Raghavendra Sumanth Pudupakam
Full Text Available Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3 gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV. This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2 to elucidate its genetic relationship to global BTV isolates. Materials and Methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.
Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu
Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.
Narender S Maan
Full Text Available Bluetongue (BT is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT are slow (taking weeks, depend on availability of reference virus-strains or antisera and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2 encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and
Full Text Available Abstract Background Dengue (DEN is an infectious disease caused by the DEN virus (DENV, which belongs to the Flavivirus genus in the family Flaviviridae. It has a (+ sense RNA genome and is mainly transmitted to humans by the vector mosquito Aedes aegypti. Dengue fever (DF and dengue hemorrhagic fever (DHF are caused by one of four closely related virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4. Epidemiological and evolutionary studies have indicated that host and viral factors are involved in determining disease outcome and have proved the importance of viral genotype in causing severe epidemics. Host immune status and mosquito vectorial capacity are also important influences on the severity of infection. Therefore, an understanding of the relationship between virus variants with altered amino acids and high pathogenicity will provide more information on the molecular epidemiology of DEN. Accordingly, knowledge of the DENV serotypes and genotypes circulating in the latest DEN outbreaks around the world, including Mexico, will contribute to understanding DEN infections. Results 1. We obtained 88 isolates of DENV, 27 from Oaxaca and 61 from Veracruz. 2. Of these 88 isolates, 16 were serotype 1; 62 serotype 2; 7 serotype 3; and 2 serotype 4. One isolate had 2 serotypes (DENV-2 and -1. 3. Partial nucleotide sequences of the genes encoding C- prM (14 sequences, the NS3 helicase domain (7 sequences, the NS5 S-adenosyl methionine transferase domain (7 sequences and the RNA-dependent RNA polymerase (RdRp domain (18 sequences were obtained. Phylogenetic analysis showed that DENV-2 isolates belonged to the Asian/American genotype. In addition, the Asian/American genotype was divided into two clusters, one containing the isolates from 2001 and the other the isolates from 2005–2006 with high bootstrap support of 94%. Conclusion DENV-2 was the predominant serotype in the DF and DHF outbreak from 2005 to 2006 in Oaxaca State as well as in the 2006
Perez-Ramirez, Gerardo; Diaz-Badillo, Alvaro; Camacho-Nuez, Minerva; Cisneros, Alejandro; Munoz, Maria de Lourdes
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
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
Mahapatra, Mana; Yuvaraj, S; Madhanmohan, M; Subramaniam, S; Pattnaik, B; Paton, D J; Srinivasan, V A; Parida, Satya
Foot-and-mouth disease (FMD) virus serotype O is the most common cause of FMD outbreaks in India and three of the six lineages that have been described are most frequently detected, namely Ind2001, PanAsia and PanAsia 2. We report the full capsid sequence of 21 serotype O viruses isolated from India between 2002 and 2012. All these viruses belong to the Middle East-South Asia (ME-SA) topotype. The serological cross-reactivity of a bovine post-vaccination serum pool raised against the current Indian vaccine strain, O/IND/R2/75,was tested by virus neutralisation test with the 23 Indian field isolates, revealing a good match between the vaccine and the field isolates. The cross reactivity of the O/IND/R2/75 vaccine with 19 field isolates from other countries (mainly from Asia and Africa) revealed a good match to 79% of the viruses indicating that the vaccine strain is broadly cross-reactive and could be used to control FMD in other countries. Comparison of the capsid sequences of the serologically non-matching isolates with the vaccine strain sequence identified substitutions in neutralising antigenic sites 1 and 2, which could explain the observed serological differences. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
Xu, Wanhong; Zhang, Zhidong; Nfon, Charles; Yang, Ming
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.
Sangula, Abraham K.; Belsham, Graham; Muwanika, Vincent B.
Background: In East Africa, foot-and-mouth disease virus serotype SAT 1 is responsible for occasional severe outbreaks in livestock and is known to be maintained within the buffalo populations. Little is known about the evolutionary forces underlying its epidemiology in the region. To enhance our...... 1 FMD viruses from East Africa has been determined and compared with known sequences derived from other SAT 1 viruses from sub-Saharan Africa. Purifying (negative) selection and low substitution rates characterized the SAT 1 virus isolates in East Africa. Two virus groups with probable independent...... appreciation of the epidemiological status of serotype SAT 1 virus in the region, we inferred its evolutionary and phylogeographic history by means of genealogy-based coalescent methods using 53 VP1 coding sequences covering a sampling period from 1948-2007. Results: The VP1 coding sequence of 11 serotype SAT...
Oludele, John; Lesko, Birgitta; Mahumane Gundane, Isabel; de Bruycker-Nogueira, Fernanda; Muianga, Argentina; Ali, Sadia; Mula, Flora; Chelene, Imelda; Falk, Kerstin I; Barreto Dos Santos, Flávia; Gudo, Eduardo Samo
After the report of an outbreak of dengue virus serotype 2 in 2014 in Nampula and Pemba cities, northern Mozambique, a surveillance system was established by the National Institute of Health. A study was performed during 2015-2016 to monitor the trend of the outbreak and confirm the circulating serotype of dengue virus (DENV). After the inclusion of consenting patients who met the case definition, samples from 192 patients were tested for the presence of nonstructural protein 1 antigen, and 60/192 (31%) samples were positive. Further analysis included DENV IgM antibodies, with 39 (20%) IgM positive cases. Reverse transcriptase (RT) PCR was performed for identification of the prevailing DENV serotype; 21/23 tested samples were DENV-2 positive, with DENV-2 present in both affected cities. When sequencing DENV, phenotype Cosmopolitan was identified. The surveillance indicates ongoing spread of DENV-2 in northern Mozambique 2 years after the first report of the outbreak.
N.L.P Indi Dharmayanti
Full Text Available Infectious Bronchitis is a contagious and acute respiratory disease in chickens caused by infectious bronchitis virus (IBV.Antigenic differences in IBV are associated with changes in the sequence of the spike glycoprotein (S. The subunit S1 which demonstrates more sequence variability than S-2 have been identified as hypervariable region (HVR-1 and 2. There were several IB virus field isolates included I-37 have been identified in Indonesia by serum neutralization method. However, gene sequence variation in HVR subunit S-1 had not yet been identified. Isolate I-37 was close to the serotype Connecticut 46 (Conn 46. The aim of this study is to identify sequence variation of HVR subunit S-1 gene of isolate I-37 produced by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR and sequencing. Several procedures were carried out in the study including virus titration, propagation and was concentrated from the allantoic fluid infected with IBV. Then, RNA was extracted for RTPCR. urther the product was sequnced and its homology with IBV references from GenBank was compared by GenMac version 8.0. Result showed that isolate I-37 produced 515 bp of amplification product. Isolate I-37 and Conn 46 are same serotype, yet their HVR subunit S-1 nucleotides and amino acids (protein differ by 6.9% and 15.6% respectively. It might be concluded that isolate I-37 was variant of Conn 46.
