Plasma Epstein-Barr virus and Hepatitis B virus in non-Hodgkin lymphomas: Two lymphotropic, potentially oncogenic, latently occurring DNA viruses.

Science.gov (United States)

Sinha, Mahua; Rao, Clementina Rama; Premalata, C S; Shafiulla, Mohammed; Lakshmaiah, K C; Jacob, Linu Abraham; Babu, Govind K; Viveka, B K; Appaji, L; Subramanyam, Jayshree R

2016-01-01

There is a need to study potential infective etiologies in lymphomas. Lymphocyte-transforming viruses can directly infect lymphocytes, disrupt normal cell functions, and promote cell division. Epstein-Barr virus (EBV) is known to be associated with several lymphomas, especially Hodgkin lymphomas (HLs). And recently, the lymphocyte-transforming role of hepatitis B virus (HBV) has been emphasized. The aim of this study was to elucidate the association of two potentially oncogenic, widely prevalent latent DNA viruses, EBV and HBV, in non-HL (NHL). In this prospective study, we estimated plasma EBV and HBV DNA in NHL patients. Peripheral blood was obtained from newly diagnosed, treatment na ïve, histologically confirmed NHL patients. Plasma EBV DNA was quantified by real-time polymerase chain reaction (PCR) targeting Epstein-Barr Nucleic acid 1 while the plasma HBV DNA was detected using nested PCR targeting HBX gene. In a small subset of patients, follow-up plasma samples post-anticancer chemotherapy were available and retested for viral DNA. Of the 110 NHL patients, ~79% were B-cell NHL and ~21% were T-cell NHL. Plasma EBV-DNA was detected in 10% NHLs with a higher EBV association in Burkitt lymphoma (33.3%) than other subtypes. Pretherapy HBV DNA was detected in 21% NHLs; most of them being diffuse large B-cell lymphoma (DLBCL). Moreover, 42% of DLBCL patients had HBV DNA in plasma. Since all patients were HBV surface antigen seronegative at diagnosis, baseline plasma HBV-DNAemia before chemotherapy was indicative of occult hepatitis B infection. Our findings indicate a significant association of HBV with newly diagnosed DLBCL.

Susceptibility testing of fish cell lines for virus isolation

DEFF Research Database (Denmark)

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

2009-01-01

and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... 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...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

Isolation and characterization of H3N2 influenza A virus from turkeys.

Science.gov (United States)

Tang, Y; Lee, C W; Zhang, Y; Senne, D A; Dearth, R; Byrum, B; Perez, D R; Suarez, D L; Saif, Y M

2005-06-01

Five 34-wk-old turkey breeder layer flocks in separate houses of 2550 birds each in a single farm in Ohio experienced a drop in egg production from late January to early February 2004. Tracheal swabs (n = 60), cloacal swabs (n = 50), and convalescent sera (n = 110) from the flocks were submitted to the laboratory for diagnostics. Virus isolation was attempted in specific-pathogen free embryonating chicken eggs and Vero and MDCK cells. Virus characterization was performed using agar gel immunodiffusion, the hemagglutination test, the hemagglutination inhibition test, the virus neutralization test, reverse transcription-polymerase chain reaction, sequencing, and phylogenetic analysis. A presumptive influenza virus was successfully propagated and isolated on the first passage in MDCK cells, but initially not in Vero cells or specific-pathogen free chicken embryos. After two passages in MDCK cells, it was possible to propagate the isolate in specific-pathogen free chicken embryos. Preliminary sequence analysis of the isolated virus confirmed that it was influenza A virus with almost 100% (235/236) identity with the matrix gene of a swine influenza A virus, A/Swine/Illinois/100084/01 (H1N2). However, it was not possible to subtype the virus using conventional serotyping methods. The results of genetic characterization of the isolated virus showed that it was the H3N2 subtype and was designated as A/Turkey/OH/313053/04 (H3N2). Phylogenetic analysis of the eight gene segments of the virus showed that A/Turkey/OH/313053/04 (H3N2) isolate was most closely related to the triple-reassortant H3N2 swine viruses [A/Swine/WI/14094/99 (H3N2)] that have been circulating among pigs in the United States since 1998, which contains gene segments from avian, swine, and human viruses. The A/Turkey/OH/313053/04 (H3N2) isolated from turkeys in this study was classified as a low pathogenic avian influenza A virus because it only caused a drop in egg production with minor other clinical

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

Science.gov (United States)

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

Integration of antibody by surface functionalization of graphite-encapsulated magnetic beads using ammonia gas plasma technology for capturing influenza A virus.

Science.gov (United States)

Sakudo, Akikazu; Chou, Han; Ikuta, Kazuyoshi; Nagatsu, Masaaki

2015-05-01

Antibody-integrated magnetic beads have been functionalized for influenza A virus capture. First, ammonia plasma produced by a radio frequency power source was reacted with the surface of graphite-encapsulated magnetic beads to introduce amino groups. Anti-influenza A virus hemagglutinin antibody was then anchored by its surface sulfide groups to the amino groups on the beads via N-succinimidyl 3-(2-pyridyldithio) propionate. After incubation with influenza A virus, adsorption of the virus to the beads was confirmed by immunochromatography, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and inoculation of chicken embryonated eggs, indicating that virus infectivity is maintained and that the proposed method is useful for the enhanced detection and isolation of influenza A virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Studies on antigenic and genomic properties of Brazilian rabies virus isolates

NARCIS (Netherlands)

Schaefer, R.; Batista, H.B.; Franco, A.C.; Rijsewijk, F.A.M.; Roehe, P.M.

2005-01-01

Despite the recognized stability of rabies virus, differences among isolates from different species have been found. This work was carried out with the aim to identify antigenic and genomic differences in Brazilian rabies virus isolates and to verify whether such alterations would bear any

Virus fitness differences observed between two naturally occurring isolates of Ebola virus Makona variant using a reverse genetics approach.

Science.gov (United States)

Albariño, César G; Guerrero, Lisa Wiggleton; Chakrabarti, Ayan K; Kainulainen, Markus H; Whitmer, Shannon L M; Welch, Stephen R; Nichol, Stuart T

2016-09-01

During the large outbreak of Ebola virus disease that occurred in Western Africa from late 2013 to early 2016, several hundred Ebola virus (EBOV) genomes have been sequenced and the virus genetic drift analyzed. In a previous report, we described an efficient reverse genetics system designed to generate recombinant EBOV based on a Makona variant isolate obtained in 2014. Using this system, we characterized the replication and fitness of 2 isolates of the Makona variant. These virus isolates are nearly identical at the genetic level, but have single amino acid differences in the VP30 and L proteins. The potential effects of these differences were tested using minigenomes and recombinant viruses. The results obtained with this approach are consistent with the role of VP30 and L as components of the EBOV RNA replication machinery. Moreover, the 2 isolates exhibited clear fitness differences in competitive growth assays. Published by Elsevier Inc.

Isolation of H13N2 influenza A virus from turkeys and surface water.

Science.gov (United States)

Sivanandan, V; Halvorson, D A; Laudert, E; Senne, D A; Kumar, M C

1991-01-01

This is the first report of the isolation of H13N2 avian influenza virus (AIV) subtype from domestic turkeys. This subtype was also isolated from nearby surface water. The observation of large numbers of gulls in close association with turkeys on range before the virus isolations suggests that this virus subtype was transmitted from gulls to range turkeys. Turkey flocks infected by this virus subtype did not show any clinical signs of the disease, although seroconversion did occur. The H13N2 isolates were found to be non-pathogenic in chickens.

Avian influenza virus and Newcastle disease virus (NDV) surveillance in commercial breeding farm in China and the characterization of Class I NDV isolates.

Science.gov (United States)

Hu, Beixia; Huang, Yanyan; He, Yefeng; Xu, Chuantian; Lu, Xishan; Zhang, Wei; Meng, Bin; Yan, Shigan; Zhang, Xiumei

2010-07-29

In order to determine the actual prevalence of avian influenza virus (AIV) and Newcastle disease virus (NDV) in ducks in Shandong province of China, extensive surveillance studies were carried out in the breeding ducks of an intensive farm from July 2007 to September 2008. Each month cloacal and tracheal swabs were taken from 30 randomly selected birds that appeared healthy. All of the swabs were negative for influenza A virus recovery, whereas 87.5% of tracheal swabs and 100% cloacal swabs collected in September 2007, were positive for Newcastle disease virus isolation. Several NDV isolates were recovered from tracheal and cloacal swabs of apparently healthy ducks. All of the isolates were apathogenic as determined by the MDT and ICPI. The HN gene and the variable region of F gene (nt 47-420) of four isolates selected at random were sequenced. A 374 bp region of F gene and the full length of HN gene were used for phylogenetic analysis. Four isolates were identified as the same isolate based on nucleotide sequences identities of 99.2-100%, displaying a closer phylogenetic relationship to lentogenic Class I viruses. There were 1.9-9.9% nucleotide differences between the isolates and other Class I virus in the variable region of F gene (nt 47-420), whereas there were 38.5-41.2% nucleotide difference between the isolates and Class II viruses. The amino acid sequences of the F protein cleavage sites in these isolates were 112-ERQERL-117. The full length of HN gene of these isolates was 1851 bp, coding 585 amino acids. The homology analysis of the nucleotide sequence of HN gene indicated that there were 2.0-4.2% nucleotide differences between the isolates and other Class I viruses, whereas there were 29.5-40.9% differences between the isolates and Class II viruses. The results shows that these isolates are not phylogenetically related to the vaccine strain (LaSota). This study adds to the understanding of the ecology of influenza viruses and Newcastle disease viruses in

Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China

NARCIS (Netherlands)

Luo, Ya-Kun; Liang, Lin; Tang, Qing-Hai; Zhou, Ling; Shi, Li-Jun; Cong, Ying-Ying; Lin, Wen-Cheng; Cui, Shang-Jin

2017-01-01

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, we describe an EMC virus isolate, identified as EMCV C15, which was successfully obtained from the serum of dogs from animal hospitals. Virus production in cell culture was confirmed by EMCV-specific

Isolation and genetic characterization of avian influenza viruses and a Newcastle disease virus from wild birds in Barbados: 2003-2004.

Science.gov (United States)

Douglas, Kirk O; Lavoie, Marc C; Kim, L Mia; Afonso, Claudio L; Suarez, David L

2007-09-01

Zoonotic transmission of an H5N1 avian influenza A virus to humans in 2003-present has generated increased public health and scientific interest in the prevalence and variability of influenza A viruses in wild birds and their potential threat to human health. Migratory waterfowl and shorebirds are regarded as the primordial reservoir of all influenza A viral subtypes and have been repeatedly implicated in avian influenza outbreaks in domestic poultry and swine. All of the 16 hemagglutinin and nine neuraminidase influenza subtypes have been isolated from wild birds, but waterfowl of the order Anseriformes are the most commonly infected. Using 9-to-11-day-old embryonating chicken egg culture, virus isolation attempts were conducted on 168 cloacal swabs from various resident, imported, and migratory bird species in Barbados during the months of July to October of 2003 and 2004. Hemagglutination assay and reverse transcription-polymerase chain reaction were used to screen all allantoic fluids for the presence of hemagglutinating agents and influenza A virus. Hemagglutination positive-influenza negative samples were also tested for Newcastle disease virus (NDV), which is also found in waterfowl. Two influenza A viruses and one NDV were isolated from Anseriformes (40/168), with isolation rates of 5.0% (2/40) and 2.5% (1/40), respectively, for influenza A and NDV. Sequence analysis of the influenza A virus isolates showed them to be H4N3 viruses that clustered with other North American avian influenza viruses. This is the first report of the presence of influenza A virus and NDV in wild birds in the English-speaking Caribbean.

Virus isolation for diagnosing dengue virus infections in returning travelers

NARCIS (Netherlands)

Teichmann, D.; Göbels, K.; Niedrig, M.; Sim-Brandenburg, J.-W.; Làge-Stehr, J.; Grobusch, M. P.

2003-01-01

Dengue fever is recognized as one of the most frequent imported acute febrile illnesses affecting European tourists returning from the tropics. In order to assess the value of virus isolation for the diagnosis of dengue fever, 70 cases of dengue fever confirmed in German travelers during the period

Report of isolations of unusual lyssaviruses (rabies and Mokola virus identified retrospectively from Zimbabwe : short communication

Directory of Open Access Journals (Sweden)

J. Bingham

2001-07-01

Full Text Available Rabies isolates that had been stored between 1983 and 1997 were examined with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies. Out of 56 isolates from cats and various wild carnivore species, 1 isolate of Mokola virus and 5 other non-typical rabies viruses were identified. The Mokola virus isolate was diagnosed as rabies in 1993 from a cat. Genetic analysis of this isolate suggests that it falls in a distinct subgroup of the Mokola virus genotype. The 5 non-typical rabies viruses were isolated from honey badgers (Mellivora capensis, African civets (Civettictis civetta and an unidentified mongoose (Herpestidae. These isolates are representatives of rarely-reported wildlife-associated strains of rabies, probably maintained by the slender mongoose (Galerella sanguinea. These findings indicate that both Mokola virus and the mongoose-associated variant may be more common in Zimbabwe than is apparent from routine surveillance.

Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

Directory of Open Access Journals (Sweden)

Xiaozhen Liang

2009-11-01

Full Text Available Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68 gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners that do not directly participate in virus replication, but rather facilitate virus

Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

Science.gov (United States)

Liang, Xiaozhen; Collins, Christopher M; Mendel, Justin B; Iwakoshi, Neal N; Speck, Samuel H

2009-11-01

Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68) gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency) account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners) that do not directly participate in virus replication, but rather facilitate virus reactivation by

Isolation and molecular characterization of Newcastle disease viruses from raptors.

Science.gov (United States)

Jindal, Naresh; Chander, Yogesh; Primus, Alexander; Redig, Patrick T; Goyal, Sagar M

2010-12-01

The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry.

Pepino mosaic virus isolates and differential symptomatology in tomato

NARCIS (Netherlands)

Hanssen, I.M.; Paeleman, A.; Vandewoestijne, E.; Bergen, Van L.; Bragard, C.; Lievens, B.; Vanachter, A.C.R.C.; Thomma, B.P.H.J.

2009-01-01

Based on a survey conducted in commercial tomato production in Belgium in 2006, four Pepino mosaic virus (PepMV) isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in four separate

Highly leukemogenic radiation leukemia virus isolate is a thymotropic, immunosuppressive retrovirus with a unique RNA structure

Energy Technology Data Exchange (ETDEWEB)

Ben David, Y.; Kotler, M.; Yefenof, E.

1987-04-15

Clones of N-, B- and NB-fibrotropic viruses were isolated from weakly (D-RadLV) and strongly (A-RadLV) leukomogenic RadLV preparations. A highly leukemogenic, thymotropic virus (TV) was isolated by ex-vivo infection of thymocytes with A-RadLV. This virus could not be isolated from D-RadLV. Two-dimensional fingerprint analysis suggested that TV recombines unique RNA sequences with RNA genomic material derived from a B-tropic endogenous virus. C57BL/6 (B6) mice injected with B- or NB-fibrotropic clones, but not with TV or N-tropic viral clones, developed reactive T lymphocytes (Tr), capable of differentiating into anti-tumor cytotoxic cells. The N-tropic virus isolates were non-immunogenic in B6 mice whereas the TV isolate induced suppressor T lymphocytes (Ts) that abrogated a potential Tr response. These results suggest that emergence of highly leukemogenic RadLV involves activation of endogenous fibrotropic virus which is immunogenic in its natural host strain (B6). This virus can further recombine with other retroviral genetic sequences, resulting in a suppressogenic and thymotropic, highly leukemogenic virus.

Characterization of a siberian virus isolated from a patient with progressive chronic tick-borne encephalitis.

Science.gov (United States)

Gritsun, T S; Frolova, T V; Zhankov, A I; Armesto, M; Turner, S L; Frolova, M P; Pogodina, V V; Lashkevich, V A; Gould, E A

2003-01-01

A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T(277)-->V and E(279)-->G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

Variability in alternanthera mosaic virus isolates from different hosts

Science.gov (United States)

We have determined the complete genome sequences of Alternanthera mosaic virus phlox isolate PA (AltMV-PA) and four infectious clone variants derived from AltMV-SP, as well as partial sequences of other isolates from various types of phlox, and from portulaca, nandina, and cineraria. Phylogenetic co...

[Phylogenetic analysis of rabies viruses isolated from animals in Tokyo in the 1950s].

Science.gov (United States)

Hatakeyama, Kaoru; Sadamasu, Kenji; Kai, Akemi

2011-05-01

Molecular epidemiological analysis of 96 rabies viruses isolated from animals in Tokyo in the 1950s involves Japanese fixed virus, Komatsugawa, Takamen, and Nishigahara strains. Strains isolated in Tokyo were divided into Tokyo 1 and Tokyo 2, and grouped into a worldwide distribution cluster differing from Takamen and Nishigahara. Tokyo 1 was grouped into the same cluster as viruses isolated from United States west coast dogs in the 1930s and 1940s. Tokyo 2 was grouped into the same cluster as the Komatsugawa strain, also known as a cluster of viruses from the Khabarovsk raccoon dog, and the Lake Baikal stepped fox in Russia. These findings suggest that 1950s Tokyo rabies viruses were related to those in Russia and the USA.

Extensive sequence divergence among bovine respiratory syncytial viruses isolated during recurrent outbreaks in closed herds

DEFF Research Database (Denmark)

Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

2000-01-01

and veal calf production units) in different years and from all confirmed outbreaks in Denmark within a short period. The results showed that identical viruses were isolated within a herd during outbreaks and that viruses from recurrent infections varied by up to 11% in sequence even in closed herds......The nucleotides coding for the extracellular part of the G glycoprotein and the full SH protein of bovine respiratory syncytial virus (BRSV) were sequenced from viruses isolated from numerous outbreaks of BRSV infection. The isolates included viruses isolated from the same herd (closed dairy farms....... It is possible that a quasispecies variant swarm of BRSV persisted in some of the calves in each herd and that a new and different highly fit virus type (master and consensus sequence) became dominant and spread from a single animal in connection with each new outbreak. Based on the high level of diversity...

Isolation of Irkut virus from a Murina leucogaster bat in China.

Directory of Open Access Journals (Sweden)

Ye Liu

Full Text Available BACKGROUND AND OBJECTIVES: Bats are recognized as a major reservoir of lyssaviruses; however, no bat lyssavirus has been isolated in Asia except for Aravan and Khujand virus in Central Asia. All Chinese lyssavirus isolates in previous reports have been of species rabies virus, mainly from dogs. Following at least two recent bat-associated human rabies-like cases in northeast China, we have initiated a study of the prevalence of lyssaviruses in bats in Jilin province and their public health implications. A bat lyssavirus has been isolated and its pathogenicity in mice and genomic alignment have been determined. RESULTS: We report the first isolation of a bat lyssavirus in China, from the brain of a northeastern bat, Murina leucogaster. Its nucleoprotein gene shared 92.4%/98.9% (nucleotide and 92.2%/98.8% (amino acid identity with the two known Irkut virus isolates from Russia, and was designated IRKV-THChina12. Following intracranial and intramuscular injection, IRKV-THChina12 produced rabies-like symptoms in adult mice with a short inoculation period and high mortality. Nucleotide sequence analysis showed that IRKV-THChina12 has the same genomic organization as other lyssaviruses and its isolation provides an independent origin for the species IRKV. CONCLUSIONS: We have identified the existence of a bat lyssavirus in a common Chinese bat species. Its high pathogenicity in adult mice suggests that public warnings and medical education regarding bat bites in China should be increased, and that surveillance be extended to provide a better understanding of Irkut virus ecology and its significance for public health.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus.

Science.gov (United States)

Field, H J; Darby, G; Wildy, P

1980-07-01

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

The lipidomes of vesicular stomatitis virus, semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry

DEFF Research Database (Denmark)

Kalvodova, Lucie; Sampaio, Julio L; Cordo, Sandra

2009-01-01

kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid...

Loss of aphid transmissibility of plum pox virus isolates

NARCIS (Netherlands)

Kamenova, I.; Lohuis, H.; Peters, D.

2002-01-01

The aphid transmissibility of seven Plum pox virus (PPV) isolates and the amino acid sequences of their coat proteins were analysed Two aphid transmissible isolates PPV-A and PPV-P contained the DAG amino triplet, while DAL or NAG replaced this triplet in the coat proteins of non-aphid transmissible

Genetic characterization of epizootic hemorrhagic disease virus strains isolated from cattle in Israel

Science.gov (United States)

Epizootic hemorrhagic disease virus (EHDV), an Orbivirus not previously reported in Israel, was isolated from Israeli cattle during a “bluetongue like” disease outbreak in 2006. To ascertain the origin of this new virus, three isolates from the outbreak were fully sequenced and compared with availab...

Avian influenza virus isolation, propagation and titration in embryonated chicken eggs

Science.gov (United States)

Avian influenza (AI) virus is usually isolated, propagated, and titrated in embryonated chickens eggs (ECE). Most any sample type can be accommodated for culture with appropriate processing. Isolation may also be accomplished in cell culture particularly if mammalian lineage isolates are suspected, ...

An improved method for isolating viruses from asymptomatic carrier fish

Science.gov (United States)

Amend, Donald F.; Pietsch, John P.

1972-01-01

This paper describes a method using elevated levels of penicillin, streptomycin, and nystatin instead of filters to control bacteria and mold contaminants in specimens processed for virus isolation. Filters were shown to significantly reduce the virus concentration. Virus and tissue cultures were not affected by this procedure. In field tests nearly three times more specimens were positive for virus with this method than with the widely used filter technique. Moreover, the cost of materials was less. This method is recommended for inspection and certification purposes.

Characterization of Brugmansia mosaic virus Isolated from Brugmansia spp. in Korea

Directory of Open Access Journals (Sweden)

Chung Youl Park

2014-12-01

Full Text Available In May 2013, an angel’s trumpet leaves showing mosaic and malformation symptoms were collected from Suwon city, Gyeonggi-do. An analysis of the collected sample by transmission electron microscopy observation showed filamentous rod particles of 720-800 nm in length. On the basis of the these observations, we performed PCR against three reported Potyviruses (Brugmansia mosaic virus, Colombian datura virus and Brugmansia suaveolens mottle virus, and the sample was positive for BruMV. Pathogenicity and host range test of BruMV was determined by mechanical inoculation. Solanaceae (tobacco, tomato and eggplant and Amaranthaceae (ground cherry appeared typical virus symptoms. To determine coat protein of this virus, we designed specific primer pairs, and performed PCR amplification, cloning, and sequencing. Phylogenetic analysis showed that BruMV-SW was most closely related to BruMV isolate SK. Comparison of the BruMV-SW coat protein nucleotide sequences showed 92% to 99% identities to the other BruMV isolates.

Differentiation among isolates of prunus necrotic ringspot virus by transcript conformation polymorphism.

Science.gov (United States)

Rosner, A; Maslenin, L; Spiegel, S

1998-09-01

A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among virus isolates. A T7 RNA polymerase promoter was attached to amplified prunus necrotic ringspot virus (PNRSV) sequences by PCR. The PCR products then served as a template for transcription. Single-stranded transcripts originated from different PNRSV isolates varied in electrophoretic mobility in polyacrylamide gels, presumably because of transcript conformation polymorphism (TCP). This procedure was applied for the differentiation of PNRSV isolates.

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

Science.gov (United States)

Bande, Faruku; Arshad, Siti Suri; Omar, Abdul Rahman

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.

Genomic Variability of Citrus tristeza virus (CTV Isolates Introduced into Morocco

Directory of Open Access Journals (Sweden)

Bouabid Lbida

2004-08-01

Full Text Available Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single stranded conformation polymorphism (SSCP and sequencing. Each isolate consisted of several related genomic variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates characterised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat protein gene sequence is useful to trace the origin of the isolates.

Isolation of Dobrava Virus from Apodemus flavicollis in Greece

Science.gov (United States)

Papa, Anna; Nemirov, Kirill; Henttonen, Heikki; Niemimaa, Jukka; Antoniadis, Antonis; Vaheri, Antti; Plyusnin, Alexander; Vapalahti, Olli

2001-01-01

Dobrava virus (DOBV) carried by Apodemus flavicollis is the causative agent of severe hemorrhagic fever with renal syndrome (HFRS). DOBV was isolated from an A. flavicollis mouse trapped in northeastern Greece. This is the third DOBV cell culture isolate in the world, clustering together with other Greek DOBV sequences from HFRS patients and rodents. PMID:11376073

Full Genomic Characterization of a Saffold Virus Isolated in Peru

Directory of Open Access Journals (Sweden)

Mariana Leguia

2015-11-01

Full Text Available While studying respiratory infections of unknown etiology we detected Saffold virus in an oropharyngeal swab collected from a two-year-old female suffering from diarrhea and respiratory illness. The full viral genome recovered by deep sequencing showed 98% identity to a previously described Saffold strain isolated in Japan. Phylogenetic analysis confirmed the Peruvian Saffold strain belongs to genotype 3 and is most closely related to strains that have circulated in Asia. This is the first documented case report of Saffold virus in Peru and the only complete genomic characterization of a Saffold-3 isolate from the Americas.

High-throughput isolation of giant viruses in liquid medium using automated flow cytometry and fluorescence staining.

Directory of Open Access Journals (Sweden)

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.

Sunguru virus: a novel virus in the family Rhabdoviridae isolated from a chicken in north-western Uganda.

Science.gov (United States)

Ledermann, Jeremy P; Zeidner, Nord; Borland, Erin M; Mutebi, John-Paul; Lanciotti, Robert S; Miller, Barry R; Lutwama, Julius J; Tendo, Joseph M; Andama, Vincent; Powers, Ann M

2014-07-01

Sunguru virus (SUNV), a novel virus belonging to the highly diverse Rhabdoviridae family, was isolated from a domestic chicken in the district of Arua, Uganda, in 2011. This is the first documented isolation of a rhabdovirus from a chicken. SUNV is related to, but distinct from, Boteke virus and other members of the unclassified Sandjimba group. The genome is 11056 nt in length and contains the five core rhabdovirus genes plus an additional C gene (within the ORF of a phosphoprotein gene) and a small hydrophobic protein (between the matrix and glycoprotein genes). Inoculation of vertebrate cells with SUNV resulted in significant viral growth, with a peak titre of 7.8 log10 p.f.u. ml(-1) observed in baby hamster kidney (BHK) cells. Little to no growth was observed in invertebrate cells and in live mosquitoes, with Anopheles gambiae mosquitoes having a 47.4% infection rate in the body but no dissemination of the virus to the salivary glands; this suggests that this novel virus is not arthropod borne as some other members of the family Rhabdoviridae.

Sunguru virus: a novel virus in the family Rhabdoviridae isolated from a chicken in north-western Uganda

Science.gov (United States)

Ledermann, Jeremy P.; Zeidner, Nord; Borland, Erin M.; Mutebi, John-Paul; Lanciotti, Robert S.; Miller, Barry R.; Lutwama, Julius J.; Tendo, Joseph M.; Andama, Vincent; Powers, Ann M.

2017-01-01

Sunguru virus (SUNV), a novel virus belonging to the highly diverse Rhabdoviridae family, was isolated from a domestic chicken in the district of Arua, Uganda, in 2011. This is the first documented isolation of a rhabdovirus from a chicken. SUNV is related to, but distinct from, Boteke virus and other members of the unclassified Sandjimba group. The genome is 11 056 nt in length and contains the five core rhabdovirus genes plus an additional C gene (within the ORF of a phosphoprotein gene) and a small hydrophobic protein (between the matrix and glycoprotein genes). Inoculation of vertebrate cells with SUNV resulted in significant viral growth, with a peak titre of 7.8 log10 p.f.u. ml−1 observed in baby hamster kidney (BHK) cells. Little to no growth was observed in invertebrate cells and in live mosquitoes, with Anopheles gambiae mosquitoes having a 47.4 % infection rate in the body but no dissemination of the virus to the salivary glands; this suggests that this novel virus is not arthropod borne as some other members of the family Rhabdoviridae. PMID:24718834

Isolation and identification of citrus psorosis virus Egyptian isolate (CPsV-EG).

Science.gov (United States)

Ghazal, S A; El-Dougdoug, Kh A; Mousa, A A; Fahmy, H; Sofy, A R

2008-01-01

Citrus psorosis ophiovirus (CPsV), is considered to be of the most serious and deter mental virus pathogen's citrus species trees in Egypt. CPsV-EG was isolated from infected citrus grapefruit (C. paradisi Macf.) at Agric. Res. Centre (ARC). The grapefruit which used for CPsV-EG isolate was found to be free from CTV, CEVd and Spiroplasma citri where as gave -ve results with DTBIA, tissue print hybridization and Diene's stain respectively. CPsV-EG was detected on the basis of biological indexing by graft inoculation which gave oak leaf pattern (OLP) on Dweet tangor and serological assay by DAS-ELISA using Mab specific CPsV. CPsV-EG was reacted with variable responses on 16 host plants belonging to 6 families. Only 8 host plants are susceptible and showed visible external symptoms which appeared as local, systemic and local followed by systemic infections. CPsV-EG isolate was transmitted from infected citrus to citrus by syringe and grafting and herbaceous plants by forefinger inoculation and syringe. The woody indicators and rootstocks were differed in response to CPsV-EG isolate which appeared as no-response, response, sensitivity and hypersensitivity. The serological characters represented as the antigenic determinants of CPsV-EG isolate related to monoclonal antibodies specific CPsV strain where as appeared precipitation reaction by DAS-ELISA and DTBIA. The partial fragment of RNA3 (coat protein gene) of CPsV-EG (-1140bp and -571bp) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from grapefruit tissues using two sets primers specific CPsV (CPV3 and CPV4) and (PS66 and PS65) respectively. The virus under study was identified as CPsV-EG isolate according to biological, serological and molecular characters.

Full-Genome Characterization and Genetic Evolution of West African Isolates of Bagaza Virus

Directory of Open Access Journals (Sweden)

Martin Faye

2018-04-01

Full Text Available Bagaza virus is a mosquito-borne flavivirus, first isolated in 1966 in Central African Republic. It has currently been identified in mosquito pools collected in the field in West and Central Africa. Emergence in wild birds in Europe and serological evidence in encephalitis patients in India raise questions on its genetic evolution and the diversity of isolates circulating in Africa. To better understand genetic diversity and evolution of Bagaza virus, we describe the full-genome characterization of 11 West African isolates, sampled from 1988 to 2014. Parameters such as genetic distances, N-glycosylation patterns, recombination events, selective pressures, and its codon adaptation to human genes are assessed. Our study is noteworthy for the observation of N-glycosylation and recombination in Bagaza virus and provides insight into its Indian origin from the 13th century. Interestingly, evidence of Bagaza virus codon adaptation to human house-keeping genes is also observed to be higher than those of other flaviviruses well known in human infections. Genetic variations on genome of West African Bagaza virus could play an important role in generating diversity and may promote Bagaza virus adaptation to other vertebrates and become an important threat in human health.

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

Directory of Open Access Journals (Sweden)

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.

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.

A simple and rapid characterization of influenza virus isolates by monoclonal antibodies in radioimmunoassay

International Nuclear Information System (INIS)

Kostolansky, F.; Styk, B.; Russ, G.

1986-01-01

Radioimmunoassay is described with infectious allantoic fluid directly bound to solid phase, suitable for the detection and further characterization of influenza virus isolates. This simple and rapid method was applied for the description of isolates obtained from different regions of Czechoslovakia during the influenza epidemic in 1983. The results confirmed that all 13 examined isolates represented influenza A viruses possessing H3 subtype haemagglutinin very similar to haemagglutinin of influenza viruses A/Bangkok/1/79 (H3N2), A/Belgium/2/81 (H3N2) and A/Philippines/2/82 (H3N2). (author)

Altered plasma concentrations of sex hormones in cats infected by feline immunodeficiency virus or feline leukemia virus.

Science.gov (United States)

Tejerizo, G; Doménech, A; Illera, J-C; Silván, G; Gómez-Lucía, E

2012-02-01

Gender differences may affect human immunodeficiency virus (HIV) infection in humans and may be related to fluctuations in sex hormone concentration. The different percentage of male and female cats observed to be infected by feline leukemia virus (FeLV) or feline immunodeficiency virus (FIV) has been traditionally explained through the transmission mechanisms of both viruses. However, sexual hormones may also play a role in this different distribution. To study this possibility, 17β-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA) concentrations were analyzed using a competitive enzyme immunoassay in the plasma of 258 cats naturally infected by FIV (FIV(+)), FeLV (FeLV(+)), or FeLV and FIV (F(-)F(+)) or negative for both viruses, including both sick and clinically healthy animals. Results indicated that the concentrations of 17β-estradiol and testosterone were significantly higher in animals infected with FIV or FeLV (P < 0.05) than in negative cats. Plasma concentrations of DHEA in cats infected by either retrovirus were lower than in negative animals (P < 0.05), and F(-)F(+) cats had significantly lower plasma values than monoinfected cats (P < 0.05). No significant differences were detected in the plasma concentration of progesterone of the four groups. No relevant differences were detected in the hormone concentrations between animal genders, except that FIV(+) females had higher DHEA concentrations than the corresponding males (P < 0.05). In addition, no differences were observed in the hormone concentrations between retrovirus-infected and noninfected animals with and without clinical signs. These results suggest that FIV and FeLV infections are associated with an important deregulation of steroids, possibly from early in the infection process, which might have decisive consequences for disease progression. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Plasma temperature during methylene blue/light treatment influences virus inactivation capacity and product quality.

