) Animal experimental results showed development of lethal MD in 57% and MD tumor in 23% of the specific pathogen-free chickens inoculated with SX1301, with tumors mainly distributed in spleen, liver, and kidney. CVI988/Rispens protected 83% of chickens upon challenge with SX1301, with a mortality rate and tumor incidence of 10% and 7%, respectively. These results implicated SX1301 as a virulent MDV strain, with commercial MDV vaccine CVI988/Rispens unable to confer adequate protection against SX1301. There have been no reports of very virulent (vv) plus MDV in China, but frequently occurring virulent MDV may account for the repeated outbreaks of MD. Vaccines with greater efficacy are needed to protect against MDV.

  16. Phylogenetic study on the 5'-untranslated region of bovine viral diarrhoea virus isolates from Iran

    Directory of Open Access Journals (Sweden)

    Majid Esmaelizad

    2014-09-01

    Full Text Available Bovine viral diarrhoea virus is a pathogen of bovids associated with reproduction system, causing in infected animals a range of ailments, from abortion to congenital defects. In this article, the nucleotide structure of the 5'-untranslated region (5-UTR from 7 Iranian bovine diarrhoea virus (BVDV isolates was characterized and subjected to comparative analysis against a panel of BVDV isolates from different sources. To this end, a 288 bp-long stretch of the internal ribosome entry site was amplified by RT-PCR. The PCR products subsequently cloned into PTZ57T vector and sequenced using T7 promoter primers. This resulted in detection of 3 new point mutations G→A and G→T in 2 isolates. When these findings were phylogenetically assessed, all the examined Iranian isolates were deemed to belong to the type1 of BVDV. Besides, 2 subtypes were identified among these isolates. In group A, a high level of similarity (99.2% between Iranian isolates with a cytopathic Australian strain of BVDV-1c was detected; while in group B, the 4 Iranian isolates proved to be very similar to NADL-like BVDV-1a strains. We believe that the surprisingly high level of similarity between group A Iranian isolates and their corresponding Australian strain is likely to be an indication of a shared common ancestor. If correct, the most likely explanation of this observation is the introduction of such strains from Australia to Iran, possibly through exportation of infected live animals or animal productions (e.g. semen and meat at some points in the past. Nevertheless, this hypothesis remains to be proved as further epidemiological work at genomic level is required to understand population of BVDV in Iran.

  17. Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    L.C. Kreutz

    2000-12-01

    Full Text Available Three Brazilian isolates of bovine viral diarrhea virus (BVDV, antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs. Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11, were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid and 1:100 (hybridoma culture supernatant in IFA and immunoperoxidase (IPX staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

  18. Molecular and antigenic characteristics of Newcastle disease virus isolates from domestic ducks in China.

    Science.gov (United States)

    Wu, Wei; Liu, Huairan; Zhang, Tingting; Han, Zongxi; Jiang, Yanyu; Xu, Qianqian; Shao, Yuhao; Li, Huixin; Kong, Xiangang; Chen, Hongyan; Liu, Shengwang

    2015-06-01

    Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192 nt, and 15,198 nt, which follow the "rule of six" that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don't form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs.

  19. Complete Genome Sequence and Phylogenetic Relatedness of Hepatitis B Virus Isolates in Papua, Indonesia▿ †

    OpenAIRE

    Utsumi, Takako; Lusida, Maria Inge; Yano, Yoshihiko; Nugrahaputra, Victor Eka; Amin, Mochamad; Juniastuti,; Soetjipto; Hayashi, Yoshitake; Hotta, Hak

    2009-01-01

    Each hepatitis B virus (HBV) genotype and subgenotype is associated with a particular geographic distribution, ethnicity, and anthropological history. Our previous study showed the novel HBV subgenotypes C6 (HBV/C6) and D6 (HBV/D6), based on the S gene sequences of isolates in Papua, Indonesia. The present study investigated the complete genome sequence of 22 strains from Papua and subjected them to molecular evolutionary analysis. A phylogenetic analysis revealed that 9 out of 22 strains wer...

  20. Genetic structure and molecular variability of Cucumber mosaic virus isolates in the United States.

    Directory of Open Access Journals (Sweden)

    Shahideh Nouri

    Full Text Available Cucumber mosaic virus (CMV has a worldwide distribution and the widest host range of any known plant virus. From 2000 to 2012, epidemics of CMV severely affected the production of snap bean (Phaseulos vulgaris L. in the Midwest and Northeastern United States. Virus diversity leading to emergence of new strains is often considered a significant factor in virus epidemics. In addition to epidemics, new disease phenotypes arising from genetic exchanges or mutation can compromise effectiveness of plant disease management strategies. Here, we captured a snapshot of genetic variation of 32 CMV isolates collected from different regions of the U.S including new field as well as historic isolates. Nucleotide diversity (π was low for U.S. CMV isolates. Sequence and phylogenetic analyses revealed that CMV subgroup I is predominant in the US and further showed that the CMV population is a mixture of subgroups IA and IB. Furthermore, phylogenetic analysis suggests likely reassortment between subgroups IA and IB within five CMV isolates. Based on phylogenetic and computational analysis, recombination between subgroups I and II as well as IA and IB in RNA 3 was detected. This is the first report of recombination between CMV subgroups I and II. Neutrality tests illustrated that negative selection was the major force operating upon the CMV genome, although some positively selected sites were detected for all encoded proteins. Together, these data suggest that different regions of the CMV genome are under different evolutionary constraints. These results also delineate composition of the CMV population in the US, and further suggest that recombination and reassortment among strain subgroups does occur but at a low frequency, and point towards CMV genomic regions that differ in types of selection pressure.

  1. Isolation of dual-tropic murine leukemia virus from radiation-induced thymoma in RF mouse

    Energy Technology Data Exchange (ETDEWEB)

    Okumoto, M.; Nishikawa, R.; Takamori, Y.; Iwai, Y.; Iwai, M. (Radiation Center of Osaka Prefecture, Sakai (Japan))

    1981-03-01

    Two different murine leukemia viruses (MuLV) were isolated from radiation-induced thymoma in RF mouse by co-cultivation method with mink lung cells. One is ecotropic MuLV, which replicates efficiently in NIH Swiss mouse embryo (NIH ME) and SC-1 cells and was able to form XC plaques. The other was dualtropic MuLV, which replicates in both mink lung cells and NIH ME cells but was unable to form XC plaques.

  2. Isolation of Hepatitis C Virus in Norjizac Vials

    Directory of Open Access Journals (Sweden)

    Zary Nokhodian

    2010-01-01

    Full Text Available Hepatitis C virus (HCV has been recognized as a major health problem worldwide. The estimated number of infected individuals is over 170 million people worldwide (1. After hepatitis B, it is the most important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Therefore, prevention of HCV infection is one of the most important public health concerns, because the majority of infected people would experience chronic hepatitis (2, 3. HCV can be easily transmitted through blood products transfusion and infected syringes. No surprise, the infection rate is remarkably high among injecting drug abusers (IDUs (4, 5.HCV has been identified as the most common viral infection affecting IDUs (6. Sharing contaminated needles and syringes, going to shooting galleries, using cocaine, unprotected sex, and sharing shaving equipments, are all among the major causes of contaminating IDUs with HCV (7, 8. In some countries such as India, Pakistan, Indonesia, and Thailand, the prevalence of anti-HCV antibody among the IDUs is reported to be at least 90% (9. The reported prevalence from Iran ranged from 38% to 47% (10 and in another study it is about 60% (11."Norjizac," also known as "hand-made Temgizac," and "Ab Crack (Crack solution," is a slang name for a drug which is abused by a number of IDUs in Iran since five years ego. It is usually used as intravenous injections, although some of drug abusers use Norjizac intramuscularly and/or subcutaneously. Norjizac is one of the most hard drugs in Iran and accompanying several complications like abscess formation, development of septic emboli and soft tissue infections in IDUs. Based on reports on probable human contamination in Norjizac vials, we conducted this study to find out why some of IDUs who used the drug have developed HCV infection without any history of sharing in injection tools. In a case series reported from Isfahan conducted in 2008, 14 vials of Norjizac bought from smugglers from various

  3. A cytoplasmic polyhedrosis virus isolated from the pine processionary caterpillar, Thaumetopoea pityocampa.

    Science.gov (United States)

    Ince, Ikbal Agah; Demir, Ismail; Demirbag, Zihni; Nalcacioglu, Remziye

    2007-04-01

    A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 microm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae.

  4. Infectivity analysis of a blackgram isolate of Mungbean yellow mosaic virus and genetic assortment with MYMIV in selective hosts.

    Science.gov (United States)

    Haq, Q M I; Rouhibakhsh, A; Ali, Arif; Malathi, V G

    2011-06-01

    Yellow mosaic disease in grain legumes in Indian subcontinent is caused by two important virus species viz. Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV), belonging to the genus Begomovirus of the family Geminiviridae. The genomic components of a begomovirus causing yellow mosaic disease in blackgram in southern India were cloned and sequenced. Nucleotide sequence comparison of DNA A component shows the virus isolate to be a variant of Mungbean yellow mosaic virus:-(MYMV-[IN:Vam:05]). However, DNA B component of the present virus isolate has greater similarity (92%) to Mungbean yellow mosaic India virus. Agroinoculations of the viral clones produced typical yellow mosaic symptoms in blackgram and mungbean, severe leaf curl and stunting in French bean, similar to blackgram isolate of MYMIV. Blackgram isolates of both the virus species were only mildly infectious on cowpea, produced atypical leaf curl symptoms and not yellow or golden mosaic. In agroinoculations done by exchanging genomic components, symptom expression was seen only in French bean. In cowpea, blackgram and mungbean there was no visible symptoms though viral DNA could be detected by PCR.

  5. Excretion of bovine herpesvirus 1 in semen is detected much longer by PCR than by virus isolation

    NARCIS (Netherlands)

    Engelenburg, van F.A.C.; Schie, van F.W.; Rijsewijk, F.A.M.; Oirschot, van J.T.

    1995-01-01

    To compare the sensitivities of PCR and virus isolation and to examine the course of virus excretion in semen, we intrapreputially inoculated eight bulls with bovine herpesvirus 1 (BHV1) and used two bulls as sentinels. From these bulls, we collected a large panel of semen samples during 65 days pos

  6. Full-Genome Sequence of a Reassortant H1N1 Swine Influenza Virus Isolated from Pigs in Italy.

    Science.gov (United States)

    Chiapponi, Chiara; Baioni, Laura; Luppi, Andrea; Moreno, Ana; Castellan, Alberto; Foni, Emanuela

    2013-10-03

    In this study, the full-genome sequence of a novel reassortant H1N1 swine influenza virus (SIV) is reported. The isolate has a hemagglutinin (HA) gene of the pandemic H1N1 influenza virus, but it carries the seven genome segments of the avian-origin H1N1 SIV currently circulating in European pig farms.

  7. Newcastle disease viruses causing recent outbreaks worldwide show unexpectedly high genetic similarity with historical virulent isolates from the 1940s

    Science.gov (United States)

    Virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), a devastating disease of poultry and wild birds. Phylogenetic analyses clearly distinguish historical isolates (obtained prior to 1960) from currently circulating viruses of class II genotypes V, VI, VII, and XII throug...

  8. Blueberry fruit drop associated virus: A new member of the family Caulimoviridae isolated from blueberry exhibiting fruit drop symptoms

    Science.gov (United States)

    This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from ‘Bluecrop’ blueberry plants named Blueberry fruit drop associated virus (BFDaV). Infected bushes lose nearly 100% of their fruit prior to harvest, and in springtime young leaves and flowers develop ...

  9. A complete sequence and comparative analysis of a SARS-associated virus (Isolate BJ01)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-associated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs. We report the complete genome sequence and comparative analysis of an isolate (BJ01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 2 ORFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS- associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymousmutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host. Two amino acid changes have been detected in the M protein, which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).

  10. Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions

    DEFF Research Database (Denmark)

    Mansfield, K.L.; Racloz, V.; McElhinney, L.M.

    2006-01-01

    We report a Molecular epidemiological study of rabies in Arctic Countries by comparing a panel of novel Greenland isolates to a larger cohort of viral sequences from both Arctic and Baltic regions. Rabies Virus isolates originating from wildlife (Arctic/red foxes, raccoon-dogs and reindeer), from...... Was differentiated into two lineages, Arctic 1 and Arctic 2, with good bootstrap Support. Arctic I is mainly comprised of Canadian isolates with a single fox isolate front Maine in the USA. Arctic 2 was further divided into sub-lineages: 2a/2b. Arctic 2a comprises isolates from the Arctic regions of Yakutia...