Drolet, B.S.; Reister, L.M.; Mecham, J.O.; Wilson, W.C.; Nol, P.; Vercauteren, K.C.; Rijn, van P.A.; Bowen, R.A.
Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild ruminants. Although only five of the 26 reported bluetongue virus (BTV) serotypes are considered endemic to the USA, 10 exotic serotypes have been isolated primarily in the southeastern region of the
Lloyd-Jones, Katie; Mahapatra, Mana; Upadhyaya, Sasmita; Paton, David J; Babu, Aravindh; Hutchings, Geoff; Parida, Satya
Foot-and-mouth disease (FMD) is endemic in Eastern Africa with circulation of multiple serotypes of the virus in the region. Most of the outbreaks are caused by serotype O followed by serotype A. The lack of concerted FMD control programmes in Africa has provided little incentive for vaccine producers to select vaccines that are tailored to circulating regional isolates creating further negative feedback to deter the introduction of vaccine-based control schemes. In this study a total of 80 serotype O FMD viruses (FMDV) isolated from 1993 to 2012 from East and North Africa were characterized by virus neutralisation tests using bovine antisera to three existing (O/KEN/77/78, O/Manisa and O/PanAsia-2) and three putative (O/EA/2002, O/EA/2009 and O/EA/2010) vaccine strains and by capsid sequencing. Genetically, these viruses were grouped as either of East African origin with subdivision into four topotypes (EA-1, 2, 3 and 4) or of Middle-East South Asian (ME-SA) topotype. The ME-SA topotype viruses were mainly detected in Egypt and Libya reflecting the trade links with the Middle East countries. There was good serological cross-reactivity between the vaccine strains and most of the field isolates analysed, indicating that vaccine selection should not be a major constraint for control of serotype O FMD by vaccination, and that both local and internationally available commercial vaccines could be used. The O/KEN/77/78 vaccine, commonly used in the region, exhibited comparatively lower percent in vitro match against the predominant topotypes (EA-2 and EA-3) circulating in the region whereas O/PanAsia-2 and O/Manisa vaccines revealed broader protection against East African serotype O viruses, even though they genetically belong to the ME-SA topotype. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.
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.
Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.
Namatovu, Alice; Tjørnehøj, Kirsten; Belsham, Graham J.; Dhikusooka, Moses T.; Wekesa, Sabenzia N.; Muwanika, Vincent B.; Siegismund, Hans R.; Ayebazibwe, Chrisostom
To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012–2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered. PMID:25664876
encephalitis(TBH-28), West Nile(E-101), Yellow fever(French neurotropic and 17D strains), and Zika . Two Sandfly Fever viruses (213452 and Candiru) were...were provided as first passage isolates ( Aedes pseudoscutellaris cells, AP-61) or human serum from recent dengue virus patients. African isolates... viruses of the Phlebovirus genus (Table 1). Several monoclonal antibody preparations reacted solely with dengue virus serotypes. Two preparations (13E7 and
Balinda Sheila N
Full Text Available Abstract Background Foot-and-mouth disease (FMD is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced. Results Phylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L, the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/1964 and A23/KEN/1965 and serotype O viral isolate (K/117/1999 revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O. Conclusions Sequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2, a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A
Salvador, Felipe Scassi; Amorim, Jaime Henrique; Alves, Rubens Prince Santos; Pereira, Sara A.; Ferreira, Luis Carlos Souza
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
M.H. Chew; M.M. Rahman; J. Jelip; M.R. Hassan; I. Isahak
Dengue is a severe disease caused by dengue virus (DENV), transmitted to human being by infected Aedes mosquitoes. It is a major public health concern in Southeast Asia due to its fatality in the form of hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The objective of the study was to isolate and identify dengue virus serotypes prevalent in endemic areas of Kuala Lumpur and Selangor in Malaysia by virus culture, indirect immunoflurecent assay and molecular techniques. A total number ...
Subramaniam, Saravanan; Mohapatra, Jajati K; Das, Biswajit; Sharma, Gaurav K; Biswal, Jitendra K; Mahajan, Sonalika; Misri, Jyoti; Dash, Bana B; Pattnaik, Bramhadev
Foot-and-mouth disease virus (FMDV) serotype Asia1 was first reported in India in 1951, where three major genetic lineages (B, C and D) of this serotype have been described until now. In this study, the capsid protein coding region of serotype Asia1 viruses (n = 99) from India were analyzed, giving importance to the viruses circulating since 2007. All of the isolates (n = 50) recovered during 2007-2013 were found to group within the re-emerging cluster of lineage C (designated as sublineage C(R)). The evolutionary rate of sublineage C(R) was estimated to be slightly higher than that of the serotype as a whole, and the time of the most recent common ancestor for this cluster was estimated to be approximately 2001. In comparison to the older isolates of lineage C (1993-2001), the re-emerging viruses showed variation at eight amino acid positions, including substitutions at the antigenically critical residues VP279 and VP2131. However, no direct correlation was found between sequence variations and antigenic relationships. The number of codons under positive selection and the nature of the selection pressure varied widely among the structural proteins, implying a heterogeneous pattern of evolution in serotype Asia1. While episodic diversifying selection appears to play a major role in shaping the evolution of VP1 and VP3, selection pressure acting on codons of VP2 is largely pervasive. Further, episodic positive selection appears to be responsible for the early diversification of lineage C. Recombination events identified in the structural protein coding region indicates its probable role in adaptive evolution of serotype Asia1 viruses.