Science.gov (United States)

Gravemann, U; Handke, W; Sumian, C; Alvarez, I; Reichenberg, S; Müller, T H; Seltsam, A

2018-02-27

Photodynamic treatment using methylene blue (MB) and visible light is in routine use for pathogen inactivation of human plasma in different countries. Ambient and product temperature conditions for human plasma during production may vary between production sites. The influence of different temperature conditions on virus inactivation capacity and plasma quality of the THERAFLEX MB-Plasma procedure was investigated in this study. Plasma units equilibrated to 5 ± 2°C, room temperature (22 ± 2°C) or 30 ± 2°C were treated with MB/light and comparatively assessed for the inactivation capacity for three different viruses, concentrations of MB and its photoproducts, activity of various plasma coagulation factors and clotting time. Reduced solubility of the MB pill was observed at 5 ± 2°C. Photocatalytic degradation of MB increased with increasing temperature, and the greatest formation of photoproducts (mainly azure B) occurred at 30 ± 2°C. Inactivation of suid herpesvirus, bovine viral diarrhoea virus and vesicular stomatitis virus was significantly lower at 5 ± 2°C than at higher temperatures. MB/light treatment affected clotting times and the activity of almost all investigated plasma proteins. Factor VIII (-17·7 ± 8·3%, 22 ± 2°C) and fibrinogen (-14·4 ± 16·4%, 22 ± 2°C) showed the highest decreases in activity. Increasing plasma temperatures resulted in greater changes in clotting time and higher losses of plasma coagulation factor activity. Temperature conditions for THERAFLEX MB-Plasma treatment must be carefully controlled to assure uniform quality of pathogen-reduced plasma in routine production. Inactivation of cooled plasma is not recommended. © 2018 International Society of Blood Transfusion.

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

Science.gov (United States)

Sailaja, B; Anjum, Najreen; Patil, Yogesh K; Agarwal, Surekha; Malathi, P; Krishnaveni, D; Balachandran, S M; Viraktamath, B C; Mangrauthia, Satendra K

2013-12-01

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

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...... infecting such hosts. In addition to the isolation procedures, methods for life cycle characterization (one-step growth experiments) of bacteriophages and cyanophages are described. Finally, limitations and drawbacks of the proposed methods are assessed and discussed...

Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva

Energy Technology Data Exchange (ETDEWEB)

Mirza, S.F. [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Staniewski, M.A. [Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (Canada); Short, C.M. [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Long, A.M. [Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (Canada); Chaban, Y.V. [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Short, S.M., E-mail: steven.short@utoronto.ca [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (Canada)

2015-12-15

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145 nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485 kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva's abundance in Lake Ontario was relatively stable, yet CpV-BQ1's abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history. - Highlights: • A virus infecting the algae C. parva was isolated from Lake Ontario. • Virus characteristics demonstrated that this novel virus is an NCLDV. • The virus's polB sequence suggests taxonomic affiliation with the Phycodnaviridae. • The virus's capsid protein sequences also suggest Mimiviridae ancestry. • Surveys of host and virus natural abundances revealed complex host–virus dynamics.

Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva

International Nuclear Information System (INIS)

Mirza, S.F.; Staniewski, M.A.; Short, C.M.; Long, A.M.; Chaban, Y.V.; Short, S.M.

2015-01-01

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145 nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485 kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva's abundance in Lake Ontario was relatively stable, yet CpV-BQ1's abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history. - Highlights: • A virus infecting the algae C. parva was isolated from Lake Ontario. • Virus characteristics demonstrated that this novel virus is an NCLDV. • The virus's polB sequence suggests taxonomic affiliation with the Phycodnaviridae. • The virus's capsid protein sequences also suggest Mimiviridae ancestry. • Surveys of host and virus natural abundances revealed complex host–virus dynamics.

First isolation of dengue virus from the 2010 epidemic in Nepal.

Science.gov (United States)

Pandey, Basu D; Nabeshima, Takeshi; Pandey, Kishor; Rajendra, Saroj P; Shah, Yogendra; Adhikari, Bal R; Gupta, Govinda; Gautam, Ishan; Tun, Mya M N; Uchida, Reo; Shrestha, Mahendra; Kurane, Ichiro; Morita, Kouichi

2013-09-01

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified Aedes aegypti in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.

Isolation of a virus with rhabdovirus morphology from a white-beaked dolphin (Lagenorhynchus albirostris).

NARCIS (Netherlands)

A.D.M.E. Osterhaus (Albert); H.W.J. Broeders; K.S. Teppema; T. Kuiken (Thijs); J.A. House; H.W. Vos (Helma); I.K.G. Visser (Ilona)

1993-01-01

textabstractA virus with rhabdovirus morphology which proved to be antigenically distinct from rabies virus and vesicular stomatitis virus was isolated from a dolphin that had beached on the Dutch coast. Neutralizing antibodies to this virus were found in several European marine mammal species.

High similarity between isolates of pepino mosaic virus suggests a common origin

NARCIS (Netherlands)

Verhoeven, J.Th.J.; Vlugt, van der R.A.A.; Roenhorst, J.W.

2003-01-01

The almost simultaneous outbreaks of Pepino mosaic virus in tomato crops in different European and non-European countries, was reason to have a closer look at the relationship between these isolates and the original isolate from pepino. Fifteen isolates from tomato from different locations and the

Isolation of Chandipura virus (Vesiculovirus: Rhabdoviridae) from Sergentomyia species of sandflies from Nagpur, Maharashtra, India.

Science.gov (United States)

Sudeep, A B; Bondre, V P; Gurav, Y K; Gokhale, M D; Sapkal, G N; Mavale, M S; George, R P; Mishra, A C

2014-05-01

An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra s0 tate, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE). The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission.

Characterisation of influenza A viruses isolated from turkeys in England during March-May 1979.

Science.gov (United States)

Alexander, D J; Spackman, D

1981-07-01

During the early spring of 1979 turkeys on at least twelve sites in England became infected with influenza A viruses. On five of these sites no virus was isolated but birds were shown to have antibodies to Havl (four sites) and Hav2 antigenic subtypes of influenza A viruses. The eight viruses isolated were typed: A/turkey/England/192-328/79 (Havl Nav2/3), A/turkey/England/192-329/79 (Hav1 N2), A/turkey/England/199/79 (Hav1 Neq1), A/turkey/ England/214/79 (Hav1 Neq1), A/turkey/England/250/79 (Hsw1 N1), A/turkey/England/262/79 (Hav1 Nav2/3), A/turkey/England/272/79 (Havl Neq1), A/turkey/England/384/79 (Hav2 Nav4). Pathogenicity index tests in 6-week-old chickens agreed with the clinical signs seen in turkeys in the field. Three of the isolates: 199, 214 and 272 were of extremely high virulence, 384 showed intermediate virulence, while the other isolates were of low virulence.

Assessing Human Immunodeficiency Virus Type 1 Tropism: Comparison of Assays Using Replication-Competent Virus versus Plasma-Derived Pseudotyped Virions ▿

Science.gov (United States)

Hosoya, Noriaki; Su, Zhaohui; Wilkin, Timothy; Gulick, Roy M.; Flexner, Charles; Hughes, Michael D.; Skolnik, Paul R.; Giguel, Françoise; Greaves, Wayne L.; Coakley, Eoin; Kuritzkes, Daniel R.

2009-01-01

Detection of CXCR4-using human immunodeficiency virus by the Trofile assay was compared to that by assays using virus isolates or replication-competent recombinants. Concordance with the Trofile assay was good, but assays using replicating viruses did not increase substantially the ability to detect the presence of CXCR4-using virus. PMID:19494074

Genetic Characterization of H1N2 Influenza A Viruses Isolated from Pigs throughout the United States

OpenAIRE

Karasin, Alexander I.; Landgraf, John; Swenson, Sabrina; Erickson, Gene; Goyal, Sagar; Woodruff, Mary; Scherba, Gail; Anderson, Gary; Olsen, Christopher W.

2002-01-01

An H1N2 influenza A virus was isolated from a pig in the United States for the first time in 1999 (A. I. Karasin, G. A. Anderson, and C. W. Olsen, J. Clin. Microbiol. 38:2453-2456, 2000). H1N2 viruses have been isolated subsequently from pigs in many states. Phylogenetic analyses of eight such viruses isolated from pigs in Indiana, Illinois, Minnesota, Ohio, Iowa, and North Carolina during 2000 to 2001 showed that these viruses are all of the same reassortant genotype as that of the initial H...

Characterization of Sri Lanka rabies virus isolates using nucleotide sequence analysis of nucleoprotein gene.

Science.gov (United States)

Arai, Y T; Takahashi, H; Kameoka, Y; Shiino, T; Wimalaratne, O; Lodmell, D L

2001-01-01

Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used

DEFF Research Database (Denmark)

Fregeneda-Grandes, J.M.; Olesen, Niels Jørgen

2007-01-01

with VHS but with no clinical signs of infection. When the sera were examined by 50%PNT using the VHSV reference isolate DK-F1 or the heat attenuated DK-F25 mutant strain, no neutralizing antibodies were found. In contrast, when one of the virus isolates from the farm (homologous virus) was used in the 50...

Fitness and virulence of an ancestral White Spot Syndrome Virus isolate from shrimp

NARCIS (Netherlands)

Marks, H.; Duijse, J.J.A.; Zuidema, D.; Hulten, van M.C.W.; Vlak, J.M.

2005-01-01

White Spot Syndrome Virus, the type species of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustaceans. Genomic analysis of three completely sequenced WSSV isolates identified two major polymorphic loci, ¿variable region ORF14/15¿ and ¿variable region ORF23/24¿.

Correlation of virus load in plasma and lymph node tissue in human immunodeficiency virus infection. INCAS Study Group. Italy, Netherlands, Canada, Australia, and (United) States.

Science.gov (United States)

Harris, M; Patenaude, P; Cooperberg, P; Filipenko, D; Thorne, A; Raboud, J; Rae, S; Dailey, P; Chernoff, D; Todd, J; Conway, B; Montaner, J S

1997-11-01

The impact of long-term changes in plasma viremia, produced by effective combination antiretroviral therapy, on human immunodeficiency virus (HIV) burden within tissue reservoirs is unknown. Fifteen patients who had received at least 1 year of therapy with two or three drug combinations of zidovudine, didanosine, and nevirapine had suitable samples of lymph node tissue obtained by ultrasound-guided core needle biopsy. HIV RNA was extracted from homogenized tissue samples and quantitated using a modified branched DNA assay. Results were correlated with antiretroviral treatment effect on the basis of plasma virus load measurements over the preceding 12-18 months. A statistically significant negative correlation was observed between magnitude of treatment effect on plasma viremia and lymph node virus load. These data suggest that combinations of antiretroviral drugs that produce sustained suppression of plasma HIV RNA may also be able to reduce the virus burden in lymphoid tissues.

Variability of geographically distinct isolates of maize rayado fino virus in Latin America.

Science.gov (United States)

Hammond, R W; Kogel, R; Ramirez, P

1997-12-01

We have examined the molecular epidemiology of the leafhopper-borne maize rayado fino virus (MRFV) in Latin America. The coat protein gene and 3' non-translated region of 14 isolates of MRFV collected from Latin America and the United States were sequenced and phylogenetic relationships examined. The nucleotide sequence revealed remarkable conservation, with a sequence similarity of 88-99%. Phylogenetic analysis of sequence data obtained from a 633 bp fragment showed that MRFV has diverged into three main clusters, i.e. the geographically distinct northern and southern isolates and the Colombian isolates. Significant differences between the isolates collected from Colombia, previously named maize rayado colombiana virus, based upon differences in symptomatology and serological relationships to MRFV, and the other MRFV isolates, provides additional evidence supporting its designation as a unique strain of MRFV.

Isolation of Chandipura virus (Vesiculovirus: Rhabdoviridae from Sergentomyia species of sandflies from Nagpur, Maharashtra, India

Directory of Open Access Journals (Sweden)

A B Sudeep

2014-01-01

Full Text Available Background & objectives: An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra s0 tate, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. Methods: Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE. The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. Results: CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. Interpretation & conclusions: This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission.

Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c

International Nuclear Information System (INIS)

Ellis, R.W.; Hopkins, N.; Fleissner, E.

1980-01-01

Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three to four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral RNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded G/sub IX/ cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype

RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

Science.gov (United States)

Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

2014-01-01

The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

Swine influenza viruses isolated in 1983, 2002 and 2009 in Sweden exemplify different lineages

Directory of Open Access Journals (Sweden)

Metreveli Giorgi

2010-12-01

Full Text Available Abstract Swine influenza virus isolates originating from outbreaks in Sweden from 1983, 2002 and 2009 were subjected to nucleotide sequencing and phylogenetic analysis. The aim of the studies was to obtain an overview on their potential relatedness as well as to provide data for broader scale studies on swine influenza epidemiology. Nonetheless, analyzing archive isolates is justified by the efforts directed to the comprehension of the appearance of pandemic H1N1 influenza virus. Interestingly, this study illustrates the evolution of swine influenza viruses in Europe, because the earliest isolate belonged to 'classical' swine H1N1, the subsequent ones to Eurasian 'avian-like' swine H1N1 and reassortant 'avian-like' swine H1N2 lineages, respectively. The latter two showed close genetic relatedness regarding their PB2, HA, NP, and NS genes, suggesting common ancestry. The study substantiates the importance of molecular surveillance for swine influenza viruses.

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.

Newcastle Disease Viruses Causing Recent Outbreaks Worldwide Show Unexpectedly High Genetic Similarity to Historical Virulent Isolates from the 1940s

Science.gov (United States)

Dimitrov, Kiril M.; Lee, Dong-Hun; Williams-Coplin, Dawn; Olivier, Timothy L.; Miller, Patti J.

2016-01-01

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 through XVIII. Here, partial and complete genomic sequences of recent virulent isolates of genotypes II and IX from China, Egypt, and India were found to be nearly identical to those of historical viruses isolated in the 1940s. Phylogenetic analysis, nucleotide distances, and rates of change demonstrate that these recent isolates have not evolved significantly from the most closely related ancestors from the 1940s. The low rates of change for these virulent viruses (7.05 × 10−5 and 2.05 × 10−5 per year, respectively) and the minimal genetic distances existing between these and historical viruses (0.3 to 1.2%) of the same genotypes indicate an unnatural origin. As with any other RNA virus, Newcastle disease virus is expected to evolve naturally; thus, these findings suggest that some recent field isolates should be excluded from evolutionary studies. Furthermore, phylogenetic analyses show that these recent virulent isolates are more closely related to virulent strains isolated during the 1940s, which have been and continue to be used in laboratory and experimental challenge studies. Since the preservation of viable viruses in the environment for over 6 decades is highly unlikely, it is possible that the source of some of the recent virulent viruses isolated from poultry and wild birds might be laboratory viruses. PMID:26888902

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...... sequences from the Arctic and Arctic-like viruses, which were distinct from rabies isolates originating ill the Baltic region of Europe, the Steppes in Russia and from North America. The Arctic-like group consist of isolates from India, Pakistan, southeast Siberia and Japan. The Arctic group...... in northeast Siberia and Alaska. Arctic 2b isolates represent a biotype, which is dispersed throughout the Arctic region. The broad distribution of rabies in the Arctic regions including Greenland, Canada and Alaska provides evidence for the movement of rabies across borders....

Genetic Characterization of Influenza A (H1N1) Pandemic 2009 Virus Isolates from Mumbai.

Science.gov (United States)

Gohil, Devanshi; Kothari, Sweta; Shinde, Pramod; Meharunkar, Rhuta; Warke, Rajas; Chowdhary, Abhay; Deshmukh, Ranjana

2017-08-01

Pandemic influenza A (H1N1) 2009 virus was first detected in India in May 2009 which subsequently became endemic in many parts of the country. Influenza A viruses have the ability to evade the immune response through its ability of antigenic variations. The study aims to characterize influenza A (H1N1) pdm 09 viruses circulating in Mumbai during the pandemic and post-pandemic period. Nasopharyngeal swabs positive for influenza A (H1N1) pdm 09 viruses were inoculated on Madin-Darby canine kidney cell line for virus isolation. Molecular and phylogenetic analysis of influenza A (H1N1) pdm 09 isolates was conducted to understand the evolution and genetic diversity of the strains. Nucleotide and amino acid sequences of the HA gene of Mumbai isolates when compared to A/California/07/2009-vaccine strain revealed 14 specific amino acid differences located at the antigenic sites. Amino acid variations in HA and NA gene resulted in changes in the N-linked glycosylation motif which may lead to immune evasion. Phylogenetic analysis of the isolates revealed their evolutionary position with vaccine strain A/California/07/2009 but had undergone changes gradually. The findings in the present study confirm genetic variability of influenza viruses and highlight the importance of continuous surveillance during influenza outbreaks.

Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

Directory of Open Access Journals (Sweden)

Camila Zanluca

Full Text Available Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV detection through the production and characterization of 22 monoclonal antibodies (mAbs against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3 and dengue serotype-specific (DENV-2 or -3. Additionally, some mAbs cross-reacted with yellow fever virus (YFV, West Nile virus (WNV and Saint Louis encephalitis virus (SLEV. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV. Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.

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.

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.

Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 in peafowl (Pavo cristatus).

Science.gov (United States)

Ismail, Mahmoud Moussa; Khan, Owais Ahmed; Cattoli, Giovanni; Lu, Huaguang

2010-03-01

An outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first diagnosed in a "backyard" flock of peafowl (Pavo cristatus) raised on palace premises in the Kingdom of Saudi Arabia in December 3, 2007. The flock consisted of 40 peafowl, and their ages ranged from 3 to 5 years old. Affected birds suffered from depression, anorexia, and white diarrhea. Four dead birds were submitted for HPAI diagnosis at the Central Veterinary Diagnostic Laboratory in Riyadh. Brain and liver tissues and tracheal and cloacal swabs were taken from the dead birds and processed for a real-time reverse transcriptase (RT)-PCR test and virus isolation in specific-pathogen-free embryonating chicken eggs. The H5N1 subtype of avian influenza virus was isolated from the four dead birds and identified by a real-time RT-PCR before and after egg inoculation. The virus isolates were characterized as HPAI H5N1 virus by sequencing analysis. Phylogenetic comparisons revealed that the H5N1 viruses isolated from peafowl belong to the genetic clade 2.2 according to the World Health Organization nomenclature. The peafowl H5N1 virus falls into 2.2.2 sublineage II and clusters with the H5N1 viruses isolated from poultry in Saudi Arabia in 2007-08.

Whole genomes of Chandipura virus isolates and comparative analysis with other rhabdoviruses.

Science.gov (United States)

Cherian, Sarah S; Gunjikar, Rashmi S; Banerjee, Arpita; Kumar, Satyendra; Arankalle, Vidya A

2012-01-01

The Chandipura virus (CHPV) belonging to the Vesiculovirus genus and Rhabdoviridae family, has recently been associated with a number of encephalitis epidemics, with high mortality in children, in different parts of India. No full length genome sequences of CHPV isolates were available in GenBank and little is known about the molecular markers for pathogenesis. In the present study, we provide the complete genomic sequences of four isolates from epidemics during 2003-2007. These sequences along with the deduced sequence of the prototype isolate of 1965 were analysed using phylogeny, motif search, homology modeling and epitope prediction methods. Comparison with other rhaboviruses was also done for functional extrapolations. All CHPV isolates clustered with the Isfahan virus and maintained several functional motifs of other rhabdoviruses. A notable difference with the prototype vesiculovirus, Vesicular Stomatitis Virus was in the L-domain flanking sequences of the M protein that are known to be crucial for interaction with host proteins. With respect to the prototype isolate, significant additional mutations were acquired in the 2003-2007 isolates. Several mutations in G mapped onto probable antigenic sites. A mutation in N mapped onto regions crucial for N-N interaction and a putative T-cell epitope. A mutation in the Casein kinase II phosphorylation site in P may attribute to increased rates of phosphorylation. Gene junction comparison revealed changes in the M-G junction of all the epidemic isolates that may have implications on read-through and gene transcription levels. The study can form the basis for further experimental verification and provide additional insights into the virulence determinants of the CHPV.

Whole genomes of Chandipura virus isolates and comparative analysis with other rhabdoviruses.

Directory of Open Access Journals (Sweden)

Sarah S Cherian

Full Text Available The Chandipura virus (CHPV belonging to the Vesiculovirus genus and Rhabdoviridae family, has recently been associated with a number of encephalitis epidemics, with high mortality in children, in different parts of India. No full length genome sequences of CHPV isolates were available in GenBank and little is known about the molecular markers for pathogenesis. In the present study, we provide the complete genomic sequences of four isolates from epidemics during 2003-2007. These sequences along with the deduced sequence of the prototype isolate of 1965 were analysed using phylogeny, motif search, homology modeling and epitope prediction methods. Comparison with other rhaboviruses was also done for functional extrapolations. All CHPV isolates clustered with the Isfahan virus and maintained several functional motifs of other rhabdoviruses. A notable difference with the prototype vesiculovirus, Vesicular Stomatitis Virus was in the L-domain flanking sequences of the M protein that are known to be crucial for interaction with host proteins. With respect to the prototype isolate, significant additional mutations were acquired in the 2003-2007 isolates. Several mutations in G mapped onto probable antigenic sites. A mutation in N mapped onto regions crucial for N-N interaction and a putative T-cell epitope. A mutation in the Casein kinase II phosphorylation site in P may attribute to increased rates of phosphorylation. Gene junction comparison revealed changes in the M-G junction of all the epidemic isolates that may have implications on read-through and gene transcription levels. The study can form the basis for further experimental verification and provide additional insights into the virulence determinants of the CHPV.

Whole Genomes of Chandipura Virus Isolates and Comparative Analysis with Other Rhabdoviruses

Science.gov (United States)

Cherian, Sarah S.; Kumar, Satyendra; Arankalle, Vidya A.

2012-01-01

The Chandipura virus (CHPV) belonging to the Vesiculovirus genus and Rhabdoviridae family, has recently been associated with a number of encephalitis epidemics, with high mortality in children, in different parts of India. No full length genome sequences of CHPV isolates were available in GenBank and little is known about the molecular markers for pathogenesis. In the present study, we provide the complete genomic sequences of four isolates from epidemics during 2003–2007. These sequences along with the deduced sequence of the prototype isolate of 1965 were analysed using phylogeny, motif search, homology modeling and epitope prediction methods. Comparison with other rhaboviruses was also done for functional extrapolations. All CHPV isolates clustered with the Isfahan virus and maintained several functional motifs of other rhabdoviruses. A notable difference with the prototype vesiculovirus, Vesicular Stomatitis Virus was in the L-domain flanking sequences of the M protein that are known to be crucial for interaction with host proteins. With respect to the prototype isolate, significant additional mutations were acquired in the 2003–2007 isolates. Several mutations in G mapped onto probable antigenic sites. A mutation in N mapped onto regions crucial for N-N interaction and a putative T-cell epitope. A mutation in the Casein kinase II phosphorylation site in P may attribute to increased rates of phosphorylation. Gene junction comparison revealed changes in the M-G junction of all the epidemic isolates that may have implications on read-through and gene transcription levels. The study can form the basis for further experimental verification and provide additional insights into the virulence determinants of the CHPV. PMID:22272333

Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses.

Science.gov (United States)

Chiapponi, Chiara; Zanni, Irene; Garbarino, Chiara; Barigazzi, Giuseppe; Foni, Emanuela

2010-01-01

Influenza A virus isolation is undertaken routinely in embryonated chicken eggs, but to improve virus detection various cell lines can be used. The CACO-2 cell line was compared to the MDCK cell line and embryonated chicken eggs for the isolation of H1N1, H1N2, H3N2 swine influenza A virus subtypes from clinical specimens. From 2006 to 2008, 104 influenza A samples found positive by PCR from 42 respiratory outbreaks in Italian swine farms were examined by virus isolation. Sixty swine influenza A viruses were isolated (16 H1N1, 28 H1N2 and 16 H3N2) and their growth behaviour on the different substrates was examined. 16/16 H1N1, 28/28 H1N2 and 8/16 of H3N2 viruses were isolated from the CACO-2 cell line, while 7/16 H1N1, 3/28 H1N2 and 16/16 H3N2 viruses were isolated using embryonated chicken eggs. Only 9/16 H1N1, 1/28 H1N2 and 6/16 H3N2 viruses replicated in MDCK cells. A link was found between viral hemagglutinin and the isolation rate on the various substrates. The CACO-2 line was statistically more sensitive (Fisher's exact test, pH1N2 subtypes. In contrast influenza A H3N2 virus was isolated more readily in embryonated chicken eggs than in cultured cells (Fisher's exact test, p<0.01).

Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

Science.gov (United States)

Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

2003-08-01

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

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.

Phylogenetic and evolutionary analyses of dengue viruses isolated in Jakarta, Indonesia.

Science.gov (United States)

Lestari, C S Whinie; Yohan, Benediktus; Yunita, Anisa; Meutiawati, Febrina; Hayati, Rahma Fitri; Trimarsanto, Hidayat; Sasmono, R Tedjo

2017-12-01

Dengue has affected Indonesia for the last five decades and become a major health problem in many cities in the country. Jakarta, the capital of Indonesia, reports dengue cases annually, with several outbreaks documented. To gain information on the dynamic and evolutionary history of dengue virus (DENV) in Jakarta, we conducted phylogenetic and evolutionary analyses of DENV isolated in 2009. Three hundred thirty-three dengue-suspected patients were recruited. Our data revealed that dengue predominantly affected young adults, and the majority of cases were due to secondary infection. A total of 171 virus isolates were successfully serotyped. All four DENV serotypes were circulating in the city, and DENV-1 was the predominant serotype. The DENV genotyping of 17 isolates revealed the presence of Genotypes I and IV in DENV-1, while DENV-2 isolates were grouped into the Cosmopolitan genotype. The grouping of isolates into Genotype I and II was seen for DENV-3 and DENV-4, respectively. Evolutionary analysis revealed the relatedness of Jakarta isolates with other isolates from other cities in Indonesia and isolates from imported cases in other countries. We revealed the endemicity of DENV and the role of Jakarta as the potential source of imported dengue cases in other countries. Our study provides genetic information regarding DENV from Jakarta, which will be useful for upstream applications, such as the study of DENV epidemiology and evolution and transmission dynamics.

New highly divergent Plum pox virus isolates infecting sour cherry in Russia.

Science.gov (United States)

Chirkov, Sergei; Ivanov, Peter; Sheveleva, Anna; Zakubanskiy, Alexander; Osipov, Gennady

2017-02-01

Unusual Plum pox virus (PPV) isolates (named Tat isolates) were discovered on sour cherry (Prunus cerasus) in Russia. They failed to be recognized by RT-PCR using commonly employed primers specific to the strains C or CR (the only ones that proved able to infect sour cherry) as well as to the strains M and W. Some of them can be detected by RT-PCR using the PPV-D-specific primers P1/PD or by TAS-ELISA with the PPV-C-specific monoclonal antibody AC. Phylogenetic analysis of the 3'-terminal genomic region assigned the Tat isolates into the cluster of cherry-adapted strains. However, they grouped separately from the C and CR strains and from each other as well. The sequence divergence of the Tat isolates is comparable to the differences between the known PPV strains. They may represent new group(s) of cherry-adapted isolates which do not seem to belong to any known strain of the virus. Copyright © 2016. Published by Elsevier Inc.

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009.

Science.gov (United States)

Takakuwa, Hiroki; Yamashiro, Tetsu; Le, Mai Q; Phuong, Lien S; Ozaki, Hiroichi; Tsunekuni, Ryota; Usui, Tatsufumi; Ito, Hiroshi; Yamaguchi, Tsuyoshi; Ito, Toshihiro; Murase, Toshiyuki; Ono, Etsuro; Otsuki, Koichi

2013-12-01

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam. Copyright © 2013 Elsevier Ltd. All rights reserved.

Deep sequencing as a method of typing bluetongue virus isolates.

Science.gov (United States)

Rao, Pavuluri Panduranga; Reddy, Yella Narasimha; Ganesh, Kapila; Nair, Shreeja G; Niranjan, Vidya; Hegde, Nagendra R

2013-11-01

Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E. Copyright © 2013 Elsevier B.V. All rights reserved.

Transmission of human immunodeficiency virus Type-1 by fresh-frozen plasma treated with methylene blue and light.

Science.gov (United States)

Álvarez, Manuel; Luis-Hidalgo, Mar; Bracho, María Alma; Blanquer, Amando; Larrea, Luis; Villalba, José; Puig, Nieves; Planelles, Dolores; Montoro, José; González-Candelas, Fernando; Roig, Roberto

2016-04-01

The risk of transfusion-transmitted infection (TTI) has been minimized by introduction of nucleic acid testing (NAT) and pathogen inactivation (PI). This case report describes transmission of human immunodeficiency virus Type 1 (HIV-1) to two recipients despite these measures. In March 2009 a possible TTI of HIV-1 was identified in a patient that had received pooled buffy coat platelet concentrate (BC-PLT) in November 2005. The subsequent lookback study found two more patients who had received methylene blue (MB)-treated fresh-frozen plasma (FFP) and red blood cells (RBCs) from the same donation. In November 2005 the donor had tested negative for both HIV antibodies and HIV-1 RNA by 44 minipool (44 MP) NAT. Repository samples of this donation and samples from the recipients were used for viral load (VL) and sequence analysis. HIV-1 RNA was detectable by individual donation (ID)-NAT in the repository sample from the 2005 window period donation and a VL of 135 copies/mL was measured. HIV-1 infection was confirmed in both recipients of both BC-PLT (65 mL of plasma) and MB-FFP (261 mL of plasma), but not in the patient that had received 4-week-old RBCs (20 mL of plasma). The sequence analysis revealed a close phylogenetic relationship between the virus strains isolated from the donor and recipients, compatible with TTI. Approximately 17,600 and 4400 virions in the MB-FFP and BC-PLT were infectious, but 1350 virions in the RBCs were not. ID-NAT would have prevented this transmission, but the combination of MP-NAT and MB-PI did not. © 2015 AABB.

Genomic characterization of Zika virus isolated from Indonesia.

Science.gov (United States)

Yudhaputri, Frilasita A; Trimarsanto, Hidayat; Perkasa, Aditya; Yohan, Benediktus; Haryanto, Sotianingsih; Wiyatno, Ageng; Soebandrio, Amin; Myint, Khin Saw; Ledermann, Jeremy P; Rosenberg, Ronald; Powers, Ann M; Sasmono, R Tedjo

2017-10-01

Zika virus (ZIKV) JMB-185 strain was isolated from a febrile patient in Jambi, Indonesia in 2014. To understand its genetic characteristics, we performed whole genome sequencing using the Ion Torrent PGM platform on the supernatant of the first passage. The phylogenetic analysis showed that the isolate was not closely related to the Brazilian ZIKV associated with microcephaly or isolates from the recent Singapore Zika outbreak. Molecular evolution analysis indicated that JMB-185 strain may have been circulating in the Southeast Asia region, including Indonesia since 2000. We observed high nucleotide sequence identity between Indonesia, Thailand, Singapore, and American strains although unique amino acid substitutions were also observed. This report provides information on the genomic characteristics of Indonesian ZIKV which may be used for further studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phylogenetic analysis of Newcastle disease viruses isolated from commercial poultry in Mozambique, 2011 to 2016

International Nuclear Information System (INIS)

Mapaco, L.P.; Monjane, I.V.A.; Nhamusso, A.E.; Viljoen, G.J; Dundon, W.G.; Achá, S.J.

2016-01-01

Full text: The complete sequence of the fusion (F) protein gene from eleven Newcastle disease viruses (NDV) isolated from commercial poultry in Mozambique between 2011 and 2016 has been generated. The F gene cleavage site motif for all eleven isolates was 112RRRKRF117 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 to 1995 and 2005. The characterization of these new NDVs has important implications for Newcastle disease management and control in Mozambique. (author)

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. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by

Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil

Directory of Open Access Journals (Sweden)

Betania Paiva Drumond

2016-03-01

Full Text Available Abstract 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.

Iguana Virus, a Herpes-Like Virus Isolated from Cultured Cells of a Lizard, Iguana iguana

Science.gov (United States)

Clark, H. Fred; Karzon, David T.

1972-01-01

An agent cytopathic for Terrapene and Iguana cell cultures was isolated from spontaneously degenerating cell cultures prepared from a green iguana (Iguana iguana). The agent, designated iguana virus, caused a cytopathic effect (CPE) of a giant cell type, with eosinophilic inclusions commonly observed within giant cell nuclei. Incubation temperature had a marked effect on CPE and on virus release from infected cells. Within the range of 23 to 36 C, low temperatures favored CPE characterized by cytolysis and small giant cell formation, and significant virus release was observed. At warmer temperatures, a purely syncytial type of CPE and total absence of released virus were noted. A unique type of hexagonal eosinophilic cytoplasmic inclusion was observed within syncytia of infected Terrapene cell cultures incubated at 36 C. In vivo studies revealed no evidence of pathogenicity of iguana virus for suckling mice, embryonated hen's eggs, or several species of reptiles and amphibians. Inoculation of iguana virus into young iguanas consistently caused infection that was “unmasked” only when cell cultures were prepared directly from the infected animal. Filtration studies revealed a virion size of >100 nm and Iguana virus is ether-sensitive and, as presumptively indicated by studies of inhibition by bromodeoxyuridine, possesses a deoxyribonucleic type of nucleic acid. The virus characteristics described, as well as electron microscopy observations described in a separate report, indicate that iguana virus is a member of the herpesvirus group. Images PMID:4344303

Phylogenetic analysis of dengue virus types 1 and 3 isolated in Jakarta, Indonesia in 1988.