  11. Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

    Directory of Open Access Journals (Sweden)

    Cisneros Alejandro

    2009-12-01

    Full Text Available Abstract Background Dengue (DEN is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating. Results To study whether there are isolates from Oaxaca having recombination, we obtained the sequence of 6 different isolates of DENV-2 Asian/American genotype from the outbreak 2005-6, one clone of the C(91-prM-E-NS1(2400 structural genes, and 10 clones of the E gene from the isolate MEX_OAX_1656_05. Evidence of recombination was found by using different methods along with two softwares: RDP3 and GARD. The Oaxaca MEX_OAX_1656_05 and MEX_OAX_1038_05 isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the Cosmopolitan genotype. Furthermore, the clone of the E gene namely MEX_OAX_165607_05 from this study was also recombinant, incorporating genome sequence from the American genotype. Conclusions This is the first report of recombination in DENV-2 in Mexico. Given such a recombinant activity new genomic combinations were produced, this could play a significant role in the DENV evolution and must be considered as a potentially important mechanism generating genetic variation in this virus with serious implications for

  12. Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

    Science.gov (United States)

    Trucco, Verónica; de Breuil, Soledad; Bejerman, Nicolás; Lenardon, Sergio; Giolitti, Fabián

    2014-06-01

    The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

  13. Lassa virus isolates from Mali and the Ivory Coast represent an emerging fifth lineage.

    Science.gov (United States)

    Manning, John T; Forrester, Naomi; Paessler, Slobodan

    2015-01-01

    Previous imported cases of Lassa fever (LF) into the United Kingdom from the Ivory Coast and Mali, as well as the detection of Lassa virus (LASV) among the Mastomys natalensis population within Mali has led to the suggestion that the endemic area for LF is expanding. Initial phylogenetic analyses arrange isolates from Mali and the Ivory Coast separately from the classical lineage IV isolates taken from Sierra Leone, Guinea, and Liberia. The availability of full genome sequences continues to increase, allowing for a more complete phylogenetic comparison of the isolates from Mali and the Ivory Coast to the other existing isolates. In this study, we utilized a Bayesian approach to infer the demographic histories of each LASV isolate for which the full sequence was available. Our results indicate that the isolates from Mali and the Ivory Coast group separately from the isolates of lineage IV, comprising a distinct fifth lineage. The split between lineages IV and V is estimated to have occurred around 200-300 years ago, which coincides with the colonial period of West Africa.

  14. Identification of a new Newcastle disease virus isolate from Indonesia represents an ancestral lineage of class II genotype XIII.

    Science.gov (United States)

    Forrester, Naomi L; Widen, Steve G; Wood, Thomas G; Travassos da Rosa, Amelia P; Ksiazek, Thomas G; Vasilakis, Nikos; Tesh, Robert B

    2013-08-01

    An unknown virus was isolated from a mosquito pool collected in Jakarta during routine surveillance in 1979. Analysis of the sample using the Illumina platform resulted in the identification of a Newcastle disease virus (NDV) isolate. The sequence of the isolate indicated that it is an ancestral lineage of class II, genotype XIII. The source of the isolate is unusual, as newcastle disease virus is not believed to be vector-borne, although this mosquito pool was processed in a laboratory also handling samples for avian influenza surveillance and it is possible that this resulted in cross-contamination. This NDV isolate is still ancestral to most extant genotype XIII strains and provides a useful insight into historic NDV evolution.

  15. Isolation and characterization of a variant porcine epidemic diarrhea virus in China

    Directory of Open Access Journals (Sweden)

    Pan Yongfei

    2012-09-01

    Full Text Available Abstract An outbreak of diarrhea in pigs started in Guangdong, South China in January 2011. Cases were characterized by watery diarrhea, dehydration and vomiting, with 80–100% morbidity and 50–90% mortality in suckling piglets. The causative agent of the diarrhea was ultimately identified as porcine epidemic diarrhea virus (PEDV. In this study, we isolated a PEDV strain designated CHGD-01 from piglet intestines using Vero cell cultures, and its specific cytopathic effects were confirmed in susceptible cells by direct immunofluorescence testing and electron microscopy. The complete genome of CHGD-01 was shown to be 28,035 nucleotides in length, with a similar structure to that of PEDV reference strains. Phylogenetic analyses based on the whole genome revealed that CHGD-01 shared nucleotide sequence identities of 98.2–98.4% with two other Chinese isolates reported in the same year, thus constituting a new cluster. Amino acid sequence analysis based on individual virus genes indicated a close relationship between the spike protein gene of CHGD-01 and the field strain KNU0802 in Korea. Its ORF3 and nucleoprotein genes, however, were divergent from all other sequenced PEDV isolate clusters and therefore formed a new group, suggesting a new variant PEDV isolate in China. Further studies will be required to determine the immunogenicity and pathogenicity of this new variant.

  16. Molecular Typing of Field Isolates from two outbreaks of Infectious Bursal Disease Virus from Pakistan

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    Q. M. Khan and M. J. Arshed

    Full Text Available A reverse transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR/RFLP technique was used for the identification and characterization of Pakistani field isolates of infectious bursal disease virus (IBDV. A total of 8 bursa samples were collected from two outbreaks during September and October 2003 from Tehsil Sumandri, Dist. Faisalabad with 40-50% mortality in commercially reared broiler chicken flocks experiencing signs typical of infectious bursal disease (IBD. Four samples were found to contain IBDV genome by One Step RTPCR using VP2 gene specific primers. The assay amplified a 743 bp fragment from 701-1444 nucleotides. RT-PCR product was further subjected to restriction digestion using MboI and MvaI restriction enzymes. A third enzyme SspI was used to identify the very virulent phenotype. The RFLP profile was found similar for all four isolates with MvaI enzyme but different for one isolate when digested with MboI. All three MvaI-positive viruses were further found positive for SspI digestion and yielded RFLP profile similar to vvIBDV in Europe whereas one isolate was SspI negative and had a RFLP profile similar to classic IBDV strains. The clinical history of high mortality and SspI restriction enzyme positivity revealed that vvIBDV strains exist in Pakistan. [Vet. World 2011; 4(7.000: 297-300

  17. Propagation of Asian isolates of canine distemper virus (CDV in hamster cell lines

    Directory of Open Access Journals (Sweden)

    Yamaguchi Ryoji

    2009-10-01

    Full Text Available Abstract Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains and by observing the development of cytopathic effect (CPE in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

  18. Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan

    Directory of Open Access Journals (Sweden)

    N.A. Elmardi

    2016-06-01

    Full Text Available A reverse transcription-polymerase chain reaction (RT-PCR was used to amplify 1412 bp of the fusion protein gene (F gene of four Newcastle disease virus (NDV isolates; two velogenic (TY-1/90 and DIK-90 and two lentogenic isolates (Dongla 88/1 and GD.S.1. Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1 suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

  19. Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

    Science.gov (United States)

    Aravind, S; Kamble, Nitin M; Gaikwad, Satish S; Shukla, Sanjeev Kumar; Dey, Sohini; Mohan, C Madhan

    2015-01-01

    Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

  20. Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

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    Gaya Prasad

    2013-06-01

    Full Text Available Aim: The study was conducted to develop ns1 gene based sensitive real-time RT-PCR assay for diagnosis of India isolates of bluetongue virus (BTV. Materials and Methods: The BTV serotype 21 isolate (KMNO7 was isolated from Andhra Pradesh and propagated in BHK-21 cell line in our laboratory. The Nucleic acid (dsRNA of virus was extracted using Trizol method and cDNA was prepared using a standard protocol. The cDNA was allowed to ns1 gene based group specific PCR to confirm the isolate as BTV. The viral RNA was diluted 10 folds and the detection limit of ns1 gene based RT-PCR was determined. Finally the tenfold diluted viral RNA was subjected to real-time RT-PCR using ns1 gene primer and Taq man probe to standardized the reaction and determine the detection limit. Results: The ns1 gene based group specific PCR showed a single 366bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The ns1 gene RT-PCR using tenfold diluted viral RNA showed the detection limit of 70.0 fg in 1%agarose gel electrophoresis. The ns1 gene based real time RT-PCR was successfully standardized and the detection limit was found to be 7.0 fg. Conclusion: The ns1 gene based real-time RT-PCR was successfully standardized and it was found to be 10 times more sensitive than conventional RT-PCR. Key words: bluetongue, BTV21, RT-PCR, Real time RT-PCR, ns1 gene [Vet World 2013; 6(8.000: 554-557

  1. Complete nucleotide sequence analysis of Cymbidium mosaic virus Indian isolate: further evidence for natural recombination among potexviruses

    Indian Academy of Sciences (India)

    Ang Rinzing Sherpa; Vipin Hallan; Promila Pathak; Aijaz Asghar Zaidi

    2007-06-01

    The complete nucleotide sequence of an Indian strain of Cymbidium mosaic virus (CymMV) was determined and compared with other potexviruses. Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV) and Potato aucuba mosaic virus (PAMV). Different sets of primers were used for the amplification of different regions of the genome through RT-PCR and the amplified genes were cloned in a suitable vector. The full genome of the Indian isolate of CymMV from Phaius tankervilliae shares 96–97% similarity with isolates reported from other countries. It was found that the CP gene of CymMV shares a high similarity with each other and other potexviruses. One of the Indian isolates seems to be a recombinant formed by the intermolecular recombination of two other CymMV isolates. The phylogenetic analyses, Recombination Detection Program (RDP2) analyses and sequence alignment survey provided evidence for the occurrence of a recombination between an Indian isolate (AM055720) as the major parent, and a Korean type-2 isolate (AF016914) as the minor parent. Recombination was also observed between a Singapore isolate (U62963) as the major parent, and a Taiwan CymMV (AY571289) as the minor parent.

  2. The complete genome sequence of a Crimean-Congo Hemorrhagic Fever virus isolated from an endemic region in Kosovo

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    Dedushaj Iusuf

    2008-01-01

    Full Text Available Abstract The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01. This suggests that distinct virus strains can coexist in highly endemic areas.

  3. Isolation and complete genome sequencing of Mimivirus bombay, a Giant Virus in sewage of Mumbai, India

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    Anirvan Chatterjee

    2016-09-01

    Full Text Available We report the isolation and complete genome sequencing of a new Mimiviridae family member, infecting Acanthamoeba castellanii, from sewage in Mumbai, India. The isolated virus has a particle size of about 435 nm and a 1,182,200-bp genome. A phylogeny based on the DNA polymerase sequence placed the isolate as a new member of the Mimiviridae family lineage A and was named as Mimivirus bombay. Extensive presence of Mimiviridae family members in different environmental niches, with remarkably similar genome size and genetic makeup, point towards an evolutionary advantage that needs to be further investigated. The complete genome sequence of Mimivirus bombay was deposited at GenBank/EMBL/DDBJ under the accession number KU761889.

  4. Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

    Institute of Scientific and Technical Information of China (English)

    Andrey Bogoyavlenskiy; Vladimir Berezin; Alexey Prilipov; Eugeniy Usachev; Ilya Korotetskiy; Irina Zaitceva; Aydyn Kydyrmanov; Marat Sayatov

    2012-01-01

    Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene.Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequenceswere compared phylogenetically with those from strains previously reported in other geographic regions.Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype Ⅵ or 4bii.To our knowledge,this is the first reported Ⅵ isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein.The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

  5. Isolation and characterization of Mayaro virus from a human in Acre, Brazil.

    Science.gov (United States)

    Terzian, Ana Carolina B; Auguste, Albert J; Vedovello, Danila; Ferreira, Marcelo U; da Silva-Nunes, Mônica; Sperança, Márcia A; Suzuki, Rodrigo B; Juncansen, Camila; Araújo, João P; Weaver, Scott C; Nogueira, Maurício L

    2015-02-01

    Mayaro virus (MAYV) is widely distributed throughout South America and is the etiologic agent of Mayaro fever, an acute febrile illness often presenting with arthralgic manifestations. The true incidence of MAYV infection is likely grossly underestimated because the symptomatic presentation is very similar to that of dengue fever and other acute febrile tropical diseases. We report the complete genome sequence of a MAYV isolate detected from an Acrelândia patient presenting with fever, chills, and sweating, but with no arthralgia. Results show that this isolate belongs to genotype D and is closely related to Bolivian strains. Our results suggest that the Acre/Mayaro strain is closely related to the progenitor of these Bolivian strains that were isolated between 2002 and 2006.

  6. Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes

    DEFF Research Database (Denmark)

    Skall, Helle Frank; Slierendrecht, W.J.; King, J.A.