Tang, Y; Lee, C W; Zhang, Y; Senne, D A; Dearth, R; Byrum, B; Perez, D R; Suarez, D L; Saif, Y M
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
Gcwalisile B. Zulu
Full Text Available Bluetongue (BT is a non-contagious disease of sheep and other domestic and wild ruminants caused by the bluetongue virus (BTV. Currently 26 serotypes of the virus have been identified. In South Africa, 22 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze-dried live attenuated polyvalent BTV vaccine. The vaccine is constituted of 15 BTV serotypes divided into three separate bottles and the aim is to develop a vaccine using fewer serotypes without compromising the immunity against the disease. This study is based on previously reported cross-neutralisation of specific BTV serotypes in in vitro studies. Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control, 1 (unrelated serotype, 9, 10 and 11 in sheep using the serum neutralisation test. The purpose of the study was to determine possible cross-protection of different serotypes in sheep. Of those vaccinated with BTV-4 and challenged with BTV-1, which is not directly related to BTV-4, 20% were completely protected and 80% showed clinical signs, but the reaction was not as severe as amongst the unvaccinated animals. In the group challenged with BTV-10, some showed good protection and some became very sick. Those challenged with BTV-9 and BTV-11 had good protection. The results showed that BTV-4 does not only elicit a specific immune response but can also protect against other serotypes.
Full Text Available Abstract Parainfluenza virus is an important pathogen threatening the health of animals and human, which brings human many kinds of disease, especially lower respiratory tract infection involving infants and young children. In order to control the virus, it is necessary to fully understand the molecular basis resulting in the genetic diversity of the virus. Homologous recombination is one of mechanisms for the rapid change of genetic diversity. However, as a negative-strand virus, it is unknown whether the recombination can naturally take place in human PIV. In this study, we isolated and identified a mosaic serotype 3 human PIV (HPIV3 from in China, and also provided several putative PIV mosaics from previous reports to reveal that the recombination can naturally occur in the virus. In addition, two swine PIV3 isolates transferred from cattle to pigs were found to have mosaic genomes. These results suggest that homologous recombination can promote the genetic diversity and potentially bring some novel biologic characteristics of HPIV.
Full Text Available Objective: Streptococcus agalactiae (Group B streptococci, GBS are frequently responsible for sepsis and meningitis seen in the early weeks of life. GBS may cause perinatal infection and premature birth in pregnant women. The aim of this study was to serotype GBS strains isolated from clinical samples and evaluate their serotype distribution according to their susceptibilities to antibiotics and isolation sites. Material and Methods: One hundred thirty one S. agalactiae strains isolated from the clinical samples were included in the study. Of the strains, 99 were isolated from urine, 20 from soft tissue, 10 from blood and 2 from vaginal swab. Penicillin G and ceftriaxone susceptibilities of GBS were determined by the agar dilution method. Susceptibilities to erythromycin, clindamycin, vancomycin and tetracycline were determined by the Kirby-Bauer method according to CLSI criteria. Serotyping was performed using the latex aglutination method using specific antisera (Ia, Ib, II-VIII. Results: While in 131 GBS strains, serotypes VII and VIII were not detected, the most frequently isolated serotypes were types Ia (36%, III (30.5% and II (13% respectively. Serotype Ia was the most frequently seen serotype in all samples. All GBS isolates were susceptible to penicilin G, ceftriaxone and vancomycin. Among the strains, tetracycline, erythromycin and clindamycin resistance rates were determined as 90%, 14.5%, and 13% respectively. Conclusion: Penicillin is still the first choice of treatment for the infections with all serotypes of S. agalactiae in Turkey.
Chen, Nan; Wang, Lin-Lin; Xue, Juan; Ma, Xiang-Bo; Zhao, Sheng; Rong, Rui-Xue; Li, Hong-Quan; Ding, Liang; Zheng, Ming-Zhi; Chen, Ying-Ying; Duan, Fei; Shen, Yue-Liang
K1 or K2 serotype Klebsiella pneumoniae isolate caused clinical pyogenic liver abscess (KLA) infection is prevalent in many areas. It has been identified that K1 or K2 serotype K. pneumoniae isolates caused KLA infection in mice by oral inoculation. In our study, K1 serotype K. pneumoniae isolate Kp1002 with hypermucoviscosity (HV)-positive phenotype caused KLA infection in C57BL/6 mice by oral inoculation. Simultaneously, non-serotype K1 and K2 isolate Kp1014 with HV-negative phenotype failed to cause KLA infection in the same manner. It seems that gastrointestinal tract translocation is the pathway by which K1 or K2 serotype K. pneumoniae caused KLA infection. Liquid chromatography-tandem mass spectrometry was used to further analyze metabolic profile changes in mice with KLA infection. Data showed that after Kp1002 or Kp1014 oral inoculation, serum Phosphatidylcholine (PC) and Lysophosphatidylcholine (LPC) levels significantly changed in mice. Some PC and LPC molecules showed changes both in the Kp1002 KLA group and the Kp1014 no-KLA group compared with the control group. The level of 18:1/18:2-PC significantly changed in the Kp1002 KLA group compared with the control group, but showed no change between the Kp1014 no-KLA group and the control group. The level of 18:1/18:2-PC might have been particularly affected by KLA infection caused by K1 serotype K. pneumoniae Kp1002. It may be a potential biomarker for KLA infection. Copyright © 2014 Elsevier Ltd. All rights reserved.
Full Text Available Purpose: To standardize and apply a polymerase chain reaction (PCR on the glycoprotein D gene to differentiate Herpes simplex virus (HSV 1 & 2 serotypes in culture negative intraocular specimens. Methods: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV and varicella zoster virus (VZV by nucleic acid amplification methods. Results: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens, and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. Conclusions: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.
Verdezoto, J; Breard, E; Viarouge, C; Quenault, H; Lucas, P; Sailleau, C; Zientara, S; Augot, D; Zapata, S
Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador. © 2017 Blackwell Verlag GmbH.