Science.gov (United States)

Sjatha, Fithriyah; Takizawa, Yamato; Yamanaka, Atsushi; Konishi, Eiji

2012-12-01

Dengue viruses are mosquito-borne viruses that cause dengue fever and dengue hemorrhagic fever, both of which are globally important diseases. These viruses have evolved in a transmission cycle between human hosts and mosquito vectors in various tropical and subtropical environments. We previously isolated three strains of dengue type 1 virus (DENV1) and 14 strains of dengue type 3 virus (DENV3) during an outbreak of dengue fever and dengue hemorrhagic fever in Jakarta, Indonesia in 1988. Here, we compared the nucleotide sequences of the entire envelope protein-coding region among these strains. The isolates were 97.6-100% identical for DENV1 and 98.8-100% identical for DENV3. All DENV1 isolates were included in two different clades of genotype IV and all DENV3 isolates were included in a single clade of genotype I. For DENV1, three Yap Island strains isolated in 2004 were the only strains closely related to the present isolates; the recently circulated Indonesian strains were in different clades. Molecular clock analyses estimated that ancestors of the genotype IV strains of DENV1 have been indigenous in Indonesia since 1948. We predict that they diverged frequently around 1967 and that their offspring distributed to Southeast Asia, the Western Pacific, and Africa. For DENV3, the clade containing all the present isolates also contained strains isolated from other Indonesian regions and other countries including Malaysia, Singapore, China, and East Timor from 1985-2010. Molecular clock analyses estimated that the common ancestor of the genotype I strains of DENV3 emerged in Indonesia around 1967 and diverged frequently until 1980, and that their offspring distributed mainly in Southeast Asia. The first dengue outbreak in 1968 and subsequent outbreaks in Indonesia might have influenced the divergence and distribution of the DENV1 genotype IV strains and the DENV3 genotype I strains in many countries. Copyright © 2012 Elsevier B.V. All rights reserved.

Diagnostic efficacy of molecular assays for the viral haemorrhagic septicaemia virus isolates from the Czech Republic

Directory of Open Access Journals (Sweden)

Ľubomír Pojezdal

2017-01-01

Full Text Available The diagnostic properties of the one-step real-time reverse-transcription polymerase chain reaction assay for viral haemorrhagic septicaemia virus detection were compared to methods currently in use in the Czech Republic, namely, virus isolation using the cell culture and conventional reverse-transcription polymerase chain reaction followed by the nested polymerase chain reaction. The assays were tested on a panel of 25 archived viral haemorrhagic septicaemia isolates and 8 archived infectious haematopoietic necrosis isolates obtained from monitoring and/or outbreaks of the diseases among farmed salmonids in the Czech Republic. The ability to detect the presence of the virus in the tissues of fish was tested on additional 32 field samples collected from the rainbow trout (Oncorhynchus mykiss, brown trout (Salmo trutta and brook trout (Salvelinus fontinalis. The real-time assay showed the highest analytic sensitivity by detecting the presence of viral nucleic acid in samples with 10-7 dilution, whereas the sensitivity of the conventional polymerase chain reaction peaked at 10-5. Diagnostic specificity of both molecular assays was confirmed by absence of cross-reactivity with the infectious haematopoietic necrosis virus isolates. This, along with consistent results in the detection of the virus in the fish tissues, confirms that the one-step real-time reverse-transcription polymerase chain reaction is currently an optimal stand-alone diagnostic method for the detection of the viral haemorrhagic septicaemia virus.

Evolutionary genetics of highly pathogenic H5N1 avian influenza viruses isolated from whooper swans in northern Japan in 2008.

Science.gov (United States)

Usui, Tatsufumi; Yamaguchi, Tsuyoshi; Ito, Hiroshi; Ozaki, Hiroichi; Murase, Toshiyuki; Ito, Toshihiro

2009-12-01

In April and May 2008, highly pathogenic avian influenza viruses subtype H5N1 were isolated from dead or moribund whooper swans in Aomori, Akita and Hokkaido prefectures in northern Japan. To trace the genetic lineage of the isolates, the nucleotide sequences of all eight genes were determined and phylogenetically analyzed. The Japanese strains were nearly identical to chicken viruses isolated in Russia in April 2008 and closely related to viruses isolated from dead wild birds in Hong Kong in 2007-2008. Their HA genes clustered in clade 2.3.2. On the other hand, NA and the other internal genes were closely related to those of clade 2.3.4 viruses (genotype V) whose NP genes originated from an HA clade 2.3.2 virus. In conclusion, the H5N1 viruses isolated in Japan, Russia and Hong Kong were derived from a common ancestor virus belonging to genotype V that was generated from genetic reassortment events between viruses of HA clades 2.3.2 and 2.3.4.

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

OpenAIRE

Miagostovich, M.P.; Santos, F.B. dos; Simone, T.S. de; Costa, E.V.; Filippis, A.M.B.; Schatzmayr, H.G.; Nogueira, R.M.R.

2002-01-01

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 gen...

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...... production. Phylogenetic homogeneity of the Shetland isolates of PVS and PVV was apparent. PVX was more heterogeneous, and Shetland isolates cluster with the Scottish isolates in a group which includes Asian and European isolates. For PVA, the majority of the Shetland and Scottish mainland isolates formed...... a predominantly Scottish grouping, with the remaining Shetland and Scottish mainland isolates clustering with a previously characterized Scottish isolate. There were three main groups of PVA, of which the Scottish grouping was the only one which did not have a fully characterized representative. To extend...

Molecular screening and isolation of Newcastle disease virus from ...

African Journals Online (AJOL)

Molecular screening and isolation of Newcastle disease virus from live poultry markets and chickens from commercial poultry farms in Zaria, Kaduna state, Nigeria. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader).

Comparison of some storage and isolation methods to recover bluetongue virus from bovine blood.

OpenAIRE

Thomas, F C

1984-01-01

Bovine blood containing bluetongue virus was stored as whole blood with EDTA (4 degrees C), as washed cellular components (4 degrees C), and as washed cellular components with 10% DMSO (-70 degrees C). Periodic isolation attempts were made over a period of 330 days in four cell lines and embryonating chicken eggs (intravenous inoculation). Bluetongue virus was successfully isolated in all systems from most samples throughout the test period. There appeared to be more variation amongst days of...

Characterization of velogenic Newcastle disease viruses isolated from dead wild birds in Serbia during 2007.

Science.gov (United States)

Vidanović, Dejan; Sekler, Milanko; Asanin, Ruzica; Milić, Nenad; Nisavić, Jakov; Petrović, Tamas; Savić, Vladimir

2011-04-01

Avian paramyxoviruses type 1 or Newcastle disease viruses (NDV) are frequently recovered from wild birds and such isolates are most frequently of low virulence. Velogenic NDV are usually recovered from poultry and only occasionally from wild birds. Five NDV isolates were obtained from carcasses of four wild bird species during 2007 in Serbia: Mallard (Anas platyrhynchos), Eurasian Sparrowhawk (Accipiter nisus), feral Rock Pigeon (Columba livia), and Eurasian Collared Dove (Streptopelia decaocto). All the isolates have a typical fusion protein cleavage site motif of velogenic viruses ((112)R-R-Q-K-R-F(117)). The highest homology (99%) for the nucleotide sequences spanning the M and F gene of the studied isolates was with the genotype VII NDV isolate Muscovy duck/China(Fujian)/FP1/02. Phylogenetic analysis based on a partial F gene sequence showed that the isolates from wild birds cluster together with concurrent isolates from poultry in Serbia within the subgenotype VIId, which is the predominant pathogen involved currently in Newcastle disease outbreaks in poultry worldwide. It is unlikely that the wild birds played an important role in primary introduction or consequent spread of the velogenic NDV to domestic poultry in Serbia, and they probably contracted the virus from locally infected poultry.

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

Science.gov (United States)

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

2011-02-25

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. 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. 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.

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

Directory of Open Access Journals (Sweden)

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.

Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006.

Science.gov (United States)

Abolnik, C; Gerdes, G H; Kitching, J; Swanepoel, S; Romito, M; Bisschop, S P R

2008-06-01

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 into 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 hornbill (Bucorvus leadbeateri) which became acutely 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 ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

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.

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.

Phylogeny of isolates of Prunus necrotic ringspot virus from the Ilarvirus Ringtest and identification of group-specific features.

Science.gov (United States)

Hammond, R W

2003-06-01

Isolates of Prunus necrotic ringspot virus (PNRSV) were examined to establish the level of naturally occurring sequence variation in the coat protein (CP) gene and to identify group-specific genome features that may prove valuable for the generation of diagnostic reagents. Phylogenetic analysis of a 452 bp sequence of 68 virus isolates, 20 obtained from the European Union Ilarvirus Ringtest held in October 1998, confirmed the clustering of the isolates into three distinct groups. Although no correlation was found between the sequence and host or geographic origin, there was a general trend for severe isolates to cluster into one group. Group-specific features have been identified for discrimination between virus strains.

Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico

OpenAIRE

Garc?s-Ayala, Fabiola; Rodr?guez-Castillo, Araceli; Ortiz-Alc?ntara, Joanna Mar?a; Gonzalez-Dur?n, Elizabeth; Segura-Candelas, Jos? Miguel; P?rez-Ag?eros, Sandra Ivette; Escobar-Escamilla, No?; M?ndez-Tenorio, Alfonso; Diaz-Qui?onez, Jos? Alberto; Ramirez-Gonz?lez, Jos? Ernesto

2015-01-01

Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico.

Characteristics of primary infection of a European human immunodeficiency virus type 1 clade B isolate in chimpanzees

NARCIS (Netherlands)

Bogers, W. M.; Koornstra, W. H.; Dubbes, R. H.; ten Haaft, P. J.; Verstrepen, B. E.; Jhagjhoorsingh, S. S.; Haaksma, A. G.; Niphuis, H.; Laman, J. D.; Norley, S.; Schuitemaker, H.; Goudsmit, J.; Hunsmann, G.; Heeney, J. L.; Wigzell, H.

1998-01-01

The aim of the study was to select, from a panel of candidate European human immunodeficiency virus type 1 (HIV-1) clade B primary virus isolates, one isolate based on replication properties in chimpanzee peripheral blood mononuclear cells (PBMC). Secondly, to evaluate the in vivo kinetics of

Measles Virus Neutralizing Antibodies in Intravenous Immunoglobulins: Is an Increase by Revaccination of Plasma Donors Possible?

Science.gov (United States)

Modrof, Jens; Tille, Björn; Farcet, Maria R; McVey, John; Schreiner, Jessica A; Borders, Charles M; Gudino, Maria; Fitzgerald, Peter; Simon, Toby L; Kreil, Thomas R

2017-11-15

We report a screen of plasma donors confirming that widespread use of childhood measles vaccination since 1963 resulted in a decrease in average measles virus antibody titers among plasma donors, which is reflected in intravenous immunoglobulins (IVIGs). The measles virus antibody titer, however, is a potency requirement for IVIGs, as defined in a Food and Drug Administration regulation. To mitigate the decline in measles virus antibody titers in IVIGs and to ensure consistent product release, revaccination of plasma donors was investigated as a means to boost titers. However, revaccination-induced titer increases were only about 2-fold and short-lived. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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.

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant

Directory of Open Access Journals (Sweden)

Renström Lena HM

2009-10-01

Full Text Available Abstract The European swine influenza viruses (SIVs show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA of the human-like H1N2 SIV viruses and the neuraminidase (NA of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early '80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain. The description of this H1N2 SIV adds further information to influenza epidemiology and supports the necessity of surveillance for influenza viruses in pigs.

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant.

Science.gov (United States)

Bálint, Adám; Metreveli, Giorgi; Widén, Frederik; Zohari, Siamak; Berg, Mikael; Isaksson, Mats; Renström, Lena Hm; Wallgren, Per; Belák, Sándor; Segall, Thomas; Kiss, István

2009-10-28

The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA) of the human-like H1N2 SIV viruses and the neuraminidase (NA) of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early '80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain.The description of this H1N2 SIV adds further information to influenza epidemiology and supports the necessity of surveillance for influenza viruses in pigs.

Rapid and specific detection of Asian- and African-lineage Zika viruses.

Science.gov (United States)

Chotiwan, Nunya; Brewster, Connie D; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M; Fauver, Joseph R; Andre, Barb; Gray, Meg; Black, William C; Kading, Rebekah C; Ebel, Gregory D; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T A; Brault, Aaron C; Harris, Eva; Foy, Brian D; Quackenbush, Sandra L; Perera, Rushika; Rovnak, Joel

2017-05-03

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. Copyright © 2017, American Association for the Advancement of Science.

Rapid and specific detection of Asian- and African-lineage Zika viruses

Science.gov (United States)

Chotiwan, Nunya; Brewster, Connie D.; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K.; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M.; Fauver, Joseph R.; Andre, Barb; Gray, Meg; Black, William C.; Kading, Rebekah C.; Ebel, Gregory D.; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T. A.; Brault, Aaron C.; Harris, Eva; Foy, Brian D.; Quackenbush, Sandra L.; Perera, Rushika; Rovnak, Joel

2017-01-01

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. PMID:28469032

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

Isolation and Genetic Characterization of Avian Influenza Viruses Isolated from Wild Birds in the Azov-Black Sea Region of Ukraine (2001-2012).

Science.gov (United States)

Muzyka, Denys; Pantin-Jackwood, Mary; Spackman, Erica; Smith, Diane; Rula, Oleksandr; Muzyka, Nataliia; Stegniy, Borys

2016-05-01

Wild bird surveillance for avian influenza virus (AIV) was conducted from 2001 to 2012 in the Azov - Black Sea region of the Ukraine, considered part of the transcontinental wild bird migration routes from northern Asia and Europe to the Mediterranean, Africa, and southwest Asia. A total of 6281 samples were collected from wild birds representing 27 families and eight orders for virus isolation. From these samples, 69 AIVs belonging to 15 of the 16 known hemagglutinin (HA) subtypes and seven of nine known neuraminidase (NA) subtypes were isolated. No H14, N5, or N9 subtypes were identified. In total, nine H6, eight H1, nine H5, seven H7, six H11, six H4, five H3, five H10, four H8, three H2, three H9, one H12, one H13, one H15, and one H16 HA subtypes were isolated. As for the NA subtypes, twelve N2, nine N6, eight N8, seven N7, six N3, four N4, and one undetermined were isolated. There were 27 HA and NA antigen combinations. All isolates were low pathogenic AIV except for eight highly pathogenic (HP) AIVs that were isolated during the H5N1 HPAI outbreaks of 2006-08. Sequencing and phylogenetic analysis of the HA genes revealed epidemiological connections between the Azov-Black Sea regions and Europe, Russia, Mongolia, and Southeast Asia. H1, H2, H3, H7, H8, H6, H9, and H13 AIV subtypes were closely related to European, Russian, Mongolian, and Georgian AIV isolates. H10, H11, and H12 AIV subtypes were epidemiologically linked to viruses from Europe and Southeast Asia. Serology conducted on serum and egg yolk samples also demonstrated previous exposure of many wild bird species to different AIVs. Our results demonstrate the great genetic diversity of AIVs in wild birds in the Azov-Black Sea region as well as the importance of this region for monitoring and studying the ecology of influenza viruses. This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.

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

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...... 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...

Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico

Science.gov (United States)

Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto

2015-01-01

Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. PMID:26159533

Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys.

Science.gov (United States)

Berhane, Yohannes; Ojkic, Davor; Neufeld, James; Leith, Marsha; Hisanaga, Tamiko; Kehler, Helen; Ferencz, Arpad; Wojcinski, Helen; Cottam-Birt, Colleen; Suderman, Matthew; Handel, Katherine; Alexandersen, Soren; Pasick, John

2010-12-01

Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.

Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

Science.gov (United States)

Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

2017-01-01

microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

Virulence and molecular polymorphism of Prunus necrotic ringspot virus isolates.

Science.gov (United States)

Hammond, R W; Crosslin, J M

1998-07-01

Prunus necrotic ringspot virus (PNRSV) occurs as numerous strains or isolates that vary widely in their pathogenic, biophysical and serological properties. Prior attempts to distinguish pathotypes based upon physical properties have not been successful; our approach was to examine the molecular properties that may distinguish these isolates. The nucleic acid sequence was determined from 1.65 kbp RT-PCR products derived from RNA 3 of seven distinct isolates of PNRSV that differ serologically and in pathology on sweet cherry. Sequence comparisons of ORF 3a (putative movement protein) and ORF 3b (coat protein) revealed single nucleotide and amino acid differences with strong correlations to serology and symptom types (pathotypes). Sequence differences between serotypes and pathotypes were also reflected in the overall phylogenetic relationships between the isolates.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus.

Science.gov (United States)

Teixeira, B M; Logan, N; Samman, A; Miyashiro, S I; Brandão, P E; Willett, B J; Hosie, M J; Hagiwara, M K

2011-09-01

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in São Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline × human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of FIV. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. Copyright © 2011 Elsevier B.V. All rights reserved.

Complete Genome Sequence of Frog virus 3, Isolated from a Strawberry Poison Frog (Oophaga pumilio) Imported from Nicaragua into the Netherlands.

Science.gov (United States)

Saucedo, Bernardo; Hughes, Joseph; van Beurden, Steven J; Suárez, Nicolás M; Haenen, Olga L M; Voorbergen-Laarman, Michal; Gröne, Andrea; Kik, Marja J L

2017-08-31

Frog virus 3 was isolated from a strawberry poison frog ( Oophaga pumilio ) imported from Nicaragua via Germany to the Netherlands, and its complete genome sequence was determined. Frog virus 3 isolate Op /2015/Netherlands/UU3150324001 is 107,183 bp long and has a nucleotide similarity of 98.26% to the reference Frog virus 3 isolate. Copyright © 2017 Saucedo et al.

Xiburema Virus, a Hitherto Undescribed Virus within the Family Rhabdoviridae Isolated in the Brazilian Amazon Region

OpenAIRE

Wanzeller, Ana Lucia M.; Martins, Lívia C.; Diniz Júnior, José Antonio P.; de Almeida Medeiros, Daniele Barbosa; Cardoso, Jedson F.; da Silva, Daisy E. A.; de Oliveira, Layanna F.; de Vasconcelos, Janaina M.; Nunes, Márcio R. T.; Vianez Júnior, João Lídio da S. G.; Vasconcelos, Pedro F. C.

2014-01-01

We report here the first complete open reading frame (ORF) genome sequence of Xiburema virus (XIBV), that of strain BE AR362159, isolated from mosquitoes (Sabethes intermedius) in Sena Madureira, Acre state, northern Brazil. All genes showed similarities with those belonging to members of the family Rhabdoviridae.

Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil.

OpenAIRE

FAJARDO, T. V. M.; NASCIMENTO, M. B.; EIRAS, M.; NICKEL, O.; PIO-RIBEIRO, G.

2016-01-01

ABSTRACT: There is no molecular characterization of Brazilian isolates of Prunus necrotic ringspot virus (PNRSV), except for those infecting peach. In this research, the causal agent of rose mosaic was determined and the movement (MP) and coat (CP) protein genes of a PNRSV isolate from rose were molecularly characterized for the first time in Brazil. The nucleotide and deduced amino acid sequences of MP and CP complete genes were aligned and compared with other isolates. Molecular analysis of...

Molecular characterization and experimental host range of an isolate of Wissadula golden mosaic St. Thomas virus.

Science.gov (United States)

Collins, A M; Mujaddad-ur-Rehman, Malik; Brown, J K; Reddy, C; Wang, A; Fondong, V; Roye, M E

2009-12-01

Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field

Virus movements on the plasma membrane support infection and transmission between cells.

Directory of Open Access Journals (Sweden)

Christoph J Burckhardt

2009-11-01

Full Text Available How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus-host interactions upstream of infectious entry offer new perspectives for anti-viral interference.

Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico.

Science.gov (United States)

Garcés-Ayala, Fabiola; Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto; Ramirez-González, José Ernesto

2015-07-09

Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. Copyright © 2015 Garcés-Ayala et al.

Exosome-associated hepatitis C virus in cell cultures and patient plasma

International Nuclear Information System (INIS)

Liu, Ziqing; Zhang, Xiugen; Yu, Qigui; He, Johnny J.

2014-01-01

Highlights: • HCV occurs in both exosome-free and exosome-associated forms. • Exosome-associated HCV is infectious and resistant to neutralizing antibodies. • More exosome-associated HCV than exosome-free HCV is present in patient plasma. - Abstract: Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission

Exosome-associated hepatitis C virus in cell cultures and patient plasma

Energy Technology Data Exchange (ETDEWEB)

Liu, Ziqing [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Zhang, Xiugen [Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Yu, Qigui [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); He, Johnny J., E-mail: johnny.he@unthsc.edu [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

2014-12-12

Highlights: • HCV occurs in both exosome-free and exosome-associated forms. • Exosome-associated HCV is infectious and resistant to neutralizing antibodies. • More exosome-associated HCV than exosome-free HCV is present in patient plasma. - Abstract: Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

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

International Nuclear Information System (INIS)

Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.; Katsafanas, G.; Roffman, E.; Danovich, R.M.; June, C.H.

1990-01-01

A new human herpes virus has been isolated from CD4 + 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

Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China

Directory of Open Access Journals (Sweden)

Tan Bin

2011-11-01

Full Text Available Abstract A new isolate of canine distemper virus (CDV, named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N, phosphoprotein (P and receptor binding haemagglutinin (H gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China.

Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China.

Science.gov (United States)

Tan, Bin; Wen, Yong-Jun; Wang, Feng-Xue; Zhang, Shu-Qin; Wang, Xiu-Dong; Hu, Jia-Xin; Shi, Xin-Chuan; Yang, Bo-Chao; Chen, Li-Zhi; Cheng, Shi-Peng; Wu, Hua

2011-11-16

A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China.

Xiburema Virus, a Hitherto Undescribed Virus within the Family Rhabdoviridae Isolated in the Brazilian Amazon Region

Science.gov (United States)

Martins, Lívia C.; Diniz Júnior, José Antonio P.; de Almeida Medeiros, Daniele Barbosa; Cardoso, Jedson F.; da Silva, Daisy E. A.; de Oliveira, Layanna F.; de Vasconcelos, Janaina M.; Vianez Júnior, João Lídio da S. G.; Vasconcelos, Pedro F. C.

2014-01-01

We report here the first complete open reading frame (ORF) genome sequence of Xiburema virus (XIBV), that of strain BE AR362159, isolated from mosquitoes (Sabethes intermedius) in Sena Madureira, Acre state, northern Brazil. All genes showed similarities with those belonging to members of the family Rhabdoviridae. PMID:24948755

Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Domber, E.A.

1986-01-01

These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

Hepatitis C virus infection influences the S-methadone metabolite plasma concentration.

Directory of Open Access Journals (Sweden)

Shiow-Ling Wu

Full Text Available Heroin-dependent patients typically contract hepatitis C virus (HCV at a disproportionately high level due to needle exchange. The liver is the primary target organ of HCV infection and also the main organ responsible for drug metabolism. Methadone maintenance treatment (MMT is a major treatment regimen for opioid dependence. HCV infection may affect methadone metabolism but this has rarely been studied. In our current study, we aimed to test the hypothesis that HCV may influence the methadone dosage and its plasma metabolite concentrations in a MMT cohort from Taiwan.A total of 366 MMT patients were recruited. The levels of plasma hepatitis B virus (HBV, HCV, human immunodeficiency virus (HIV antibodies (Ab, liver aspartate aminotransferase (AST and alanine aminotransferase (ALT, as well as methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP were measured along with the urine morphine concentration and amphetamine screening.Of the 352 subjects in our cohort with HCV test records, 95% were found to be positive for plasma anti-HCV antibody. The liver functional parameters of AST (Wilcoxon Rank-Sum test, P = 0.02 and ALT (Wilcoxon Rank-Sum test, P = 0.04, the plasma methadone concentrations (Wilcoxon Rank-Sum test, P = 0.043 and the R-enantiomer of methadone concentrations (Wilcoxon Rank-Sum test, P = 0.032 were significantly higher in the HCV antibody-positive subjects than in the HCV antibody-negative patients, but not the S-EDDP/methadone dose ratio. The HCV levels correlated with the methadone dose (β= 14.65 and 14.13; P = 0.029 and 0.03 and the S-EDDP/methadone dose ratio (β= -0.41 and -0.40; P = 0.00084 and 0.002 in both univariate and multivariate regression analyses.We conclude that HCV may influence the methadone dose and plasma S-EDDP/methadone dose ratio in MMT patients in this preliminary study.

Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico.

Science.gov (United States)

Perez-Ramirez, Gerardo; Diaz-Badillo, Alvaro; Camacho-Nuez, Minerva; Cisneros, Alejandro; Munoz, Maria de Lourdes

2009-12-15

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. 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. 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 the vaccines and drugs formulation as occurs for other

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

Inactivation of Zika virus by solvent/detergent treatment of human plasma and other plasma-derived products and pasteurization of human serum albumin.

Science.gov (United States)

Kühnel, Denis; Müller, Sebastian; Pichotta, Alexander; Radomski, Kai Uwe; Volk, Andreas; Schmidt, Torben

2017-03-01

In 2016 the World Health Organization declared the mosquito-borne Zika virus (ZIKV) a "public health emergency of international concern." ZIKV is a blood-borne pathogen, which therefore causes concerns regarding the safety of human plasma-derived products due to potential contamination of the blood supply. This study investigated the effectiveness of viral inactivation steps used during the routine manufacturing of various plasma-derived products to reduce ZIKV infectivity. Human plasma and intermediates from the production of various plasma-derived products were spiked with ZIKV and subjected to virus inactivation using the identical techniques (either solvent/detergent [S/D] treatment or pasteurization) and conditions used for the actual production of the respective products. Samples were taken and the viral loads measured before and after inactivation. After S/D treatment of spiked intermediates of the plasma-derived products Octaplas(LG), Octagam, and Octanate, the viral loads were below the limit of detection in all cases. The mean log reduction factor (LRF) was at least 6.78 log for Octaplas(LG), at least 7.00 log for Octagam, and at least 6.18 log for Octanate after 60, 240, and 480 minutes of S/D treatment, respectively. For 25% human serum albumin (HSA), the mean LRF for ZIKV was at least 7.48 log after pasteurization at 60°C for 120 minutes. These results demonstrate that the commonly used virus inactivation processes utilized during the production of human plasma and plasma-derived products, namely, S/D treatment or pasteurization, are effective for inactivation of ZIKV. © 2016 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Complete Genome Sequence of an Atypical Dengue Virus Serotype 2 Lineage Isolated in Brazil

Science.gov (United States)

Salvador, Felipe Scassi; Amorim, Jaime Henrique; Alves, Rubens Prince Santos; Pereira, Sara A.; Ferreira, Luis Carlos Souza

2015-01-01

Here, we report the complete polyprotein sequence of a dengue virus 2 strain isolated in Brazil. This virus belongs to the American genotype and has the ability to cause neurovirulence in immunocompetent adult mice. The data presented here may help understand the genetic determinants responsible for neurovirulence. PMID:26184939

Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

International Nuclear Information System (INIS)

Mochalova, Larisa; Gambaryan, Alexandra; Romanova, Julia; Tuzikov, Alexander; Chinarev, Alexander; Katinger, Dietmar; Katinger, Herman; Egorov, Andrej; Bovin, Nicolai

2003-01-01

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac α2-6Galβ1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acα2-6Galβ1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses

A novel monopartite dsRNA virus isolated from the entomopathogenic and nematophagous fungus Purpureocillium lilacinum.

Science.gov (United States)

Herrero, Noemi

2016-12-01

Purpureocillium lilacinum is a ubiquitous saprophytic fungus commonly isolated from soils and widely known as a biological control agent against phytopathogenic nematodes and pest insects. Mycoviruses infect a wide number of fungal species, but the study of viruses infecting entomopathogenic fungi is still quite recent. In this study, a total of 86 P. lilacinum isolates collected from soil in natural and cultivated habitats throughout the Czech Republic were analyzed; 22 % of the isolates harbored double-stranded RNA (dsRNA) elements with viral characteristics. These results suggest that mycoviruses are common in P. lilacinum. One of the most common dsRNA elements detected in the survey was completely sequenced and corresponded to the 2,864-bp genome of a previously undescribed mycovirus, designated Purpureocillium lilacinum nonsegmented virus 1 (PlNV-1). Phylogenetic analysis of the RNA-dependent RNA polymerase of PlNV-1 indicated that this virus might belong to a new taxon related to the family Partitiviridae.

Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes

OpenAIRE

Dutra, Heverton Leandro Carneiro; Rocha, Marcele Neves; Dias, Fernando Braga Stehling; Mansur, Simone Brutman; Caragata, Eric Pearce; Moreira, Luciano Andrade

2016-01-01

Summary The recent association of Zika virus with cases of microcephaly has sparked a global health crisis and highlighted the need for mechanisms to combat the Zika vector, Aedes aegypti mosquitoes. Wolbachia pipientis, a bacterial endosymbiont of insect, has recently garnered attention as a mechanism for arbovirus control. Here we report that Aedes aegypti harboring Wolbachia are highly resistant to infection with two currently circulating Zika virus isolates from the recent Brazilian epide...

Dengue and chikungunya viruses in plasma are effectively inactivated after treatment with methylene blue and visible light.

Science.gov (United States)

Fryk, Jesse J; Marks, Denese C; Hobson-Peters, Jody; Prow, Natalie A; Watterson, Daniel; Hall, Roy A; Young, Paul R; Reichenberg, Stefan; Sumian, Chryslain; Faddy, Helen M

2016-09-01

Arboviruses, such as dengue viruses (DENV) and chikungunya virus (CHIKV), pose a risk to the safe transfusion of blood components, including plasma. Pathogen inactivation is an approach to manage this transfusion transmission risk, with a number of techniques being used worldwide for the treatment of plasma. In this study, the efficacy of the THERAFLEX MB-Plasma system to inactivate all DENV serotypes (DENV-1, DENV-2, DENV-3, DENV-4) or CHIKV in plasma, using methylene blue and light illumination at 630 nm, was investigated. Pooled plasma units were spiked with DENV-1, DENV-2, DENV-3 DENV-4, or CHIKV and treated with the THERAFLEX MB-Plasma system at four light illumination doses: 20, 40, 60, and 120 (standard dose) J/cm(2) . Pre- and posttreatment samples were collected and viral infectivity was determined. The reduction in viral infectivity was calculated for each dose. Treatment of plasma with the THERAFLEX MB-Plasma system resulted in at least a 4.46-log reduction in all DENV serotypes and CHIKV infectious virus. The residual infectivity for each was at the detection limit of the assay used at 60 J/cm(2) , with dose dependency also observed. Our study demonstrated the THERAFLEX MB-Plasma system can reduce the infectivity of all DENV serotypes and CHIKV spiked into plasma to the detection limit of the assay used at half of the standard illumination dose. This suggests this system has the capacity to be an effective option for managing the risk of DENV or CHIKV transfusion transmission in plasma. © 2016 AABB.

Xiburema Virus, a Hitherto Undescribed Virus within the Family Rhabdoviridae Isolated in the Brazilian Amazon Region.

Science.gov (United States)

Wanzeller, Ana Lucia M; Martins, Lívia C; Diniz Júnior, José Antonio P; de Almeida Medeiros, Daniele Barbosa; Cardoso, Jedson F; da Silva, Daisy E A; de Oliveira, Layanna F; de Vasconcelos, Janaina M; Nunes, Márcio R T; Vianez Júnior, João Lídio da S G; Vasconcelos, Pedro F C

2014-06-19

We report here the first complete open reading frame (ORF) genome sequence of Xiburema virus (XIBV), that of strain BE AR362159, isolated from mosquitoes (Sabethes intermedius) in Sena Madureira, Acre state, northern Brazil. All genes showed similarities with those belonging to members of the family Rhabdoviridae. Copyright © 2014 Wanzeller et al.

The plasma virome of febrile adult Kenyans shows frequent parvovirus B19 infections and a novel arbovirus (Kadipiro virus).

Science.gov (United States)

Ngoi, Carolyne N; Siqueira, Juliana; Li, Linlin; Deng, Xutao; Mugo, Peter; Graham, Susan M; Price, Matt A; Sanders, Eduard J; Delwart, Eric

2016-12-01

Viral nucleic acids present in the plasma of 498 Kenyan adults with unexplained fever were characterized by metagenomics analysis of 51 sample pools. The highest to lowest fraction of plasma pools was positive for parvovirus B19 (75 %), pegivirus C (GBV-C) (67 %), alpha anellovirus (59 %), gamma anellovirus (55 %), beta anellovirus (41 %), dengue virus genotype 2 (DENV-2) (16 %), human immunodeficiency virus type 1 (6 %), human herpesvirus 6 (6 %), HBV (4 %), rotavirus (4 %), hepatitis B virus (4 %), rhinovirus C (2 %), Merkel cell polyomavirus (MCPyV; 2 %) and Kadipiro virus (2 %). Ranking by overall percentage of viral reads yielded similar results. Characterization of viral nucleic acids in the plasma of a febrile East African population showed a high frequency of parvovirus B19 and DENV infections and detected a reovirus (Kadipiro virus) previously reported only in Asian Culex mosquitoes, providing a baseline to compare with future virome studies to detect emerging viruses in this region.

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....

Plasma membrane phosphatidylinositol 4,5 bisphosphate is required for internalization of foot-and-mouth disease virus and vesicular stomatitis virus.