    2004-01-01

    The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in Europ......The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes...... in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion...... while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates...

  7. Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa.

    Science.gov (United States)

    Kimbi, Gerald C; Kramvis, Anna; Kew, Michael C

    2004-05-01

    Phylogenetic analysis of hepatitis B virus (HBV) has led to its classification into eight genotypes, A to H. The dominant genotype in South Africa is genotype A, which consists of two subgenotypes, A1 and A2. Subgenotype A1 (previously subgroup A') predominates over subgenotype A2 (previously subgroup A minus A'). The complete genome of HBV isolated from 18 asymptomatic carriers of the virus and five acute hepatitis B patients was amplified; the resulting amplicons were cloned and sequenced. All acute hepatitis isolates belonged to subgenotype A1 and had no distinguishing mutations relative to the isolates from asymptomatic carriers, which had a distribution of ten subgenotype A1, two subgenotype A2 and six genotype D. The presence of the previously described amino acid residues that distinguish subgenotype A1 (subgroup A') from the remainder of genotype A in the S and polymerase genes was confirmed. Moreover, the large number of subgenotype A1 isolates sequenced allowed identification in the other open reading frames of additional nucleotide and amino acid changes that are characteristic of subgenotype A1. In particular, nucleotide mutations at positions 1809-1812 that alter the Kozak sequence of the precore/core open reading frame, and A(1888) in the precore region, were found exclusively in subgenotype A1 isolates. Unique sequence alterations of the transcriptional regulatory elements were also found in subgenotype A1 isolates. The mean nucleotide divergence of subgenotype A1 was greater than that of subgenotype A2, suggesting that this subgenotype has been endemic for a longer time in the South African black population than had subgenotype A2.

  8. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail.

    Science.gov (United States)

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Hung, Vu-Khac; Nguyen, Duc Tan; Kim, Wonyong

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010) and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014), were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010) and clade 2.3.2.1c (CVVI-50/2014), which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail.

  9. Sinu virus, a novel and divergent orthomyxovirus related to members of the genus Thogotovirus isolated from mosquitoes in Colombia.

    Science.gov (United States)

    Contreras-Gutiérrez, María Angélica; Nunes, Marcio R T; Guzman, Hilda; Uribe, Sandra; Gallego Gómez, Juan Carlos; Suaza Vasco, Juan David; Cardoso, Jedson F; Popov, Vsevolod L; Widen, Steven G; Wood, Thomas G; Vasilakis, Nikos; Tesh, Robert B

    2017-01-15

    The genome and structural organization of a novel insect-specific orthomyxovirus, designated Sinu virus, is described. Sinu virus (SINUV) was isolated in cultures of C6/36 cells from a pool of mosquitoes collected in northwestern Colombia. The virus has six negative-sense ssRNA segments. Genetic analysis of each segment demonstrated the presence of six distinct ORFs encoding the following genes: PB2 (Segment 1), PB1, (Segment 2), PA protein (Segment 3), envelope GP gene (Segment 4), the NP (Segment 5), and M-like gene (Segment 6). Phylogenetically, SINUV appears to be most closed related to viruses in the genus Thogotovirus.

  10. Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

    Institute of Scientific and Technical Information of China (English)

    YI Long; ZHOU Chang-yong

    2014-01-01

    The genetic variation and phylogenetic relationships of Citrus tristeza virus (CTV) isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3´ proximal region was relatively conserved, and the 5´ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.

  11. Isolation of Highly Pathogenic Avian Influenza H5N1 Virus from Saker Falcons (Falco cherrug in the Middle East

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    Henju Marjuki

    2009-01-01

    Full Text Available There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses. However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them.

  12. Isolation and characterization of ZH14 with antiviral activity against Tobacco mosaic virus.

    Science.gov (United States)

    Zhou, Wen-Wen; Zhang, Li-Xiang; Zhang, Bin; Wang, Fei; Liang, Zhi-Hong; Niu, Tian-Gui

    2008-06-01

    A large number of bacteria were isolated from plant samples and screened for antiviral activity against the Tobacco mosaic virus (TMV). The bacterium ZH14, which was isolated from Chinese Anxi oolong tea, secreted the antiviral substances, having 94.2% virus inhibition when the bacterial culture filtrate and TMV extract were mixed at a ratio of 1:1. The ZH14 strain is a gram-positive, spore-forming rod and has the ability to degrade ribonucleic acid. Based on its effectiveness on virus inhibition, ZH14 was selected for characterization and was identified as a strain of the Bacillus cereus group based on phenotypic tests and comparative analysis of its 16S rDNA sequence. At the same time, we determined the antiviral product of ZH14 as an extracellular protein with high molecular mass, having an optimum temperature of 15-60 degrees C and an optimum pH of 6-10. Hence, the ZH14 strain and its culture filtrate have potential application in controlling plant diseases caused by TMV.

  13. Molecular characteristics and pathogenicity of an avian leukosis virus isolated from avian neurofibrosarcoma.

    Science.gov (United States)

    Ochi, Akihiro; Ochiai, Kenji; Nakamura, Sayuri; Kobara, Akiko; Sunden, Yuji; Umemura, Takashi

    2012-03-01

    Peripheral nerve sheath tumors (PNSTs) are rare in chickens and their etiology remains to be elucidated. In this study, a naturally occurring PNST in a Japanese native fowl (Gallus gallus domesticus) was pathologically examined and the strain of avian leukosis virus (ALV) isolated from the neoplasm was characterized by molecular biological analysis. The fowl presented with a firm subcutaneous mass in the neck. The mass, connected to the adjacent spinal cord (C9-14), was microscopically composed of highly cellular tissue of spindle cells arranged in interlacing bundles, streams, and palisading patterns with Verocay bodies and less cellular tissue with abundant collagen. Immunohistochemically, neoplastic cells were divided into two types: perineurial cells positive for vimentin, glucose transporter 1 (GLUT1), and claudin1; and Schwann cells positive for vimentin, occasionally positive for S-100 alpha/beta but negative for GLUT1. Based on these findings, a diagnosis of neurofibrosarcoma was made. The complete nucleotide sequence of an ALV strain, CTS_5371, isolated from the neoplasm was determined and phylogenetic analysis indicated that the strain was a novel recombinant virus from avian leukosis/sarcoma viruses previously reported. Additionally, experimental infection revealed that CTS_5371 induced the proliferation of Schwann cells and perineurial cells. These results suggest that this ALV strain has the ability to induce PNSTs in chickens.

  14. Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

    Directory of Open Access Journals (Sweden)

    María de los Angeles Ribas Antúnez

    2013-04-01

    Full Text Available Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV procedure using 24-well tissue culture plates with the mouse inoculation test (MIT, which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36% were positive at 24 h, 10 (40% were positive at 48 h and six (24% were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

  15. The Development of Pathogenicity of Avian Influenza Virus Isolated from Indonesia

    Directory of Open Access Journals (Sweden)

    Michael Haryadi Wibowo

    2015-11-01

    Full Text Available Highly pathogenic avian infl uenza outbreak in Indonesia has been reported in various poultry due toH5N1 subtype. The presence of multiple basic amino acids within the cleavage site of HA glycoprotein hasbeen identifi ed to be associated with the pathogenicity of avian infl uenza virus. The study was retrospectivestudy which was designed to characterize the cleavage site and fusion site region of haemagglutinin gene ofAIV isolated from various poultry in 2003 to 2013. Isolation, Identifi cation and propagation were carried outto collect viral stock. For virus detection, reverse transcriptase PCR (RT-PCR method on H5 and N1 genefragment was performed. All of RT-PCR HA gene positive products were sequenced for further nucleotideanalysis and to determine the nucleotide composition at the targeted fragment. The results are all AIV isolateswere identifi ed as H5N1 subtype. The sequence analyses revealed some motives of basic amino acid motivethat were classifi ed as highly pathogenic avian infl uenza virus. Further analyses on fusion domain of all AIVisolated during the period 2003 to 2013 showed conserved amino acid.Keywords: avian infl uenza, haemagglutinin, cleavage site, basic amino acid, fusion site

  16. Potato virus X isolation and purification and coat protein gene cloning

    Directory of Open Access Journals (Sweden)

    L. Duplat

    2011-12-01

    Full Text Available In this work we describe the construction of potato virus X coat protein (PVX-CP gene clones from a Colombian isolate. Virus was isolated from field potato plants cultured at páramo de San Jorge. PVX particles were purified from Nicotiana tabacum leaves infected with a local lesion taken from Datura stramonium plants. The genomic RNA present in the virus particles consisted of a single species of about 6 Kb. cDNA synthesis representing genomic RNA was followed by Digoxigenin incorporation after priming with oligonucleotide 4DT/KPN. PCR amplification of CP gene sequence was made by using oligonucleotides OX6 and L/Kpn. An expected fragment of 870 bp, besides some 600-123 bp fragments, was obtained after PCR. The blunt-ended fragments were clonned into the PCR cloning pMOSblue plasmid. The insert size of the selected recombinant cDNA clones was determined by restriction analysis of the plasmidDNAwith Sma I and Hind III enzymes. Positive clones were also screened by direct colony PCR screening. The selected recombinant cDNA clones were stored in glycerol at .70 °C.

  17. Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection.

    Directory of Open Access Journals (Sweden)

    Nao Sugiyama

    Full Text Available The hepatitis C virus (HCV culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.

  18. Detection and some properties of cowpea mild mottle virus isolated from soybean in Iran.

    Science.gov (United States)

    Tavassoli, M; Shahraeen, N; Ghorbani, S

    2008-12-01

    During 2006-2007 growing seasons, survey were carried to identify a virus disease causing mosaic of soybean in the field in Southern region (Khozestan Province) of Iran. To detect the viral infection, diseased leaf samples showing mild mosaic and leaf malformation were collected from soybean fields in Dezful, located in Khozestan Province. Infected samples were carried to the lab in a proper condition on ice packages. TPIA and DAS-ELISA serological tests were applied to identify the viral agent. To investigate the host-range, several indicator plants were mechanically inoculated under green-house condition. Seed transmission of CPMMV was examined using the seeds obtained from infected plants. The virus isolate was not found to be seed-borne in Clark variety of soybean. Different steps of ultracentrifugation including sucrose density gradient (10-40%) were carried out in order to obtain partial purified virus. On the basis of biological, serological and EM results, CPMMV-Carla virus was identified in the infected soybean samples. This is the first report of CPMMV infection of soybean in Iran.

  19. Complete Genome Sequence of a Newcastle Disease Virus Isolated from a Rock Dove (Columba livia) in the Russian Federation.

    Science.gov (United States)

    Yurchenko, Kseniya S; Sivay, Mariya V; Glushchenko, Alexandra V; Alkhovsky, Sergey V; Shchetinin, Alexey M; Shchelkanov, Michail Y; Shestopalov, Alexander M

    2015-01-29

    We report here the complete genome sequence of a Newcastle disease virus (NDV) isolate, NDV/Altai/pigeon/770/2011, isolated from a rock dove in the Russian Federation. On the basis of phylogenetic analysis, this strain was clustered into genotype VIb class II.

  20. Characterization of avian influenza virus isolates submitted to the National Centre for Foreign Animal Disease between 1997 and 2001.

    Science.gov (United States)

    Pasick, J; Weingartl, H; Clavijo, A; Riva, J; Kehler, H; Handel, K; Watkins, E; Hills, K

    2003-01-01

    The National Centre for Foreign Animal Disease (NCFAD) in Winnipeg, Manitoba, is the Canadian Food Inspection Agency's (CFIA) newest high biocontainment laboratory. One of the functions of the NCFAD is to serve as a national reference laboratory for avian influenza. Between 1997 and 2001, 15 avian influenza virus isolates were characterized. These isolates originated from domestic poultry, imported caged birds held in quarantine, and wild birds. Diagnostic specimens were submitted to the NCFAD by CFIA field veterinarians, provincial veterinary diagnostic laboratories, and veterinary colleges. Characterization of isolates included the determination of H and N subtypes: H1, H6, H7, and H10 subtypes were isolated from domestic poultry; H3, H4, and three H13 viruses were isolated from water fowl, and six H3 viruses were isolated from caged birds being held in import quarantine. Selected isolates were characterized with respect to their pathogenic potential by intravenous inoculation of 4-to-6-wk-old chickens. A molecular-based protocol was used to assess the pathogenicity of one H7 isolate. During this period, work was also carried out toward validating our molecular pathotyping protocol for avian influenza viruses with H5 and H7 hemagglutinin subtypes.