Gupta, Birendra Prasad; Singh, Sneha; Kurmi, Roshan; Malla, Rajani; Sreekumar, Easwaran; Manandhar, Krishna Das
Background & objectives: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. Methods: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. Results: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. Interpretations & conclusions: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region. PMID:26905233
Firacative, Carolina; Torres, Germán; Rodríguez, María Claudia; Escandón, Patricia
In Cúcuta, Cryptococcus gattii serotype B is commonly recovered from immunocompetent patients with cryptococcosis, but it has not been recovered from the environment in spite of its high incidence which is 77% out of reported cases. The aim of this work was to carry out an extensive environmental sampling in Cúcuta, in an attempt to isolate C. gattii serotype B and to expand our knowledge about the ecology and epidemiology of this important yeast. Samples associated with 3,634 trees from 40 zones of Cúcuta were collected and processed with 28 samples collected near the houses of four patients with cryptococcosis caused by C. gattii serotype B. The serotype of the recovered isolates was done using multiplex PCR, molecular patterns were determined by RFLP of the URA5 gene and mating type was determined using the primers MfÎ±U, MfÎ±L, MFa2U and MFa2L. In total, 4,389 samples were processed and one isolate of C. gattii serotype B (VGI/a), two isolates of C. gattii serotype C (VGIII/Î±) and three isolates of C. neoformans var. grubii, serotype A (VNI/Î±), were recovered. The density of the recovered isolates varied from 50 to 350 cfu/g of soil. This is the first report on the environmental isolation of C. gattii serotype B from Cúcuta. However, because of the low rate of recovery of isolates from soil only, the environmental niche of C. gattii has not been established and further environmental studies in Cúcuta are necessary, owing that this serotype is not only causing cryptococcosis but also has shown a higher virulence after the Vancouver outbreak.
Dhikusooka, Moses Tefula; Tjørnehøj, Kirsten; Ayebazibwe, Chrisostom
After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closest...
Sharma, K; Hair-Bejo, M; Omar, A R; Aini, I
Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.
Horsington, Jacquelyn; Hartley, Carol A; Gilkerson, James R
Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50-80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74-86%); ERBV-3 antibodies were most prevalent (58-62%) and ERBV-2 antibodies were least prevalent (10-16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.
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
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.
Namatovu, Alice; Tjørnehøj, Kirsten; Belsham, Graham
To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012-2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples...... were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK® FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45...... Kiruhura, Isingiro and Ntungamo districts. Consistent with the detection of high levels of neutralising antibodies against SAT 2, was the isolation of a SAT 2 FMDV from Isingiro; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently...
Carolina de la Guardia
Full Text Available Dengue virus causes dengue fever, a debilitating disease with an increasing incidence in many tropical and subtropical territories. So far, there are no effective antivirals licensed to treat this virus. Here we describe the synthesis and antiviral activity evaluation of two compounds based on the quinoline scaffold, which has shown potential for the development of molecules with various biological activities. Two of the tested compounds showed dose-dependent inhibition of dengue virus serotype 2 in the low and sub micromolar range. The compounds 1 and 2 were also able to impair the accumulation of the viral envelope glycoprotein in infected cells, while showing no sign of direct virucidal activity and acting possibly through a mechanism involving the early stages of the infection. The results are congruent with previously reported data showing the potential of quinoline derivatives as a promising scaffold for the development of new antivirals against this important virus.
Ruder, Mark G; Howerth, Elizabeth W; Stallknecht, David E; Allison, Andrew B; Carter, Deborah L; Drolet, Barbara S; Klement, Eyal; Mead, Daniel G
Culicoides sonorensis (Diptera: Ceratopogonidae) is a vector of epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD) and other wild ruminants. Although historically rare, reports of clinical EHDV infection in cattle have increased in some parts of the world over the past decade. In 2006, an EHDV-7 epizootic in cattle resulted in economic loss for the Israeli dairy industry. White-tailed deer are susceptible to EHDV-7 infection and disease; however, this serotype is exotic to the US and the susceptibility of C. sonorensis to this cattle-virulent EHDV is not known. The objective of the study was to determine if C. sonorensis is susceptible to EHDV-7 infection and is a competent vector. To evaluate the susceptibility of C. sonorensis, midges were fed on EHDV-7 infected WTD, held at 22 ± 1°C, and processed individually for virus isolation and titration on 4-16 days post feeding (dpf). Midges with a virus titer of ≥ 10(2.7) median tissue culture infective doses (TCID(50))/midge were considered potentially competent. To determine if infected C. sonorensis were capable of transmitting EHDV-7 to a host, a susceptible WTD was then fed on by a group of 14-16 dpf midges. From 4-16 dpf, 45% (156/350) of midges that fed on WTD with high titer viremia (>10(7) TCID(50)/ml) were virus isolation-positive, and starting from 10-16 dpf, 32% (35/109) of these virus isolation-positive midges were potentially competent (≥ 10(2.7) TCID(50)/midge). Midges that fed on infected deer transmitted the virus to a susceptible WTD at 14-16 dpf. The WTD developed viremia and severe clinical disease. This study demonstrates that C. sonorensis is susceptible to EHDV-7 infection and can transmit the virus to susceptible WTD, thus, C. sonorensis should be considered a potential vector of EHDV-7. Together with previous work, this study demonstrates that North America has a susceptible ruminant and
Lestari, C S Whinie; Yohan, Benediktus; Yunita, Anisa; Meutiawati, Febrina; Hayati, Rahma Fitri; Trimarsanto, Hidayat; Sasmono, R Tedjo
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.
Maneerat, K; Yongkiettrakul, S; Kramomtong, I; Tongtawe, P; Tapchaisri, P; Luangsuk, P; Chaicumpa, W; Gottschalk, M; Srimanote, P
Isolates of Streptococcus suis from different Western countries as well as those from China and Vietnam have been previously well characterized. So far, the genetic characteristics and relationship between S. suis strains isolated from both humans and pigs in Thailand are unknown. In this study, a total of 245 S. suis isolates were collected from both human cases (epidemic and sporadic) and pigs (diseased and asymptomatic) in Thailand. Bacterial strains were identified by biochemical tests and PCR targeting both, the 16S rRNA and gdh genes. Thirty-six isolates were identified as serotype 2 based on serotyping and the cps2-PCR. These isolates were tested for the presence of six virulence-associated genes: an arginine deiminase (arcA), a 38-kDa protein and protective antigen (bay046), an extracellular factor (epf), an hyaluronidase (hyl), a muramidase-released protein (mrp) and a suilysin (sly). In addition, the genetic diversities of these isolates were studied by RAPD PCR and multilocus sequence typing (MLST) analysis. Four virulence-associated gene patterns (VAGP 1 to 4) were obtained, and the majority of isolates (32/36) carried all genes tested (VAGP1). Each of the three OPB primers used provided 4 patterns designated RAPD-A to RAPD-D. Furthermore, MLST analysis could also distinguish the 36 isolates into four sequence types (STs): ST1 (n = 32), ST104 (n = 2), ST233 (n = 1) and a newly identified ST, ST336 (n = 1). Dendrogram constructions based on RAPD patterns indicated that S. suis serotype 2 isolates from Thailand could be divided into four groups and that the characteristics of the individual groups were in complete agreement with the virulence gene profiles and STs. The majority (32/36) of isolates recovered from diseased pigs, slaughterhouse pigs or human patients could be classified into a single group (VAGP1, RAPD-A and ST1). This genetic information strongly suggests the transmission of S. suis isolates from pigs to humans in Thailand. Our findings are
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
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
Betania Paiva Drumond
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.