Directory of Open Access Journals (Sweden)

Angela Vázquez-Calvo

Full Text Available Phosphatidylinositol-4,5-bisphosphate, PI(4,5P(2, is a phospholipid which plays important roles in clathrin-mediated endocytosis. To investigate the possible role of this lipid on viral entry, two viruses important for animal health were selected: the enveloped vesicular stomatitis virus (VSV - which uses a well characterized clathrin mediated endocytic route - and two different variants of the non-enveloped foot-and-mouth disease virus (FMDV with distinct receptor specificities. The expression of a dominant negative dynamin, a PI(4,5P(2 effector protein, inhibited the internalization and infection of VSV and both FMDV isolates. Depletion of PI(4,5P(2 from plasma membrane using ionomycin or an inducible system, and inhibition of its de novo synthesis with 1-butanol revealed that VSV as well as FMDV C-S8c1, which uses integrins as receptor, displayed a high dependence on PI(4,5P(2 for internalization. Expression of a kinase dead mutant (KD of phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K-Iα, an enzyme responsible for PI(4,5P(2 synthesis that regulates clathrin-dependent endocytosis, also impaired entry and infection of VSV and FMDV C-S8c1. Interestingly FMDV MARLS variant that uses receptors other than integrins for cell entry was less sensitive to PI(4,5P(2 depletion, and was not inhibited by the expression of the KD PIP5K-Iα mutant suggesting the involvement of endocytic routes other than the clathrin-mediated on its entry. These results highlight the role of PI(4,5P(2 and PIP5K-Iα on clathrin-mediated viral entry.

Genetic characterization and molecular epidemiology of foot-and-mouth disease viruses isolated from Afghanistan in 2003-2005.

Science.gov (United States)

Schumann, Kate R; Knowles, Nick J; Davies, Paul R; Midgley, Rebecca J; Valarcher, Jean-Francois; Raoufi, Abdul Quader; McKenna, Thomas S; Hurtle, William; Burans, James P; Martin, Barbara M; Rodriguez, Luis L; Beckham, Tammy R

2008-04-01

Foot-and-mouth disease virus (FMDV) isolates collected from various geographic locations in Afghanistan between 2003 and 2005 were genetically characterized, and their phylogeny was reconstructed utilizing nucleotide sequences of the complete VP1 coding region. Three serotypes of FMDV (types A, O, and Asia 1) were identified as causing clinical disease in Afghanistan during this period. Phylogenetic analysis revealed that the type A viruses were most closely related to isolates collected in Iran during 2002-2004. This is the first published report of serotype A in Afghanistan since 1975, therefore indicating the need for inclusion of serotype A in vaccine formulations that will be used to control disease outbreaks in this country. Serotype O virus isolates were closely related to PanAsia strains, including those that originated from Bhutan and Nepal during 2003-2004. The Asia 1 viruses, collected along the northern and eastern borders of Afghanistan, were most closely related to FMDV isolates collected in Pakistan during 2003 and 2004. Data obtained from this study provide valuable information on the FMDV serotypes circulating in Afghanistan and their genetic relationship with strains causing FMD in neighboring countries.

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

Directory of Open Access Journals (Sweden)

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.

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

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

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

NARCIS (Netherlands)

Wahyuningsih, R.; SahBandar, IN; Theelen, B.; Hagen, F.; Poot, G.; Meis, J.F.; Rozalyani, A.; Sjam, R.; Widodo, D.; Djauzi, S.; Boekhout, T.

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 isavuconazole and

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.

Genetic characterization of H1N2 swine influenza virus isolated in China and its pathogenesis and inflammatory responses in mice.

Science.gov (United States)

Zhang, Yan; Wang, Nan; Cao, Jiyue; Chen, Huanchun; Jin, Meilin; Zhou, Hongbo

2013-09-01

In 2009, two H1N2 influenza viruses were isolated from trachea swabs of pigs in Hubei in China. We compared these sequences with the other 18 complete genome sequences of swine H1N2 isolates from China during 2004 to 2010 and undertook extensive analysis of their evolutionary patterns. Six different genotypes - two reassortants between triple reassortant (TR) H3N2 and classical swine (CS) H1N1 virus, three reassortants between TR H1N2, Eurasian avian-like H1N1 swine virus and H9N2 swine virus, and one reassortant between H1N1, H3N2 human virus and CS H1N1 virus - were observed in these 20 swine H1N2 isolates. The TR H1N2 swine virus is the predominant genotype, and the two Hubei H1N2 isolates were located in this cluster. We also used a mouse model to examine the pathogenesis and inflammatory responses of the two isolates. The isolates replicated efficiently in the lung, and exhibited a strong inflammatory response, serious pathological changes and mortality in infected mice. Given the role that swine can play as putative "genetic mixing vessels" and the observed transmission of TR H1N2 in ferrets, H1N2 influenza surveillance in pigs should be increased to minimize the potential threat to public health.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Science.gov (United States)

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

Temperature-Sensitive Mutants of Mouse Hepatitis Virus Strain A59: Isolation, Characterization and Neuropathogenic Properties.

NARCIS (Netherlands)

M.J.M. Koolen (Marck); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert); M.C. Horzinek; B.A.M. van der Zeijst (Ben)

1983-01-01

textabstractTwenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the

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. © Her Majesty in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. Published by Oxford University Press on behalf of Entomological Society of America.

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009.

Science.gov (United States)

Kanehira, Katsushi; Takemae, Nobuhiro; Uchida, Yuko; Hikono, Hirokazu; Saito, Takehiko

2014-06-01

In 2013, three reassortant swine influenza viruses (SIVs)-two H1N2 and one H3N2-were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human-like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human-like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human-lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human-lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Genetic characterization of the influenza A pandemic (H1N1 2009 virus isolates from India.

Directory of Open Access Journals (Sweden)

Varsha A Potdar

Full Text Available BACKGROUND: The Influenza A pandemic H1N1 2009 (H1N1pdm virus appeared in India in May 2009 and thereafter outbreaks with considerable morbidity and mortality have been reported from many parts of the country. Continuous monitoring of the genetic makeup of the virus is essential to understand its evolution within the country in relation to global diversification and to track the mutations that may affect the behavior of the virus. METHODS: H1N1pdm viruses were isolated from both recovered and fatal cases representing major cities and sequenced. Phylogenetic analyses of six concatenated whole genomes and the hemagglutinin (HA gene of seven more isolates from May-September 2009 was performed with reference to 685 whole genomes of global isolates available as of November 24, 2009. Molecular characterization of all the 8 segments was carried out for known pathogenic markers. RESULTS: The first isolate of May 2009 belonged to clade 5. Although clade 7 was the dominant H1N1pdm lineage in India, both clades 6 and 7 were found to be co-circulating. The neuraminidase of all the Indian isolates possessed H275, the marker for sensitivity to the neuraminidase inhibitor Oseltamivir. Some of the mutations in HA are at or in the vicinity of antigenic sites and may therefore be of possible antigenic significance. Among these a D222G mutation in the HA receptor binding domain was found in two of the eight Indian isolates obtained from fatal cases. CONCLUSIONS: The majority of the 13 Indian isolates grouped in the globally most widely circulating H1N1pdm clade 7. Further, correlations of the mutations specific to clade 7 Indian isolates to viral fitness and adaptability in the country remains to be understood. The D222G mutation in HA from isolates of fatal cases needs to be studied for pathogenicity.

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.

Novel reassortant influenza A(H1N2) virus derived from A(H1N1)pdm09 virus isolated from swine, Japan, 2012.

Science.gov (United States)

Kobayashi, Miho; Takayama, Ikuyo; Kageyama, Tsutomu; Tsukagoshi, Hiroyuki; Saitoh, Mika; Ishioka, Taisei; Yokota, Yoko; Kimura, Hirokazu; Tashiro, Masato; Kozawa, Kunihisa

2013-12-01

We isolated a novel influenza virus A(H1N2) strain from a pig on January 13, 2012, in Gunma Prefecture, Japan. Phylogenetic analysis showed that the strain was a novel type of double-reassortant virus derived from the swine influenza virus strains H1N1pdm09 and H1N2, which were prevalent in Gunma at that time.

Mechanism and scaling for convection of isolated structures in nonuniformly magnetized plasmas

DEFF Research Database (Denmark)

Garcia, O.E.; Bian, N.H.; Naulin, V.

2005-01-01

Large-scale radial advection of isolated structures in nonuniformly magnetized plasmas is investigated. The underlying mechanism considered is due to the nonlinear evolution of interchange motions, without any presumption of plasma sheaths. Theoretical arguments supported by numerical simulations...

Identification and phylogenetic analysis of a sheep pox virus isolated from the Ningxia Hui Autonomous Region of China.

Science.gov (United States)

Zhu, X L; Yang, F; Li, H X; Dou, Y X; Meng, X L; Li, H; Luo, X N; Cai, X P

2013-05-14

An outbreak of sheep pox was investigated in the Ningxia Hui Autonomous Region in China. Through immunofluorescence testing, isolated viruses, polymerase chain reaction identification, and electron microscopic examination, the isolated strain was identified as a sheep pox virus. The virus was identified through sequence and phylogenetic analysis of the P32 gene, open reading frame (ORF) 095, and ORF 103 genes. This study is the first to use the ORF 095 and ORF 103 genes as candidate genes for the analysis of sheep pox. The results showed that the ORF 095 and ORF 103 genes could be used for the genotyping of the sheep pox virus.

First Complete Genome Sequence of a Watermelon Mosaic Virus Isolated from Watermelon in the United States

OpenAIRE

Rajbanshi, Naveen; Ali, Akhtar

2016-01-01

Watermelon mosaic virus was first reported in 1965 from the Rio Grande Valley, TX. We report here the first complete genome sequence of a watermelon mosaic virus isolate from watermelon collected from the Rio Grande Valley of Texas.

Experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccine-derived virus

DEFF Research Database (Denmark)

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

2002-01-01

The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foctuses, stillborn pigs, and dead: piglets, indicating that the l......The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foctuses, stillborn pigs, and dead: piglets, indicating...... than 99.6% identity to the attenuated vaccine virus, originated from the lungs of a stillborn pig from a swine herd with a sudden high level of stillborn pigs and increased piglet mortality in the nursing period. Intranasal inoculation of sows with the virus isolate resulted in congenital infection......, foetal death, and preweaning pig mortality. As such, the present study showed that vaccine-derived PRRSV can cause disease in swine consistent with PRRS....

Molecular characterization of two prunus necrotic ringspot virus isolates from Canada.

Science.gov (United States)

Cui, Hongguang; Hong, Ni; Wang, Guoping; Wang, Aiming

2012-05-01

We determined the entire RNA1, 2 and 3 sequences of two prunus necrotic ringspot virus (PNRSV) isolates, Chr3 from cherry and Pch12 from peach, obtained from an orchard in the Niagara Fruit Belt, Canada. The RNA1, 2 and 3 of the two isolates share nucleotide sequence identities of 98.6%, 98.4% and 94.5%, respectively. Their RNA1- and 2-encoded amino acid sequences are about 98% identical to the corresponding sequences of a cherry isolate, CH57, the only other PNRSV isolate with complete RNA1 and 2 sequences available. Phylogenetic analysis of the coat protein and movement protein encoded by RNA3 of Pch12 and Chr3 and published PNRSV isolates indicated that Chr3 belongs to the PV96 group and Pch12 belongs to the PV32 group.

Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein

OpenAIRE

Scheifele, Lisa Z.; Rhoads, Jonathan D.; Parent, Leslie J.

2003-01-01

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affin...

Isolation, preliminary characterization, and full-genome analyses of tick-borne encephalitis virus from Mongolia.

Science.gov (United States)

Frey, Stefan; Mossbrugger, Ilona; Altantuul, Damdin; Battsetseg, Jigjav; Davaadorj, Rendoo; Tserennorov, Damdindorj; Buyanjargal, Tsoodol; Otgonbaatar, Dashdavaa; Zöller, Lothar; Speck, Stephanie; Wölfel, Roman; Dobler, Gerhard; Essbauer, Sandra

2012-12-01

Tick-borne encephalitis virus (TBEV) causes one of the most important inflammatory diseases of the central nervous system, namely severe encephalitis in Europe and Asia. Since the 1980s tick-borne encephalitis is known in Mongolia with increasing numbers of human cases reported during the last years. So far, however, data on TBEV strains are still sparse. We herein report the isolation of a TBEV strain from Ixodes persulcatus ticks collected in Mongolia in 2010. Phylogenetic analysis of the E-gene classified this isolate as Siberian subtype of TBEV. The Mongolian TBEV strain showed differences in virus titers, plaque sizes, and growth properties in two human neuronal cell-lines. In addition, the 10,242 nucleotide long open-reading frame and the corresponding polyprotein sequence were revealed. The isolate grouped in the genetic subclade of the Siberian subtype. The strain Zausaev (AF527415) and Vasilchenko (AF069066) had 97 and 94 % identity on the nucleotide level. In summary, we herein describe first detailed data regarding TBEV from Mongolia. Further investigations of TBEV in Mongolia and adjacent areas are needed to understand the intricate dispersal of this virus.

Guiding-center models for edge plasmas and numerical simulations of isolated plasma filaments

International Nuclear Information System (INIS)

Madsen, Jens

2010-09-01

The work presented in this thesis falls into two categories: development of reduced dynamical models applicable to edge turbulence in magnetically confined fusion plasmas and numerical simulations of isolated plasma filaments in the scrape-off layer region investigating the influence of finite Larmor radius effects on the radial plasma transport. The coexistence of low-frequency fluctuations, having length scales comparable to the ion gyroradius, steep pressure gradients and strong E x B flows in the edge region of fusion plasmas violates the standard gyrokinetic ordering. In this thesis two models are presented that overcome some of the difficulties associated with the development of reduced dynamical models applicable to the edge. Second order guiding-center coordinates are derived using the phasespace Lie transform method. Using a variational principle the corresponding Vlasov-Maxwell equations expressed in guiding-center coordinates are derived including a local energy theorem. The second order terms describe lowest order finite Larmor radius effects. This set of equations might be relevant for edge plasmas due to the capability of capturing strong E x B flows and lowest order finite Larmor radius effects self-consistently. Next, an extension of the existing gyrokinetic formalism with strong flows is presented. In this work the background electric fields is dynamical, whereas earlier contributions did only incorporate a stationary electric field. In an ordering relevant for edge plasma turbulence, fully electromagnetic second order gyrokinetic coordinates and the corresponding gyrokinetic Vlasov-Maxwell equations are derived, including a local energy theorem. By taking the polarization and magnetization densities in the drift kinetic limit, we present the gyrokinetic Vlasov-Maxwell equations in a more tractable form, which could be relevant for direct numerical simulations of edge plasma turbulence. Finally, an investigation of the influence of finite Larmor

Guiding-center models for edge plasmas and numerical simulations of isolated plasma filaments

Energy Technology Data Exchange (ETDEWEB)

Madsen, Jens

2010-09-15

The work presented in this thesis falls into two categories: development of reduced dynamical models applicable to edge turbulence in magnetically confined fusion plasmas and numerical simulations of isolated plasma filaments in the scrape-off layer region investigating the influence of finite Larmor radius effects on the radial plasma transport. The coexistence of low-frequency fluctuations, having length scales comparable to the ion gyroradius, steep pressure gradients and strong E x B flows in the edge region of fusion plasmas violates the standard gyrokinetic ordering. In this thesis two models are presented that overcome some of the difficulties associated with the development of reduced dynamical models applicable to the edge. Second order guiding-center coordinates are derived using the phasespace Lie transform method. Using a variational principle the corresponding Vlasov-Maxwell equations expressed in guiding-center coordinates are derived including a local energy theorem. The second order terms describe lowest order finite Larmor radius effects. This set of equations might be relevant for edge plasmas due to the capability of capturing strong E x B flows and lowest order finite Larmor radius effects self-consistently. Next, an extension of the existing gyrokinetic formalism with strong flows is presented. In this work the background electric fields is dynamical, whereas earlier contributions did only incorporate a stationary electric field. In an ordering relevant for edge plasma turbulence, fully electromagnetic second order gyrokinetic coordinates and the corresponding gyrokinetic Vlasov-Maxwell equations are derived, including a local energy theorem. By taking the polarization and magnetization densities in the drift kinetic limit, we present the gyrokinetic Vlasov-Maxwell equations in a more tractable form, which could be relevant for direct numerical simulations of edge plasma turbulence. Finally, an investigation of the influence of finite Larmor

Mallard or chicken? Comparing the isolation of avian influenza A viruses in embryonated Mallard and chicken eggs

Directory of Open Access Journals (Sweden)

Josef D. Järhult

2015-09-01

Full Text Available Background: To date, the most efficient and robust method for isolating avian influenza A viruses (IAVs is using embryonated chicken eggs (ECEs. It is known that low-pathogenic avian IAVs undergo rapid genetic changes when introduced to poultry holdings, but the factors driving mutagenesis are not well understood. Despite this, there is limited data on the effects of the standard method of virus isolation of avian-derived viruses, that is, whether isolation in ECEs causes adaptive changes in avian IAVs. Eggs from a homologous species could potentially offer an isolation vessel less prone to induce adaptive changes. Methods: We performed eight serial passages of two avian IAVs isolated from fecal samples of wild Mallards in both ECEs and embryonated Mallard eggs, and hemagglutination assay titers and hemagglutinin sequences were compared. Results: There was no obvious difference in titers between ECEs and embryonated Mallard eggs. Sequence analyses of the isolates showed no apparent difference in the rate of introduction of amino acid substitutions in the hemagglutinin gene (three substitutions in total in embryonated Mallard eggs and two substitutions in ECEs. Conclusion: Embryonated Mallard eggs seem to be good isolation vessels for avian IAVs but carry some practical problems such as limited availability and short egg-laying season of Mallards. Our study finds isolation of Mallard-derived avian IAVs in ECEs non-inferior to isolation in embryonated Mallard eggs, but more research in the area may be warranted as this is a small-scale study.

Molecular Variability Among Isolates of Prunus Necrotic Ringspot Virus from Different Prunus spp.

Science.gov (United States)

Aparicio, F; Myrta, A; Di Terlizzi, B; Pallás, V

1999-11-01

ABSTRACT Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5' untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of the CP, the amino acid residues in this region, which were previously thought to be functionally important in the replication cycle of the virus, were strictly conserved. No clear correlation with the type of symptom or host specificity could be observed. The validity of this grouping was confirmed when other isolates recently characterized by other authors were included in these analyses.

Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil

Directory of Open Access Journals (Sweden)

Thor Vinícius Martins Fajardo

2015-12-01

Full Text Available ABSTRACT: There is no molecular characterization of Brazilian isolates of Prunus necrotic ringspot virus (PNRSV, except for those infecting peach. In this research, the causal agent of rose mosaic was determined and the movement (MP and coat (CP protein genes of a PNRSV isolate from rose were molecularly characterized for the first time in Brazil. The nucleotide and deduced amino acid sequences of MP and CP complete genes were aligned and compared with other isolates. Molecular analysis of the MP and CP nucleotide sequences of a Brazilian PNRSV isolate from rose and others from this same host showed highest identities of 96.7% and 98.6%, respectively, and Rose-Br isolate was classified in PV32 group.

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea.

Science.gov (United States)

Ghedin, Elodie; Rogers, Matthew B; Widen, Steven G; Guzman, Hilda; Travassos da Rosa, Amelia P A; Wood, Thomas G; Fitch, Adam; Popov, Vsevolod; Holmes, Edward C; Walker, Peter J; Vasilakis, Nikos; Tesh, Robert B

2013-12-01

Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae.

Isolation, propagation, and titration of human immunodeficiency virus type 1 from peripheral blood of infected individuals

NARCIS (Netherlands)

Schuitemaker, Hanneke; Kootstra, Neeltje A.

2005-01-01

HIV-1 can be isolated from peripheral blood mononuclear cells and is easily propagated on primary cells in vitro. Here we describe the method for bulk isolation of the HIV-1 quasispecies and a limiting dilution virus isolation protocol by which single coexisting clones can be obtained. In addition,

Estandarización del método de centrifugación en placa para el aislamiento del virus dengue Rapid centrifugation assay standarization for dengue virus isolation

Directory of Open Access Journals (Sweden)

Miryam Palomino

2010-03-01

Full Text Available Se estandarizó el método de centrifugación en placa, para el aislamiento del virus dengue a partir de muestras de suero humano. Se utilizó la línea celular C6/36-HT determinándose los valores óptimos de velocidad de centrifugación, volumen de inóculo, dilución de suero y tiempo de incubación. Posteriormente, 22 muestras de suero con aislamiento viral positivo y cepas referenciales de los cuatro serotipos del virus dengue, fueron procesadas simultáneamente por el método de centrifugación en placa y el método convencional de cultivo en tubo, los aislamientos fueron tipificados mediante inmunofluorescencia indirecta empleando anticuerpos monoclonales. Se optimizó el método de centrifugación en placa inoculando 200 μL de diluciσn de suero 1/20, centrifugaciσn a 1600 rpm/30 min, presentando sensibilidad de 95,5% a cinco dνas postinoculación. Se concluye que el método de centrifugación en placa mejora el porcentaje de aislamiento, con significativa reducción en tiempo de aislamiento del virus dengue.The plate centrifugation assay was standardized for dengue virus isolation from serum samples. C6/36-HT cells were used determining the optimal values for centrifugation spin speed, inoculum, sera dilution, and incubation time. Then, 22 positive serum samples with viral isolation and viral strains of the four reference dengue virus serotypes were tested simultaneously by the standardized plate centrifugation method and the conventional tube culture. The isolations were typified by indirect immunofluorescent test using monoclonal antibodies. The plate centrifugation method was optimized to 200 μL of inoculum, dilution of sera 1/20, centrifugation speed at 1600 rpm/30 min, and sensitivity of 95,5% after 5 days post-inoculation. We concluded that the plate centrifugation method increased dengue virus isolation, with a significant reduction of the time of isolation for dengue virus.

West Nile virus isolated from a Virginia opossum (Didelphis virginiana) in northwestern Missouri, USA, 2012.

Science.gov (United States)

Bosco-Lauth, Angela; Harmon, Jessica R; Lash, R Ryan; Weiss, Sonja; Langevin, Stanley; Savage, Harry M; Godsey, Marvin S; Burkhalter, Kristen; Root, J Jeffrey; Gidlewski, Thomas; Nicholson, William L; Brault, Aaron C; Komar, Nicholas

2014-10-01

We describe the isolation of West Nile virus (WNV; Flaviviridae, Flavivirus) from blood of a Virginia opossum (Didelphis virginiana) collected in northwestern Missouri, USA in August 2012. Sequencing determined that the virus was related to lineage 1a WNV02 strains. We discuss the role of wildlife in WNV disease epidemiology.

Phylogenetic analysis of West Nile virus isolated in Italy in 2008.

Science.gov (United States)

Savini, G; Monaco, F; Calistri, P; Lelli, R

2008-11-27

In Italy the first occurrence of West Nile virus (WNV) infection was reported in Tuscany region during the late summer of 1998. In August 2008, the WNV infection re-emerged in Italy, in areas surrounding the Po river delta, and involving three regions Lombardy, Emilia Romagna and Veneto. WNV was isolated from blood and organs samples of one horse, one donkey, one pigeon (Columba livia) and three magpies (Pica pica). The phylogenetic analysis of the isolates, conducted on 255 bp in the region coding for the E protein, indicates that these isolates belong to the lineage I among the European strains. According to the analysis, both the 1998 and 2008 Italian strains as well as isolates from Romania, Russia, Senegal and Kenya fell in the same sub-cluster.

Molecular characterization and phylogenetic analysis of Sugarcane yellow leaf virus isolates from China.

Science.gov (United States)

Gao, San-Ji; Lin, Yi-Hua; Pan, Yong-Bao; Damaj, Mona B; Wang, Qin-Nan; Mirkov, T Erik; Chen, Ru-Kai

2012-10-01

Sugarcane yellow leaf virus (SCYLV) (genus Polerovirus, family Luteoviridae), the causal agent of sugarcane yellow leaf disease (YLD), was first detected in China in 2006. To assess the distribution of SCYLV in the major sugarcane-growing Chinese provinces, leaf samples from 22 sugarcane clones (Saccharum spp. hybrid) showing YLD symptoms were collected and analyzed for infection by the virus using reverse transcription PCR (RT-PCR), quantitative RT-PCR, and immunological assays. A complete genomic sequence (5,879 nt) of the Chinese SCYLV isolate CHN-FJ1 and partial genomic sequences (2,915 nt) of 13 other Chinese SCYLV isolates from this study were amplified, cloned, and sequenced. The genomic sequence of the CHN-FJ1 isolate was found to share a high identity (98.4-99.1 %) with those of the Brazilian (BRA) genotype isolates and a low identity (86.5-86.9 %) with those of the CHN1 and Cuban (CUB) genotype isolates. The genetic diversity of these 14 Chinese SCYLV isolates was assessed along with that of 29 SCYLV isolates of worldwide origin reported in the GenBank database, based on the full or partial genomic sequence. Phylogenetic analysis demonstrated that all the 14 Chinese SCYLV isolates clustered into one large group with the BRA genotype and 12 other reported SCYLV isolates. In addition, five reported Chinese SCYLV isolates were grouped with the Peruvian (PER), CHN1 and CUB genotypes. We therefore speculated that at least four SCYLV genotypes, BRA, PER, CHN1, and CUB, are associated with YLD in China. Interestingly, a 39-nt deletion was detected in the sequence of the CHN-GD3 isolate, in the middle of the ORF1 region adjacent to the overlap between ORF1 and ORF2. This location is known to be one of the recombination breakpoints in the Luteoviridae family.

Quantification of Epstein-Barr virus-DNA load in lung transplant recipients : A comparison of plasma versus whole blood

NARCIS (Netherlands)

Bakker, Nicolaas A.; Verschuuren, Erik A.; Veeger, Nic J.; van der Bij, Wim; van Imhoff, Gustaaf W.; Kallenberg, Cees G.; Hepkema, Bouke G.

2008-01-01

Background: Monitoring of the Epstein-Barr virus-DNA load is frequently used to identify patients at risk for post-transplant lymphoproliferative disease (PTLD). Epstein-Barr virus DNA can be measured in the plasma and whole blood serum compartments. Methods: We compared levels of Epstein-Barr virus

Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

Directory of Open Access Journals (Sweden)

Munir Iqbal

2009-06-01

Full Text Available The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84, a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP, and matrix (M genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

Science.gov (United States)

Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

2014-07-01

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

DEFF Research Database (Denmark)

Knüsel, R.; Bergmann, S. M.; Einer-Jensen, Katja

2007-01-01

in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus......This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools...... (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and...

Characterization of sida golden mottle virus isolated from Sida santaremensis Monteiro in Florida.

Science.gov (United States)

Al-Aqeel, H A; Iqbal, Zafar; Polston, J E

2018-06-21

The genome of sida golden mottle virus (SiGMoV) (GU997691 and GU997692) isolated from Sida santaremensis Monteiro in Manatee County, Florida, was sequenced and characterized. SiGMoV was determined to be a bipartite virus belonging to the genus Begomovirus with a genome organization typical of the New World viruses in the genus. SiGMoV DNA-A had the highest identity scores (89%) and showed the closest evolutionary relationships to sida golden mosaic Buckup virus (SiGMBuV) (JX162591 and HQ008338). However, SiGMoV DNA-B had the highest identity scores (93%) and showed the closest evolutionary relationship to corchorus yellow spot virus (DQ875869), SiGMBuV (JX162592) and sida golden mosaic Florida virus (SiGMFlV) (HE806443). There was extensive recombination in the SiGMoV DNA-A and much less in DNA-B. Full-length clones of SiGMoV were infectious and were able to infect and cause symptoms in several plant species.

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

International Nuclear Information System (INIS)

Lamb, Kristen; Lokesh, G.L.; Sherman, Michael; Watowich, Stanley

2010-01-01

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

Isolation and characterization of virus of highly pathogenic avian influenza H5 subtype of chicken from outbreaks in Indonesia

Directory of Open Access Journals (Sweden)

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

Increased Plasma Cell-Free DNA Level during HTNV Infection: Correlation with Disease Severity and Virus Load

Directory of Open Access Journals (Sweden)

Jing Yi

2014-07-01

Full Text Available Cell-free DNA (cf-DNA in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS and Hantavirus cardiopulmonary syndrome (HCPS, depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.

Pathotyping and Phylogenetic Characterization of Newcastle Disease Viruses Isolated in Peru: Defining Two Novel Subgenotypes Within Genotype XII.

Science.gov (United States)

Chumbe, Ana; Izquierdo-Lara, Ray; Tataje, Luis; Gonzalez, Rosa; Cribillero, Giovana; González, Armando E; Fernández-Díaz, Manolo; Icochea, Eliana

2017-03-01

Infections of poultry with virulent strains of avian paramyxovirus 1 (APMV-1), also known as Newcastle disease viruses (NDVs), cause Newcastle disease (ND). This highly contagious disease affects poultry and many other species of birds worldwide. In countries where the disease is prevalent, constant monitoring and characterization of isolates causing outbreaks are necessary. In this study, we report the results of pathogenicity testing and phylogenetic analyses of seven NDVs isolated from several regions of Peru between 2004 and 2015. Six viruses had intracerebral pathogenicity indices (ICPIs) of between 1.75 and 1.88, corresponding to a velogenic pathotype. The remaining virus had an ICPI of 0.00, corresponding to a lentogenic pathotype. These results were consistent with amino acid sequences at the fusion protein (F) cleavage site. All velogenic isolates had the polybasic amino acid sequence 112 RRQKR↓F 117 at the F cleavage site. Phylogenetic analyses of complete F gene sequences showed that all isolates are classified in class II of APMV-1. The velogenic viruses are classified in genotype XII, while the lentogenic virus is classified in genotype II, closely related to the LaSota vaccine strain. Moreover, tree topology, bootstrap values, and genetic distances observed within genotype XII resulted in the identification of novel subgenotypes XIIa (in South America) and XIIb (in China) and possibly two clades within genotype XIIa. All velogenic Peruvian viruses belonged to subgenotype XIIa. Overall, our results confirm the presence of genotype XII in Peru and suggest that it is the prevalent genotype currently circulating in our country. The phylogenetic characterization of these isolates helps to characterize the evolution of NDV and may help with the development of vaccines specific to our regional necessities.

[Isolation and characterization of exosomes from blood plasma of breast cancer and colorectal cancer patients].

Science.gov (United States)

Tamkovich, S N; Yunusova, N V; Stakheeva, М N; Somov, A K; Frolova, А Y; Kirushina, N А; Afanasyev, S G; Grigoryeva, А E; Laktionov, P P; Kondakova, I V

2017-03-01

A simple approach for isolation of exosomes from the blood plasma, which allows to obtain highly purified preparations of microvesicles no larger than 100 nm has been proposed. The presence of different subpopulations of exosomes in the blood plasma of healthy donors and cancer patients has been recognized. We found the presence of the universal markers CD9, CD24 and CD81 on exosomes isolated from blood plasma that can be used to their routine typing.

Serological and molecular characterization of Syrian Tomato spotted wilt virus isolates

Directory of Open Access Journals (Sweden)

Faiz ISMAEIL

2015-04-01

Full Text Available Thirty four Syrian isolates of Tomato spotted wilt virus (TSWV collected from tomato and pepper were tested against five specific monoclonal antibodies using TAS-ELISA. The isolates were in two serogroups. Fourteen tomato and sixteen pepper isolates were similar in their reaction with MAb-2, MAb-4, MAb-5 and MAb-6, but did not react with MAb-7 (Serogroup 1. Meanwhile, four isolates collected from pepper reacted with all the MAbs used (Serogroup 2. The expected 620 bp DNA fragment was obtained by RT-PCR from six samples using a specific primer pair designed to amplify the nucleocapsid protein (NP gene of TSWV. The PCR products were sequenced and a phylogenetic tree was constructed. Sequence analysis revealed that the Syrian TSWV isolates were very similar at the nucleotide (97.74 to 99.84% identity and amino acid (96.17 to 99.03% identity sequences levels. The phylogenetic tree showed high similarity of Syrian TSWV isolates with many other representative isolates from different countries.

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

Directory of Open Access Journals (Sweden)

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.

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

Science.gov (United States)

Uchida, Yuko; Mase, Masaji; Yoneda, Kumiko; Kimura, Atsumu; Obara, Tsuyoshi; Kumagai, Seikou; Saito, Takehiko; Yamamoto, Yu; Nakamura, Kikuyasu; Tsukamoto, Kenji; Yamaguchi, Shigeo

2008-09-01

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.

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.

[Explantation method of isolating a persistent tick-borne encephalitis virus from the organs of infected monkeys].

Science.gov (United States)

Levina, L S; Pogodina, V V

1981-01-01

The method of explantation was used to examine 63 organs from M. rhesus monkeys 92-783 days after intracerebral and subcutaneous inoculation with the Vasilchenko, Aina/1448 and 41/65 strains of tick-borne encephalitis virus. The optimal time for examination of the explants by tests of the hemagglutinating, cytopathogenic activity of the virus and its pathogenicity for mice was found to be the 15th day of cultivation. A comparative study of the properties of 3 isolates obtained from explants of the spleen, liver and subcortical cerebral ganglia 202 and 307 days after inoculation of monkeys was carried out. The isolates differed from the parental TBE virus strains by their capacity to form small plaques in PEKV cell cultures (pig embryo kidney cells in versen medium).

The requirements for herpes simplex virus type 1 cell-cell spread via nectin-1 parallel those for virus entry.

Science.gov (United States)

Even, Deborah L; Henley, Allison M; Geraghty, Robert J

2006-08-01

Herpes simplex virus type 1 (HSV-1) spreads from an infected cell to an uninfected cell by virus entry, virus-induced cell fusion, and cell-cell spread. The three forms of virus spread require the viral proteins gB, gD, and gH-gL, as well as a cellular gD receptor. The mutual requirement for the fusion glycoproteins and gD receptor suggests that virus entry, cell fusion, and cell-cell spread occur by a similar mechanism. The goals of this study were to examine the role of the nectin-1alpha transmembrane domain and cytoplasmic tail in cell-cell spread and to obtain a better understanding of the receptor-dependent events occurring at the plasma membrane during cell-cell spread. We determined that an intact nectin-1alpha V-like domain was required for cell-cell spread, while a membrane-spanning domain and cytoplasmic tail were not. Chimeric forms of nectin-1 that were non-functional for virus entry did not mediate cell-cell spread regardless of whether they could mediate cell fusion. Also, cell-cell spread of syncytial isolates was dependent upon nectin-1alpha expression and occurred through a nectin-1-dependent mechanism. Taken together, our results indicate that nectin-1-dependent events occurring at the plasma membrane during cell-cell spread were equivalent to those for virus entry.