  1. Characterization and Sequencing of a Genotype XII Newcastle Disease Virus Isolated from a Peacock (Pavo cristatus) in Peru

    Science.gov (United States)

    Tataje-Lavanda, Luis; Figueroa, Aling; Segovia, Karen; Gonzalez, Rosa; Cribillero, Giovana; Montalvan, Angela; Fernández-Díaz, Manolo; Icochea, Eliana

    2015-01-01

    Here, we report the first complete sequence and biological characterization of a Newcastle disease virus (NDV) isolated from a peacock in South America (NDV/peacock/Peru/2011). This isolate, classified as genotype XII in class II, highlights the need for increased surveillance of noncommercial avian species. PMID:26227592

  2. Complete Genome Sequence of an American Avian Leukosis Virus Subgroup J Isolate That Causes Hemangiomas and Myeloid Leukosis

    OpenAIRE

    Malhotra, Sanandan; Justice, James; De Lee, Nathan; Li, Yingying; Zavala, Guillermo; Ruano, Miguel; Morgan, Robin; Beemon, Karen

    2015-01-01

    We report the complete genome sequence of avian leukosis virus subgroup J (ALV-J) isolate PDRC-59831, which causes myeloid leukosis and hemangiomas in chickens. This is an American ALV-J isolate, which was found in a 38-week-old broiler breeder chicken on a farm in Georgia in 2007.

  3. Complete Genome Sequences of Two Chikungunya Viruses Isolated in the Central African Republic in the 1970s and 1980s

    Science.gov (United States)

    Desdouits, Marion; Nakouné, Emmanuel; Gessain, Antoine; Kazanji, Mirdad; Berthet, Nicolas

    2017-01-01

    ABSTRACT Some arboviruses threaten human global health with potentially explosive emergence. Analysis of whole-genome sequences of decades-old isolates might contribute to the understanding of the complex dynamics which drive their circulation and emergence. Here, we report the whole-genome sequences of two Chikungunya viruses isolated in the Central African Republic in the 1970s and 1980s. PMID:28254965

  4. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates

    NARCIS (Netherlands)

    Souza, M.T.; Níckel, O.; Gonsalves, D.

    2005-01-01

    Line 63-1 is a 'Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R, plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thai

  5. Cross-protection or enhanced symptom display in greenhouse tomato co-infected with different Pepino mosaic virus isolates

    NARCIS (Netherlands)

    Hanssen, I.M.; Gutiérrez-Aguirre, I.; Paeleman, A.; Goen, K.; Wittemans, L.; Lievens, B.; Vanachter, A.C.R.C.; Ravnikar, M.; Thomma, B.P.H.J.

    2010-01-01

    The potential of three mild Pepino mosaic virus (PepMV) isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato (Solanum lycopersicum) crop against an aggressive challenge isolate of the CH2 genotype was assessed in greenhouse trials and PepMV symptoms were rated at regular time poi

  6. [Isolation and identification of avian leukosis virus-B from layer chickens infected with avian leukosis virus-J].

    Science.gov (United States)

    Liu, Gong-Zhen; Zhang, Hong-Hai; Liu, Qing; Qiu, Bo; Wang, Feng; Wang, Xiao-Wei; Chen, Hong-Bo; Cheng, Zi-Qiang

    2009-11-01

    Two strains of Avian leukosis virus subgroup B (ALV-B) were isolated for the first time in China Hy-line White on the cultured DF-1 cells which were inoculated tissue samples from by an ELISA assay, a histopathology examination and a PCR-based diagnosis. The results from the ELISA assay indicated that the positive rate of serum antibodies to ALV-B and ALV-J virus were 16.3% (15/92) and 13% (12/92), respectively. The histopathological examination indicated that two types of tumor cells existed at same focus in liver and spleen, which mainly were myelocytoma cells and lymphosarcoma cells. The PCR-based diagnosis were performed as follows: the cellular DNA was extracted from the inoculated DF-1 cells; the specific fragments of 1100 bp and 924 bp were obtained by a PCR system with the diagnostic primers of ALV-B and ALV-J; and the PCR results for ALV-A, MDV and REV were all negative. Then, the amplified fragments of the two ALV-B stains were partially sequenced and shown an identity of 92.8%,94.7% with the prototype strain of ALV-B (RSV Schmidt-ruppin B). The identities of two ALV-J strains with the prototype strain HPRS-103 at 96.9%, 91.5%; The identities of two ALV-J strains with the American prototype strain at 85.9%, 81.5%. Our study had shown that ALV-B was isolated for the first time from the ALV-J infected commercial layer flocks in China. It also indicated that the chance of genetic recombination among various subgroups of ALV was increased.

  7. [Taxonomy of the Sokuluk virus (SOKV) (Flaviviridae, Flavivirus, Entebbe bat virus group) isolated from bats (Vespertilio pipistrellus Schreber, 1774), ticks (Argasidae Koch, 1844), and birds in Kyrgyzstan].

    Science.gov (United States)

    L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Shchetinin, A M; Deriabin, P G; Gitel'man, A K; Samokhvalov, E I; Botikov, A G

    2014-01-01

    Complete genome sequencing of the Sokuluk virus (SOKV) isolated in Kyrgyzstan from bats Vespertilio pipistrellus and their obligatory parasites--Argasidae Koch, 1844, ticks was carried out. SOKV was classified as attributed to the Flaviviridae family, Flavivirus genus. The maximum homology (71% for nucleotide and 79% for amino acid sequences) was detected with respect to the Entebbe bat virus (ENTV). ENTV and SOKV form a group joining to the yellow fever virus (YFV) within the limits of the mosquito flavivirus branch. Close relation of SOKV with bat covers and human housings permits to assume SOKV potentially patogenic to human health.

  8. Evolutionary and Ecological Characterization of Mayaro Virus Strains Isolated during an Outbreak, Venezuela, 2010.

    Science.gov (United States)

    Auguste, Albert J; Liria, Jonathan; Forrester, Naomi L; Giambalvo, Dileyvic; Moncada, Maria; Long, Kanya C; Morón, Dulce; de Manzione, Nuris; Tesh, Robert B; Halsey, Eric S; Kochel, Tadeusz J; Hernandez, Rosa; Navarro, Juan-Carlos; Weaver, Scott C

    2015-10-01

    In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions.

  9. Different pattern of haemagglutinin immunoreactivity of equine influenza virus strains isolated in Poland

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    Kwaśnik Małgorzata

    2015-12-01

    Full Text Available The immunoreactivity of haemagglutinin (HA polypeptides of equine influenza virus was compared among the strains isolated in Poland, using H3 monoclonal antibody. A stronger signal in immunoblot reaction was observed for A/equi/Pulawy/2008 HA polypeptides compared to A/equi/Pulawy/2006, despite the fact that both strains are phylogenetically closely related and belong to Florida clade 2 of American lineage. The strongest signal, observed in the case of A/equi/Pulawy/2008, seemed to be connected with the presence of G135, I213, E379, and/or V530 instead of R135, M213, G379, and I530 present in A/equi/Pulawy/2006 HA sequence. This implies that point mutations within amino acid sequences of HA polypeptides of equine influenza virus may change their immunoreactivity even when they are not located within five basic antigenic sites.

  10. [Identification of a new subgroup of avian leukosis virus isolated from Chinese indigenous chicken breeds].

    Science.gov (United States)

    Wang, Xin; Zhao, Peng; Cui, Zhi-Zhong

    2012-11-01

    In order to clarify Avian leukosis virus (ALV) characteristics from Chinese native chicken breeds, three ALV JS11C1, JS11C2 and JS11C3 were isolated from Chinese native breed "luhua" by inoculation of DF1 cell culture and detection of p27 antigen. Using PCR amplification of env gene, the amplified gp85 genes were analyzed and compared to all six chicken ALV subgroups reported. The gp85 genes of these three viruses were 1 005bp in length and encoded 335 amino acids, and the gp37 genes were 609bp and encoded 203 amino acids. The homology of gp85 among these three isolated strains was 91.9%-97.0%. Comparing to 18 stains of subgroup A, B, C, D, E published in GenBank, the homology was only in the range of 77.7%-84.6%, significantly lower than the gp85 homology observed within the common chicken subgroups A (88.2%-98.5%), B (91.6%-98.8%), and E (97.9%-99.4%). The gp85 homology compared with subgroup J was only 34.2%-36.5%. These results suggested that three isolated strains from Chinese native breed "luhua" belong to a new subgroup different from all six known subgroups from Chickens, and thus designated as subgroup K.

  11. Complete Genomic Sequence of a Chinese Isolate of Duck Hepatitis Virus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The complete genomic sequence of Duck hepatitis virus 1 (DHV-1) ZJ-V isolate was sequenced and determined to be 7 691 nucleotides (nt) in length with a 5'-terminal un-translated region (UTR) of 626 nt and a 3'-terminal UTR of 315 nt (not including the poly(A) tail). One large open reading frame (ORF) was found within the genome (nt 627 to 7 373) coding for a polypeptide of 2 249amino acids. Our data also showed that the poly (A) tail of DHV-1 has at least 22 A's. Sequence comparison revealed significant homology (from 91.9% to 95.7%) between the protein sequences of the virus in the Picornaviridae family, its genome showed some unique characteristics. DHV-1 contains 3copies of the 2A gene and only 1 copy of the 3B gene, and its 3'-NCR is longer than those of other picornaviruses. Phylogenetic analysis to do sequence homology based on the VP1 protein sequences showed that the ZJ-V isolate shares high sequence homology with the reported DHV-1 isolates (from 92.9% to 99.2%), indicating that DHV-1 is genetically stable.

  12. Complete nucleotide sequence of a Spanish isolate of Parietaria mottle virus infecting tomato.

    Science.gov (United States)

    Galipienso, Luis; Rubio, Luis; López, Luis; Soler, Salvador; Aramburu, José

    2009-10-01

    The genome of a Spanish isolate of Parietaria mottle virus (PMoV) obtained from tomato (strain PMoV-T) was completely sequenced. Protein motifs conserved for RNA viruses were identified: the p1 protein contained a metyltransferase domain in its N-terminal half and a triphosphatase/ helicase domain in its C-terminal half, the p2 protein contained a RNA polymerase domain; the 3a protein contained a RNA-binding domain with α-helix and β-sheet secondary structures. In addition, stem-loop structures with potential capacity of protein interactions were predicted on the untranslated terminal regions. Comparison with the other sequenced PMoV isolate showed nucleotide identities of 93, 90, and 93% for genomic RNAs 1, 2 and 3, respectively, and amino acid identities ranging from 88 to 97% for the different proteins. A cytosine deletion was detected at position 1,366 of RNA 3, involving a start codon for the coat protein (CP) gene different from the other PMoV isolate, resulting in a CP 16 amino acids shorter. Comparison of synonymous and nonsynonymous mutations revealed different selective constraints along the genome.

  13. Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

    Science.gov (United States)

    Rhee, Sun-Ju; Hong, Jin-Sung; Lee, Gung Pyo

    2014-07-01

    Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

  14. Characteristics of a Lettuce mosaic virus Isolate Infecting Lettuce in Korea

    Directory of Open Access Journals (Sweden)

    Seungmo Lim

    2014-06-01

    Full Text Available Lettuce mosaic virus (LMV causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMVRoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup than to LMV-E (LMV-RoW group but not LMV-Common subgroup even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group.

  15. Genetic diversity of bovine viral diarrhea virus 1: Italian isolates clustered in at least seven subgenotypes.

    Science.gov (United States)

    Giammarioli, Monica; Pellegrini, Claudia; Casciari, Cristina; Rossi, Elisabetta; De Mia, Gian Mario

    2008-11-01

    Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. Two approved species are recognized, namely BVDV-1 and BVDV-2. To date, only 4 subgenotypes of BVDV-2 are known, and at least 11 distinct subgenotypes have been detected for BVDV-1. In a previous study, the genetic characteristics of 38 field isolates of BVDV from northern Italy were investigated, and all 38 isolates were classified as BVDV-1 and could be assigned to 5 different subgenotypes, namely BVDV-1b, BVDV-1d, BVDV-1e, BVDV-1h, and BVDV-1f. However, the circulation of BVDV-2 has been reported in Italy as well. The aim of the current study was to type 88 BVD viruses found throughout Italy. Genetic study was based on the 5'-UTR, supported by select comparison within the N(pro) coding region. Phylogenetic analysis showed that 5 isolates could be typed as BVDV-2a. The remaining 83 isolates were typed as BVDV-1 and were found to belong to 7 distinct subgenotypes, namely BVDV-1a (n = 8), BVDV-1b (n = 37), BVDV-1d (n = 3), BVDV-1e (n = 22), BVDV-1f (n = 4), BVDV-1g (n = 4), and BVDV-1h (n = 5). The majority of cattle farms in the current study were predominantly infected by BVDV-1b and BVDV-1e isolates, whereas the other BVDV subgenotypes occurred only sporadically. The results also provided evidence for circulation of additional subgenotypes BVDV-1a and BVDV-1g. The occurrence of BVDV-2 was also reconfirmed.