Full Text Available ABSTRACT The aim of this work was to study the prevalence of Listeria monocytogenes in foods obtained in retail shops and food industries located in Montevideo-Uruguay, and to identify the serogroups of the obtained isolates. Three-thousand one-hundred and seventy-five food samples (frozen, deli meats, ready-to-eat and cheese were analyzed. The obtained isolates were serogrouped by multiplex PCR and serotyped by conventional procedure. Genetic comparisons were performed using pulsed-field gel electrophoresis on a sub-set of isolates belonging to the same serotype successively recovered from the same establishment. L. monocytogenes was isolated from 11.2% of samples. The highest prevalence was observed in frozen foods (38%, followed by cheese (10%. 1/2b and 4b were the most frequently identified serotypes. In six of 236 analyzed establishments we successively recovered L. monocytogenes isolates belonging to the same serotype. Most of them corresponded to serotype 1/2b. Pulsed-field gel electrophoresis profiles suggest that at least 33% of L. monocytogenes 1/2b isolates are genetically related and that may remain viable for prolonged periods. The observed prevalence of L. monocytogenes was lower than reported in neighboring countries. Our findings highlight the role that frozen foods may play in the spread of this pathogen, and the relevance of serotypes 1/2b and 4b.
Le, Van Phan; Vu, Thi Thu Hang; Duong, Hong-Quan; Than, Van Thai; Song, Daesub
Foot-and-mouth disease virus (FMDV) is one of the highest risk factors that affects the animal industry of the country. The virus causes production loss and high ratio mortality in young cloven-hoofed animals in Vietnam. The VP1 coding gene of 80 FMDV samples (66 samples of the serotype O and 14 samples of the serotype A) collected from endemic outbreaks during 2006-2014 were analyzed to investigate their phylogeny and genetic relationship with other available FMDVs globally. Phylogenetic analysis indicated that the serotype O strains were clustered into two distinct viral topotypes (the SEA and ME-SA), while the serotype A strains were all clustered into the genotype IX. Among the study strains, the amino acid sequence identities were shared at a level of 90.1-100, 92.9-100, and 92.8-100% for the topotypes SEA, ME-SA, and genotype IX, respectively. Substitutions leading to changes in the amino acid sequence, which are critical for the VP1 antigenic sites were also identified. Our results showed that the studied strains are most closely related to the recent FMDV isolates from Southeast Asian countries (Myanmar, Thailand, Cambodia, Malaysia, and Laos), but are distinct from the earlier FMDV isolates within the genotypes. This study provides important evidence of recent movement of FMDVs serotype O and A into Vietnam within the last decade and their genetic accumulation to be closely related to strains causing FMD in surrounding countries.
Mahapatra, Mana; Upadhyaya, Sasmita; Aviso, Sharie; Babu, Aravindh; Hutchings, Geoff; Parida, Satya
Foot-and-mouth disease (FMD) is endemic in Southeast Asia (SEA) and East Asia with circulation of multiple serotypes and multiple genotypes within each serotype of the virus. Although countries like Japan and South Korea in the Far East were free of FMD, in 2010 FMD serotype O (O/Mya-98) outbreaks were recorded and since then South Korea has experienced several FMD outbreaks despite regular vaccination. In this study a total of 85 serotype O FMD viruses (FMDV) isolated from 2007 to 2012 from SEA, East Asia and Far East were characterized by virus neutralisation tests using antisera to four existing (O/HKN/6/83, O/IND/R2/75, O/SKR/2010 and O/PanAsia-2) and one putative (O/MYA/2009) vaccine strains, and by full capsid sequencing. Serological studies revealed broad cross-reactivity with the vaccine strains; O/PanAsia-2 exhibited a good match with 95.3%, O/HKN/6/83 with 91.8%, O/IND/R2/75 with 80%, and the putative strain O/MYA/2009 with 89.4% isolates employed in this study. Similarly O/PanAsia-2 and O/IND/R2/75 vaccines showed a good match with all eight viruses belonging to O-Ind-2001d sublineage whereas the vaccines of O/Mya-98 lineage, O/MYA/2009 and O/SKR/2010 exhibited the lowest match indicating their unsuitability to protect infections from O-Ind-2001d viruses. A Bayesian analysis of the capsid sequence data indicated these circulating viruses (n = 85) to be of either SEA or Middle East-South Asian (ME-SA) topotype. The ME-SA topotype viruses were mainly detected in Lao PDR, Vietnam, Myanmar and Thailand reflecting the trade links with the Indian subcontinent, and also within the SEA countries. Implications of these results in the context of FMD control in SEA and East Asian countries are discussed. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.
Wekesa, S N; Muwanika, V B; Siegismund, H R; Sangula, A K; Namatovu, A; Dhikusooka, M T; Tjørnehøj, K; Balinda, S N; Wadsworth, J; Knowles, N J; Belsham, G J
Foot-and-mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these viruses were determined to ascertain whether these were independent outbreaks or one single strain spreading throughout the country. The sequences of the complete VP1-coding region were analysed from viruses sampled within different areas of Kenya during 2010 and 2011. The results indicated that the 2010 to 2011 outbreaks in Kenya were caused by four independent strains. By comparison with earlier type O isolates from Eastern Africa, it was apparent that the outbreaks were caused by viruses from three different lineages of topotype EA-2 and a fourth virus strain belonging to topotype EA-4. The topotypes EA-1 and EA-3 were not detected from these outbreaks. Implications of these results for FMD control in Eastern Africa are discussed. © 2013 Blackwell Verlag GmbH.
Bachanek-Bankowska, Katarzyna; Mero, Herieth R; Wadsworth, Jemma; Mioulet, Valerie; Sallu, Raphael; Belsham, Graham J; Kasanga, Christopher J; Knowles, Nick J; King, Donald P
Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce C T values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
Full Text Available Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011-2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011-2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.