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea

Science.gov (United States)

Ghedin, Elodie; Rogers, Matthew B.; Widen, Steven G.; Guzman, Hilda; Travassos da Rosa, Amelia P. A.; Wood, Thomas G.; Fitch, Adam; Popov, Vsevolod; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

2013-01-01

Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae. PMID:24062532

Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

Directory of Open Access Journals (Sweden)

Guus Koch

2013-04-01

Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

Phylogenetic analysis of partial RNA-polymerase blocks II and III of Rabies virus isolated from the main rabies reservoirs in Brazil.

Science.gov (United States)

Carnieli, Pedro; de Novaes Oliveira, Rafael; de Oliveira Fahl, Willian; de Carvalho Ruthner Batista, Helena Beatriz; Scheffer, Karin Corrêa; Iamamoto, Keila; Castilho, Juliana Galera

2012-08-01

This study describes the results of the sequencing and analysis of segments of Blocks II and III of the RNA polymerase L gene of Rabies virus isolates from different reservoir species of Brazil. The phylogenetic relations of the virus were determined and a variety of species-specific nucleotides were found in the analyzed areas, but the majority of these mutations were found to be synonymous. However, an analysis of the putative amino acid sequences were shown to have some characteristic mutations between some reservoir species of Brazil, indicating that there was positive selection in the RNA polymerase L gene of Rabies virus. On comparing the putative viral sequences obtained from the Brazilian isolates and other Lyssavirus, it was determined that amino acid mutations occurred in low-restriction areas. This study of the L gene of Rabies virus is the first to be conducted with samples of virus isolates from Brazil, and the results obtained will help in the determination of the phylogenetic relations of the virus.

African and Asian Zika virus isolates display phenotypic differences both in vitro and in vivo

Science.gov (United States)

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently in the Americas. Here we utilized isolate history, as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African ...

Characterization of Newcastle disease virus isolates obtained from Eurasian collared doves (Streptopelia decaocto) in Italy.

Science.gov (United States)

Terregino, Calogero; Cattoli, Giovanni; Grossele, Barbara; Bertoli, Elena; Tisato, Ernesto; Capua, Ilaria

2003-02-01

Eurasian collared doves (Streptopelia decaocto) are thought to originate from India and they have colonized, throughout the centuries, the Middle East and, more recently, Mediterranean countries such as Italy and Spain. In the present paper we report of the isolation and characterization of Newcastle disease viruses (NDV) obtained from Eurasian collared doves during 2000-2001, and compare them to isolates obtained from feral pigeons (Columba livia) during the same period. All isolates could be classified as avian paramyxovirus type 1 (APMV1) and belonged to the pigeon variant group (PPMV1), as their haemagglutinating activity was inhibited by mAb 161/617 which is specific for PPMV1. The intracerebral pathogenicity indices ranged from 0.68 to 1.38 and all isolates contained multiple basic amino acids at the deduced cleavage site of the fusion protein, which is a typical feature of virulent viruses. Phylogenetic analysis of the isolates indicate that 18/20 of these form a separate cluster from the isolates obtained from pigeons in the same period. These findings suggest that different lineages are circulating in feral pigeon populations, and that a separate lineage affects Eurasian collared doves.

Isolation and complete genome analysis of neurotropic dengue virus serotype 3 from the cerebrospinal fluid of an encephalitis patient.

Directory of Open Access Journals (Sweden)

Rama Dhenni

2018-01-01

Full Text Available Although neurological manifestations associated with dengue viruses (DENV infection have been reported, there is very limited information on the genetic characteristics of neurotropic DENV. Here we describe the isolation and complete genome analysis of DENV serotype 3 (DENV-3 from cerebrospinal fluid of an encephalitis paediatric patient in Jakarta, Indonesia. Next-generation sequencing was employed to deduce the complete genome of the neurotropic DENV-3 isolate. Based on complete genome analysis, two unique and nine uncommon amino acid changes in the protein coding region were observed in the virus. A phylogenetic tree and molecular clock analysis revealed that the neurotropic virus was a member of Sumatran-Javan clade of DENV-3 genotype I and shared a common ancestor with other isolates from Jakarta around 1998. This is the first report of neurotropic DENV-3 complete genome analysis, providing detailed information on the genetic characteristics of this virus.

Avian Influenza Virus Isolated in Wild Waterfowl in Argentina: Evidence of a potentially unique phylogenetic lineage in South America

Science.gov (United States)

Pereda, Ariel J.; Uhart, Marcela; Perez, Alberto A.; Zaccagnini, Maria E.; La Sala, Luciano; Decarre, Julieta; Goijman, Andrea; Solari, Laura; Suarez, Romina; Craig, Maria I.; Vagnozzi, Ariel; Rimondi, Agustina; König, Guido; Terrera, Maria V.; Kaloghlian, Analia; Song, Haichen; Sorrell, Erin M.; Perez, Daniel R.

2008-01-01

Avian Influenza (AI) viruses have been sporadically isolated in South America. The most recent reports are from an outbreak in commercial poultry in Chile in 2002 and its putative ancestor from a wild bird in Bolivia in 2001. Extensive surveillance in wild birds was carried out in Argentina during 2006-2007. Using RRT-PCR, 12 AI positive detections were made from cloacal swabs. One of those positive samples yielded an AI virus isolated from a wild kelp gull (Larus dominicanus) captured in the South Atlantic coastline of Argentina. Further characterization by nucleotide sequencing reveals that it belongs to the H13N9 subtype. Phylogenetic analysis of the 8 viral genes suggests that the 6 internal genes are related to the isolates from Chile and Bolivia. The analysis also indicates that a cluster of phylogenetically related AI viruses from South America may have evolved independently, with minimal gene exchange, from influenza viruses in other latitudes. The data produced from our investigations are valuable contributions to the study of AI viruses in South America. PMID:18632129

Recombination between vaccine and field strains of canine parvovirus is revealed by isolation of virus in canine and feline cell cultures.

Science.gov (United States)

Mochizuki, Masami; Ohshima, Takahisa; Une, Yumi; Yachi, Akiko

2008-12-01

Canine parvovirus type 2 (CPV) is a pathogen that causes severe hemorrhagic gastroenteritis with a high fatality rate in pups worldwide. Since CPV emerged in the late 1970s, its origin has been explored with the conclusion that CPV originated from feline panleukopenia virus or a closely related virus. Both high mutation rate and recombination are assumed to be key factors in the evolution of parvoviruses. Here we provide evidence for natural recombination in CPV isolated from dogs in cell culture. Antigenic and genetic properties of isolates from 10 diseased pups were elucidated. Six pups had been vaccinated beforehand with live combined vaccine containing original antigenic type CPV (CPV-2). Six isolates recovered from 4 vaccinated pups in cell cultures were found to contain either CPV-2 or CPV-2-like viruses. The other isolates, including all those from non-vaccinated pups, were CPV-2b viruses. Antigenic typing of two CPV-2-like isolates, 03-029/M and 1887/f, with a monoclonal antibody panel suggested they were a mixture of CPV-2 and CPV-2a (03-029/M) and a recombinant of CPV-2 and CPV-2b (1887/f). Genetic analysis of the VP1 gene indicated that isolate 03-029/M was a mixture of CPV-2, CPV-2a and a recombinant of CPV-2 and CPV-2a viruses, while isolate 1887/f was composed of a recombinant of CPV-2 and CPV-2b viruses. This is the first demonstration of natural CPV recombination in the field and suggests that recombination in the evolution of CPV is a more frequent and important process than previously believed.

Isolation of Biologically Active Exosomes from Plasma of Patients with Cancer.

Science.gov (United States)

Hong, Chang-Sook; Funk, Sonja; Whiteside, Theresa L

2017-01-01

A method for exosome isolation from human plasma was developed for rapid, high-throughput processing of plasma specimens obtained from patients with cancer. This method removes the bulk of plasma proteins associated with exosomes and can be used for comparative examinations of exosomes and their content in serial specimens of patients' plasma, allowing for monitoring changes in exosome numbers, profiles, and functions in the course of cancer progression or during therapy. The plasma-derived exosomes can be recovered in quantities sufficient for the characterization of their morphology by transmission electron microscopy (TEM), size and concentration by qNano, protein/lipid ratios, nucleic acid extraction, molecular profiling by Western blots or immune arrays, and functional assays.

Complete Genome Sequences of Getah Virus Strains Isolated from Horses in 2016 in Japan.

Science.gov (United States)

Nemoto, Manabu; Bannai, Hiroshi; Ochi, Akihiro; Niwa, Hidekazu; Murakami, Satoshi; Tsujimura, Koji; Yamanaka, Takashi; Kokado, Hiroshi; Kondo, Takashi

2017-08-03

Getah virus is mosquito-borne and causes disease in horses and pigs. We sequenced and analyzed the complete genomes of three strains isolated from horses in Ibaraki Prefecture, eastern Japan, in 2016. They were almost identical to the genomes of strains recently isolated from horses, pigs, and mosquitoes in Japan. Copyright © 2017 Nemoto et al.

Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes.

Science.gov (United States)

Dutra, Heverton Leandro Carneiro; Rocha, Marcele Neves; Dias, Fernando Braga Stehling; Mansur, Simone Brutman; Caragata, Eric Pearce; Moreira, Luciano Andrade

2016-06-08

The recent association of Zika virus with cases of microcephaly has sparked a global health crisis and highlighted the need for mechanisms to combat the Zika vector, Aedes aegypti mosquitoes. Wolbachia pipientis, a bacterial endosymbiont of insect, has recently garnered attention as a mechanism for arbovirus control. Here we report that Aedes aegypti harboring Wolbachia are highly resistant to infection with two currently circulating Zika virus isolates from the recent Brazilian epidemic. Wolbachia-harboring mosquitoes displayed lower viral prevalence and intensity and decreased disseminated infection and, critically, did not carry infectious virus in the saliva, suggesting that viral transmission was blocked. Our data indicate that the use of Wolbachia-harboring mosquitoes could represent an effective mechanism to reduce Zika virus transmission and should be included as part of Zika control strategies. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Optimized exosome isolation protocol for cell culture supernatant and human plasma

Directory of Open Access Journals (Sweden)

Richard J. Lobb

2015-07-01

Full Text Available Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a

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.

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.

Influenza virus epidemiological surveillance in Argentina, 1987-1993, with molecular characterization of 1990 and 1993 isolates

Directory of Open Access Journals (Sweden)

Osvaldo C. Uez

1998-12-01

Full Text Available This report describes findings from epidemiological surveillance of influenza virus in two cities in Argentina (Mar del Plata and Córdoba from 1987 to 1993. It includes information on reporting and serologic characterization of isolated influenza viruses. In addition, determination was made of the nucleotide sequences of the HA1 subunits of five type A (subtype H3 viral strains isolated in the epidemics of 1990 and 1993. The incidence of illness, type of viruses isolated, and H gene sequences were similar to what has been reported from other parts of the world during the same period. The H3 strains isolated in the 1990 and 1993 seasons were somewhat removed in their molecular characteristics from the strains the World Health Organization recommended for vaccines for those years, and appeared closer to the strains recommended for vaccination in subsequent seasonsEn este informe se describen los resultados de la vigilancia epidemiológica de virus de gripe en dos ciudades de la Argentina (Mar del Plata y Córdoba de 1987 a 1993. Se incluye información acerca de la notificación y la caracterización serológica de los virus aisaldos. Además, se determinaron las secuencias de nucleótidos de las subunidades HA1 de cinco cepas tipo A (subtipo H3 aisladas durante las epidemias de 1990 y 1993. La incidencia de enfermedad, los tipos de virus aislados y las secuencias genéticas H fueron similares a las notificaciones del mismo período en otras partes del mundo. En sus características moleculares, las cepas H3 aisladas en las estaciones de 1990 y 1993 se distinguían un poco de las cepas que la Organización Mundial de la Salud recomendó incluir en las vacunas de esos años y se parecían más a las cepas recomendadas para vacunación en estaciones subsecuentes.

ISOLATION OF EGG DROP SYNDROME VIRUS AND ITS MOLECULAR CHARACTERIZATION USING SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS

Directory of Open Access Journals (Sweden)

M. H. Rasool, S. U. Rahman and M. K. Mansoor

2005-10-01

Full Text Available Six isolates of egg drop syndrome (EDS virus were recovered from five different outbreaks of EDS in commercial laying hens in and around Faisalabad. The aberrant eggs were fed to the susceptible laying hens for experimental induction of infection. The samples from infected birds (egg washing, cloacal swabs, oviducts and spleens were collected, processed and inoculated into 11-day old duck embryos. The presence of virus in harvested allanto-amniotic fluid was monitored by spot and microhaemagglutination tests and confirmed by haemagglutination inhibition and agar gel precipitation tests. The EDS virus grew well in duck embryos and agglutinated only avian but not mammalian red blood cells. These isolates were purified through velocity density gradient centrifugation. Protein concentration was determined through Lowry method and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE was conducted by loading 300 µg protein concentration on 12.5% gel using discontinuous buffer system. All the six isolates showed 13 polypeptides, which were identical to those described in the referral EDS-76 virus (strain-127. The molecular weights of the polypeptides ranged from 6.5 KDa to 126 KDa.

A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

Directory of Open Access Journals (Sweden)

Rui eZhang

2014-07-01

Full Text Available Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10 of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1. A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A tail. The genome possesses two non-overlapping open reading frames (ORFs: a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1. Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1and FgV1.

Isolation of avian influenza H5N1 virus from vaccinated commercial layer flock in Egypt

Directory of Open Access Journals (Sweden)

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.

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

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 11...

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.

Complete Genome Sequence of Genotype VI Newcastle Disease Viruses Isolated from Pigeons in Pakistan

OpenAIRE

Wajid, Abdul; Rehmani, Shafqat Fatima; Sharma, Poonam; Goraichuk, Iryna V.; Dimitrov, Kiril M.; Afonso, Claudio L.

2016-01-01

Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI.

Isolation and characterization of gelatin-binding proteins from goat seminal plasma

Directory of Open Access Journals (Sweden)

Lazure Claude

2003-04-01

Full Text Available Abstract A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation and destabilization (capacitation process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins. Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in

Isolated corneal papilloma-like lesion associated with human papilloma virus type 6.

Science.gov (United States)

Park, Choul Yong; Kim, Eo-Jin; Choi, Jong Sun; Chuck, Roy S

2011-05-01

To report a case of a corneal papilloma-like lesion associated with human papilloma virus type 6. A 48-year-old woman presented with a 2-year history of ocular discomfort and gradual visual deterioration in her right eye. Ophthalmic examination revealed an elevated, semitranslucent, well-defined vascularized mass approximately 4 × 2.5 mm in size localized to the right cornea. The surface of the mass appeared smooth and many small, shallow, and irregular elevations were noted. An excisional biopsy was performed. The underlying cornea was markedly thinned, and fine ramifying vasculature was also noted on the exposed corneal stroma. Typical koilocytic change was observed on the histopathologic examination. Polymerase chain reaction revealed the existence of human papilloma virus type 6 DNA. Here we describe a case of an isolated corneal papilloma-like lesion. Although the corneal extension of the limbal or the conjunctival papillomas has been commonly observed, an isolated corneal papilloma-like lesion with underlying stromal destruction has only rarely been reported.

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

Science.gov (United States)

Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

2017-01-01

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

A low pathogenic H5N2 influenza virus isolated in Taiwan acquired high pathogenicity by consecutive passages in chickens.

OpenAIRE

Soda, Kosuke; Cheng, Ming-Chu; Yoshida, Hiromi; Endo, Mayumi; Lee, Shu-Hwae; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Wang, Ching-Ho; Kida, Hiroshi

2011-01-01

H5N2 viruses were isolated from cloacal swab samples of apparently healthy chickens in Taiwan in 2003 and 2008 during surveillance of avian influenza. Each of the viruses was eradicated by stamping out. The official diagnosis report indicated that the Intravenous Pathogenicity Indexes (IVPIs) of the isolates were 0.00 and 0.89, respectively, indicating that these were low pathogenic strains, although the hemagglutinin of the strain isolated in 2008 (Taiwan08) had multibasic amino acid residue...

[Molecular-biologic analysis of avian influenza virus isolates which caused epizootics on the south of West Siberia and in Crimea].

Science.gov (United States)

Onishchenko, G G; Berezhnov, S P; Shestopalov, A M; Alekseev, A Iu; Ternovoĭ, V A; Khaĭtovich, A B; Kroviakova, M T; Netesov, S V; Drozdov, I G

2007-01-01

The objective of the study was to determine reasons of poultry deaths in Crimea Republic in December 2005 as well as isolation, identification, and comparative analysis of pathogens, which caused epizootics in Siberia and Crimea. During epizootic in poultry in North-East Crimea highly pathogenic avian influenza virus H5N1 was isolated. Phylogenetic analysis of RNA sequences revealed that they belong to one big cluster. Isolated strain was close to viruses, which caused epizootic in July-August 2005 in the south of West Siberia. Conclusion about the high importance of the south of West Siberia in spreading of highly pathogenic influenza viruses H5N1 in Eurasia was made.

Genetic characterization of highly pathogenic avian influenza A H5N8 viruses isolated from wild birds in Egypt.

Science.gov (United States)

Kandeil, Ahmed; Kayed, Ahmed; Moatasim, Yassmin; Webby, Richard J; McKenzie, Pamela P; Kayali, Ghazi; Ali, Mohamed A

2017-07-01

A newly emerged H5N8 influenza virus was isolated from green-winged teal in Egypt during December 2016. In this study, we provide a detailed characterization of full genomes of Egyptian H5N8 viruses and some virological features. Genetic analysis demonstrated that the Egyptian H5N8 viruses are highly pathogenic avian influenza viruses. Phylogenetic analysis revealed that the genome of the Egyptian H5N8 viruses was related to recently characterized reassortant H5N8 viruses of clade 2.3.4.4 isolated from different Eurasian countries. Multiple peculiar mutations were characterized in the Egyptian H5N8 viruses, which probably permits transmission and virulence of these viruses in mammals. The Egyptian H5N8 viruses preferentially bound to avian-like receptors rather than human-like receptors. Also, the Egyptian H5N8 viruses were fully sensitive to amantadine and neuraminidase inhibitors. Chicken sera raised against commercial inactivated avian influenza-H5 vaccines showed no or very low reactivity with the currently characterized H5N8 viruses in agreement with the genetic dissimilarity. Surveillance of avian influenza in waterfowl provides early warning of specific threats to poultry and human health and hence should be continued.

Rapid differentiation of closely related isolates of two plant viruses by polymerase chain reaction and restriction fragment length polymorphism analysis.

Science.gov (United States)

Barbara, D J; Morton, A; Spence, N J; Miller, A

1995-09-01

Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the product has been shown to be an effective procedure for discriminating serologically indistinguishable isolates of two plant viruses, raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For both viruses, only limited sequence information was available at the time of primer design, but most of the isolates which were tested could be amplified (the one exception being a serologically quite distinct isolate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs were readily identified. Each of two isolates of ZYMV could be detected in the presence of the other and the relative proportions approximately quantified by visual estimation of the relative intensity of the appropriate bands. A range of isolates of different RBDV pathotypes were compared; isolates were grouped in ways that accorded with their known history. Computer analysis of the published sequence from which the primers had been derived showed the sequenced isolate to be identical with an isolate imported from the USSR. The PCR/RFLP procedure is rapid (it can be completed in less than 2 days), effective and will probably be generally applicable to distinguishing closely related virus isolates, even where little sequence information is available.

Genome characterization, antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south China.

Science.gov (United States)

Jiang, Lei; Zhao, Wenjun; Han, Zongxi; Chen, Yuqiu; Zhao, Yan; Sun, Junfeng; Li, Huixin; Shao, Yuhao; Liu, Liangliang; Liu, Shengwang

2017-10-01

In 2014, three infectious bronchitis virus (IBV) strains, designated as γCoV/ck/China/I0111/14, γCoV/ck/China/I0114/14 and γCoV/ck/China/I0118/14, were isolated and identified from chickens suspected to be infected with IBV in Guangxi province, China. Based upon data arising from S1 sequence and phylogenetic analyses, the three IBV isolates were genetically different from other known IBV types, which represented a novel genotype (GI-29). Virus cross-neutralization tests, using γCoV/ck/China/I0111/14 as a representative, showed that genotype GI-29 was antigenically different from all other known IBV types, thus representing a novel serotype. Complete genomic analysis showed that GI-29 type viruses were closely related to and might originate from a GX-YL5-like virus by accumulation of substitutions in multiple genes. These GI-29 viral genomes are still evolving and diverging, particularly in the 3' region, although we cannot rule out the possibility of recombination events occurring. For isolate γCoV/ck/China/I0114/14, we found that recombination events had occurred between nsps 2 and 3 in gene 1 which led to the introduction of a 4/91 gene fragment into the γCoV/ck/China/I0114/14 viral genome. In addition, we found that the GI-29 type γCoV/ck/China/I0111/14 isolate was a nephropathogenic strain and high pathogenic to 1-day-old specific pathogen-free (SPF) chickens although cystic oviducts were not observed in the surviving layer chickens challenged with γCoV/ck/China/I0111/14 isolate. Copyright © 2017 Elsevier B.V. All rights reserved.

Complete genome sequencing and phylogenetic analysis of dengue type 1 virus isolated from Jeddah, Saudi Arabia.

Science.gov (United States)

Azhar, Esam I; Hashem, Anwar M; El-Kafrawy, Sherif A; Abol-Ela, Said; Abd-Alla, Adly M M; Sohrab, Sayed Sartaj; Farraj, Suha A; Othman, Norah A; Ben-Helaby, Huda G; Ashshi, Ahmed; Madani, Tariq A; Jamjoom, Ghazi

2015-01-16

Dengue viruses (DENVs) are mosquito-borne viruses which can cause disease ranging from mild fever to severe dengue infection. These viruses are endemic in several tropical and subtropical regions. Multiple outbreaks of DENV serotypes 1, 2 and 3 (DENV-1, DENV-2 and DENV-3) have been reported from the western region in Saudi Arabia since 1994. Strains from at least two genotypes of DENV-1 (Asia and America/Africa genotypes) have been circulating in western Saudi Arabia until 2006. However, all previous studies reported from Saudi Arabia were based on partial sequencing data of the envelope (E) gene without any reports of full genome sequences for any DENV serotypes circulating in Saudi Arabia. Here, we report the isolation and the first complete genome sequence of a DENV-1 strain (DENV-1-Jeddah-1-2011) isolated from a patient from Jeddah, Saudi Arabia in 2011. Whole genome sequence alignment and phylogenetic analysis showed high similarity between DENV-1-Jeddah-1-2011 strain and D1/H/IMTSSA/98/606 isolate (Asian genotype) reported from Djibouti in 1998. Further analysis of the full envelope gene revealed a close relationship between DENV-1-Jeddah-1-2011 strain and isolates reported between 2004-2006 from Jeddah as well as recent isolates from Somalia, suggesting the widespread of the Asian genotype in this region. These data suggest that strains belonging to the Asian genotype might have been introduced into Saudi Arabia long before 2004 most probably by African pilgrims and continued to circulate in western Saudi Arabia at least until 2011. Most importantly, these results indicate that pilgrims from dengue endemic regions can play an important role in the spread of new DENVs in Saudi Arabia and the rest of the world. Therefore, availability of complete genome sequences would serve as a reference for future epidemiological studies of DENV-1 viruses.

Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

Science.gov (United States)

Schneider, William L; Damsteegt, Vernon D; Gildow, Fred E; Stone, Andrew L; Sherman, Diana J; Levy, Laurene E; Mavrodieva, Vessela; Richwine, Nancy; Welliver, Ruth; Luster, Douglas G

2011-05-01

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011.

Science.gov (United States)

Liu, Di; Zhang, Xiang-Bin; Yan, Zhuan-Qiang; Chen, Feng; Ji, Jun; Qin, Jian-Ping; Li, Hai-Yan; Lu, Jun-Peng; Xue, Yu; Liu, Jia-Jia; Xie, Qing-Mei; Ma, Jing-Yun; Xue, Chun-Yi; Bee, Ying-Zuo

2013-06-01

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that causes immunosuppressive disease in young chickens. Thousands of cases of IBDV infection are reported each year in South China, and these infections can result in considerable economic losses to the poultry industry. To monitor variations of the virus during the outbreaks, 30 IBDVs were identified from vaccinated chicken flocks from nine provinces in South China in 2011. VP2 fragments from different virus strains were sequenced and analyzed by comparison with the published sequences of IBDV strains from China and around the world. Phylogenetic analysis of hypervariable regions of the VP2 (vVP2) gene showed that 29 of the isolates were very virulent (vv) IBDVs, and were closely related to vvIBDV strains from Europe and Asia. Alignment analysis of the deduced amino acid (aa) sequences of vVP2 showed the 29 vv isolates had high uniformity, indicated low variability and slow evolution of the virus. The non-vvIBDV isolate JX2-11 was associated with higher than expected mortality, and had high deduced aa sequence similarity (99.2 %) with the attenuated vaccine strain B87 (BJ). The present study has demonstrated the continued circulation of IBDV strains in South China, and emphasizes the importance of reinforcing IBDV surveillance.

Isolation and Characterization of Avian Influenza Viruses, Including Highly Pathogenic H5N1, from Poultry in Live Bird Markets in Hanoi, Vietnam, in 2001

Science.gov (United States)

Nguyen, Doan C.; Uyeki, Timothy M.; Jadhao, Samadhan; Maines, Taronna; Shaw, Michael; Matsuoka, Yumiko; Smith, Catherine; Rowe, Thomas; Lu, Xiuhua; Hall, Henrietta; Xu, Xiyan; Balish, Amanda; Klimov, Alexander; Tumpey, Terrence M.; Swayne, David E.; Huynh, Lien P. T.; Nghiem, Ha K.; Nguyen, Hanh H. T.; Hoang, Long T.; Cox, Nancy J.; Katz, Jacqueline M.

2005-01-01

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia. PMID:15767421

Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

International Nuclear Information System (INIS)

Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

1990-01-01

The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

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

Science.gov (United States)

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.

2014-01-01

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. PMID:24421116

Circular viral DNA detection and junction sequence analysis from PBMC of SHIV-infected cynomolgus monkeys with undetectable virus plasma RNA

International Nuclear Information System (INIS)

Cara, Andrea; Maggiorella, Maria Teresa; Bona, Roberta; Sernicola, Leonardo; Baroncelli, Silvia; Negri, Donatella R.M.; Leone, Pasqualina; Fagrouch, Zahra; Heeney, Jonathan; Titti, Fausto; Cafaro, Aurelio; Ensoli, Barbara

2004-01-01

Extrachromosomal forms of human immunodeficiency virus (HIV)-1 can be detected in peripheral blood mononuclear cell (PBMC) from HIV-infected patients in the absence of detectable viral replication and are thought to be a sign of active but cryptic virus replication. No information, however, are available on whether these forms are also present in animal models for acquired immunodeficiency syndrome (AIDS) and on their relation with other methods of detection of virus replication. To this aim, a polymerase chain reaction (PCR) approach was used to detect and analyze unintegrated circular 2-LTR-containing forms in PBMC of simian human immunodeficiency virus (SHIV)89.6P infected cynomolgus monkeys with RNA levels ranging between 1.8x10 6 and less than 50 copies/ml of plasma. 2-LTR forms were detected in 96.5% of monkeys' samples above 50 copies/ml of plasma, whereas they were present in 75.8% of monkeys' samples below 50 copies/ml of plasma. Persistence of unintegrated viral DNA in monkeys with undetectable plasma RNA could indicate either stability in non-dividing cells or ongoing low levels of viral replication in dividing cells

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

Science.gov (United States)

Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

2012-10-01

This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

Thermal de-isolation of silicon microstructures in a plasma etching environment

International Nuclear Information System (INIS)

Lee, Yong-Seok; Jang, Yun-Ho; Kim, Yong-Kweon; Kim, Jung-Mu

2013-01-01

This paper presents a theoretical and experimental strategy for thermal de-isolation of silicon microstructures during a plasma etching process. Heat sinking blocks and thin metal layers are implemented around a thermally isolated mass to avoid severe spring width losses by a steep temperature rise. Thermal de-isolation significantly reduces the fabrication errors from −51.0% to −9.0% and from −39.5% to −6.7% for spring widths and resonant frequencies, respectively. Thermal de-isolation also reduces the standard deviation of resonant frequencies from 8.7% to 1.5% across a wafer, which clearly demonstrates the proposed method. (paper)

Complete Genome Sequence of Frog virus 3, Isolated from a Strawberry Poison Frog (Oophaga pumilio) Imported from Nicaragua into the Netherlands

NARCIS (Netherlands)

Saucedo, Bernardo; Hughes, Joseph; van Beurden, Steven J; Suárez, Nicolás M; Haenen, Olga L M; Voorbergen-Laarman, Michal A; Gröne, Andrea; Kik, Marja J L

2017-01-01

Frog virus 3 was isolated from a strawberry poison frog (Oophaga pumilio) imported from Nicaragua via Germany to the Netherlands, and its complete genome sequence was determined. Frog virus 3 isolate Op/2015/Netherlands/UU3150324001 is 107,183 bp long and has a nucleotide similarity of 98.26% to the

Complete genome sequence of frog virus 3, isolated from a strawberry poison frog (Oophaga pumilio) imported from nicaragua into the Netherlands

NARCIS (Netherlands)

Saucedo, Bernardo; Hughes, Joseph; Beurden, van Steven J.; Suárez, Nicolás M.; Haenen, Olga L.M.; Voorbergen-Laarman, Michal; Gröne, Andrea; Kika, Marja J.L.

2017-01-01

Frog virus 3 was isolated from a strawberry poison frog (Oophaga pumilio) imported from Nicaragua via Germany to the Netherlands, and its complete genome sequence was determined. Frog virus 3 isolate Op/2015/Netherlands/UU3150324001 is 107,183 bp long and has a nucleotide similarity of 98.26% to the

Restriction analysis of genetic variability of Polish isolates of Tomato black ring virus.

Science.gov (United States)

Jończyk, Magdalena; Borodynko, Natasza; Pospieszny, Henryk

2004-01-01

Several different isolates of Tomato black ring virus (TBRV) have been collected in Poland from cucumber, tomato, potato and black locust plants. Biological tests showed some differences in the range of infected plants and the type of symptoms, which was the basis for selection of seven the most biologically different TBRV isolates. According to the sequence of TBRV-MJ, several primer pairs were designed and almost the entire sequence of both genomic RNAs was amplified. The RT-PCR products derived from all tested TBRV isolates were digested by restriction enzymes. On the basis of the restriction patterns, the variable and the conserved regions of the TBRV genome were defined and the relationships between the Polish TBRV isolates established.

Characterization of Apricot pseudo-chlorotic leaf spot virus, A Novel Trichovirus Isolated from Stone Fruit Trees.

Science.gov (United States)

Liberti, D; Marais, A; Svanella-Dumas, L; Dulucq, M J; Alioto, D; Ragozzino, A; Rodoni, B; Candresse, T

2005-04-01

ABSTRACT A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only approximately 30 to 40 nt of the 5' terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only approximately 65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.

Complete Genome Sequence of Genotype VI Newcastle Disease Viruses Isolated from Pigeons in Pakistan

Science.gov (United States)

Wajid, Abdul; Rehmani, Shafqat Fatima; Sharma, Poonam; Goraichuk, Iryna V.; Dimitrov, Kiril M.

2016-01-01

Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI. PMID:27540069

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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...

Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains

International Nuclear Information System (INIS)

Bedows, E.; Payne, F.E.; Kohne, D.E.; Tourtellotte, W.W.

1982-01-01

A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs. 70-100% of the measles virus-specific RNA present in these SSPE brain samples were recovered in this enriched fraction. (Auth.)

Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains

Energy Technology Data Exchange (ETDEWEB)

Bedows, E; Payne, F E [Michigan Univ., Ann Arbor (USA). School of Public Health; Kohne, D E [Center for Neurologic Study, San Diego, CA, USA; Tourtellotte, W W [Neurology Service, V.A. Wadsworth Hospital Center, Los Angeles, CA, USA

1982-02-01

A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs. 70-100% of the measles virus-specific RNA present in these SSPE brain samples were recovered in this enriched fraction.

Genomic and biological characterization of Newcastle disease viruses isolated from migratory mallards (Anas platyrhynchos).

Science.gov (United States)

Habib, Momena; Yaqub, Tahir; Nazir, Jawad; Shehzad, Wasim; Aziz-Ul-Rahman; Sohail, Tayyebah; Mukhtar, Nadia; Mehboob, Arsalan; Munir, Muhammad; Shabbir, Muhammad Zubair

2018-04-30

Given the global evolutionary dynamics of Newcastle disease viruses (NDVs), it is imperative to continue extensive surveillance, routine monitoring and characterization of isolates originating from natural reservoirs (waterfowls). In this report, we isolated and characterized two virulent NDV strains from clinically healthy mallard (Anas platyrhynchos). Both isolates had a genome of 15,192 nucleotides encoding six genes in an order of 3´-NP-P-M-F-HN-L-5´. The biological characteristics (mean death time: 49.5-50 hr, EID 50 10 8.5  ml -1 ) and presence of a typical cleavage site in the fusion (F) protein (112R-R-Q-K-R↓F117) confirmed the velogenic nature of these isolates. Phylogenetic analysis classified both isolates as members of genotype VII within class-II. Furthermore, based upon the hypervariable region of the F gene (375 nt), isolates showed clustering within sub-genotype VIIi. Similarity index and parallel comparison revealed a higher nucleotide divergence from commonly used vaccine strains; LaSota (21%) and Mukteswar (17%). A comparative residues analysis with representative strains of different genotypes, including vaccine strains, revealed a number of substitutions at important structural and functional domains within the F and hemagglutinin-neuraminidase (HN) proteins. Together, the results highlight consistent evolution among circulating NDVs supporting extensive surveillance of the virus in waterfowl to better elucidate epidemiology, evolutionary relationships and their impacts on commercial and backyard poultry.