  16. Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil

    Science.gov (United States)

    da Silva, Kely A. B.; de Castro, Marcia G.; Gerber, Alexandra L.; de Almeida, Luiz G. P.; Lourenço-de-Oliveira, Ricardo; Vasconcelos, Ana Tereza R.

    2016-01-01

    Background Zika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil. Methodology/Principal Findings Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak. Conclusions/Significance The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological

  17. Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

    Directory of Open Access Journals (Sweden)

    Myrna C Bonaldo

    2016-06-01

    Full Text Available Zika virus (ZIKV is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil.Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak.The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution

  18. Genotypic lineages and restriction fragment length polymorphism of canine distemper virus isolates in Thailand.

    Science.gov (United States)

    Radtanakatikanon, Araya; Keawcharoen, Juthatip; Charoenvisal, Na Taya; Poovorawan, Yong; Prompetchara, Eakachai; Yamaguchi, Ryoji; Techangamsuwan, Somporn

    2013-09-27

    Canine distemper virus (CDV) is known to cause multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used to control CDV in a variety of species for many decades, yet a number of CDV infections in vaccinated dogs are still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phosphoprotein (P), hemagglutinin (H) and fusion protein (F) genes and to develop the restriction fragment length polymorphism (RFLP) technique for effective differentiation among individual wild-type and vaccine lineages in Thailand. Four commercial vaccine products, thirteen conjunctival swabs and various tissues from 9 necropsied dogs suspected of having CDV infections were included. Virus isolation was performed using Vero cell expressing canine signaling lymphocyte activation molecules (Vero-DST cells). Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions from the dog derived specimens and the vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other CDV lineages in Genbank. Phylogenetic relationships revealed that CDV field isolates were separated from the vaccine lineage and could be divided into two clusters; one of which belonged to the Asia-1 lineage and another, not related to any previous recognized lineages was proposed as 'Asia-4'. RFLP patterns demonstrating concordance with phylogenetic trees of the distemper virus allowed for differentiation between the Asia-1, Asia-4 and vaccine lineages. Thus, RFLP technique is able to effectively distinguish individual wild-type canine distemper virus from vaccine lineages in Thailand.

  19. Vaccine Potential of Two Previously Uncharacterized African Swine Fever Virus Isolates from Southern Africa and Heterologous Cross Protection of an Avirulent European Isolate.

    Science.gov (United States)

    Souto, R; Mutowembwa, P; van Heerden, J; Fosgate, G T; Heath, L; Vosloo, W

    2016-04-01

    African swine fever (ASF) is a mostly fatal viral infection of domestic pigs for which there is no vaccine available. The disease is endemic to most of sub-Saharan Africa, causes severe losses and threatens food security in large parts of the continent. Naturally occurring attenuated ASF viruses have been tested as vaccine candidates, but protection was variable depending on the challenge virus. In this study, the virulence of two African isolates, one from a tick vector and the other from an indigenous pig, was determined in domestic pigs to identify a potential vaccine strain for southern Africa. Neither isolate was suitable as the tick isolate was moderately virulent and the indigenous pig virus was highly virulent. The latter was subsequently used as heterologous challenge in pigs first vaccinated with a naturally attenuated isolate previously isolated in Portugal. Although a statistically significant reduction in death rate and virus load was observed compared with unvaccinated pigs post-challenge, all pigs succumbed to infection and died.

  20. The first evidence of subgroup IB isolates of Cucumber mosaic virus in Ukraine

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    Shevchenko T. P.

    2015-02-01

    Full Text Available Aim. In current work, we proceeded with the strain attribution of Ukrainian isolates CMV based on the phylogenetic analysis of the partial sequences of the coat protein gene. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. Cucumber mosaic virus (CMV is widespread among the variety of crops from the Cucurbitaceae and Solanaceae families in Ukraine. The symptomatic samples from different regions of Ukraine were collected and tested for the presence of CMV. The coat protein (CP gene of two isolates was amplified and sequenced. The partial nucleotide sequences of CP gene were determined and compared to those of other CMV strains belonging to the IA, IB and II subgroups. Comparison of the nucleotide sequences of Ukrainian isolates showed their similar identity percentages and close relationships with the subgroup IB strains from other countries. The highest nucleotide homology was shared with the strains ABI (Korea and SD (China. Conclusions. Based on the highest identities of the coat protein gene sequences and close phylogenetic relationships with the subgroup IB members of CMV, the Ukrainian isolates under study were identified as belonging to the subgroup IB. Our findings show for the first time an occurrence of the IB subgroup isolates of CMV in Ukraine.

  1. Genetic analysis of ORF5 porcine reproductive and respiratory syndrome virus isolated in Vietnam.

    Science.gov (United States)

    Thuy, Nguyen Thi Dieu; Thu, Nguyen Thi Dieu; Son, Nguyen Giang; Ha, Le Thi Thu; Hung, Vo Khanh; Nguyen, Nguyen Thao; Khoa, Do Vo Anh

    2013-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008-2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8-100.0% nt identity and 94.0-100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR-2332; nt 87.8-89.3%, aa 87.5-90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1-61.9%, aa 55.1-57.0%). There was greater similarity with QN07 (nt 96.5-98.5%, aa 96.0-99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH-1a (nt 93.2-95.1%, 91.5-93.5%) isolated in China in 1995 and JXA1 (nt 96.5-98.6%, aa 95.0-98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1-R (nt 96.5-98.6%, aa 95.0-98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL-PS (nt 87.2-89.0%, aa 86.0-89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub-lineage 8.7. This sub-lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam.

  2. Characterization of a new dog isolate of canine distemper virus from China.

    Science.gov (United States)

    Qiao, J; Meng, Q; Chen, C; Xia, X; Cai, X; Ren, Y; Zhang, H

    2011-01-01

    Canine distemper virus (CDV) is a highly contagious pathogen of dogs. Vaccination is an effective way to protect dogs from CDV infection, but occasionally fails. In the present study, a wild type (wt) CDV, named XJ2, was isolated from a dead vaccinated dog. The hemagglutinin (H) gene of the XJ2 was amplified and analyzed for the molecular characteristics including N-glycosylation sites, phylogenesis, hydrophobicity and epitopes. The data indicated that XJ2 was a genetic variant strain of CDV. CDV-sero-negative dogs were inoculated intranasally with XJ2, developed severe clinical symptoms and died, suggesting high virulence.

  3. Genome sequence analysis of in vitro and in vivo phenotypes of Bunyamwera and Ngari virus isolates from northern Kenya.

    Directory of Open Access Journals (Sweden)

    Collins Odhiambo

    Full Text Available Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP and large plaque (LP and determined in vitro growth properties and in vivo pathogenesis in suckling mice. We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies.

  4. Genome Sequence Analysis of In Vitro and In Vivo Phenotypes of Bunyamwera and Ngari Virus Isolates from Northern Kenya

    Science.gov (United States)

    Odhiambo, Collins; Venter, Marietjie; Limbaso, Konongoi; Swanepoel, Robert; Sang, Rosemary

    2014-01-01

    Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP) and large plaque (LP) and determined in vitro growth properties and in vivo pathogenesis in suckling mice. We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I) that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies. PMID:25153316

  5. A spatial simulation model for the dispersal of the bluetongue vector Culicoides brevitarsis in Australia.

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    Joel K Kelso

    Full Text Available The spread of Bluetongue virus (BTV among ruminants is caused by movement of infected host animals or by movement of infected Culicoides midges, the vector of BTV. Biologically plausible models of Culicoides dispersal are necessary for predicting the spread of BTV and are important for planning control and eradication strategies.A spatially-explicit simulation model which captures the two underlying population mechanisms, population dynamics and movement, was developed using extensive data from a trapping program for C. brevitarsis on the east coast of Australia. A realistic midge flight sub-model was developed and the annual incursion and population establishment of C. brevitarsis was simulated. Data from the literature was used to parameterise the model.The model was shown to reproduce the spread of C. brevitarsis southwards along the east Australian coastline in spring, from an endemic population to the north. Such incursions were shown to be reliant on wind-dispersal; Culicoides midge active flight on its own was not capable of achieving known rates of southern spread, nor was re-emergence of southern populations due to overwintering larvae. Data from midge trapping programmes were used to qualitatively validate the resulting simulation model.The model described in this paper is intended to form the vector component of an extended model that will also include BTV transmission. A model of midge movement and population dynamics has been developed in sufficient detail such that the extended model may be used to evaluate the timing and extent of BTV outbreaks. This extended model could then be used as a platform for addressing the effectiveness of spatially targeted vaccination strategies or animal movement bans as BTV spread mitigation measures, or the impact of climate change on the risk and extent of outbreaks. These questions involving incursive Culicoides spread cannot be simply addressed with non-spatial models.

  6. Spatial analysis of bluetongue cases and vaccination of Swiss cattle in 2008 and 2009

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    Katriina J. E. Willgert

    2011-05-01

    Full Text Available Bluetongue (BT is a vector-borne viral disease of ruminants. The infection is widespread globally with major implications for international animal trade and production. In 2006, BT virus serotype 8 (BTV-8 was encountered in Europe for the first time, causing extensive production losses and death in susceptible livestock. Following the appearance of BTV- 8 in Switzerland in 2007, a compulsory vaccination programme was launched in the subsequent year. Due to social factors and difficulties to reach animals on high pasture, the regional vaccination coverage varied across the country in both 2008 and 2009. In this study, the effect of vaccination on the spatial occurrence of BTV-8 and the associated relative disease risk in Switzerland in 2008 and 2009 were investigated by a spatial Bayesian hierarchical approach. Bayesian posterior distributions were obtained by integrated nested Laplace approximations, a promising alternative to commonly used Markov chain Monte Carlo methods. The number of observed BTV-8 outbreaks in Switzerland decreased notably from 2008 to 2009. However, only a non-significant association between vaccination coverage and the probability of a spatial unit being infected with BTV-8 was identified using the model developed for this study. The relative disease risk varied significantly across the country, with a higher relative risk of BTV-8 infection in western and north-western Switzerland where environmental conditions are more suitable for vector presence and viral transmission. Examination of the spatial correlation between disease occurrence, control measures and associated ecological factors can be valuable in the evaluation and development of disease control programmes, allowing prioritisation of areas with a high relative risk of disease.

  7. Hepatitis B virus subgenotype A1: evolutionary relationships between Brazilian, African and Asian isolates.

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    Bárbara V Lago

    Full Text Available Brazil is a country of low hepatitis B virus (HBV endemicity in which the genotype A of HBV (HBV/A is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase, and one (Phe17 in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an 'Asian-American' clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300-400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

  8. Hepatitis B virus subgenotype A1: evolutionary relationships between Brazilian, African and Asian isolates.

    Science.gov (United States)

    Lago, Bárbara V; Mello, Francisco C; Kramvis, Anna; Niel, Christian; Gomes, Selma A

    2014-01-01

    Brazil is a country of low hepatitis B virus (HBV) endemicity in which the genotype A of HBV (HBV/A) is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase), and one (Phe17) in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an 'Asian-American' clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300-400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

  9. Partial biological and molecular characterization of a Cucumber mosaic virus isolate naturally infecting Cucumis melo in Iran.

    Science.gov (United States)

    Rasoulpour, Rasoul; Afsharifar, Alireza; Izadpanah, Keramat

    2016-06-01

    Melon seedlings showing systemic chlorotic spots and mosaic symptoms were collected in central part of Iran, and a virus was isolated from diseased plants by mechanical inoculation. The virus systemically infected the most inoculated test plants by inducing mosaic symptoms, while, in the members of Fabaceae family and Chenopodium quinoa induced local lesions. Agar gel diffusion test using a polyclonal antiserum against a squash Cucumber mosaic virus (CMV) isolate showed the presence of CMV in the mechanically inoculated plants (designated CMV-Me). The virus was purified by polyethylene glycol precipitation and differential centrifugation. A polyclonal antiserum was produced against the virus that reacted specifically with virus antigen in ELISA and agar gel diffusion tests. The virus was molecularly characterized by PCR amplification of the full length of the coat protein gene using cucumovirus genus specific primer pair CPTALL-3/CPTALL-5 and sequence analysis of the resulting product. No RNA satellite was detected using the primer pair CMVsat3H/sat5T7P. Phylogenetic analysis based on the coat protein amino acid sequences showed that CMV-Me belongs to Subgroup IB. These results may be helpful in melon breeding programs, focusing on plant resistance to plant viruses including CMV.