Nielsen, Eva Møller; Nielsen, Niels Ladefoged
to study the serotype distribution of C. jejuni and C. coli isolated from different food products of poultry origin sampled from retail outlets in Denmark. A total of 156 isolates were serotyped, 85% of these were C. jejuni and 15% were C. coli. The most common C. jejuni serotypes were O:2 (30%), O:1...... nontypable. This rate of nontypable isolates is significantly higher than experienced for isolates from other sources than food products, i.e faecal samples from animals and humans. Subculturing and re-typing of the nontypable isolates improved the typability. After two, five and 10 subcultures 16, six...
Venter, G J; Paweska, J T; Van Dijk, A A; Mellor, P S; Tabachnick, W J
The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis. The concentration of the virus per millilitre of bloodmeal was 10(5.0) and 10(6.0)TCID50 for BLU 4 and 10(7.2)TCID50 for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log10 TCID50 titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola. The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola. However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.
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.
Ehizibolo, D O; Haegeman, A; De Vleeschauwer, A R; Umoh, J U; Kazeem, H M; Okolocha, E C; Van Borm, S; De Clercq, K
The knowledge of foot-and-mouth disease virus (FMDV) dynamics and epidemiology in Nigeria and the West Africa subregion is important to support local and regional control plans and international risk assessment. Foot-and-mouth disease virus serotype South African territories (SAT)1 was isolated, identified and characterized from an FMD outbreak in cattle in Nigeria in 2015, 35 years after the last report of FMDV SAT1 in West Africa. The VP1 coding sequence of the Nigerian 2015 SAT1 isolates diverges from reported SAT1 topotypes resulting in a separate topotype. The reporting of a novel FMDV SAT1 strain in the virus pool 5 (West and Central Africa) highlights the dynamic and complex nature of FMDV in this region of Africa. Sustained surveillance is needed to understand the origin, the extent and distribution of this novel SAT1 topotype in the region as well as to detect and monitor the occurrence of (re-)emerging FMDV strains. © 2017 Blackwell Verlag GmbH.
Goraichuk, Iryna V.; Dimitrov, Kiril M.; Sharma, Poonam; Miller, Patti J.; Swayne, David E.; Suarez, David L.; Afonso, Claudio L.
ABSTRACT The first complete genome sequences of four avian paramyxovirus serotype 10 (APMV-10) isolates are described here. The viruses were isolated from rockhopper penguins on the Falkland Islands, sampled in 2007. All four genomes are 15,456 nucleotides in length, and phylogenetic analyses show them to be closely related.
Ruder Mark G
Full Text Available Abstract Background Culicoides sonorensis (Diptera: Ceratopogonidae is a vector of epizootic hemorrhagic disease virus (EHDV serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD and other wild ruminants. Although historically rare, reports of clinical EHDV infection in cattle have increased in some parts of the world over the past decade. In 2006, an EHDV-7 epizootic in cattle resulted in economic loss for the Israeli dairy industry. White-tailed deer are susceptible to EHDV-7 infection and disease; however, this serotype is exotic to the US and the susceptibility of C. sonorensis to this cattle-virulent EHDV is not known. The objective of the study was to determine if C. sonorensis is susceptible to EHDV-7 infection and is a competent vector. Methods To evaluate the susceptibility of C. sonorensis, midges were fed on EHDV-7 infected WTD, held at 22 ± 1°C, and processed individually for virus isolation and titration on 4–16 days post feeding (dpf. Midges with a virus titer of ≥102.7 median tissue culture infective doses (TCID50/midge were considered potentially competent. To determine if infected C. sonorensis were capable of transmitting EHDV-7 to a host, a susceptible WTD was then fed on by a group of 14–16 dpf midges. Results From 4–16 dpf, 45% (156/350 of midges that fed on WTD with high titer viremia (>107 TCID50/ml were virus isolation-positive, and starting from 10–16 dpf, 32% (35/109 of these virus isolation-positive midges were potentially competent (≥102.7 TCID50/midge. Midges that fed on infected deer transmitted the virus to a susceptible WTD at 14–16 dpf. The WTD developed viremia and severe clinical disease. Conclusion This study demonstrates that C. sonorensis is susceptible to EHDV-7 infection and can transmit the virus to susceptible WTD, thus, C. sonorensis should be considered a potential vector of EHDV-7. Together with previous work, this study demonstrates
Kusmintarsih, Endang S; Hayati, Rahma F; Turnip, Oktaviani N; Yohan, Benediktus; Suryaningsih, Suhestri; Pratiknyo, Hery; Denis, Dionisius; Sasmono, R Tedjo
Dengue is hyper-endemic in Indonesia. Purwokerto city in Central Java province is routinely ravaged by the disease. Despite the endemicity of dengue in this city, there is still no data on the virological aspects of dengue in the city. We conducted a molecular surveillance study of the circulating dengue viruses (DENV) in Purwokerto city to gain information on the virus origin, serotype and genotype distribution, and phylogenetic characteristics of DENV. A cross-sectional dengue molecular surveillance study was conducted in Purwokerto. Sera were collected from dengue-suspected patients attending three hospitals in the city. Diagnosis was performed using dengue NS1 antigen and IgG/IgM antibodies detection. DENV serotyping was performed using Simplexa Dengue real-time RT-PCR. Sequencing was conducted to obtain full-length DENV Envelope (E) gene sequences, which were then used in phylogenetic and genotypic analyses. Patients' clinical and demographic data were collected and analyzed. A total of 105 dengue-suspected patients' sera were collected, in which 80 (76.2%) were positive for IgM and/or IgG, and 57 (54.2%) were confirmed as dengue by NS1 antigen and/or DENV RNA detection using RT-PCR. Serotyping was successful for 47 isolates. All four serotypes circulated in the area with DENV-3 as the predominant serotype. Phylogenetic analyses grouped the isolates into Genotype I for DENV-1, Cosmopolitan genotype for DENV-2, and Genotype I and II for DENV-3 and -4, respectively. The analyses also revealed the close relatedness of Purwokerto isolates to other DENV strains from Indonesia and neighboring countries. We reveal the molecular and virological characteristics of DENV in Purwokerto, Banyumas regency, Central Java. The genotype and phylogenetic analyses indicate the endemicity of the circulating DENV in the city. Our serotype and genotype data provide references for future dengue molecular epidemiology studies and disease management in the region. Copyright © 2017 The
Goraichuk, Iryna V.; Dimitrov, Kiril M.; Sharma, Poonam; Miller, Patti J.; Swayne, David E.; Suarez, David L.