Characterization of low pathogenicity avian influenza viruses isolated from wild birds in Mongolia 2005 through 2007

Directory of Open Access Journals (Sweden)

Sodnomdarjaa Ruuragchaa

2009-11-01

Full Text Available Abstract Background Since the emergence of H5N1 high pathogenicity (HP avian influenza virus (AIV in Asia, numerous efforts worldwide have focused on elucidating the relative roles of wild birds and domestic poultry movement in virus dissemination. In accordance with this a surveillance program for AIV in wild birds was conducted in Mongolia from 2005-2007. An important feature of Mongolia is that there is little domestic poultry production in the country, therefore AIV detection in wild birds would not likely be from spill-over from domestic poultry. Results During 2005-2007 2,139 specimens representing 4,077 individual birds of 45 species were tested for AIV by real time RT-PCR (rRT-PCR and/or virus isolation. Bird age and health status were recorded. Ninety rRT-PCR AIV positive samples representing 89 individual birds of 19 species including 9 low pathogenicity (LP AIVs were isolated from 6 species. A Bar-headed goose (Anser indicus, a Whooper swan (Cygnus cygnus and 2 Ruddy shelducks (Tadorna ferruginea were positive for H12N3 LP AIV. H16N3 and H13N6 viruses were isolated from Black-headed gulls (Larus ridibundus. A Red-crested pochard (Rhodonessa rufina and 2 Mongolian gulls (Larus vagae mongolicus were positive for H3N6 and H16N6 LP AIV, respectively. Full genomes of each virus isolate were sequenced and analyzed phylogenetically and were most closely related to recent European and Asian wild bird lineage AIVs and individual genes loosely grouped by year. Reassortment occurred within and among different years and subtypes. Conclusion Detection and/or isolation of AIV infection in numerous wild bird species, including 2 which have not been previously described as hosts, reinforces the wide host range of AIV within avian species. Reassortment complexity within the genomes indicate the introduction of new AIV strains into wild bird populations annually, however there is enough over-lap of infection for reassortment to occur. Further work is

Phylogenetic relationships and pathogenicity variation of two Newcastle disease viruses isolated from domestic ducks in Southern China.

Science.gov (United States)

Kang, Yinfeng; Li, Yanling; Yuan, Runyu; Li, Xianwei; Sun, Minhua; Wang, Zhaoxiong; Feng, Minsha; Jiao, Peirong; Ren, Tao

2014-08-12

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which is enzootic and causes large economic losses in the poultry sector. Genotype VII and genotype IX NDV viruses were the predominant circulating genotype in China, which may possibly be responsible for disease outbreaks in chicken flocks in recent years. While ducks and geese usually have exhibited inapparent infections. In the present study, we investigate the complete genome sequence, the clinicopathological characterization and transmission of two virulent Newcastle disease viruses, SS-10 and NH-10, isolated from domestic ducks in Southern China in 2010. F, and the complete gene sequences based on phylogenetic analysis demonstrated that SS-10 (genotype VII) and NH-10 (genotype IX) belongs to class II. The deduced amino acid sequence was (112)R-R-Q-K/R-R-F(117) at the fusion protein cleavage site. Animal experiment results showed that the SS-10 virus isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks. It could be detected from spleen, lung, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. Moreover, it could transmit to chickens, ducks and geese by naive contact. However, the NH-10 virus isolated from ducks could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to the naive contact chickens, ducks, and geese. The two NDV isolates exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. Therefore, no species-preference exists for chicken, duck or goose viruses and more attention should be paid to the trans-species transmission of VII NDVs between ducks, geese and chickens for the control and eradication of ND.

Genetic and antigenic relationship of foot-and-mouth disease virus serotype O isolates with the vaccine strain O1/BFS.

Science.gov (United States)

Xu, Wanhong; Zhang, Zhidong; Nfon, Charles; Yang, Ming

2018-05-15

Foot-and-mouth disease serotype O viruses (FMDV/O) are responsible for the most outbreaks in FMD endemic countries. O1/BFS is one of the recommended FMD/O vaccine strains by World Reference Laboratory for FMD. In the current study, FMDV/O1 BFS vaccine strain and serotype O field isolates (45) were analyzed phylogenetically and antigenically to gain more insight into the genetic and antigenic characteristics of the vaccine strain and field isolates. O1/BFS showed similarity with 89% of the field isolates using a virus neutralization test (VNT). The P1 region encoding the FMDV capsid was sequenced and analysed for 46 strains of FMDV/O. Phylogenetic analysis showed these viruses originated from five continents and covered eight of 11 reported topotypes. Five isolates that demonstrated low antigenic similarities with O1/BFS were analyzed for their antigenic variation at the known neutralizing antigenic sites. Three of the five isolates demonstrated unique amino acid substitutions at various antigenic sites. No unique amino acid substitutions were observed for the other two unmatched isolates. Positively selected residues were identified on the surface of the FMD virus capsid supporting that it is important to continuously monitor field isolates for their antigenic and phenotypic changes. In conclusion, the vaccine strain O1/BFS is likely to confer protection against 89% of the 45 FMDV/O isolates based on VNT. Thus O1/BFS vaccine strain is still suitable for use in global FMD serotype O outbreak control. Combining data from phylogenetic, molecular and antigenic analysis can provide improvements in the process of vaccine selection. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

Foot-and-mouth disease virus: A first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods.

NARCIS (Netherlands)

Ferris, N.P.; King, D.P.; Reid, S.M.; Hutchings, G.H.; Shawa, A.E.; Paton, D.J.; Goris, N.; Haas, B.; Hoffmann, B.; Brocchi, E.; Bugnetti, M.; Dekker, A.; Clerq, De K.

2006-01-01

Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10

Susceptibility of peach GF 305 seedlings and selected herbaceous plants to plum pox virus isolates from western Slovakia.

Science.gov (United States)

Glasa, M; Matisová, J; Hricovský, I; Kúdela, O

1997-12-01

The susceptibility of peach GF 305 seedlings and herbaceous plants to five plum pox virus (PPV) isolates from orchards of western Slovakia was investigated. PPV was isolated from diseased plum, apricot and peach trees, and transmitted by chip-budding to peach GF 305. The herbaceous plants were infected by mechanical inoculation. The transmission was analysed by symptomatology and double sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Infected peaches developed leaf distortion, tissue clearing along the veins and small chlorotic spots (isolate BOR-3). With exception of BOR-3, the PPV isolates transmitted from peach caused local chlorotic spots on Chenopodium foetidum. The character of symptoms changed when a sap from PPV-infected Nicotiana benthamiana was used as virus inoculum. From N. benthamiana, the PPV isolates could be transmitted to Pisum sativum, cv. Colmo (light green mosaic), N. clevelandii and N. clevelandii x N. glutinosa hybrid (latent infection or chlorotic spots).

Characterization of Seasonal Influenza Virus Type and Subtypes Isolated from Influenza Like Illness Cases of 2012.

Science.gov (United States)

Upadhyay, B P; Ghimire, P; Tashiro, M; Banjara, M R

Background Seasonal influenza is one of the increasing public health burdens in Nepal. Objective The objective of this study was to isolate and characterize the influenza virus type and subtypes of Nepal. Method A total of 1536 throat swab specimens were collected from January to December 2012. Total ribonucleic acid was extracted using Qiagen viral nucleic acid extraction kit and polymerase chain reaction assay was performed following the US; CDC Real-time PCR protocol. Ten percent of positive specimens were inoculated onto Madin-Darby Canine Kidney cells. Isolates were characterized by using reference ferret antisera. Result Of the total specimens (n=1536), influenza virus type A was detected in 196 (22%) cases; of which 194 (99%) were influenza A (H1N1) pdm09 and 2 (1 %) were influenza A/H3 subtype. Influenza B was detected in 684 (76.9%) cases. Influenza A (H1N1) pdm09, A/H3 and influenza B virus were antigenically similar to the recommended influenza virus vaccine candidate of the year 2012. Although sporadic cases of influenza were observed throughout the year, peak was observed during July to November. Conclusion Similar to other tropical countries, A (H1N1) pdm09, A/H3 and influenza B viruses were co-circulated in Nepal.

Complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of HIV-1 infection.

Directory of Open Access Journals (Sweden)

Michael Huber

2006-11-01

Full Text Available BACKGROUND: To explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus. METHODS AND FINDINGS: Sera from two groups of patients-25 with acute HIV-1 infection and 31 with chronic infection-were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies. CONCLUSIONS: Collectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.

Biological and molecular characterization of Brazilian isolates of Zucchini yellow mosaic virus

Directory of Open Access Journals (Sweden)

David Marques de Almeida Spadotti

2015-02-01

Full Text Available Zucchini yellow mosaic virus (ZYMV causes substantial economic losses in cucurbit crops. Although ZYMV has been present in Brazil for more than 20 years, there is little information about the biological and molecular characteristics of the isolates found in the country. This study aimed to characterize the experimental hosts, pathotypes and genetic diversity of a collection of eleven Brazilian ZYMV isolates within the coat protein gene. For biological analysis, plant species from Amaranthaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Pedaliaceae were mechanically inoculated and pathotypes were identified based on the reaction of a resistant Cucumis melo, accession PI414723. All of the cucurbit species/varieties and Sesamum indicum were systemically infected with all isolates. The nucleotide sequence variability of the coat protein gene ranged from 82 % to 99 % compared to the corresponding sequences of ZYMV isolates from different geographical locations. No recombination event was detected in the coat protein gene of the isolates.

First Complete Genome Sequence of Papaya ringspot virus-W Isolated from a Gourd in the United States.

Science.gov (United States)

Ali, Akhtar

2017-01-12

In the United States, the Papaya ringspot virus was first reported from papaya in Florida in 1949. Here, we determined the first complete genome sequence (10,302 nucleotides) of a Papaya ringspot virus-W isolate, which was collected from a commercial field of gourd in Tulsa, OK. Copyright © 2017 Ali.

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus.

Science.gov (United States)

Hammond, R W; Crosslin, J M

1995-04-01

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Sequence-based comparative study of classical swine fever virus genogroup 2.2 isolate with pestivirus reference strains.

Science.gov (United States)

Kumar, Ravi; Rajak, Kaushal Kishor; Chandra, Tribhuwan; Muthuchelvan, Dhanavelu; Saxena, Arpit; Chaudhary, Dheeraj; Kumar, Ajay; Pandey, Awadh Bihari

2015-09-01

This study was undertaken with the aim to compare and establish the genetic relatedness between classical swine fever virus (CSFV) genogroup 2.2 isolate and pestivirus reference strains. The available complete genome sequences of CSFV/IND/UK/LAL-290 strain and other pestivirus reference strains were retrieved from GenBank. The complete genome sequence, complete open reading frame, 5' and 3' non-coding region (NCR) sequences were analyzed and compared with reference pestiviruses strains. Clustal W model in MegAlign program of Lasergene 6.0 software was used for analysis of genetic heterogeneity. Phylogenetic analysis was carried out using MEGA 6.06 software package. The complete genome sequence alignment of CSFV/IND/UK/LAL-290 isolate and reference pestivirus strains showed 58.9-72% identities at the nucleotide level and 50.3-76.9% at amino acid level. Sequence homology of 5' and 3' NCRs was found to be 64.1-82.3% and 22.9-71.4%, respectively. In phylogenetic analysis, overall tree topology was found similar irrespective of sequences used in this study; however, whole genome phylogeny of pestivirus formed two main clusters, which further distinguished into the monophyletic clade of each pestivirus species. CSFV/IND/UK/LAL-290 isolate placed with the CSFV Eystrup strain in the same clade with close proximity to border disease virus and Aydin strains. CSFV/IND/UK/LAL-290 exhibited the analogous genomic organization to those of all reference pestivirus strains. Based on sequence identity and phylogenetic analysis, the isolate showed close homology to Aydin/04-TR virus and distantly related to Bungowannah virus.

Characterization of a low pathogenic avian influenza H5N2 virus isolated from a turkey breeder flock in Manitoba, Canada.

Science.gov (United States)

Berhane, Y; Joseph, T; Kehler, H; Hisanaga, T; Embury-Hyatt, C; Diederich, S; McGreevy, K Hooper; Handel, K; Cottam-Birt, C; Pasick, J

2014-03-01

In November 2010, an outbreak of avian influenza (AI) due to the H5N2 subtype virus occurred in a turkey breeder farm in northern Manitoba, Canada. The only clinical signs observed were depression, decrease in food consumption, and loss of egg production. The hemagglutinin (HA) cleavage (HA(0)) site of the isolated H5N2 virus was PQRETR/GLF, consistent with low pathogenic AI viruses. The intravenous pathogenicity index of this virus was zero. Whole-genome sequencing of two isolates that originated from two different barns was performed, and both isolates had 100% identical protein sequence in PB2, HA, NP, M1, M2, NS1, and NS2. The remaining gene segments (PB1, PA, and NA) had a single amino-acid difference when compared with each other. The nucleotide and protein sequences of eight gene segments from both isolates showed 99 or greater identity with other AI viruses that have been circulating in free-living aquatic birds in Canada and the United States within the last 10 yr. Phylogenetic analysis of the HA and neuraminidase (NA) gene segments showed that these viruses are closely related to other H5 strains that have been isolated from Manitoba and other parts of Canada. Serologic testing of archived serum samples collected from these turkeys a week before the outbreak showed no evidence of AI infection. In addition, other farms that were located within 3 km radius from the infected farm and farms that had epidemiologic connection with the farm also tested negative for the presence of H5N2 AI virus or antibody. This indicates that the virus might have been introduced to the farm from wild aquatic birds only a short time before detection. Results of this study highlight the importance of early detection and the significance of ongoing Canada-wide surveillance of AI in domestic poultry as well as in wild aquatic birds/ducks.

Molecular characterization of Banana streak virus isolate from Musa Acuminata in China.

Science.gov (United States)

Zhuang, Jun; Wang, Jian-Hua; Zhang, Xin; Liu, Zhi-Xin

2011-12-01

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, ORF1 with a non-AUG start codon 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 Vietnam (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.

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

OpenAIRE

Gao, Yugang; Wang, Shijie; Du, Rui; Wang, Quankai; Sun, Changjiang; Wang, Nan; 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...

Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient.

Directory of Open Access Journals (Sweden)

Mirella Biava

2017-01-01

Full Text Available An unprecedented Ebola virus (EBOV epidemic occurred in 2013-2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA and the presence of positive sense RNA (pos-RNA, including both replication intermediate (antigenomic-RNA and messenger RNA (mRNA molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases "Lazzaro Spallanzani" (INMI, Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract.

Comparison of Nucleotide Sequence of P2C Region in Diabetogenic and Non-Diabetogenic Coxsackie Virus B5 Isolates

Directory of Open Access Journals (Sweden)

Cheng-Chong Chou

2004-11-01

Full Text Available Enteroviruses are environmental triggers in the pathogenesis of type 1 diabetes mellitus (DM. A sequence of six identical amino acids (PEVKEK is shared by the 2C protein of Coxsackie virus B and the glutamic acid decarboxylase (GAD molecules. Between 1995 and 2002, we investigated 22 Coxsackie virus B5 (CVB5 isolates from southern Taiwan. Four of these isolates were obtained from four new-onset type 1 DM patients with diabetic ketoacidosis. We compared a 300 nucleotide sequence in the 2C protein gene (p2C in 24 CVB5 isolates (4 diabetogenic, 18 non-diabetogenic and 2 prototype. We found 0.3-10% nucleotide differences. In the four isolates from type 1 DM patients, there was only 2.4-3.4% nucleotide difference, and there was only 1.7-7.1% nucleotide difference between type 1 DM isolates and non-diabetogenic isolates. Comparison of the nucleotide sequence between prototype virus and 22 CVB5 isolates revealed 18.4-24.1% difference. Twenty-one CVB5 isolates from type 1 DM and non-type 1 DM patients contained the PEVKEK sequence, as shown by the p2C nucleotide sequence. Our data showed that the viral p2C sequence with homology with GAD is highly conserved in CVB5 isolates. There was no difference between diabetogenic and non-diabetogenic CVB5 isolates. All four type 1 DM patients had at least one of the genetic susceptibility alleles HLA-DR, DQA1, DQB1. Other genetic and autoimmune factors such as HLA genetic susceptibility and GAD may also play important roles in the pathogenesis in type 1 DM.

Pathogenesis of swine influenza virus (Thai isolates in weanling pigs: an experimental trial

Directory of Open Access Journals (Sweden)

Kitikoon Pravina

2009-03-01

Full Text Available Abstract Background The objective of this study is to investigate the pathogenesis of swine influenza virus (SIV subtype H1N1 and H3N2 (Thai isolates in 22-day-old SPF pigs. Results The study found that all pigs in the infected groups developed typical signs of flu-like symptoms on 1–4 days post- infection (dpi. The H1N1-infected pigs had greater lung lesion scores than those of the H3N2-infected pigs. Histopathological lesions related to swine influenza-induced lesions consisting of epithelial cells damage, airway plugging and peribronchial and perivascular mononuclear cell infiltration were present in both infected groups. Immunofluorescence and immunohistochemistry using nucleoprotein specific monoclonal antibodies revealed positive staining cells in lung sections of both infected groups at 2 and 4 dpi. Virus shedding was detected at 2 dpi from both infected groups as demonstrated by RT-PCR and virus isolation. Conclusion The results demonstrated that both SIV subtypes were able to induce flu-like symptoms and lung lesions in weanling pigs. However the severity of the diseases with regards to lung lesions both gross and microscopic lesions was greater in the H1N1-infected pigs. Based on phylogenetic analysis, haemagglutinin gene of subtype H1N1 from Thailand clustered with the classical H1 SIV sequences and neuraminidase gene clustered with virus of avian origin, whereas, both genes of H3N2 subtype clustered with H3N2 human-like SIV from the 1970s.

Characterization of field isolates of Suid herpesvirus 1 (Aujeszky's disease virus) as derivatives of attenuated vaccine strains

DEFF Research Database (Denmark)

Christensen, Laurids Siig; Medveczky, I.; Strandbygaard, Bertel

1992-01-01

Field isolates of suid herpesvirus 1 (Aujeszky's disease virus) from Poland and Hungary were identified by restriction fragment pattern analysis as derivatives of attenuated vaccine strains. The Polish isolates were found to be related to the BUK-TK-900 strain (Suivac A) which is widely used...

Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

Science.gov (United States)

Rasmussen, N S; Nielsen, C T; Houen, G; Jacobsen, S

2016-12-01

We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients. © The Author(s) 2016.

Characterization of sour cherry isolates of plum pox virus from the Volga Basin in Russia reveals a new cherry strain of the virus.

Science.gov (United States)

Glasa, Miroslav; Prikhodko, Yuri; Predajňa, Lukáš; Nagyová, Alžbeta; Shneyder, Yuri; Zhivaeva, Tatiana; Subr, Zdeno; Cambra, Mariano; Candresse, Thierry

2013-09-01

Plum pox virus (PPV) is the causal agent of sharka, the most detrimental virus disease of stone fruit trees worldwide. PPV isolates have been assigned into seven distinct strains, of which PPV-C regroups the genetically distinct isolates detected in several European countries on cherry hosts. Here, three complete and several partial genomic sequences of PPV isolates from sour cherry trees in the Volga River basin of Russia have been determined. The comparison of complete genome sequences has shown that the nucleotide identity values with other PPV isolates reached only 77.5 to 83.5%. Phylogenetic analyses clearly assigned the RU-17sc, RU-18sc, and RU-30sc isolates from cherry to a distinct cluster, most closely related to PPV-C and, to a lesser extent, PPV-W. Based on their natural infection of sour cherry trees and genomic characterization, the PPV isolates reported here represent a new strain of PPV, for which the name PPV-CR (Cherry Russia) is proposed. The unique amino acids conserved among PPV-CR and PPV-C cherry-infecting isolates (75 in total) are mostly distributed within the central part of P1, NIa, and the N terminus of the coat protein (CP), making them potential candidates for genetic determinants of the ability to infect cherry species or of adaptation to these hosts. The variability observed within 14 PPV-CR isolates analyzed in this study (0 to 2.6% nucleotide divergence in partial CP sequences) and the identification of these isolates in different localities and cultivation conditions suggest the efficient establishment and competitiveness of the PPV-CR in the environment. A specific primer pair has been developed, allowing the specific reverse-transcription polymerase chain reaction detection of PPV-CR isolates.

Characterisation of a highly pathogenic H5N1 clade 2.3.2 influenza virus isolated from swans in Shanghai, China.

Science.gov (United States)

Zhao, Guo; Zhong, Lei; Lu, Xinlun; Hu, Jiao; Gu, Xiaobing; Kai, Yan; Song, Qingqing; Sun, Qing; Liu, Jinbao; Peng, Daxin; Wang, Xiaoquan; Liu, Xiaowen; Liu, Xiufan

2012-02-01

In spring 2009, one strain of H5N1 clade 2.3.2 virus was isolated from wild swans in Shanghai, indicating the importance of the wild swan in the ecology of this highly pathogenic avian influenza virus (HPAIV) in Eastern China. Pathogenicity experiments conducted in this study indicated that the virus was highly pathogenic for chickens but lowly pathogenic for mammalian hosts, as evidenced by reduced infection of mice. The analysis of complete genome sequences and genetic evolution showed that A/Swan/Shanghai/10/09 (SW/SH/09) may be derived from the strain A/silky chicken/Shantou/475/2004 (CK/ST/04), which is homologous to the influenza viruses isolated from chicken, duck, pika, little egret, swan, mandarin duck and bar-headed goose in China Hunan, China Qinghai, Mongolia, Russia, Japan, Korea, Laos and Hong Kong during 2007-2011, indicating that the virus has retro-infected diverse wild birds from chicken, and significant spread of the virus is still ongoing through overlapping migratory flyways. On the basis of the molecular analysis, we also found that there was a deletion of the glycosylation site (NSS) in amino acid 156 of the hemagglutinin (HA) protein when compared with that of the other Clade 2.3.2 viruses isolated between 2007 and 2011. More importantly, the sequence analysis of SW/SH/09 virus displayed the drug-resistant mutations on the matrix protein (M2) and neuraminidase (NA) genes.

Complexities in Isolation and Purification of Multiple Viruses from Mixed Viral Infections: Viral Interference, Persistence and Exclusion.

Directory of Open Access Journals (Sweden)

Naveen Kumar

Full Text Available Successful purification of multiple viruses from mixed infections remains a challenge. In this study, we investigated peste des petits ruminants virus (PPRV and foot-and-mouth disease virus (FMDV mixed infection in goats. Rather than in a single cell type, cytopathic effect (CPE of the virus was observed in cocultured Vero/BHK-21 cells at 6th blind passage (BP. PPRV, but not FMDV could be purified from the virus mixture by plaque assay. Viral RNA (mixture transfection in BHK-21 cells produced FMDV but not PPRV virions, a strategy which we have successfully employed for the first time to eliminate the negative-stranded RNA virus from the virus mixture. FMDV phenotypes, such as replication competent but noncytolytic, cytolytic but defective in plaque formation and, cytolytic but defective in both plaque formation and standard FMDV genome were observed respectively, at passage level BP8, BP15 and BP19 and hence complicated virus isolation in the cell culture system. Mixed infection was not found to induce any significant antigenic and genetic diversity in both PPRV and FMDV. Further, we for the first time demonstrated the viral interference between PPRV and FMDV. Prior transfection of PPRV RNA, but not Newcastle disease virus (NDV and rotavirus RNA resulted in reduced FMDV replication in BHK-21 cells suggesting that the PPRV RNA-induced interference was specifically directed against FMDV. On long-term coinfection of some acute pathogenic viruses (all possible combinations of PPRV, FMDV, NDV and buffalopox virus in Vero cells, in most cases, one of the coinfecting viruses was excluded at passage level 5 suggesting that the long-term coinfection may modify viral persistence. To the best of our knowledge, this is the first documented evidence describing a natural mixed infection of FMDV and PPRV. The study not only provides simple and reliable methodologies for isolation and purification of two epidemiologically and economically important groups of

Masking of the contribution of V protein to sendai virus pathogenesis in an infection model with a highly virulent field isolate

International Nuclear Information System (INIS)

Sakaguchi, Takemasa; Kiyotani, Katsuhiro; Watanabe, Hitoshi; Huang Cheng; Fukuhara, Noriko; Fujii, Yutaka; Shimazu, Yukie; Sugahara, Fumihiro; Nagai, Yoshiyuki; Yoshida, Tetsuya

2003-01-01

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted

Biological, serological and molecular typing of potato virus Y (PVY) isolates from Tunisia.

Science.gov (United States)

Tayahi, M; Gharsallah, C; Khamassy, N; Fakhfakh, H; Djilani-Khouadja, F

2016-10-17

In Tunisia, potato virus Y (PVY) currently presents a significant threat to potato production, reducing tuber yield and quality. Three hundred and eighty-five potato samples (six different cultivars) collected in autumn 2007 from nine regions in Tunisia were tested for PVY infection by DAS-ELISA. The virus was detected in all regions surveyed, with an average incidence of 80.26%. Subsequently, a panel of 82 Tunisian PVY isolates (PVY-TN) was subjected to systematic biological, serological and molecular typing using immunocapture reverse-transcription polymerase chain reaction and a series of PVY OC - and PVY N -specific monoclonal antibodies. Combined analyses revealed ~67% of PVY NTN variants of which 17 were sequenced in the 5'NTR-P1 region to assess the genetic diversity and phylogenetic relationship of PVY-TN against other worldwide PVY isolates. To investigate whether selective constraints could act on viral genomic RNA, synonymous and non-synonymous substitution rates and their ratio were analyzed. Averages of all pairwise comparisons obtained in the 5'NTR-P1 region allowed more synonymous changes, suggesting selective constraint acting in this region. Selective neutrality test was significantly negative, suggesting a rapid expansion of PVY isolates. Pairwise mismatch distribution gave a bimodal pattern and pointed to an eventually early evolution characterizing these sequences. Genetic haplotype network topology provided evidence of the existence of a distinct geographical structure. This is the first report of such genetic analyses conducted on PVY isolates from Tunisia.

First Report of Cucumber mosaic virus Isolated from Sambungai (Gynura procumbens

Directory of Open Access Journals (Sweden)

June Kwon

2017-12-01

Full Text Available In March 2016, an isolate of Cucumber mosaic virus (named Gyp-CMV was isolated from the Sambungai (Gynura procumbens showing the symptoms of mosaic and chlorosis. The isolate Gyp-CMV was characterized by disease reactions in several indicator plants, reverse transcription-polymerase chain reaction (RT-PCR, PCR-restriction fragment length polymorphism, and sequence analysis of movement protein (3a and coat protein (CP genes. Tobacco, tomato, pepper, ground cherry, and lambsquarters (Chenopodium quinoa and C. amaranticolor appeared typical CMV symptoms, but zucchini and cucumber were not infected. Phylogenetic analysis of the 3a and CP gene indicated that Gyp-CMV belongs to the CMV subgroup II. Sequence identities of the Gyp-CMV 3a and CP genes showed 99.3% and 100% to that of Hnt-CMV at amino acid level. To our knowledge, this is the first report of CMV infection in Gynura procumbens.

Characterization of Oita virus 296/1972 of Rhabdoviridae isolated from a horseshoe bat bearing characteristics of both lyssavirus and vesiculovirus.

Science.gov (United States)

Iwasaki, T; Inoue, S; Tanaka, K; Sato, Y; Morikawa, S; Hayasaka, D; Moriyama, M; Ono, T; Kanai, S; Yamada, A; Kurata, T

2004-06-01

Oita virus 296/1972 was isolated from the blood of a wild horseshoe bat, Rhinolophus cornutus (Temminck) in 1972. We investigated the pathogenicity of this virus in mice in relation to its histological, immunohistochemical and ultrastructural characteristics and the entire sequence of nucleoprotein gene. This virus caused lethal encephalitis in mice through intracerebral route. This susceptibility of mice was until 3 weeks of age. Immunohistochemical analysis using the convalescent sera obtained from survived adult mice after intracerebral inoculation revealed that many neurons were positive in the cytoplasm, besides no cross reactivity with normal and rabies virus-infected mouse brain tissues to this anti-sera. Ultrastructural analysis disclosed many bullet-shaped and enveloped virions in neurons. These morphological characteristics of the virions are consistent of that of viruses in the family Rhabdoviridae. Budding from endoplasmic membrane suggests that this virus has a similarity with lyssaviruses. Molecular analysis of cDNA coding a tentative nucleoprotein sequence revealed homology with those of viruses in the family Rhabdoviridae. Distance matrix analysis of this gene sequence with those of other rhabdoviruses isolated from mammals disclosed the discrete position of this virus in the phylogenic tree of rhabdoviridae infecting mammals and we renamed this virus as Oita rhabdovirus.

Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

Science.gov (United States)

Ramsey, Jolene; Renzi, Emily C; Arnold, Randy J; Trinidad, Jonathan C; Mukhopadhyay, Suchetana

2017-02-01

Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the

Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

Science.gov (United States)

2010-01-01

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. PMID:20398268

Isolates of Liao ning virus from wild-caught mosquitoes in the Xinjiang province of China in 2005.

Directory of Open Access Journals (Sweden)

Xinjun Lv

Full Text Available Liao ning virus (LNV is related to Banna virus, a known human-pathogen present in south-east Asia. Both viruses belong to the genus Seadornavirus, family Reoviridae. LNV causes lethal haemorrhage in experimentally infected mice. Twenty seven isolates of LNV were made from mosquitoes collected in different locations within the Xinjiang province of north-western China during 2005. These mosquitoes were caught in the accommodation of human patients with febrile manifestations, or in animal barns where sheep represent the main livestock species. The regions where LNV was isolated are affected by seasonal encephalitis, but are free of Japanese encephalitis (JE. Genome segment 10 (Seg-10 (encoding cell-attachment and serotype-determining protein VP10 and Seg-12 (encoding non-structural protein VP12 were sequenced for multiple LNV isolates. Phylogenetic analyses showed a less homogenous Seg-10 gene pool, as compared to segment 12. However, all of these isolates appear to belong to LNV type-1. These data suggest a relatively recent introduction of LNV into Xinjiang province, with substitution rates for LNV Seg-10 and Seg-12, respectively, of 2.29×10(-4 and 1.57×10(-4 substitutions/nt/year. These substitution rates are similar to those estimated for other dsRNA viruses. Our data indicate that the history of LNV is characterized by a lack of demographic fluctuations. However, a decline in the LNV population in the late 1980s-early 1990s, was indicated by data for both Seg-10 and Seg-12. Data also suggest a beginning of an expansion in the late 1990s as inferred from Seg-12 skyline plot.

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.

Molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in Egypt during 2012

Directory of Open Access Journals (Sweden)

Karim Selim

2013-12-01

Full Text Available One of the major problems of avian infectious bronchitis virus (IBV is the frequent emergence of new variants. In the present study 205 tracheal swabs and organs were collected from broilers and layers chicken farms during January to August 2012 from 19 governorates all over Egypt. The chickens demonstrated respiratory signs and mortality. Out of the examined samples, 130 of which (about 64% of suspected farms were positive for IBV with real time RT-PCR. 13 IBV-positive samples were selected for further isolation and characterization. Isolation in specific pathogen free (SPF embryos was carried out after studies three blind successive passages and the hypervariable region of spike protein1 (SP1 was amplified by RT-PCR and sequenced to study the genetic diversity between the isolated viruses. Phylogenetic analysis of the obtained sequences of 13 isolates compared with other IBV strains from the Middle East and worldwide reveled that 11 out of the 13 isolates had close relationship the Israeli variants (IS/885 and IS/1494/06 with nucleotide homology reached up to 89.9% and 82.3%, respectively. Only two isolates had close relationship with CR/88121 and 4/91 viruses with identities of 95% and 96%, respectively. This study indicates existence of two variant groups of IBV circulating in Egypt during 2012. Group I was similar but distinguishable from Israeli variant IS/885 and group II was related to 4/91 and CR/88121 vaccine strains. There was no geographical link between the 2 groups as they were distributed all over the country. These findings necessitate the need to revise the vaccination programs and control measures for IBV.

Comparison of Plasmagel with LeucoPREP-Macrodex methods for separation of leukocytes for virus isolation.

Science.gov (United States)

Woods, G L; Proffitt, M R

1987-10-01

Plasmagel (Cellular Products, Inc., Buffalo, NY), which can separate both polymorphonuclear leukocytes (PMN) and mononuclear cells from other blood components, and LeucoPREP (Becton Dickinson Immunocytometry Systems, Mountain View, CA), which can separate mononuclear cells from other blood components, were used to harvest leukocytes from whole blood for the purpose of virus isolation. Macrodex was combined with the later, in a second step, for recovery of PMN. Of 90 peripheral blood specimens examined, cytomegalovirus was recovered from 10: in six by both methods, in three from Plasmagel prepared cells only, and in one from cells from the LeucoPREP-Macrodex preparation only. Total leukocyte counts, differential counts, and leukocyte viability did not differ significantly for the two methods. Plasmagel provided an efficient, inexpensive means of harvesting leukocytes from whole blood for virus isolation.