  10. High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates.

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    Passos-Castilho, Ana Maria; Granato, Celso Francisco Hernandes

    2017-01-03

    Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

  11. Molecular characterization of double-stranded RNA virus in Trichomonas vaginalis Egyptian isolates and its association with pathogenicity.

    Science.gov (United States)

    El-Gayar, Eman K; Mokhtar, Amira B; Hassan, Wael A

    2016-10-01

    Trichomoniasis is a common human sexually transmitted infection caused by Trichomonas vaginalis. The parasite can be infected with double-stranded RNA viruses (TVV). This viral infection may have important implications on trichomonal virulence and disease pathogenesis. This study aimed to determine the prevalence of T. vaginalis virus among isolates obtained from infected (symptomatic and asymptomatic) women in Ismailia City, Egypt, and to correlate the virus-infected isolates with the clinical manifestations of patients. In addition, the pathogenicity of TVV infected isolates on mice was also evaluated. T. vaginalis isolates were obtained from symptomatic and asymptomatic female patients followed by axenic cultivation in Diamond's TYM medium. The presence of T. vaginalis virus was determined from total extraction of nucleic acids (DNA-RNA) followed by reverse transcriptase-PCR. Representative samples were inoculated intraperitoneally in female albino/BALB mice to assess the pathogenicity of different isolates. A total of 110 women were examined; 40 (36.3 %) samples were positive for T. vaginalis infection. Of these 40 isolates, 8 (20 %) were infected by TVV. Five isolates contained TVV-2 virus species, and the remaining three isolates were infected withTVV-4 variant. A significant association was found between the presence of TVV and particular clinical manifestations of trichomoniasis. Experimental mice infection showed varying degrees of pathogenicity. This is the first report on T. vaginalis infection by TVV in Egypt. The strong association detected between TVV and particular clinical features of trichomoniasis and also the degree of pathogenicity in experimentally infected mice may indicate a possible clinical significance of TVV infection of T. vaginalis isolates.

  12. Platelet aggregation responses and virus isolation from platelets in calves experimentally infected with type I or type II bovine viral diarrhea virus.

    Science.gov (United States)

    Walz, P H; Bell, T G; Grooms, D L; Kaiser, L; Maes, R K; Baker, J C

    2001-10-01

    Altered platelet function has been reported in calves experimentally infected with type II bovine viral diarrhea virus (BVDV). The purpose of the present study was to further evaluate the ability of BVDV isolates to alter platelet function and to examine for the presence of a virus-platelet interaction during BVDV infection. Colostrum-deprived Holstein calves were obtained immediately after birth, housed in isolation, and assigned to 1 of 4 groups (1 control and 3 treatment groups). Control calves (n = 4) were sham inoculated, while calves in the infected groups (n = 4 for each group) were inoculated by intranasal instillation with 10(7) TCID50 of either BVDV 890 (type II), BVDV 7937 (type II), or BVDV TGAN (type I). Whole blood was collected prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation for platelet function testing by optical aggregometry by using adenosine diphosphate and platelet activating factor. The maximum percentage aggregation and the slope of the aggregation curve decreased over time in BVDV-infected calves; however, statistically significant differences (Freidman repeated measures ANOVA on ranks, P infected with the type II BVDV isolates. Bovine viral diarrhea virus was not isolated from control calves, but was isolated from all calves infected with both type II BVDV isolates from days 4 through 12 after inoculation. In calves infected with type I BVDV, virus was isolated from 1 of 4 calves on days 4 and 12 after inoculation and from all calves on days 6 and 8 after inoculation. Altered platelet function was observed in calves infected with both type II BVDV isolates, but was not observed in calves infected with type I BVDV. Altered platelet function may be important as a difference in virulence between type I and type II BVDV infection.

  13. Sequencing and Phylogenetic Analysis of Potato virus Y Liaoning Isolate in China

    Institute of Scientific and Technical Information of China (English)

    WANG Fang; GAO Zheng-liang; AN Meng-nan; ZHOU Ben-guo; and WU Yuan-hua

    2013-01-01

    Complete genome sequence of Potato virus Y Liaoning isolate (PVY-LN) causing tobacco vein necrosis symptoms were isolated from Liaoning Province in China. Genome sequences of PVY-LN was 9 714 nucleotides in length, excluding the 3′-terminal poly (A) tail. PVY-LN encodes a single long open reading frame (ORF) of polyprotein that is predicted to be cleaved into ten mature proteins by three viral proteases. No recombination can be predicted in PVY-LN sequences compared with that of the other PVY strains using Recombination Detection Programe v. 4.16 (RDP4). Complete genome sequence comparison and phylogenetic analysis indicated that PVY-LN is closely related to PVY necrosis strain (PVYN).

  14. Cloning and Identification of S Gene from Chinese Isolate TH-98 of Transmissible Gastroenteritis Virus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle (ST)cells. Two pairs of primers were designed according to the published sequence with Oligo 4. 1 and DNasis softwares. The products of RT-PCR were named Sa and Sb ,of 2. 3kb and 2. 1kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC- S was verified and analyzed by corresponding restriction endonuclease (RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid ,which was identified as S gene from Chinese isolate of TGEV.

  15. Isolation of EEE virus from Ochlerotatus taeniorhynchus and Culiseta melanura in coastal South Carolina.

    Science.gov (United States)

    Ortiz, Diana I; Wozniak, Arthur; Tolson, Marsha W; Turner, Pearl E; Vaughan, David R

    2003-03-01

    A 1-year arbovirus study was conducted at The Wedge Plantation located in coastal South Carolina to determine the occurrence and level of arbovirus activity in mosquito species inhabiting the site. Mosquito species composition and temporal abundance were also determined. A total of 45,051 mosquitoes representing 27 species in 9 genera was collected and identified during 130 trap-nights between August, 1997, and July, 1998. The most abundant species was Culex salinarius (n = 20,954) followed by Ochlerotatus taeniorhynchus (n = 12,185). Eastern equine encephalomyelitis virus (EEE) was isolated from 2 pools collected in August, 1997; one pool of Oc. taeniorhynchus (minimum infection rate [MIR] = 0.6/1,000) and a second of Culiseta melanura (MIR = 3.8/1,000). This report represents the first record of an EEE isolation from Oc. taeniorhynchus and Cs. melanura in South Carolina.

  16. Serological and pathological studies of Newcastle disease viruses isolated from caged birds from Southeast Asia.

    Science.gov (United States)

    Kawamura, M; Nerome, K; Kodama, H; Izawa, H; Mikami, T

    1987-01-01

    Eleven isolates of Newcastle disease virus (NDV), from caged birds imported from or captured in Southeast Asia in 1979-80, were antigenically divided into five distinct groups. Most of them were distinguishable from more classical NDVs (vaccine B1 strain and Miyadera strain) on the basis of their reactivity to eight monoclonal antibodies against the HN molecule of NDV in hemagglutination-inhibition tests. However, when three representative isolates were evaluated for their biological properties and pathogenicity against 1-day-old chickens, all three were found to be velogenic types that could induce serious symptoms of Newcastle disease and which eventually killed all of the chickens, regardless of the route of infection. There was not any significant correlation between their reactivity patterns with the monoclonal antibodies and their virulence.

  17. [Detection and description of avian hepatitis E virus isolated in China--a review].

    Science.gov (United States)

    Zhao, Qin; Sun, Yani; Zhou, Enmin

    2012-03-04

    Avian hepatitis E virus (HEV), a member of Hepeviridae family, is genetically and antigenically related with human and swine HEV in the family. Since its discovery, avian HEV infection has been investigated in many countries from serology and molecular epidemiology studies. At present, five complete or near complete genomes of avian HEV isolates were reported in GenBank and were divided into three genotypes. The complete genome of avian HEV contains 3 ORFs of which ORF2 gene encodes capsid protein containing the primary epitopes of viral particles and is target gene for serodiagnostic antigen and vaccine candidate. Because avian HEV infection has significant impact on the poultry industry and potential zoonotic transmission, the researches on avian HEV have been given much attention. We here give a broad review of the research update on the aetiology, pathogenesis and the antigenicity of capsid protein of avian HEV based on identification of Chinese avian HEV isolate.

  18. Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam

    Science.gov (United States)

    NGUYEN, Dung Van; SUZUKI, Junko; MINAMI, Shohei; YONEMITSU, Kenzo; NAGATA, Nao; KUWATA, Ryusei; SHIMODA, Hiroshi; VU, Chien Kim; TRUONG, Thuy Quoc; MAEDA, Ken

    2016-01-01

    Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China. PMID:27746406

  19. Diversity of beet curly top Iran virus isolated from different hosts in Iran.

    Science.gov (United States)

    Gharouni Kardani, Sara; Heydarnejad, Jahangir; Zakiaghl, Mohammad; Mehrvar, Mohsen; Kraberger, Simona; Varsani, Arvind

    2013-06-01

    Beet curly top Iran virus (BCTIV) is a major pathogen of sugar beet in Iran. In order to study diversity of BCTIV, we sampled 68 plants in Iran during the summer of 2010 with curly top disease symptoms on beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), tomatoes (Solanum lycopersicum L.), sea beets (Beta vulgaris subsp. maritima), and sugar beets (Beta vulgaris). Plant samples showing leaf curling, yellowing, and/or swelling of veins on the lower leaf surfaces were collected from various fields in Khorasan Razavi, Northern Khorasan (north-eastern Iran), East Azarbayejan, West Azarbayejan (north-western Iran), and Fars (southern Iran) provinces. Using rolling circle amplification coupled with restriction digests, cloning, and Sanger sequencing, we determined the genomes of nine new BCTIV isolates from bean, cowpea, tomato, sea beet, and sugar beet in Iran. Our analysis reveals ~11 % diversity amongst BCTIV isolates and we detect evidence of recombination within these genomes.

  20. Vector monitoring at Belgian outbreak sites during the bluetongue epidemic of 2006.

    Science.gov (United States)

    De Deken, G; Madder, M; Deblauwe, I; De Clercq, K; Fassotte, C; Losson, B; Haubruge, E; De Deken, R

    2008-10-15

    In response to the first bluetongue outbreak in Belgium a monitoring programme was started at the end of August 2006 to identify possible vectors transmitting the disease. Black light traps were deployed at 36 outbreak sites and captured 1959 Culicoides specimens belonging to 16 different species. Eighty four percent of the biting midges captured belonged to the C. obsoletus complex, among them C. obsoletus s.s., C. dewulfi and C. scoticus, three suspected bluetongue vectors. The Veterinary and Agrochemical Research Centre detected viral RNA in pools of individuals belonging to this complex. Culicoides pulicaris, a potential bluetongue vector in Italy, should yet not be excluded as a possible vector in Belgium as this species was frequently found around outbreak sites, notwithstanding this species is not easily captured with the trapping techniques used during this survey.

  1. Molecular Characterization of Banana streak virus Isolate from Musa Acuminata in China

    Institute of Scientific and Technical Information of China (English)

    Jun Zhuang; Jian-hua Wang; Xin Zhang; Zhi-xinLiu

    2011-01-01

    Banana streak virus (BSV),a member of genus Badnavirus,is a causal agent of banana streak disease throughout the world.The genetic diversity of BSVs from different regions of banana plantations has previously been investigated,but there are relatively few reports of the genetic characteristic of episomal (non-integrated)BSV genomes isolated from China.Here,the complete genome,a total of 7722bp (GenBank accession number DQ092436),of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAcYNV) in Yunnan,China was determined.The genome organises in the typical manner of badnaviruses.The intergenic region of genomic DNA contains a large stem-loop,which may contribute to the ribosome shift into the following open reading frames (ORFs).The coding region of BSAcYNV consists of three overlapping ORFs,ORF 1 with a non-AUG start eodon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein.Besides the complete genome,a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants.Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vientam (BSAcVNV).The corresponding coding regions shared identities of 88% and ~95% at nucleotide and amino acid levels,respectively.Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.