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.

The HIV-1 V3 domain on field isolates: participation in generation of escape virus in vivo and accessibility to neutralizing antibodies

DEFF Research Database (Denmark)

Arendrup, M; Akerblom, L; Heegaard, P M

1995-01-01

The V3 domain is highly variable and induces HIV neutralizing antibodies (NA). Here we addressed the issues of 1) the participation of mutations in V3 in generation of neutralization resistant escape virus in vivo and 2) the applicability of synthetic V3 peptides corresponding to field isolates...... to induce neutralizing immune sera. Seven peptides corresponding to the V3 region of primary and escape virus from 3 HIV-1 infected patients were synthesized and used for antibody (Abs) studies and immunizations. The anti-V3 Abs titre in patient serum was generally low against peptides corresponding...... to autologous virus isolated later than the serum sample in contrast to the titre against peptides corresponding to virus isolated earlier than the serum sample. Furthermore, neutralizing anti-V3 monoclonal antibodies (MAbs) raised against V3 peptides from laboratory strains of HIV-1 showed distinct binding...

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

International Nuclear Information System (INIS)

Wheeler, J.J.; Boss, W.F.

1987-01-01

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2- 3 H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [ 3 H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2 ). An additional [ 3 H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP 2 on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitchrondrial-rich fraction

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

Science.gov (United States)

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Short communication: isolation and phylogenetic analysis of an avian-origin H3N2 canine influenza virus in dog shelter, China.

Science.gov (United States)

Su, Shuo; Yuan, Ziguo; Chen, Jidang; Xie, Jiexiong; Li, Huatao; Huang, Zhen; Zhang, Minze; Du, Guohao; Chen, Zhongming; Tu, Liqing; Zou, Yufei; Miao, Junhao; Wang, Hui; Jia, Kun; Li, Shoujun

2013-06-01

A H3N2 canine influenza virus, A/canine/Guangdong/3/2011 (H3N2), was isolated from roaming dogs in rural China. Sequence and phylogenetic analysis of eight gene segments revealed that the A/canine/Guangdong/3/2011 (H3N2) was most similar to a recent H3N2 canine influenza virus isolated in cats from South Korea, which originated from an avian strain. To our knowledge, this is the first report of an avian-origin H3N2 CIV which was isolated from roaming dogs in China. The epidemiologic information provided herein suggests that continued study is required to determine if this virus could be established in the roaming dog population in rural China and pose potential threats to public health.

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

A noda-like virus isolated from the sweetpotato pest spodoptera eridania (Cramer) (Lep.; noctuidae)

Science.gov (United States)

Zeddam; Rodriguez; Ravallec; Lagnaoui

1999-11-01

A small isometric virus has been isolated from larvae of the sweetpotato pest Spodoptera eridania (Cramer) collected near Pariacoto, Ancash province, Peru. It is designated the Pariacoto virus (PaV). In addition to its high pathogenicity on its natural host Spodoptera eridania, PaV was found to replicate in Spodoptera ochrea (Hampson) larvae but not in Spodoptera frugiperda (Smith) larvae. The size of the viral particle was estimated to be about 30 nm in diameter. Polyacrylamide gel electrophoresis showed a protein of approximately 40.5 kDa. After agarose gel electrophoresis, the viral genome appeared to be bipartite RNA. Gel immunodiffusion tests showed no serological relationship between PaV and Nodamura virus, the type species for insect nodaviruses. Electron microscopy confirmed that viral replication occurs in the cytoplasm. These properties are similar to those of other members of family Nodaviridae, to which the virus is currently assigned. Copyright 1999 Academic Press.

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.

A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods

Directory of Open Access Journals (Sweden)

Abraham A

2009-01-01

Full Text Available Background: Typing of Herpes simplex virus (HSV isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF assays and polymerase chain reaction (PCR. This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb based IF test. Study design : This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42 of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42 were HSV-2, and two (4.8% were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30, whereas the cost per MAb IF test is about Rs 1,500 (USD 35 including all overheads (reagents, instruments, personnel time, and consumables. Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers is now more widespread than fluorescence microscopes in a country like India.

Evaluation of different continuous cell lines in the isolation of mumps virus by the shell vial method from clinical samples

Science.gov (United States)

Reina, J; Ballesteros, F; Mari, M; Munar, M

2001-01-01

Aims—To compare prospectively the efficacy of the Vero, LLC-MK2, MDCK, Hep-2, and MRC-5 cell lines in the isolation of the mumps virus from clinical samples by means of the shell vial method. Methods—During an epidemic outbreak of parotiditis 48 clinical samples (saliva swabs and CSF) were studied. Two vials of the Vero, LLC-MK2, MDCK, MRC-5, and Hep-2 cell lines were inoculated with 0.2 ml of the samples by the shell vial assay. The vials were incubated at 36°C for two and five days. The vials were then fixed with acetone at -20°C for 10 minutes and stained by a monoclonal antibody against mumps virus by means of an indirect immunofluorescence assay. Results—The mumps virus was isolated from 36 samples. The Vero and LLC-MK2 cell lines showed a 100% isolation capacity, MDCK showed 77.7%, MRC-5 showed 44.4%, and Hep-2 showed 22.2%. The Vero and LLC-MK2 lines were significantly different to the other cell lines (p 5 infectious foci) were 94.4% for Vero, 97.2% for LLC-MK2, 5.5% for MDCK, 5.5% for Hep-2, and 0% for MRC-5. Conclusions—The Vero and LLC-MK2 cell lines are equally efficient at two and five days incubation for the isolation of the mumps virus from clinical samples, and the use of the shell vial method considerably shortens the time of aetiological diagnosis with higher specificity. Key Words: mumps virus • Vero cell line • LLC-MK2 cell line • MDCK cell line • Hep-2 cell line • MRC-5 cell line • isolation • shell vial PMID:11729211

Isolation and characterization of plasma membranes from guinea pig ileum

International Nuclear Information System (INIS)

Anon.

1986-01-01

A plasma membrane fraction from guinea pig ileum has been isolated by extraction of a crude microsomal fraction with a low ionic strength buffer containing ATP and Ca 2+ . The extracted microsomes were subjected to sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membranes were substantially free from contamination with contractile proteins, mitochondria and sarco-plasmic reticulum. The plasma membrane vesicles were enriched 30-to-40-fold in Na + -K + -ATPase and 5'-nucleotidase activities. The plasma membrane vesicles accumulated Ca 2+ in the presence of ATP. The addition of Ca 2+ ionophore A23187 to vesicles loaded with Ca 2+ in the presence of ATP removed Ca 2+ completely from the vesicles in one minute. The Km values for the Ca 2+ -dependent phosphorylated intermediates of Ca 2+ -Mg 2+ -ATPase and Ca 2+ uptake were approximately 0.8 μM indicating that the phosphorylated intermediates represent phosphorylation of Ca 2+ pump ATPase. The 3 H-nitrendipine binding to plasma membranes was characterized by high affinity with Kd of 185 pM and B/sub max/ 1280 fmol/mg protein. The plasma membrane vesicles prepared by these procedures can prove useful for the study of ion transport

Isolation and identification of porcine reproductive and respiratory syndrome virus in cell cultures.

Science.gov (United States)

Valícek, L; Psikal, I; Smíd, B; Rodák, L; Kubalíková, R; Kosinová, E

1997-10-01

Three strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in porcine lung macrophage (PLM) cultures from three swine herds. This has been the first successful isolation of PRRSV in the Czech Republic and the strains received the designations CAPM V-501, CAPM V-502 and CAPM V-503, respectively. All the three isolates in PLM were identified by immunofluorescence and immunoperoxidase tests and the strain CAPM V-502 also by electron microscopy using the ultrathin section technique. The strain CAPM V-502 has been adapted to the cell line MARC-145. Viral RNA in PLM cultures infected with any of the isolated PRRSV strains was demonstrated by RT-PCR targeted to the more conserved ORF 7 genomic region encoding the nucleocapsid protein. The assessment of PCR products in agarose gel revealed a uniform size of 394 bp in all the three isolates and the European prototype strain Lelystad used as positive control.

Isolation and characterization of avian influenza viruses from raw poultry products illegally imported to Japan by international flight passengers.

Science.gov (United States)

Shibata, A; Hiono, T; Fukuhara, H; Sumiyoshi, R; Ohkawara, A; Matsuno, K; Okamatsu, M; Osaka, H; Sakoda, Y

2018-04-01

The transportation of poultry and related products for international trade contributes to transboundary pathogen spread and disease outbreaks worldwide. To prevent pathogen incursion through poultry products, many countries have regulations about animal health and poultry product quarantine. However, in Japan, animal products have been illegally introduced into the country in baggage and confiscated at the airport. Lately, the number of illegally imported poultry and the incursion risk of transboundary pathogens through poultry products have been increasing. In this study, we isolated avian influenza viruses (AIVs) from raw poultry products illegally imported to Japan by international passengers. Highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIVs were isolated from raw chicken and duck products carried by flight passengers. H5 and H9 isolates were phylogenetically closely related to viruses isolated from poultry in China, and haemagglutinin genes of H5N1 and H5N6 isolates belonged to clades 2.3.2.1c and 2.3.4.4, respectively. Experimental infections of H5 and H9 isolates in chickens and ducks demonstrated pathogenicity and tissue tropism to skeletal muscles. To prevent virus incursion by poultry products, it is important to encourage the phased cleaning based on the disease control and eradication and promote the reduction in contamination risk in animal products. © 2017 Blackwell Verlag GmbH.

New Korean isolates of Pepper mild mottle virus (PMMoV) differ in symptom severity and subcellular localization of the 126 kDa protein

Science.gov (United States)

Two isolates of Pepper mild mottle virus (PMMoV) were selected from a nationwide survey of pepper fields in South Korea in 2014 and 2015, in which Cucumber mosaic virus was also detected; the two PMMoV isolates, Sangcheong 47 (S-47, KX399390) and Jeongsong 76 (J-76, KX399389), share ~99% nucleotide ...

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

Science.gov (United States)

Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

2009-11-15

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

Direct isolation of H1N2 recombinant virus from a throat swab of a patient simultaneously infected with H1N1 and H3N2 influenza A viruses.

OpenAIRE

Nishikawa, F; Sugiyama, T

1983-01-01

Two H1N2 recombinant viruses were isolated by a plaquing method from a throat swab of a patient who was simultaneously infected with H1N1 and H3N2 influenza viruses during the Tokyo epidemic of 1981. This is the first direct evidence that recombination of influenza viruses occurred in the human body.

Genetic variation of coat protein gene among the isolates of Rice tungro spherical virus from tungro-endemic states of the India.

Science.gov (United States)

Mangrauthia, Satendra K; Malathi, P; Agarwal, Surekha; Ramkumar, G; Krishnaveni, D; Neeraja, C N; Madhav, M Sheshu; Ladhalakshmi, D; Balachandran, S M; Viraktamath, B C

2012-06-01

Rice tungro disease, one of the major constraints to rice production in South and Southeast Asia, is caused by a combination of two viruses: Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). The present study was undertaken to determine the genetic variation of RTSV population present in tungro endemic states of Indian subcontinent. Phylogenetic analysis based on coat protein sequences showed distinct divergence of Indian RTSV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Coimbatore (Tamil Nadu), and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic study were further supported with the SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. In addition, sequence difference count matrix revealed 2-68 nucleotides differences among all the Indian RTSV isolates taken in this study. However, at the protein level these differences were not significant as revealed by Ka/Ks ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100% and 97-100%, respectively, among Indian isolates of RTSV. Understanding of the population structure of RTSV from tungro endemic regions of India would potentially provide insights into the molecular diversification of this virus.

Single virus genomics: a new tool for virus discovery.

Directory of Open Access Journals (Sweden)

Lisa Zeigler Allen

Full Text Available Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called 'Single Virus Genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA. The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

Full genome sequences and molecular characterization of tick-borne encephalitis virus strains isolated from human patients.

Science.gov (United States)

Formanová, Petra; Černý, Jiří; Bolfíková, Barbora Černá; Valdés, James J; Kozlova, Irina; Dzhioev, Yuri; Růžek, Daniel

2015-02-01

Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), one of the most important human neuroinfections across Eurasia. Up to date, only three full genome sequences of human European TBEV isolates are available, mostly due to difficulties with isolation of the virus from human patients. Here we present full genome characterization of an additional five low-passage TBEV strains isolated from human patients with severe forms of TBE. These strains were isolated in 1953 within Central Bohemia in the former Czechoslovakia, and belong to the historically oldest human TBEV isolates in Europe. We demonstrate here that all analyzed isolates are distantly phylogenetically related, indicating that the emergence of TBE in Central Europe was not caused by one predominant strain, but rather a pool of distantly related TBEV strains. Nucleotide identity between individual sequenced TBEV strains ranged from 97.5% to 99.6% and all strains shared large deletions in the 3' non-coding region, which has been recently suggested to be an important determinant of virulence. The number of unique amino acid substitutions varied from 3 to 9 in individual isolates, but no characteristic amino acid substitution typical exclusively for all human TBEV isolates was identified when compared to the isolates from ticks. We did, however, correlate that the exploration of the TBEV envelope glycoprotein by specific antibodies were in close proximity to these unique amino acid substitutions. Taken together, we report here the largest number of patient-derived European TBEV full genome sequences to date and provide a platform for further studies on evolution of TBEV since the first emergence of human TBE in Europe. Copyright © 2014 Elsevier GmbH. All rights reserved.

Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

Directory of Open Access Journals (Sweden)

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.

Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses.

Science.gov (United States)

Zhang, Zhenjia; Wang, Deya; Yu, Chengming; Wang, Zenghui; Dong, Jiahong; Shi, Kerong; Yuan, Xuefeng

2016-01-14

Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5' regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative

Analysis of the biological and molecular variability of the Polish isolates of Tomato black ring virus (TBRV).

Science.gov (United States)

Rymelska, N; Borodynko, N; Pospieszny, H; Hasiów-Jaroszewska, B

2013-10-01

Tomato black ring virus (TBRV) is an important pathogen infecting many plant species worldwide. The biological and molecular variability of the Polish isolates of TBRV was analyzed. The analysis was performed based on the symptoms induced by various isolates on test plant species as well as on phylogenetic relationships between isolates. Isolates differed in their host range and symptomatology. In addition, genetic variation among isolates was characterized by restriction fragment length polymorphism analysis and confirmed by sequencing. The phylogenetic analysis revealed that the Polish isolates differ from each other and do not form a monophyletic cluster. Finally, we identified and analyzed sequences of defective RNA forms arising from the TBRV genome.

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004

Directory of Open Access Journals (Sweden)

Siddique Naila

2010-06-01

Full Text Available Abstract Background Avian influenza virus (AIV infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis. Results Phylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct. Conclusions Although rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers. Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.

Characterization of Low Pathogenic Avian Influenza Virus Subtype H9N2 Isolated from Free-Living Mynah Birds (Acridotheres tristis) in the Sultanate of Oman.

Science.gov (United States)

Body, Mohammad H; Alrarawahi, Abdulmajeed H; Alhubsy, Saif S; Saravanan, Nirmala; Rajmony, Sunil; Mansoor, Muhammad Khalid

2015-06-01

A low pathogenic avian influenza virus was identified from free-living birds (mynah, Acridotheres tristis) of the starling family. Virus was isolated by inoculation of homogenized suspension from lung, tracheal, spleen, and cloacal swabs into the allantoic cavity of embryonated chicken eggs. Subtype of the isolate was characterized as H9N2 by hemagglutination inhibition test using monospecific chicken antisera to a wide range of influenza reference strain. Pathogenicity of the isolate was determined by intravenous pathogenicity index. The virus was reisolated from experimentally infected chicken. Additionally, the isolate was subjected to reverse transcriptase PCR using partial hemagglutinin (HA) gene-specific primers and yielded an amplicon of 487 bp. HA gene sequence analysis revealed 99% sequence homology among mynah and chicken isolates from Oman. On phylogenetic analysis, isolates from mynah (A/mynnah/Oman/AIVS6/2005) and chicken (A/chicken/Oman/AIVS3/2006; A/chicken/Oman/AIVS7/2006) clustered together tightly, indicating these free-flying birds may be a source of introduction of H9N2 subtype in poultry bird in Oman. Moreover, the HA gene of H9N2 isolates from Oman resembled those of viruses of the G1-like lineage and were very similar to those from United Arab Emirates.

Identification of ribonucleotide reductase mutation causing temperature-sensitivity of herpes simplex virus isolates from whitlow by deep sequencing.

Science.gov (United States)

Daikoku, Tohru; Oyama, Yukari; Yajima, Misako; Sekizuka, Tsuyoshi; Kuroda, Makoto; Shimada, Yuka; Takehara, Kazuhiko; Miwa, Naoko; Okuda, Tomoko; Sata, Tetsutaro; Shiraki, Kimiyasu

2015-06-01

Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

Identification of syncytial mutations in a clinical isolate of herpes simplex virus 2

International Nuclear Information System (INIS)

Muggeridge, Martin I.; Grantham, Michael L.; Johnson, F. Brent

2004-01-01

Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis

Vacuum laser-initiated plasma shutters for retropulse isolation in Antares

International Nuclear Information System (INIS)

Sheheen, T.W.; Czuchlewski, S.J.; Hyde, J.; Ainsworth, R.L.

1983-03-01

We have demonstrated that sintered LiF spatial filters may be used in a 10 -6 -torr vacuum environment as laser-initiated plasma shutters for retropulse isolation in the Antares high-energy laser fusion system. In our experiments, a 1.1-ns pulsed CO 2 laser, at a 10-μm wavelength and an energy of up to 3.0 J, was used for plasma initiation; a chopped probe laser tuned to a 9l6-μm wavelength was used in determining the blocking time of the plasma. We measured the 10.6- and 9.6-μm beam transmissions as a function of fluence on the aperture edge. For an 800-μm-diam aperture and a 1.2-mm-diam Gaussian beam determined at the 1/e 2 intensity points, we observed blocking times in excess of 1.0 μs

Study of the genetic variability of isolated belonging to the group B of the Respiratory Virus Human Sincicial

International Nuclear Information System (INIS)

Delfraro, A.

1998-07-01

The study allows analyzing the genetic variability of stumps belonging to the group B of the Breathing Virus Sincicial (Vrs), isolated in Uruguay among the years 1990 and 1996. They were evidenced by sequence the nucleotides changes and the changes were determined that take place at level of amino acids, the following ones were used technical: enzyme immunoassay, of extraction of viral RNA, of reverse transcription and Pcr, of purification of DNA and electrophoresis of nucleic acids. The result proven in the entirety of the isolated virus the genetic variability, enlarging and confirming the evolution pattern proposed by Sullender and collaborators, (1991) for the group B of Vrs [es

Clinical and virological characteristics of calves experimentally infected with a Brazilian isolate of bovine viral diarrhea virus type 1a

Directory of Open Access Journals (Sweden)

Luana Marchi Quadros

Full Text Available ABSTRACT: To study the pathogenicity of the Brazilian bovine viral diarrhea virus (BVDV type 1a 241.10 isolate, four calves were intranasally inoculated with a viral suspension containing 107.2 TCID50 mL-1. One calf was left uninoculated and kept in contact with the other calves to investigate viral transmissibility. After inoculation, the animals were monitored daily for clinical signs of infection. The presence of the virus in the blood and nasal secretions was confirmed by virus isolation in cell culture. White blood cells were quantified prior to and every 3 days after infection, and the presence of antibodies was checked every 7 days, starting at day 0 until day 42 post-inoculation (pi. After infection, nasal and ocular serous secretions were observed between days 1 and 5 pi, along with a mild cough from days 2 to 4 pi; however, no severe clinical signs were present. Body temperature was slightly elevated between days 4 and 6 pi. The control calf did not develop any of the signs observed in the infected animals. Cell culture-mediated virus isolation confirmed viremia between days 4 and 8 pi and the presence of the virus in the nasal secretions between days 1 and 10 pi. All infected animals showed a decrease in white blood cell count. Antibodies could be detected from day 14 pi, and these levels remained high until day 35 pi. The control calf had no viremia, viral presence in nasal secretions, or positive serology, indicating the absence of viral transmission. Thus, isolate BVDV 1a 241.10 has low pathogenicity and transmissibility but retains immunosuppressive capacity.

Partial Characterization of Tick-Borne Encephalitis Virus Isolates from Ticks of Southern Ukraine.

Science.gov (United States)

Yurchenko, Oksana O; Dubina, Dmytro O; Vynograd, Nataliya O; Gonzalez, Jean-Paul

2017-08-01

Tick-borne encephalitis (TBE) is the most common tick-borne viral infection in Eurasia; thousands of human cases are annually reported from several European countries. Several tick species are vectors of the tick-borne encephalitis virus (TBEV), while TBE appears to be spreading from the Eurasian continent westward to Europe. Fifteen study sites were chosen from five territories of southern Ukraine, including Odessa, Mykolaiv, Kherson Oblast, the Autonomous Republic of Crimea, and Sevastopol. Tick collection was performed in spring season of three consecutive years (1988-1990) using either flagging technique or direct collection of specimens feeding on cattle. A total of 15,243 tick imagoes and nymphs were collected from nine species, including Dermacentor marginatus, D. reticulatus, Haemaphysalis parva, H. punctata, Hyalomma marginatum, Ixodes ricinus, Rhipicephalus bursa, R. rossicus, and R. sanguineus, pooled in 282 monospecific samples. Supernatant of grinded pool was used for inoculation to suckling mice for virus isolation. Eight TBEV isolates were identified from ticks among six study sites. Ticks showed a minimum infection rate from 0.11% to 0.81%. Phylogenetic analysis of the envelope (E) protein gene of seven isolates, assigned all to the European subtype (TBEV-Eu) showing a maximum identity of 97.17% to the "Pan" TBEV-Eu reference strain. Compared to 104 TBEV-Eu isolates they clustered within the same clade as the Pan reference strain and distinguished from other TBEV-Eu isolates. Amino acid sequence analysis of the South Ukrainian TBEV-Eu isolates revealed the presence of four amino acid substitutions 67 (N), 266 (R), 306 (V), and 407 (R), in the ectodomains II and III and in the stem-anchor region of the E protein gene. This study confirmed TBEV-Eu subtype distribution in the southern region of Ukraine, which eventually overlaps with TBEV-FE (Far Eastern subtype) and TBEV-Sib (Siberian subtype) domains, showing the heterogeneity of TBEV circulating in

Identify-Isolate-Inform: A Tool for Initial Detection and Management of Zika Virus Patients in the Emergency Department

Directory of Open Access Journals (Sweden)

Kristi L. Koenig

2016-05-01

Full Text Available First isolated in 1947 from a monkey in the Zika forest in Uganda, and from mosquitoes in the same forest the following year, Zika virus has gained international attention due to concerns for infection in pregnant women potentially causing fetal microcephaly. More than one million people have been infected since the appearance of the virus in Brazil in 2015. Approximately 80% of infected patients are asymptomatic. An association with microcephaly and other birth defects as well as Guillain-Barre Syndrome has led to a World Health Organization declaration of Zika virus as a Public Health Emergency of International Concern in February 2016. Zika virus is a vector-borne disease transmitted primarily by the Aedes aegypti mosquito. Male to female sexual transmission has been reported and there is potential for transmission via blood transfusions. After an incubation period of 2-7 days, symptomatic patients develop rapid onset fever, maculopapular rash, arthralgia, and conjunctivitis, often associated with headache and myalgias. Emergency department (ED personnel must be prepared to address concerns from patients presenting with symptoms consistent with acute Zika virus infection, especially those who are pregnant or planning travel to Zika-endemic regions, as well as those women planning to become pregnant and their partners. The identify-isolate-inform (3I tool, originally conceived for initial detection and management of Ebola virus disease patients in the ED, and later adjusted for measles and Middle East Respiratory Syndrome, can be adapted for real-time use for any emerging infectious disease. This paper reports a modification of the 3I tool for initial detection and management of patients under investigation for Zika virus. Following an assessment of epidemiologic risk, including travel to countries with mosquitoes that transmit Zika virus, patients are further investigated if clinically indicated. If after a rapid evaluation, Zika or other

Partial characterization of Maize rayado fino virus isolates from Ecuador: phylogenetic analysis supports a Central American origin of the virus.

Science.gov (United States)

Chicas, Mauricio; Caviedes, Mario; Hammond, Rosemarie; Madriz, Kenneth; Albertazzi, Federico; Villalobos, Heydi; Ramírez, Pilar

2007-06-01

Maize rayado fino virus (MRFV) infects maize and appears to be restricted to, yet widespread in, the Americas. MRFV was previously unreported from Ecuador. Maize plants exhibiting symptoms of MRFV infection were collected at the Santa Catalina experiment station in Quito, Ecuador. RT-PCR reactions were performed on total RNA extracted from the symptomatic leaves using primers specific for the capsid protein (CP) gene and 3' non-translated region of MRFV and first strand cDNA as a template. Nucleotide sequence comparisons to previously sequenced MRFV isolates from other geographic regions revealed 88-91% sequence identity. Phylogenetic trees constructed using Maximum Likelihood, UPGMA, Minimal Evolution, Neighbor Joining, and Maximum Parsimony methods separated the MRFV isolates into four groups. These groups may represent geographic isolation generated by the mountainous chains of the American continent. Analysis of the sequences and the genetic distances among the different isolates suggests that MRFV may have originated in Mexico and/or Guatemala and from there it dispersed to the rest of the Americas.

Molecular Characterization of Geographically Different Banana bunchy top virus Isolates in India.

Science.gov (United States)

Selvarajan, R; Mary Sheeba, M; Balasubramanian, V; Rajmohan, R; Dhevi, N Lakshmi; Sasireka, T

2010-10-01

Banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV) is one of the most devastating diseases of banana and poses a serious threat for cultivars like Hill Banana (Syn: Virupakshi) and Grand Naine in India. In this study, we have cloned and sequenced the complete genome comprised of six DNA components of BBTV infecting Hill Banana grown in lower Pulney hills, Tamil Nadu State, India. The complete genome sequence of this hill banana isolate showed high degree of similarity with the corresponding sequences of BBTV isolates originating from Lucknow, Uttar Pradesh State, India, and from Fiji, Egypt, Pakistan, and Australia. In addition, sixteen coat protein (CP) and thirteen replicase genes (Rep) sequences of BBTV isolates collected from different banana growing states of India were cloned and sequenced. The replicase sequences of 13 isolates showed high degree of similarity with that of South Pacific group of BBTV isolates. However, the CP gene of BBTV isolates from Shervroy and Kodaikanal hills of Tamil Nadu showed higher amino acid sequence variability compared to other isolates. Another hill banana isolate from Meghalaya state had 23 nucleotide substitutions in the CP gene but the amino acid sequence was conserved. This is the first report of the characterization of a complete genome of BBTV occurring in the high altitudes of India. Our study revealed that the Indian BBTV isolates with distinct geographical origins belongs to the South Pacific group, except Shervroy and Kodaikanal hill isolates which neither belong to the South Pacific nor the Asian group.

Isolation and phylogenetic characterization of Canine distemper virus from India.

Science.gov (United States)

Swati; Deka, Dipak; Uppal, Sanjeev Kumar; Verma, Ramneek

2015-09-01

Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that infects a variety of carnivores. Sequence analysis of CDVs from different geographical areas has shown a lot of variation in the genome of the virus especially in haemagglutinin gene which might be one of the causes of vaccine failure. In this study, we isolated the virus (place: Ludhiana, Punjab; year: 2014) and further cloned, sequenced and analyzed partial haemagglutinin (H) gene and full length genes for fusion protein (F), phosphoprotein (P) and matrix protein (M) from an Indian wild-type CDV. Higher sequence homology was observed with the strains from Switzerland, Hungary, Germany; and lower with the vaccine strains like Ondersteport, CDV3, Convac for all the genes. The multiple sequence alignment showed more variation in partial H (45 nucleotide and 5 amino acid substitutions) and complete F (79 nucleotide and 30 amino acid substitutions) than in complete P (44 nucleotide and 22 amino acid substitutions) and complete M (22 nucleotide and 4 amino acid substitutions) gene/protein. Predicted potential N-linked glycosylation sites in H, F, M and P proteins were similar to the previously known wild-type CDVs but different from the vaccine strains. The Indian CDV formed a distinct clade in the phylogenetic tree clearly separated from the previously known wild-type and vaccine strains.

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant

OpenAIRE

Renström Lena HM; Isaksson Mats; Berg Mikael; Zohari Siamak; Widén Frederik; Metreveli Giorgi; Bálint Ádám; Wallgren Per; Belák Sándor; Segall Thomas; Kiss István

2009-01-01

Abstract The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority o...

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

Science.gov (United States)

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016-2017 in Northern Japan.

Science.gov (United States)

Hiono, Takahiro; Okamatsu, Masatoshi; Matsuno, Keita; Haga, Atsushi; Iwata, Ritsuko; Nguyen, Lam Thanh; Suzuki, Mizuho; Kikutani, Yuto; Kida, Hiroshi; Onuma, Manabu; Sakoda, Yoshihiro

2017-09-01

On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Characterization and Sequencing of a Genotype XII Newcastle Disease Virus Isolated from a Peacock (Pavo cristatus) in Peru.

Science.gov (United States)

Chumbe, Ana; Izquierdo-Lara, Ray; Tataje-Lavanda, Luis; Figueroa, Aling; Segovia, Karen; Gonzalez, Rosa; Cribillero, Giovana; Montalvan, Angela; Fernández-Díaz, Manolo; Icochea, Eliana

2015-07-30

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. Copyright © 2015 Chumbe et al.

Study of the virulence and cross-neutralization capability of recent porcine parvovirus field isolates and vaccine viruses in experimentally infected pregnant gilts.

Science.gov (United States)

Zeeuw, E J L; Leinecker, N; Herwig, V; Selbitz, H-J; Truyen, U

2007-02-01

The pathogenicity of two recent German field isolates of Porcine parvovirus (PPV-27a and PPV-143a) and two vaccine viruses [PPV-NADL-2 and PPV-IDT (MSV)], which are used for the production of inactivated vaccines, was investigated by inoculation of pregnant sows at day 40 of gestation. Post-infection sera of these sows as well as antisera prepared in rabbits by immunization with the four above-mentioned PPV isolates and with the virulent strain PPV-Challenge (Engl.) were tested for their homologous and heterologous neutralization activities. All antisera had high neutralization activity against the vaccine viruses, the PPV-Challenge (Engl.) virus and PPV-143a, but much lower activity against PPV-27a. These results suggest that PPV-27a represents a new antigenic variant or type of PPV and vaccines based on the established vaccine viruses may not be fully protective against this field isolate. PPV-27a has been characterized based on the amino acid sequences of the capsid protein as a member of a new and distinct PPV cluster (Zimmermann et al., 2006). Interestingly, the homologous neutralizing antibody titres of the sera of all three pigs and both rabbits inoculated or immunized with PPV-27a were 100- to 1000-fold lower than the heterologous titres against any of the other viruses. The low homologous neutralizing antibody titres suggest a possible, yet undefined, immune escape mechanism of this PPV isolate.

Characterization of potato and tobacco isolates of Cucumber mosaic virus from Syria and the first report on CMV satellite RNA from potato

Directory of Open Access Journals (Sweden)

Mohamad CHIKH ALI

2012-05-01

Full Text Available Cucumber mosaic virus (CMV has been reported from potato production areas in Europe, USA, Japan and more frequently in regions with warm climates such as Egypt, India, Saudi Arabia and Syria. As it is considered as an uncommon virus in potato, the characterization of potato isolates of CMV is far behind those from other hosts. In addition to potato, CMV is a common virus infecting many crops in Syria, but nothing is known about its molecular characteristics. The present study aimed to characterize Syrian CMV isolates collected from potato and neighboring tobacco fields. All potato isolates of CMV (total of four co-infected potato plants with Potato virus Y (PVY which is the most frequent potato virus in Syria. According to the sequence analyses of the coat protein (CP coding region, three potato and three tobacco CMV isolates were found to be closely related regardless of the host species or geographic origin, and all belonged to the IA strain subgroup of CMV. A potato CMV isolate, PoCMV7-5, readily infected solanaceous plants in which it induced systemic infection, but was less infectious to other hosts including those of Leguminosae and Cucurbitaceae. When inoculated on potato plants, PoCMV7-5 alone or with various PVY strains was able to cause local but not systemic infection in all potato cultivars inoculated. PoCMV7-5 contained heterogeneous variants of satellite RNA which varied in length due to A or/and T deletion/insertion at approximate nucleotide position 225‒240. This is the first report on CMV satellite RNA from potato.

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.

Isolation of avian influenza virus (H9N2 from emu in china

Directory of Open Access Journals (Sweden)

Kang Wenhua

2006-03-01

Full Text Available Abstract This is the first reported isolation of avian influenza virus (AIV from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic AIV (LPAIV, according to OIE standards. It was named A/Emu/HeNen/14/2004(H9N2(Emu/HN/2004. The HA gene (1683bp was amplified by RT-PCR and it was compared with other animal H9N2 AIV sequences in GenBank, the US National Institutes of Health genetic sequence database. The results suggested that Emu/HN/2004 may have come from an avian influenza virus (H9N2 from Southern China.

Effect of experimental influenza A virus infection on isolation of Streptococcus pneumoniae and other aerobic bacteria from the oropharynges of allergic and nonallergic adult subjects.

Science.gov (United States)

Wadowsky, R M; Mietzner, S M; Skoner, D P; Doyle, W J; Fireman, P

1995-04-01

Intranasal challenge with both influenza A virus and Streptococcus pneumoniae promotes otitis media with S. pneumoniae in chinchillas. We investigated whether influenza A virus infection promotes oropharyngeal colonization with S. pneumoniae and other middle ear pathogens by selectively inhibiting commensal bacteria. On study day 0, 12 allergic and 15 nonallergic adult subjects were intranasally inoculated with influenza A/Kawasaki (H1N1) virus. Every subject was infected with the virus as demonstrated by nasal shedding or seroconversion. Average upper respiratory symptom scores and nasal secretion weights from the entire subject group were elevated between days 2 and 6 (acute phase) and were not significantly different between allergic and nonallergic subjects. S. pneumoniae was not isolated from any subject prior to the virus challenge but was isolated in heavy density from 4 (15%) subjects on day 6 (P = 0.055). Staphylococcus aureus was isolated more frequently from the nonallergic subjects than from the allergic subjects on days 2 (80 versus 25%, respectively) 4, (67 versus 17%, respectively), and 6 (73 versus 25%, respectively) (P < 0.05). The isolation rates of other middle ear pathogens were not significantly different before virus challenge and during the acute and resolution phases (days 27 to 30) of the experimental infection for the entire subject group or either the allergic or nonallergic subgroup. Densities and isolation rates of commensal bacteria from the entire subject group were similar throughout the observational period. These results suggest that the virus infection promoted S. pneumoniae colonization of the oropharynx and that nonallergic persons may be more vulnerable to colonization with S. aureus than allergic persons. The altered colonization rates were not attributed to inhibition of commensal bacteria.

Comparison of complete genome sequences of dog rabies viruses isolated from China and Mexico reveals key amino acid changes that may be associated with virus replication and virulence.

Science.gov (United States)

Yu, Fulai; Zhang, Guoqing; Zhong, Xiangfu; Han, Na; Song, Yunfeng; Zhao, Ling; Cui, Min; Rayner, Simon; Fu, Zhen F

2014-07-01

Rabies is a global problem, but its impact and prevalence vary across different regions. In some areas, such as parts of Africa and Asia, the virus is prevalent in the domestic dog population, leading to epidemic waves and large numbers of human fatalities. In other regions, such as the Americas, the virus predominates in wildlife and bat populations, with sporadic spillover into domestic animals. In this work, we attempted to investigate whether these distinct environments led to selective pressures that result in measurable changes within the genome at the amino acid level. To this end, we collected and sequenced the full genome of two isolates from divergent environments. The first isolate (DRV-AH08) was from China, where the virus is present in the dog population and the country is experiencing a serious epidemic. The second isolate (DRV-Mexico) was taken from Mexico, where the virus is present in both wildlife and domestic dog populations, but at low levels as a consequence of an effective vaccination program. We then combined and compared these with other full genome sequences to identify distinct amino acid changes that might be associated with environment. Phylogenetic analysis identified strain DRV-AH08 as belonging to the China-I lineage, which has emerged to become the dominant lineage in the current epidemic. The Mexico strain was placed in the D11 Mexico lineage, associated with the West USA-Mexico border clade. Amino acid sequence analysis identified only 17 amino acid differences in the N, G and L proteins. These differences may be associated with virus replication and virulence-for example, the short incubation period observed in the current epidemic in China.

The HIV-1 V3 domain on field isolates: participation in generation of escape virus in vivo and accessibility to neutralizing antibodies

DEFF Research Database (Denmark)

Arendrup, M; Akerblom, L; Heegaard, P M

1995-01-01

The V3 domain is highly variable and induces HIV neutralizing antibodies (NA). Here we addressed the issues of 1) the participation of mutations in V3 in generation of neutralization resistant escape virus in vivo and 2) the applicability of synthetic V3 peptides corresponding to field isolates...... patterns against V3 peptides corresponding to sequential primary and escape field isolates, with the strongest reactivity against late isolated escape virus. These observations suggest that the neutralization epitope was influenced by the appearance of mutations. When used as immunogen in rabbits, V3...... to induce neutralizing immune sera. Seven peptides corresponding to the V3 region of primary and escape virus from 3 HIV-1 infected patients were synthesized and used for antibody (Abs) studies and immunizations. The anti-V3 Abs titre in patient serum was generally low against peptides corresponding...

Efficient neutralization of primary isolates by the plasma from HIV-1 infected Indian children.

Science.gov (United States)

Prakash, S S; Chaudhary, Alok Kumar; Lodha, Rakesh; Kabra, S K; Vajpayee, Madhu; Hazarika, Anjali; Bagga, Barun; Luthra, Kalpana

2011-10-01

We tested the plasma of 51 HIV-1-infected children (23 naïve and 28 ART treated) for neutralization against five primary isolates (PIs) generated from adult Indian HIV-1-infected patients. The plasma exhibited neutralization potential with significantly higher neutralizing antibody titers in ART-treated children than naïve children against three out of five PIs (pIndian children.

Molecular characterisation of the full-length genome of olive latent virus 1 isolated from tomato.

Science.gov (United States)

Hasiów-Jaroszewska, Beata; Borodynko, Natasza; Pospieszny, Henryk

2011-05-01

Olive latent virus 1 (OLV-1) is a species of the Necrovirus genus. So far, it has been reported to infect olive, citrus tree and tulip. Here, we determined and analysed the complete genomic sequence of an isolate designated as CM1, which was collected from tomato plant in the Wielkopolska region of Poland and represents the prevalent isolate of OLV-1. The CM1 genome consists of monopartite single-stranded positive-sense RNA genome sized 3,699 nt with five open reading frames (ORFs) and small inter-cistronic regions. ORF1 encodes a polypeptide with a molecular weight of 23 kDa and the read-through (RT) of its amber stop codon results in ORF1 RT that encodes the virus RNA-dependent RNA polymerase. ORF2 and ORF3 encode two peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in cell-to-cell movement. ORF4 is located in the 3' terminal and encodes a protein with 30 kDa identified as the viral coat protein (CP). The differences in CP region of four OLV-1 isolates whose sequences have been deposited in GenBank were observed. Nucleotide sequence identities of the CP of tomato CM1 isolate with those of olive, citrus and tulip isolates were 91.8%, 89.5% and 92.5%, respectively. In contrast to other OLV-1 isolates, CM1 induced necrotic spots on tomato plants and elicited necrotic local lesions on Nicotiana benthamiana, followed by systemic infection. This is the third complete genomic sequence of OLV-1 reported and the first one from tomato.

Investigation of the antigenic evolution of field isolates using the reverse genetics system of infectious bursal disease virus (IBDV).

Science.gov (United States)

Durairaj, Vijay; Sellers, Holly S; Linnemann, Erich G; Icard, Alan H; Mundt, Egbert

2011-10-01

The antigenic profiles of over 300 infectious bursal disease virus (IBDV) isolates were analyzed using a panel of monoclonal antibodies in a reverse genetics system. In addition, the sequences of a large portion of the neutralizing-antibody-inducing VP2 of IBDV were determined. Phylogenetic analysis of nucleotide and amino acid sequences in combination with the antigenic profiles obtained using the monoclonal antibody panel, revealed a lack of correlation between antigenicity and isolate's placement within the phylogenetic tree. In-depth analysis of amino acid exchanges revealed that changes within a certain region of the VP2 molecule resulted in differences in the antigenicity of the virus. This comprehensive analysis of VP2 sequences indicated a high selective pressure in the field that was likely due to vaccination programs, which increase the rate of evolution of the virus.

Phylogenetic characterisation of the G(L) sequences of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis in Denmark

DEFF Research Database (Denmark)

Larsen, Lars Erik; Storgaard, Torben; Holm, Elisabeth

2001-01-01

The study describes for the first time the phylogenetic relationship between equine arteritis virus (EAV) isolated from asymptomatic virus-shedding stallions and fatal cases of equine viral arteritis (EVA) in an European country. EAV was isolated from three dead foals and an aborted foetus during...

Characterization of Hungarian isolates of zucchini yellow mosaic virus (ZYMV, potyvirus) transmitted by seeds of Cucurbita pepo var Styriaca.

Science.gov (United States)

Tóbiás, István; Palkovics, László

2003-04-01

Zucchini yellow mosaic virus (ZYMV) has emerged as an important pathogen of cucurbits within the last few years in Hungary. The Hungarian isolates show a high biological variability, have specific nucleotide and amino acid sequences in the N-terminal region of coat protein and form a distinct branch in the phylogenetic tree. The virus is spread very efficiently in the field by several aphid species in a non-persistent manner. It can be transmitted by seed in holl-less seeded oil pumpkin (Cucurbita pepo (L) var Styriaca), although at a very low rate. Three isolates from seed transmission assay experiments were chosen and their nucleotide sequences of coat proteins have been compared with the available CP sequences of ZYMV. According to the sequence analysis, the Hungarian isolates belong to the Central European branch in the phylogenetic tree and, together with the ZYMV isolates from Austria and Slovenia, share specific amino acids at positions 16, 17, 27 and 37 which are characteristic only to these isolates. The phylogenetic tree suggests the common origin of distantly distributed isolates which can be attributed to widespread seed transmission.

Sequence Analysis and Phylogenetic Profiling of the Nonstructural (NS Genes of H9N2 Influenza A Viruses Isolated in Iran during 1998-2007

Directory of Open Access Journals (Sweden)

Ebrahimi, M.

2014-11-01

Full Text Available The earliest evidences on circulation of Avian Influenza (AI virus on the Iranian poultry farms date back to 1998. Great economic losses through dramatic drop in egg production and high mortality rates are characteristically attributed to H9N2 AI virus. In the present work non-structural (NS genes of 10 Iranian H9N2 chicken AI viruses collected during 1998-2007 were fully sequenced and subjected to a phylogenetic analysis. The observations proved allele A was the single-detectable type of the NS gene within the studied isolates. All the examined Iranian isolates fell into the Korean sublineage with a relatively broad sequence homology (91.6-98% in nucleotide construction of the NS genes. The motif for PDZ ligand recognition of the group one isolates was either EDEV (N=6 or ESEV (N=1 While all viruses as group two contained a PL motif “KSEV” (N=3. The present work provides useful epidemiological data at molecular level on source and contemporary evolution of H9N2 virus population in Iran.

Highly pathogenic avian influenza virus subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates.

Directory of Open Access Journals (Sweden)

Giovanni Cattoli

Full Text Available Highly pathogenic avian influenza virus A/H5N1 was first officially reported in Africa in early 2006. Since the first outbreak in Nigeria, this virus spread rapidly to other African countries. From its emergence to early 2008, 11 African countries experienced A/H5N1 outbreaks in poultry and human cases were also reported in three of these countries. At present, little is known of the epidemiology and molecular evolution of A/H5N1 viruses in Africa. We have generated 494 full gene sequences from 67 African isolates and applied molecular analysis tools to a total of 1,152 A/H5N1 sequences obtained from viruses isolated in Africa, Europe and the Middle East between 2006 and early 2008. Detailed phylogenetic analyses of the 8 gene viral segments confirmed that 3 distinct sublineages were introduced, which have persisted and spread across the continent over this 2-year period. Additionally, our molecular epidemiological studies highlighted the association between genetic clustering and area of origin in a majority of cases. Molecular signatures unique to strains isolated in selected areas also gave us a clearer picture of the spread of A/H5N1 viruses across the continent. Mutations described as typical of human influenza viruses in the genes coding for internal proteins or associated with host adaptation and increased resistance to antiviral drugs have also been detected in the genes coding for transmembrane proteins. These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent. They further stress how imperative it is to implement sustainable control strategies to improve animal and public health at a global level.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

Johansson, T.; Einer-Jensen, K.; Batts, W.; Ahrens, P.; Bjorkblom, C.; Kurath, G.; Bjorklund, H.; Lorenzen, N.

2009-01-01

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. ?? 2009 Inter-Research.

Genetic variability and evolutionary implications of RNA silencing suppressor genes in RNA1 of sweet potato chlorotic stunt virus isolates infecting sweetpotato and related wild species.

Directory of Open Access Journals (Sweden)

Arthur K Tugume

Full Text Available BACKGROUND: The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae encodes a Class 1 RNase III (RNase3, a putative hydrophobic protein (p7 and a 22-kDa protein (p22 from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied. METHODOLOGY/PRINCIPAL FINDINGS: Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b encoding an RNase3 homolog (<56% identity to SPCSV RNase3 able to suppresses sense-mediated RNA silencing was detected in I. sinensis. CONCLUSIONS/SIGNIFICANCE: SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

Directory of Open Access Journals (Sweden)

Camila Appolinário

2015-09-01

Full Text Available Rabies virus (RABV isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2, Desmodus rotundus (variant 3, crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT and real time polymerase chain reaction (qRT-PCR for quantification of rabies virus nucleoprotein gene (N gene. Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

International Nuclear Information System (INIS)

Peschek, G.A.; Wastyn, M.; Trnka, M.; Molitor, V.; Fry, I.V.; Packer, L.

1989-01-01

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [ 35 S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min -1 (mg of protein) -1 , respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa 3 -type enzyme from the properties of its redox-active and EDTA-resistant Cu 2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa 3 -type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa 3 -type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme

Stability and infectivity of novel pandemic influenza A (H1N1) virus in blood-derived matrices under different storage conditions.

Science.gov (United States)

Wang, Xue; Zoueva, Olga; Zhao, Jiangqin; Ye, Zhiping; Hewlett, Indira

2011-12-22

Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied. We examined the stability of novel pandemic influenza A (H1N1) virus RNA when the virus was stored in phosphate buffered saline (PBS), plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EID(50) assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C. Viral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low. These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be transmitted by blood or blood derived samples.

Stability and infectivity of novel pandemic influenza A (H1N1 virus in blood-derived matrices under different storage conditions

Directory of Open Access Journals (Sweden)

Wang Xue

2011-12-01

Full Text Available Abstract Background Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied. Methods We examined the stability of novel pandemic influenza A (H1N1 virus RNA when the virus was stored in phosphate buffered saline (PBS, plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EID50 assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C. Results Viral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low. Conclusion These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be

Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes.

Science.gov (United States)

Savage, H M; Ceianu, C; Nicolescu, G; Karabatsos, N; Lanciotti, R; Vladimirescu, A; Laiv, L; Ungureanu, A; Romanca, C; Tsai, T F

1999-10-01

Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic were conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, including 5 of 13 ducks (38%), 1 of 1 goose, 19 of 52 chickens (37%), 1 of 1 peahen, and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 26) from the urban Bucharest site was not significantly different (P = 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46). Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted for 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obtained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97-50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained from a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenetic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest the existence of two large lineages of viruses. Bootstrap analysis of the nucleotide alignment indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Algeria, and west, central, and east Africa, all of

LNA probe-based assay for the detection of Tomato black ring virus isolates.

Science.gov (United States)

Hasiów-Jaroszewska, Beata; Rymelska, Natalia; Borodynko, Natasza

2015-02-01

Tomato black ring virus (TBRV) infects a wide range of economically important plant species worldwide. In the present study we developed a locked nucleic acid (LNA) real-time RT-PCR assay for accurate detection of genetically diverse TBRV isolates collected from different hosts. The assay based on the LNA probe has a wide detection range, high sensitivity, stability and amplification efficiency. The assay amplified all tested TBRV isolates, but no signal was observed for the RNA from other nepoviruses and healthy plant species. Under optimum reaction conditions, the detection limit was estimated around 17 copies of the TBRV target region in total RNA. Real-time RT-PCR with the LNA probe described in this paper will serve as a valuable tool for robust, sensitive and reliable detection of TBRV isolates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of a non-pathogenic H5N1 influenza virus isolated from a migratory duck flying from Siberia in Hokkaido, Japan, in October 2009

Directory of Open Access Journals (Sweden)

Okamatsu Masatoshi

2011-02-01

Full Text Available Abstract Background Infection with H5N1 highly pathogenic avian influenza viruses (HPAIVs of domestic poultry and wild birds has spread to more than 60 countries in Eurasia and Africa. It is concerned that HPAIVs may be perpetuated in the lakes in Siberia where migratory water birds nest in summer. To monitor whether HPAIVs circulate in migratory water birds, intensive surveillance of avian influenza has been performed in Mongolia and Japan in autumn each year. Until 2008, there had not been any H5N1 viruses isolated from migratory water birds that flew from their nesting lakes in Siberia. In autumn 2009, A/mallard/Hokkaido/24/09 (H5N1 (Mal/Hok/24/09 was isolated from a fecal sample of a mallard (Anas platyrhynchos that flew from Siberia to Hokkaido, Japan. The isolate was assessed for pathogenicity in chickens, domestic ducks, and quails and analyzed antigenically and phylogenetically. Results No clinical signs were observed in chickens inoculated intravenously with Mal/Hok/24/09 (H5N1. There was no viral replication in chickens inoculated intranasally with the isolate. None of the domestic ducks and quails inoculated intranasally with the isolate showed any clinical signs. There were no multiple basic amino acid residues at the cleavage site of the hemagglutinin (HA of the isolate. Each gene of Mal/Hok/24/09 (H5N1 is phylogenetically closely related to that of influenza viruses isolated from migratory water birds that flew from their nesting lakes in autumn. Additionally, the antigenicity of the HA of the isolate was similar to that of the viruses isolated from migratory water birds in Hokkaido that flew from their northern territory in autumn and different from those of HPAIVs isolated from birds found dead in China, Mongolia, and Japan on the way back to their northern territory in spring. Conclusion Mal/Hok/24/09 (H5N1 is a non-pathogenic avian influenza virus for chickens, domestic ducks, and quails, and is antigenically and genetically

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

Science.gov (United States)

Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

Isolation and characterization of Newcastle disease virus from vaccinated commercial layer chicken

Directory of Open Access Journals (Sweden)

P. Balachandran

2014-07-01

Full Text Available Aim: Newcastle disease (ND is an infectious, highly contagious and destructive viral disease of poultry and controlled by vaccination. In spite of vaccination, incidence of ND was reported in commercial layers with gastrointestinal lesions. This study was undertaken to assess the prevalence and pathotypes of Newcastle disease virus (NDV involved in gastrointestinal tract abnormalities of vaccinated commercial layer chicken of Namakkal region for a period of three years from 2008 and 2011. Materials and Methods: Pooled tissue (trachea, lung, spleen, proventriculus, intestine and caecal tonsils samples collected from dead birds on postmortem examination from 100 layer flocks above 20 weeks of age with gastrointestinal lesions were subjected to isolation of NDV in embryonated specific pathogen free (SPF chicken eggs. Mean death time (MDT and intracerebral pathogenicity index of the isolates were characterized. Flock details were collected from NDV positive flocks to assess the prevalence and impact of NDV on vaccinated commercial layer chicken. Results: Among the 100 flocks examined Newcastle disease virus was detected in 14 flocks as a single infection and 10 flocks as combined infections with worm infestation, necrotic enteritis and coccidiosis. Chicken embryo mean death time (MDT and intracerebral pathogenicity index (ICPI values ranged from 50.4 to 96.0 hrs and from 0.650 to 1.675 respectively. Affected birds showed anorexia, diarrohea and drop in egg production. Macropathologically, matting of vent feathers, petechial haemorrhage on the tip of proventricular papilla, caecal tonsils and degeneration of ovarian follicles were noticed. The incidence of ND was most commonly noticed in 20-50 wk of age and between the months of September to November. Morbidity rate varied from 5% to 10% in the NDV alone affected flocks and 5 to 15% in NDV with other concurrent infections. Egg production drop from the expected level ranged between 3 to 7 % in ND and

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia

DEFF Research Database (Denmark)

Toplak, Ivan; Hostnik, Peter; Rihtaric, Danijela

2010-01-01

and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide...... sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis...

Molecular characterization of infectious myonecrosis virus (IMNV isolated from the shrimp Litopenaeus vannamei farmed in Ceará State, Brazil

Directory of Open Access Journals (Sweden)

Maria Verônyca Coelho-Melo

2014-07-01

Full Text Available 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 viral capsid protein was obtained by RT-PCR and confirmed by automatic sequencing. Comparison with sequences which encode the capsid protein obtained from Indonesia isolates showed a high identity.

Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis.

Science.gov (United States)

MacLachlan, N J; Nunamaker, R A; Katz, J B; Sawyer, M M; Akita, G Y; Osburn, B I; Tabachnick, W J

1994-01-01

The interval after infection when bluetongue virus (BTV) was present in the blood of calves inoculated with BTV serotype 10 (BTV 10) was evaluated by virus isolation (VI) in embryonated chicken eggs (ECE), BTV-specific polymerase chain reaction (PCR), and in vitro blood feeding of vector Culicoides variipennis (C.v.) sonorensis. BTV nucleic acid was detected by PCR in blood cells for 16 to 20 weeks after infection whereas infectious virus was detected by VI in ECE for 2 to 8 weeks. BTV was detected in calf blood by in vitro feeding of C.v. sonorensis for only 0 to 2 weeks after inoculation of calves with BTV 10. Selected bloods which were positive by PCR analysis but not by VI in ECE were not infectious for sheep. The data are consistent with the hypothesis that prolonged viremia in BTV-infected cattle results from association of the virus with blood cells, especially erythrocytes. The fact that calf blood that contained viral nucleic acid as determined by PCR analysis, but not infectious virus as determined by VI in ECE, was not infectious for either the insect vector or sheep suggests that cattle whose blood contains BTV nucleic acid but not infectious virus are unimportant to the epidemiology of BTV infection.

Porcine reproductive and respiratory syndrome virus: antigenic and molecular diversity of British isolates and implications for diagnosis.

Science.gov (United States)

Frossard, Jean-Pierre; Fearnley, Catherine; Naidu, Brindha; Errington, Jane; Westcott, David G; Drew, Trevor W

2012-08-17

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers. To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990 s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously. For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues. As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea

Directory of Open Access Journals (Sweden)

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.

Isolation and whole-genome sequencing of a Crimean-Congo hemorrhagic fever virus strain, Greece.

Science.gov (United States)

Papa, Anna; Papadopoulou, Elpida; Tsioka, Katerina; Kontana, Anastasia; Pappa, Styliani; Melidou, Ageliki; Giadinis, Nektarios D

2018-03-01

Crimean-Congo hemorrhagic fever virus (CCHFV) was isolated from a pool of two adult Rhipicephalus bursa ticks removed from a goat in 2015 in Greece. The strain clusters into lineage Europe 2 representing the second available whole-genome sequenced isolate of this lineage. CCHFV IgG antibodies were detected in 8 of 19 goats of the farm. Currently CCHFV is not associated with disease in mammals other than humans. Studies in animal models are needed to investigate the pathogenicity level of lineage Europe 2 and compare it with that of other lineages. Copyright © 2018 Elsevier GmbH. All rights reserved.

Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

Directory of Open Access Journals (Sweden)

Felipe L Assis

Full Text Available Since 1999, several Vaccinia virus (VACV isolates, the etiological agents of bovine vaccinia (BV, have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005 molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.

Genetic characterization of bank vole virus (BaVV), a new paramyxovirus isolated from kidneys of bank voles in Russia.

Science.gov (United States)

Alkhovsky, Sergey; Butenko, Alexander; Eremyan, Aykaz; Shchetinin, Alexey

2018-03-01

A genome of bank vole virus (BaVV), isolated from kidney tissues of bank voles (Myodes glareolus) in Russia in 1973, was sequenced. The genomic organization of BaVV (3'-N-P/V/C-M-F-G-L-5', 16,992 nt in length; GenBank accession number MF943130) is most similar to that of Mossman virus (MoV) and Nariva virus (NarPV), two ungrouped paramyxoviruses isolated from rodents in Australia and Trinidad, respectively. The proteins of BaVV have the highest level of sequence identity (ranging from 23-28% for G protein to 66-73% for M protein) to proteins of MoV and NarPV. The results of genetic and phylogenetic analysis suggest that BaVV represents a new species and, together with MoV and NarPV, belongs to a new, yet not established genus of the family Paramyxoviridae.

Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic.

Science.gov (United States)

Gachara, George; Symekher, Samuel; Otieno, Michael; Magana, Japheth; Opot, Benjamin; Bulimo, Wallace

2016-06-01

An influenza pandemic caused by a novel influenza virus A(H1N1)pdm09 spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally. While whole genome data on new virus enables a deeper insight in the pathogenesis, epidemiology, and drug sensitivities of the circulating viruses, there are relatively limited complete genetic sequences available for this virus from African countries. We describe herein the full genome analysis of influenza A(H1N1)pdm09 viruses isolated in Kenya between June 2009 and August 2010. A total of 40 influenza A(H1N1)pdm09 viruses isolated during the pandemic were selected. The segments from each isolate were amplified and directly sequenced. The resulting sequences of individual gene segments were concatenated and used for subsequent analysis. These were used to infer phylogenetic relationships and also to reconstruct the time of most recent ancestor, time of introduction into the country, rates of substitution and to estimate a time-resolved phylogeny. The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences but local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide. The time of the most recent common ancestor was estimated between April and June 2009, and distinct clusters circulated during the pandemic. The complete genome had an estimated rate of nucleotide substitution of 4.9×10(-3) substitutions/site/year and greater diversity in surface expressed proteins was observed. We show that two clades of influenza A(H1N1)pdm09 virus were introduced into Kenya from the UK and the pandemic was sustained as a result of importations. Several closely related but distinct clusters co-circulated locally during the peak pandemic phase but only one cluster dominated in the late phase of the pandemic suggesting that it possessed greater adaptability. Copyright © 2016 Elsevier B.V. All rights reserved.

The sequencing of the complete genome of a Tomato black ring virus (TBRV) and of the RNA2 of three Grapevine chrome mosaic virus (GCMV) isolates from grapevine reveals the possible recombinant origin of GCMV.

Science.gov (United States)

Digiaro, M; Yahyaoui, E; Martelli, G P; Elbeaino, T

2015-02-01

The complete genome of a Tomato black ring virus isolate (TBRV-Mirs) (RNA1, 7,366 nt and RNA2, 4,640 nt) and the RNA2 sequences (4,437; 4,445; and 4,442 nts) of three Grapevine chrome mosaic virus isolates (GCMV-H6, -H15, and -H27) were determined. All RNAs contained a single open reading frame encoding polyproteins of 254 kDa (p1) and 149 kDa (p2) for TBRV-Mirs RNA1 and RNA2, respectively, and 146 kDa for GCMV RNA2. p1 of TBRV-Mirs showed the highest identity with TBRV-MJ (94 %), Beet ringspot virus (BRSV, 82 %), and Grapevine Anatolian ringspot virus (GARSV, 66 %), while p2 showed the highest identity with TBRV isolates MJ (89 %) and ED (85 %), followed by BRSV (65 %), GCMV (58 %), and GARSV (57 %). The amino acid identity of RNA2 sequences of four GCMV isolates (three from this study and one from GenBank) ranged from 91 to 98 %, the homing protein being the most variable. The RDP3 program predicted putative intra-species recombination events for GCMV-H6 and recognized GCMV as a putative inter-species recombinant between GARSV and TBRV. In both cases, the recombination events were at the movement protein level.

Isolation of plasma membrane-associated membranes from rat liver.

Science.gov (United States)

Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

2014-02-01

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

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

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

[Yellow fever virus, dengue 2 and other arboviruses isolated from mosquitos, in Burkina Faso, from 1983 to 1986. Entomological and epidemiological considerations].

Science.gov (United States)

Robert, V; Lhuillier, M; Meunier, D; Sarthou, J L; Monteny, N; Digoutte, J P; Cornet, M; Germain, M; Cordellier, R

1993-01-01

An arbovirus surveillance was carried out in Burkina Faso from 1983 to 1986. It was based on crepuscular catches of mosquitoes on human bait in some wooded areas and in one town. The total collection was 228 catches with an average of 8 men per catch. The total number of mosquitoes caught was 44,956 among which 32,010 potential vector of yellow fever; all these mosquitoes were analysed for arbovirology. In the south-western part of the country (region of Bobo-Dioulasso), surveillance was conducted each year from August to November, whilst the circulation of Aedes-borne arboviruses is well known to be favoured. In 1983, 1984 and 1986, seven strains of yellow fever virus were isolated in circumstances remarkably similar. They came from selvatic areas and never from the town. They concerned only Aedes (Stegomyia) luteocephalus which is the very predominant potential vector of yellow fever in the region. They were obtained in low figure, between 1 and 4 per year. They occurred from 27th of October to 21th of November. These observations confirm that the southern portion of the Sudan savanna zone of West Africa is the setting of a customary circulation of yellow fever virus and therefore belongs to the endemic emergence zone. In 1986, two strains of dengue 2 virus were isolated. One concerned Ae. luteocephalus from the selvatic area, the other Ae. (St.) aegypti from the heart of town. These data suggest two distinct cycles for dengue 2 virus, one urban and one selvatic, which could coexist simultaneously in the same region. In the south-eastern part of the country (region of Fada-N'Gourma) a yellow fever epidemic occurred between September and December 1983; its study has enable to precise their entomological aspects. The entomological inoculation rate of yellow fever virus has been evaluated to 22 infected bites per man during the month of october, for a man living close to forest gallery. 25 strains of yellow fever virus strains was isolated from Ae. (Diceromyia

Archaeal viruses of the sulfolobales

DEFF Research Database (Denmark)

Erdmann, Susanne; Garrett, Roger Antony

2015-01-01

in CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs...... with an environmental virus mixture isolated from Yellowstone National Park (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012). Experimental studies of isolated genetic elements from this mixture revealed that SMV1 (S ulfolobus Monocauda Virus 1), a tailed spindle-shaped virus, can induce spacer acquisition...... and the techniques used both to infect laboratory strains with these virus mixtures and to obtain purified virus particles. Secondly, we present the experimental conditions required for activating SMV1-induced spacer acquisition in two different Sulfolobus species....

A molecular and antigenic survey of H5N1 highly pathogenic avian influenza virus isolates from smallholder duck farms in Central Java, Indonesia during 2007-2008

Directory of Open Access Journals (Sweden)

Junaidi Akhmad

2011-09-01

Full Text Available Abstract Background Indonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed. Results All 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1, clade 2.1.3 (80, and IDN/6/05-like viruses (3 that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA and neuraminidase (NA sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens. Conclusions The 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to

A molecular and antigenic survey of H5N1 highly pathogenic avian influenza virus isolates from smallholder duck farms in Central Java, Indonesia during 2007-2008.

Science.gov (United States)

Wibawa, Hendra; Henning, Joerg; Wong, Frank; Selleck, Paul; Junaidi, Akhmad; Bingham, John; Daniels, Peter; Meers, Joanne

2011-09-07

Indonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI) virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed. All 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1), clade 2.1.3 (80), and IDN/6/05-like viruses (3) that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA) and neuraminidase (NA) sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens. The 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to chickens over the study period suggests that ducks are

Antigenic, genetic, and pathogenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from dead whooper swans (Cygnus cygnus) found in northern Japan in 2008.

Science.gov (United States)

Okamatsu, Masatoshi; Tanaka, Tomohisa; Yamamoto, Naoki; Sakoda, Yoshihiro; Sasaki, Takashi; Tsuda, Yoshimi; Isoda, Norikazu; Kokumai, Norihide; Takada, Ayato; Umemura, Takashi; Kida, Hiroshi

2010-12-01

In April and May 2008, whooper swans (Cygnus cygnus) were found dead in Hokkaido in Japan. In this study, an adult whooper swan found dead beside Lake Saroma was pathologically examined and the identified H5N1 influenza virus isolates were genetically and antigenically analyzed. Pathological findings indicate that the swan died of severe congestive edema in the lungs. Phylogenetic analysis of the HA genes of the isolates revealed that they are the progeny viruses of isolates from poultry and wild birds in China, Russia, Korea, and Hong Kong. Antigenic analyses indicated that the viruses are distinguished from the H5N1 viruses isolated from wild birds and poultry before 2007. The chickens vaccinated with A/duck/Hokkaido/Vac-1/2004 (H5N1) survived for 14 days after challenge with A/whooper swan/Hokkaido/1/2008 (H5N1), although a small amount of the challenge virus was recovered from the tissues of the birds. These findings indicate that H5N1 highly pathogenic avian influenza viruses are circulating in wild birds in addition to domestic poultry in Asia and exhibit antigenic variation that may be due to vaccination.

Ancient ancestry of KFDV and AHFV revealed by complete genome analyses of viruses isolated from ticks and mammalian hosts.

Directory of Open Access Journals (Sweden)

Kimberly A Dodd

2011-10-01

Full Text Available BACKGROUND: Alkhurma hemorrhagic fever virus (AHFV and Kyasanur forest disease virus (KFDV cause significant human disease and mortality in Saudi Arabia and India, respectively. Despite their distinct geographic ranges, AHFV and KFDV share a remarkably high sequence identity. Given its emergence decades after KFDV, AHFV has since been considered a variant of KFDV and thought to have arisen from an introduction of KFDV to Saudi Arabia from India. To gain a better understanding of the evolutionary history of AHFV and KFDV, we analyzed the full length genomes of 16 AHFV and 3 KFDV isolates. METHODOLOGY/PRINCIPAL FINDINGS: Viral genomes were sequenced and compared to two AHFV sequences available in GenBank. Sequence analyses revealed higher genetic diversity within AHFVs isolated from ticks than human AHFV isolates. A Bayesian coalescent phylogenetic analysis demonstrated an ancient divergence of AHFV and KFDV of approximately 700 years ago. CONCLUSIONS/SIGNIFICANCE: The high sequence diversity within tick populations and the presence of competent tick vectors in the surrounding regions, coupled with the recent identification of AHFV in Egypt, indicate possible viral range expansion or a larger geographic range than previously thought. The divergence of AHFV from KFDV nearly 700 years ago suggests other AHFV/KFDV-like viruses might exist in the regions between Saudi Arabia and India. Given the human morbidity and mortality associated with these viruses, these results emphasize the importance of more focused study of these significant public health threats.