  2. Isolation and characterization of virus of highly pathogenic avian influenza H5 subtype of chicken from outbreaks in Indonesia

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    Agus Wiyono

    2004-03-01

    Full Text Available A study on the isolation and characterization of Highly Pathogenic Avian Influenza of chicken from outbreaks in Indonesia was conducted at Indonesian Research Institute for Veterinary Science. Outbreaks of avian disease had been reported in Indonesia since August 2003 affecting commercial layer, broiler, quail, and ostrich and also native chicken with showing clinical signs such as cyanosis of wattle and comb, nasal discharges and hypersalivation, subcutaneous ptechiae on foot and leg, diarre and sudden high mortality. The aim of this study is to isolate and characterize the causal agent of the disease. Samples of serum, feather follicle, tracheal swab, as well as organs of proventriculus, intestine, caecal tonsil, trachea and lungs were collected from infected animals. Serum samples were tested haemaglutination/haemaglutination inhibition to Newcastle Disease and Egg Drop Syndrome viruses. Isolation of virus of the causal agent of the outbreak was conducted from samples of feather follicle, tracheal swab, and organs using 11 days old specific pathogen free (SPF embryonated eggs. The isolated viruses were then characterised by agar gel precipitation test using swine influenza reference antisera, by haemaglutination inhibition using H1 to H15 reference antisera, and by electron microscope examination. The pathogenicity of the viruses was confirmed by intravenous pathogenicity index test and its culture in Chicken Embryo Fibroblast primary cell culture without addition of trypsin. The study revealed that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus based upon serological tests, virus isolation and characterization using swine influenza reference antisera, and electron microscope examination. While subtyping of the viruses using H1 to H15 reference antisera suggested that the virus is very likely to be an avian influenza H5N1 subtype virus. The pathogenicity test confirmed that the viruses

  3. Isolation and characterization of a single-stranded DNA virus infecting the marine diatom Chaetoceros sp. strain SS628-11 isolated from western Japan.

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    Kei Kimura

    Full Text Available Diatoms are significant organisms for primary production in the earth's aquatic environment. Hence, their dynamics are an important focus area in current studies. Viruses are a great concern as potential factors of diatom mortality, along with other physical, chemical, and biological factors. We isolated and characterized a new diatom virus (Csp07DNAV that lyses the marine planktonic diatom Chaetoceros sp. strain SS628-11. This paper examines the physiological, morphological, and genomic characteristics of Csp07DNAV. The virus was isolated from a surface water sample that was collected at Hiroshima Bay, Japan. It was icosahedral, had a diameter of 34 nm, and accumulated in the nuclei of host cells. Rod-shaped virus particles also coexisted in the host nuclei. The latent period and burst size were estimated to be <12 h and 29 infectious units per host cell, respectively. Csp07DNAV had a closed circular single-stranded DNA genome (5,552 nucleotides, which included a double-stranded region and 3 open reading frames. The monophyly of Csp07DNAV and other Bacilladnavirus group single-stranded DNA viruses was supported by phylogenetic analysis that was based on the amino acid sequence of each virus protein. On the basis of these results, we considered Csp07DNAV to be a new member of the genus Bacilladnavirus.

  4. Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

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    Martínez-de la Puente Josué

    2012-07-01

    Full Text Available Abstract Background Culicoides (Diptera: Ceratopogonidae biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD, Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i to identify the midge species in question and characterize their COI barcoding region and (ii to ascertain the source of their bloodmeals using molecular tools. Methods Biting midges were captured using CDC traps baited with a 4-W blacklight (UV bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST. Results The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST showed that both

  5. Comparison of the pathogenic, antigenic and molecular characteristics of two encephalomyocarditis virus (EMCV) isolates from Belgium and Greece.

    Science.gov (United States)

    Koenen, F; Vanderhallen, H; Papadopoulos, O; Billinis, C; Paschaleri-Papadopoulou, E; Brocchi, E; De Simone, F; Carra, E; Knowles, N J

    1997-01-01

    The pathogenicity of two porcine encephalomyocarditis virus (EMCV) isolates for sows in gestation and young piglets was studied. One virus originated from a case of reproductive failure in pigs in Belgium and the other from a case of acute myocarditis in pigs in Greece. Sows in the mid-gestation period and one- to two-month old piglets were inoculated with each isolate. The molecular relationship between both isolates was studied by determining the nucleotide sequence located across the junction of the 1C and 1D capsid-coding genes. Antigenic analysis was performed using a panel of 35 monoclonal antibodies raised against an Italian field isolate of EMCV. All three approaches revealed differences between both isolates and also confirmed that there was no link between the two outbreaks of disease.

  6. Isolation and sequence analysis of a canine distemper virus from a raccoon dog in Jilin Province, China.

    Science.gov (United States)

    Cheng, Yuening; Wang, Jianke; Zhang, Miao; Zhao, Jianjun; Shao, Xiqun; Ma, Zengjun; Zhao, Hang; Lin, Peng; Wu, Hua

    2015-10-01

    Canine distemper virus (CDV) is a major pathogen not only in raccoon dogs but also in a variety of carnivorous animals, including domesticated animals, particularly if they have not been vaccinated. In this study, a wild-type strain of CDV was isolated from lung tissue from a raccoon dog kept at a fur farm in Jilin Province, China. Cytopathic effects typical of CDV infection were observed after three blind passages in Vero cells, yielding a virus titer of 10(4.6) TCID50/mL. Virus identification was carried out by RT-PCR, immunofluorescence, electron microscopy, and genome sequencing. The results showed that the isolated virus, termed the SY strain, corresponded to the Asia-1 genotype of CDV and has a genome of 15,690 nucleotides. This represents the first complete nucleotide sequence of a CDV strain circulating in raccoon dogs in China.

  7. Hepatitis B virus genotypes circulating in Brazil: molecular characterization of genotype F isolates

    Directory of Open Access Journals (Sweden)

    Virgolino Helaine A

    2007-11-01

    Full Text Available Abstract Background Hepatitis B virus (HBV isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil. Results Genotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%, and most of these isolates were classified as subgenotype A1 (138/153; 90.2%. Genotype D was the most common genotype in the South (84.2% and Central (47.6% regions. The prevalence of genotype F was low (13% countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5% belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127. Conclusion The presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F

  8. Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS in the hemagglutinin protein (HA. Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

  9. Mus cervicolor murine leukemia virus isolate M813 belongs to a unique receptor interference group.

    Science.gov (United States)

    Prassolov, V; Hein, S; Ziegler, M; Ivanov, D; Münk, C; Löhler, J; Stocking, C

    2001-05-01

    Murine leukemia virus (MuLV) M813 was originally isolated from the Southeast Asian rodent Mus cervicolor. As with the ecotropic MuLVs derived from Mus musculus, its host range is limited to rodent cells. Earlier studies have mapped its receptor to chromosome 2, but it has not been established whether M813 shares a common receptor with any other MuLVs. In this study, we have performed interference assays with M813 and viruses from four interference groups of MuLV. The infection efficiency of M813 was not compromised in cells expressing any one of the other MuLVs, demonstrating that M813 must use a distinct receptor for cell entry. The entire M813 env coding region was molecularly cloned. Sequence analysis revealed high similarity with other MuLVs but with a unique receptor-binding domain. Substitution of M813 env sequences in Moloney MuLV resulted in a replication-competent virus with a host range and interference profile similar to those of the biological clone M813. M813 thus defines a novel receptor interference group of type C MuLVs.

  10. Genetic and antigenic characterization of H5 and H7 influenza viruses isolated from migratory water birds in Hokkaido, Japan and Mongolia from 2010 to 2014.

    Science.gov (United States)

    Hiono, Takahiro; Ohkawara, Ayako; Ogasawara, Kohei; Okamatsu, Masatoshi; Tamura, Tomokazu; Chu, Duc-Huy; Suzuki, Mizuho; Kuribayashi, Saya; Shichinohe, Shintaro; Takada, Ayato; Ogawa, Hirohito; Yoshida, Reiko; Miyamoto, Hiroko; Nao, Naganori; Furuyama, Wakako; Maruyama, Junki; Eguchi, Nao; Ulziibat, Gerelmaa; Enkhbold, Bazarragchaa; Shatar, Munkhduuren; Jargalsaikhan, Tserenjav; Byambadorj, Selenge; Damdinjav, Batchuluun; Sakoda, Yoshihiro; Kida, Hiroshi

    2015-08-01

    Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.

  11. Molecular characterization of an apoptotic strain of Newcastle disease virus isolated from an outbreak in India.

    Science.gov (United States)

    Kumar, U; Kumar, S

    2015-08-01

    Newcastle disease (ND) is a highly contagious disease of poultry. The ND virus (NDV) encodes an error-prone RNA-dependent RNA polymerase which can cause high mutation rate leading to the emergence of its new antigenic variants. Antigenic difference of NDV strains may result in massive outbreak in vaccinated and unvaccinated poultry flocks around the globe. Apart from its pathogenic potential NDV has been explored as an oncolytic agent for a broad range of human cancers. In the present study, we isolated a novel NDV strain from an outbreak in chicken flock from the eastern part of India. Molecular characterization showed the NDV strain to be virulent in nature. The complete genome sequence analysis of the newly isolated strains belongs to genotype XIII. Moreover, the newly isolated strain of NDV showed positive results in various apoptotic assays in human breast cancer cells, MCF-7. The study will be useful to explore the possibility of using a newly isolated strain of NDV for virotherapy.

  12. Isolated antibody to hepatitis B core antigen in patients with chronic hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Ahmed Helmy; Mohammed Ibrahim Al-Sebayel

    2006-01-01

    AIM: To evaluate the prevalence of isolated anti-HBc in patients with chronic hepatitis C virus (HCV) infection,and its relation to disease severity.METHODS: We screened all patients with chronic HCV infection referred to King Faisal Specialist Hospital and Research Center for hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen (antiHBs), and anti-HBc. One hundred and sixty nine patients who tested negative for both HBsAg and anti-HBs were included in this study.RESULTS: Pathologically, 59 had biopsy-proven cirrhosis and 110 had chronic active hepatitis (CAH). Of these 169 patients, 85 (50.3%) tested positive for anti-HBc.Patients with CAH had significantly higher prevalence of isolated anti-HBc than patients with cirrhosis, 71 (64.5%)and 14 (23.7%) respectively (P < 0.001). Twenty-five patients were tested for HBV DNA by qualitative PCR.The test was positive in 3 of them (12%; occult HBV infection).CONCLUSION: Isolated anti-HBc alone is common in Saudi patients with chronic HCV infection, and is significantly more common in those with CAH than those with cirrhosis. Therefore, a screening strategy that only tests for HBsAg and anti-HBs in these patients will miss a large number of individuals with isolated anti-HBc, who may be potentially infectious.

  13. Screening, isolation and optimization of anti-white spot syndrome virus drug derived from marine plants

    Institute of Scientific and Technical Information of China (English)

    Somnath Chakraborty; Upasana Ghosh; Thangavel Balasubramanian; Punyabrata Das

    2014-01-01

    Objective: To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model.Methods:ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.Results:Thirty species of marine plants were subjected to Soxhlet extraction using water, formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.Conclusions:Nine plant isolates exhibited significant survivability in host. The drug MP07X thus The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug.

  14. The feline lymphoid cell line MBM and its use for feline immunodeficiency virus isolation and quantitation.

    Science.gov (United States)

    Matteucci, D; Mazzetti, P; Baldinotti, F; Zaccaro, L; Bendinelli, M

    1995-05-01

    We report on the development of a feline T lymphoblastoid cell line obtained from the peripheral blood mononuclear cells (PBMC) of a specific pathogen free cat and designated MBM. The cells are pan-T+, CD4- and CD8- and remained interleukin-2-dependent and concanavalin A-dependent throughout the period of observation. MBM cells have proved at least as sensitive as fresh blasts to infection with cell-free stocks of three feline immunodeficiency virus (FIV) isolates. Upon infection, they exhibit a lytic cytopathic effect. Repeated attempts to establish a chronic infection have failed. Using a limiting cell dilution method, it has been shown that MBM cells may be more sensitive than fresh blasts as substrate for isolating FIV from the PBMC of infected cats. These studies have also shown that considerable individual variations exist in the virus loads present in the PBMC of naturally infected cats, and that load size does not appear to correlate with cat age, clinical status, CD4/CD8 ratio and titer of serum neutralizing antibody.

  15. Molecular Characterization of Avian-like H1N1 Swine Influenza A Viruses Isolated in Eastern China, 2011

    Institute of Scientific and Technical Information of China (English)

    Xian Qi; Yuning Pan; Yuanfang Qin; Rongqiang Zu; Fengyang Tang; Minghao Zhou; Hua Wang; Yongchun Song

    2012-01-01

    Currently,three predominant subtypes of influenza virus are prevalent in pig populations worldwide:H1N1,H3N2,and H1N2.European avian-like H1N1 viruses,which were initially detected in European pig populations in 1979,have been circulating in pigs in eastern China since 2007.In this study,six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China.Based on whole genome sequencing,molecular characteristics of two isolates were determined.Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations,especially similar to those found in China.Four potential glycosylation sites were observed at positions 13,26,198,277 in the HA1 proteins of the two isolates.Due to the presence of a stop codon at codon 12,the isolates contained truncated PB1-F2 proteins.In this study,the isolates contained 591Q,627E and 701N in the polymerase subunit PB2,which had been shown to be determinants of virulence and host adaptation.The isolates also had a D rather than E at position 92 of the NS1,a marker of mammalian adaptation.Both isolates contained the GPKV motif at the PDZ ligand domain of the 3' end of the NS1,a characteristic marker of the European avian-like swine viruses since about 1999,which is distinct from those of avian,human and classical swine viruses.The M2 proteins of the isolates have the mutation (S31N),a characteristic marker of the European avian-like swine viruses since about 1987,which may confer resistance to amantadine and rimantadine antivirals.Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs,and raise more concerns about the occurrence of cross-species transmission events.

  16. Isolation of a complete circular virus genome sequence from an Alaskan black-capped chickadee (Poecile atricapillus) gastrointestinal tract sample.

    Science.gov (United States)

    Hanna, Zachary R.; Runckel, Charles; Fuchs, Jerome; DeRisi, Joseph L.; Mindell, David P.; Van Hemert, Caroline R.; Handel, Colleen M.; Dumbacher, John P.

    2015-01-01

    We report here the genome sequence of a circular virus isolated from samples of an Alaskan black-capped chickadee (Poecile atricapillus) gastrointestinal tract. The genome is 2,152 bp in length and is most similar (30 to 44.5% amino acid identity) to the genome sequences of other single-stranded DNA (ssDNA) circular viruses belonging to the gemycircularvirus group.

  17. Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China.

    Science.gov (United States)

    Zhang, Yanjun; Mao, Haiyan; Yan, Juying; Zhang, Lei; Sun, Yi; Wang, Xinying; Chen, Yin; Lu, Yiyu; Chen, Enfu; Lv, Huakun; Gong, Liming; Li, Zhen; Gao, Jian; Xu, Changping; Feng, Yan; Ge, Qiong; Xu, Baoxiang; Xu, Fang; Yang, Zhangnv; Zhao, Guoqiu; Han, Jiankang; Guus, Koch; Li, Hui; Shu, Yuelong; Chen, Zhiping; Xia, Shichang

    2014-08-30

    In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E-3 (95% Highest posterior density: 3.8218E-3 to 7.7873E-3) while the NA gene showed 2.243E-3 (4.378E-4 to 3.79E-3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation.

  18. Molecular characterization of infectious myonecrosis virus (IMNV) isolated from the shrimp Litopenaeus vannamei farmed in Ceará State, Brazil

    OpenAIRE

    Maria Verônyca Coelho-Melo; Maria Izabel Florindo-Guedes; Sergio Rodriguez-Málaga; Lia Magalhães de Almeida; Mariana de Freitas Moreira; Tatiane Rodrigues de Oliveira

    2014-01-01

    The shrimp Litopenaeus vannamei, one of the most important species in world aquaculture, has seriously affected by infectious myonecrosis virus (IMNV) that causes up to 70% mortalities. With the aim of improving the development of new strategies for rapid and reliable diagnosis, we isolated IMNV, from L. vannamei farmed in Brazil, through a discontinuous sucrose gradient, and sequenced cDNA fragment encoding the major capsid protein from this virus. Nucleotides sequences corresponding to the ...

  19. Relationship between the immunosuppressive potential and the pathotype of Marek's disease virus isolates.

    Science.gov (United States)

    Calnek, B W; Harris, R W; Buscaglia, C; Schat, K A; Lucio, B

    1998-01-01

    Isolates of Marek's disease virus (MDV) representing three pathotypes of differing virulence were compared for relative immunosuppressive properties in genetically susceptible P2a-strain and genetically resistant N2a-strain chickens. Criteria of immunosuppression were 1) persistence of early cytolytic infection (i.e., a delay or failure to enter latency) in lymphoid organs, 2) atrophy of the bursa of Fabricius and thymus as measured by organ weight proportional to body weight at 8 and 14 days postinfection (DPI), and 3) histopathologic evidence of necrosis and atrophy in lymphoid organs. No significant differences in infection level were observed among the pathotypes during the early (4-5 DPI) period of infection. However, the extent of persistent cytolytic infection at 7-8 DPI, based on numbers of tissues positive and mean scores in immunofluorescence tests, was greater (P Md5) classified in the intermediate very virulent pathotype (very virulent MDV [vvMDV]) fell between those from the other two pathotypes. Similarly, there was a stepwise effect of viral pathotype in which the vv + MDV isolates caused the most severe damage to lymphoid organs in terms of atrophy (relative organ weights) and histopathologic changes. Organs from chickens infected with vv + MDVs showed little recovery between 8 and 14 DPI. The vMDV isolates caused the least severe damage, and lymphoid organs showed a significant return toward normal by 14 DPI; vvMDV isolates induced intermediate degrees of atrophy and recovery. The same pattern of relationship between virulence pathotype and degree of bursal and thymic atrophy was also observed in genetically resistant N2a chickens. These results suggest that the degree of immunosuppression is linked to virulence and that a simple measure of atrophic changes (relative organ weights) in the bursa of Fabricius and thymus might be useful in determining the pathotype classification of new MDV isolates. The basis for differences in immunosuppressive

  20. Cell-mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus.

    Science.gov (United States)

    Tan, X; Phillips, D M

    1996-01-01

    We have previously demonstrated that HIV-infected transformed T-cells or monocytes adhere to monolayers of CD4-negative epithelial cells. Adhesion is soon followed by budding of HIV from infected mononuclear cells onto the surface of epithelial cells. Epithelial cells subsequently take up virus and become productively infected. Based on these findings, we proposed that sexual transmission of HIV may involve cell-mediated infection of intact mucosal epithelia of the urogenital tract. However, it has become increasingly clear that primary cells and HIV strains isolated from patients are more appropriate models for HIV infection than established cell lines and lab strains of virus. In the studies described here, we infected cervix-derived epithelial monolayers with primary monocytes infected with patient isolates of non-syncytial inducing (NSI) macrophage-tropic strains of HIV. Under the culture conditions employed, HIV-infected primary monocytes do not remain adherent to the apical surface of the epithelium, as did HIV-infected transformed cells. Instead, following adherence, the primary cells migrate between epithelial cells. Virus is secreted from a pseudopod as HIV-infected primary monocytes pass between cells of the epithelium. Productive infection of the epithelium was detected by p24 ELISA and PCR Southern blot analysis. Infection can be blocked by sera from HIV-seropositive individuals or by certain sulfated polysaccharides. These findings support the supposition that transmission of HIV may occur via cell-mediated infection of intact epithelia. The observations also hint at the possibility that-HIV-infected monocyte/macrophages in semen or cervical-vaginal secretions could cross intact epithelia by passing between epithelial cells. Blocking studies suggest that it may be possible to inhibit sexual transmission of HIV either by antibodies in genital tract secretions or by a topical formulation containing certain sulfated polysaccharides.

  1. Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

    1986-10-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

  2. Molecular characterization of highly pathogenic H5N1 avian influenza A viruses isolated from raccoon dogs in China.

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    Xian Qi

    Full Text Available BACKGROUND: The highly pathogenic avian influenza H5N1 virus can infect a variety of animals and continually poses a threat to animal and human health. While many genotypes of H5N1 virus can be found in chicken, few are associated with the infection of mammals. Characterization of the genotypes of viral strains in animal populations is important to understand the distribution of different viral strains in various hosts. This also facilitates the surveillance and detection of possible emergence of highly pathogenic strains of specific genotypes from unknown hosts or hosts that have not been previously reported to carry these genotypes. METHODOLOGY/PRINCIPAL FINDINGS: Two H5N1 isolates were obtained from lung samples of two raccoon dogs that had died from respiratory disease in China. Pathogenicity experiments showed that the isolates were highly pathogenic to chicken. To characterize the genotypes of these viruses, their genomic sequences were determined and analyzed. The genetic contents of these isolates are virtually identical and they may come from the same progenitor virus. Phylogenetic analysis indicated that the isolates were genetically closely related to genotype V H5N1 virus, which was first isolated in China in 2003, and were distinct from the dominant virus genotypes (e.g. genotype Z of recent years. The isolates also contain a multibasic amino acid motif at their HA cleavage sites and have an E residue at position 627 of the PB2 protein similar to the previously-identified avian viruses. CONCLUSIONS/SIGNIFICANCE: This is the first report that genotype V H5N1 virus is found to be associated with a mammalian host. Our results strongly suggest that genotype V H5N1 virus has the ability to cross species barriers to infect mammalian animals. These findings further highlight the risk that avian influenza H5N1 virus poses to mammals and humans, which may be infected by specific genotypes that are not known to infect these hosts.

  3. Isolation, identification and evolution analysis of a novel subgroup of avian leukosis virus isolated from a local Chinese yellow broiler in South China.

    Science.gov (United States)

    Li, Xinjian; Lin, Wencheng; Chang, Shuang; Zhao, Peng; Zhang, Xinheng; Liu, Yang; Chen, Weiguo; Li, Baohong; Shu, Dingming; Zhang, Huanmin; Chen, Feng; Xie, Qingmei

    2016-10-01

    Avian leukosis virus (ALV) causes high mortality associated with tumor formation and decreased fertility, and results in major economic losses in the poultry industry worldwide. Recently, a putative novel ALV subgroup virus named ALV-K was observed in Chinese local chickens. In this study, a novel ALV strain named GD14LZ was isolated from a Chinese local yellow broiler in 2014. The proviral genome was sequenced and phylogenetically analyzed. The replication ability and pathogenicity of this virus were also evaluated. The complete proviral genome sequence of GD14LZ was 7482 nt in length, with a genetic organization typical of replication-competent type C retroviruses lacking viral oncogenes. Sequence analysis showed that the gag, pol and gp37 genes of GD14LZ have high sequence similarity to those of other ALV strains (A-E subgroups), especially to those of ALV-E. The gp85 gene of the GD14LZ isolate showed a low sequence similarity to those other ALV strains (A-E subgroups) but showed high similarity to strains previously described as ALV-K. Phylogenetic analysis of gp85 also suggested that the GD14LZ isolate was related to ALV-K strains. Further study showed that this isolate replicated more slowly and was less pathogenic than other ALV strains. These results indicate that the GD14LZ isolate belongs to the novel subgroup ALV-K and probably arose by recombination of ALV-K with endogenous viruses with low replication and pathogenicity. This virus might have existed in local Chinese chickens for a long time.

  4. Isolation, Identification, and Sequencing of a Goose-Derived Newcastle Disease Virus and Determination of Its Pathogenicity.

    Science.gov (United States)

    Chen, Xiao-Qing; Li, Zi-Bing; Hu, Gui-Xue; Gu, Song-Zhi; Zhang, Shuang; Ying, Ying; Gao, Feng-Shan

    2015-06-01

    In August 2010, geese in the Meihekou area of Jilin province in China were found to be infected by a pathogen that caused a disease similar to Newcastle disease. To determine the causative agent of the infections, a virus was isolated from liver tissues of infected geese, followed by a pathogenicity determination. The isolated virus was named NDV/White Goose/China/Jilin(Meihekou)/MHK-1/2010. Specific primers were designed to amplify the whole genome of the MHK-1 virus, followed by sequencing and splicing of the entire genome. Sequencing and phylogenetic analysis of MHK-1 showed that the isolate was a virulent strain of Newcastle disease virus. The MHK-1 genome is 15,192 nucleotides long, and it belongs to the class II branch of Newcastle disease viruses, as evidenced by the amino acid sequence (112R-R-Q-K-R-F117) of the F protein. The hemagglutinin titer was 1:128 to 1:512. The chicken embryo mean death time, the intracerebral pathogenicity index, and the median lethal dose of chicken embryos of MHK-1 were 43 hr, 1.63, and 10(9)/ml, respectively, which revealed that the newly isolated MHK-1 strain is strongly pathogenic to geese.

  5. Phylogenetic Analysis of the Non-structural (NS) Gene of Influenza A Viruses Isolated in Kazakhstan in 2002-2009

    Institute of Scientific and Technical Information of China (English)

    Andrey Bogoyavlenskiy; Marat