ABSTRACT The first complete genome sequences of four avian paramyxovirus serotype 10 (APMV-10) isolates are described here. The viruses were isolated from rockhopper penguins on the Falkland Islands, sampled in 2007. All four genomes are 15,456 nucleotides in length, and phylogenetic analyses show them to be closely related. PMID:28572332
Holman, David H; Wang, Danher; Raviprakash, Kanakatte; Raja, Nicholas U; Luo, Min; Zhang, Jianghui; Porter, Kevin R; Dong, John Y
Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.
de Castro Márcia Gonçalves
Full Text Available Summary Background Dengue, a mosquito-borne viral infection caused by one of the four dengue virus (DENV serotypes (DENV-1 to 4, replicate alternately on the mosquito vector and human host and are responsible for infections throughout tropical and subtropical regions of the world. In Brazil, the disease has become a major public health problem and the introduction of DENV-3 in 2000 in Rio de Janeiro (RJ was associated with severe dengue epidemics. The potential emergence of strains associated with severe disease highlights the need for the surveillance of DENV in human host and vectors. Methods Aiming to contribute for DENV phylogenetic and vector-virus-human host studies, we sequenced the entire genome of one DENV-3 isolated from naturally infected Aedes aegypti from RJ in 2001 and characterized the 3’ UTR from strains isolated from mosquitoes and humans. Mosquitoes were pooled and submitted to virus isolation in Ae. albopictus C6/36 cells and the infecting serotype was identified by immunofluorescence using type-specific monoclonal antibody. Sequence analysis was performed using BioEdit software, the multiple alignments were performed using CLUSTAL W and the phylogenetic analysis by MEGA 5, using the Neighbor-joining method. Secondary structure prediction was performed by using the MFOLD program. Results Exclusive substitutions and a substitution leading to a stop codon on the NS5 gene were observed in the DENV-3 isolated from a naturally infected Ae. aegypti and fully sequenced. As an 8- nucleotides deletion was observed within the 11- nucleotides (nts insertion on the variable region (VR from the 3′UTR in this isolate, we further sequenced other DENV-3 from both mosquitoes and humans. The majority of DENV-3 from RJ analyzed were characterized by the 11-nts insertion in the VR of the 3′UTR, despite the observation of strains carrying the 8-nts deletion. The latter presented similar secondary structures, however not all strains
Bari, Fufa Dawo; Parida, Satya; Asfor, Amin S.; Haydon, Daniel T.; Reeve, Richard; Paton, David J.
Epitopes on the surface of the foot-and-mouth disease virus (FMDV) capsid have been identified by monoclonal antibody (mAb) escape mutant studies leading to the designation of four antigenic sites in serotype A FMDV. Previous work focused on viruses isolated mainly from Asia, Europe and Latin America. In this study we report on the prediction of epitopes in African serotype A FMDVs and testing of selected epitopes using reverse genetics. Twenty-four capsid amino acid residues were predicted to be of antigenic significance by analysing the capsid sequences (n = 56) using in silico methods, and six residues by correlating capsid sequence with serum–virus neutralization data. The predicted residues were distributed on the surface-exposed capsid regions, VP1–VP3. The significance of residue changes at eight of the predicted epitopes was tested by site-directed mutagenesis using a cDNA clone resulting in the generation of 12 mutant viruses involving seven sites. The effect of the amino acid substitutions on the antigenic nature of the virus was assessed by virus neutralization (VN) test. Mutations at four different positions, namely VP1-43, VP1-45, VP2-191 and VP3-132, led to significant reduction in VN titre (P value = 0.05, 0.05, 0.001 and 0.05, respectively). This is the first time, to our knowledge, that the antigenic regions encompassing amino acids VP1-43 to -45 (equivalent to antigenic site 3 in serotype O), VP2-191 and VP3-132 have been predicted as epitopes and evaluated serologically for serotype A FMDVs. This identifies novel capsid epitopes of recently circulating serotype A FMDVs in East Africa. PMID:25614587
Wekesa, Sabenzia N.; Sangula, Abraham K.; Belsham, Graham
Serotype A is the most genetically and antigenically diverse of the foot-and-mouth disease virus (FMDV) serotypes. Records of its occurrence in Kenya date back to 1952 and the antigenic diversity of the outbreak viruses in this region is reflected by the current use of two different vaccine strains...... (K5/1980 and K35/1980) and previous use of two other strains (K18/66 and K179/71). This study aimed at enhancing the understanding of the patterns of genetic variation of serotype A FMDV in Kenya. The complete VP1 coding region sequences of 38 field isolates, identified as serotype A FMDV, collected...... between 1964 and 2013 were determined. Coalescent-based methods were used to infer times of divergence of the virus strains and the evolutionary rates alongside 27 other serotype A FMDV sequences from Genbank and the World Reference Laboratory (WRL). This study represents the first comprehensive genetic...
Rao, Pavuluri Panduranga; Reddy, Yella Narasimha; Ganesh, Kapila; Nair, Shreeja G; Niranjan, Vidya; Hegde, Nagendra R
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.
Sluijs, van der M.T.W.; Schroer-Joosten, D.P.H.; Fid-Fourkour, A.; Vrijenhoek, M.P.; Debyser, I.; Moulin, V.; Moormann, R.J.M.; Smit, de A.J.
The Bluetongue virus serotype 8 (BTV-8) strain, which emerged in Europe in 2006, had an unusually high ability to cause foetal infection in pregnant ruminants. Other serotypes of BTV had already been present in Europe for more than a decade, but transplacental transmission of these strains had never
Fulton, R W; Pearson, N J
The interferon inducing ability of bluetongue viruses was studied in bovine and feline monolayer cultures inoculated with each of four bluetongue virus serotypes. Interferon was assayed by a plaque reduction method in monolayer cultures with vesicular stomatitis virus as challenge virus. Interferon was produced by bovine turbinate, Georgia bovine kidney, and Crandell feline kidney monolayer cultures in response to bluetongue virus serotypes 10, 11, 13 and 17. The antiviral substances produced...
Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation
Reid, Scott M; Mioulet, Valerie; Knowles, Nick J; Shirazi, Nazeem; Belsham, Graham J; King, Donald P
Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). In countries where FMD is endemic, identification of the serotypes of the causative virus strains is important for vaccine selection and tracing the source of outbreaks. In this study, real-time reverse transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative field samples of serotype O strains belonging exclusively to the PanAsia-2 lineage, serotype A strains of the Iran-05 lineage and serotype Asia-1 viruses from three relevant sub-groups. When RNA extracted from archival and contemporary field strains was tested using one- or two-step rRT-PCR assays, all three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two copies. The results illustrate the potential of tailored rRT-PCR tools for the detection and categorization of viruses circulating in the Middle East belonging to distinct subgroups of serotypes O, A and Asia-1. These assays can also overcome the problem of serotyping samples which are found positive by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
Takano, Tomomi; Nakano, Kenta; Doki, Tomoyoshi; Hohdatsu, Tsutomu
Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. The genome of coronaviruses encodes a hydrophobic transmembrane protein, the envelope (E) protein. The E protein possesses ion channel activity. Viral proteins with ion channel activity are collectively termed "viroporins". Hexamethylene amiloride (HMA), a viroporin inhibitor, can inhibit the ion channel activity of the E protein and replication of several coronaviruses. However, it is not clear whether HMA and other viroporin inhibitors affect replication of FIPV. We examined the effect of HMA and other viroporin inhibitors (DIDS [4,4'-disothiocyano-2,2'-stilbenedisulphonic acid] and amantadine) on infection by FIPV serotypes I and II. HMA treatment drastically decreased the titers of FIPV serotype I strains Black and KU-2 in a dose-dependent manner, but it only slightly decreased the titer of FIPV serotype II strain 79-1146. In contrast, DIDS treatment decreased the titer of FIPV serotype II strain 79-1146 in dose-dependent manner, but it only slightly decreased the titers of FIPV serotype I strains Black and KU-2. We investigated whether there is a difference in ion channel activity of the E protein between viral serotypes using E. coli cells expressing the E protein of FIPV serotypes I and II. No difference was observed, suggesting that a viroporin other than the E protein influences the differences in the actions of HMA and DIDS on FIPV serotypes I and II.
Wang, Ping; Tong, Jing-jing; Ma, Xiu-hua; Song, Feng-li; Fan, Ling; Guo, Cui-mei; Shi, Wei; Yu, Sang-jie; Yao, Kai-hu; Yang, Yong-hong
To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections. All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles. In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB. S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB.
Candelora, Kristen L; Spalding, Marilyn G; Sellers, Holly S
Captive-reared Whooping Cranes (Grus americana) released into Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997-98 and 2001-02 release seasons. Exposure to infectious bursal disease virus (IBDV) serotype 2 as evidenced by seroconversion was suspected to be the factor that precipitated these mortality events. Very little is known about the incidence of IBD in wild bird populations. Before this study, natural exposure had not been documented in wild birds of North America having no contact with captive-reared cranes, and the prevalence and transmission mechanisms of the virus in wild birds were unknown. Sentinel chickens (Gallus gallus) monitored on two Whooping Crane release sites in central Florida, USA, during the 2003-04 and 2004-05 release seasons seroconverted, demonstrating natural exposure to IBDV serotype 2. Blood samples collected from Wild Turkeys (Meleagris gallopavo) and Sandhill Cranes (Grus canadensis) in eight of 21 counties in Florida, USA, and one of two counties in southern Georgia, USA, were antibody-positive for IBDV serotype 2, indicating that exposure from wild birds sharing habitat with Whooping Cranes is possible. The presence of this virus in wild birds in these areas is a concern for the resident flock of Whooping Cranes because they nest and raise their chicks in Florida, USA. However, passively transferred antibodies may protect them at this otherwise vulnerable period in their lives.
Thompson, Kimberly M; Duintjer Tebbens, Radboud J
Comparing model expectations with the experience of oral poliovirus vaccine (OPV) containing serotype 2 (OPV2) cessation can inform risk management for the expected cessation of OPV containing serotypes 1 and 3 (OPV13). We compare the expected post-OPV2-cessation OPV2-related viruses from models with the evidence available approximately 6 months after OPV2 cessation. We also model the trade-offs of use vs nonuse of monovalent OPV (mOPV) for outbreak response considering all 3 serotypes. Although too early to tell definitively, the observed die-out of OPV2-related viruses in populations that attained sufficiently intense trivalent OPV (tOPV) use prior to OPV2 cessation appears consistent with model expectations. As expected, populations that did not intensify tOPV use prior to OPV2 cessation show continued circulation of serotype 2 vaccine-derived polioviruses (VDPVs). Failure to aggressively use mOPV to respond to circulating VDPVs results in a high risk of uncontrolled outbreaks that would require restarting OPV. Ensuring a successful endgame requires more aggressive OPV cessation risk management than has occurred to date for OPV2 cessation. This includes maintaining high population immunity to transmission up until OPV13 cessation, meeting all prerequisites for OPV cessation, and ensuring sufficient vaccine supply to prevent and respond to outbreaks. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.
Balinda, Sheila; Sangula, Abraham; Heller, Rasmus
Foot-and-mouth disease (FMD) virus serotype O has been responsible for most reported outbreaks of the disease in East Africa. A sustained campaign for the past 40 years to control FMD mainly by vaccination, combined with quarantine and zoosanitary measures has been undertaken with limited success....... We investigated the genetic relationships among serotype O strains in eastern Africa using complete VP1 coding region sequences obtained from 46 FMD virus isolates collected in Kenya in the years 1964–2008 and 8 Ugandan isolates collected between 1999 and 2006. In addition, 21 selected FMDV sequences...... the dominant evolutionary force. Cross-border disease transmission within the region has been suggested with probable incursions of topotypes EA-3 and EA-4 into Kenya and Uganda from neighboring Ethiopia and Sudan. We conclude that the vaccines have probably been effective in controlling EA-1, but less so...
Sangula, Abraham; Siegismund, Hans; Belsham, Graham
Most viruses are maintained by complex processes of evolution that enable them to survive but also complicate efforts to achieve their control. In this paper, we study patterns of evolution in foot-and-mouth disease (FMD) serotype C virus isolates from Kenya, one of the few places in the world wh...
Full Text Available Shigella flexneri serotype 1b is among the most prominent serotypes in developing countries, followed by serotype 2a. However, only limited data is available on the global phenotypic and genotypic characteristics of S. flexneri 1b. In the present study, 40 S. flexneri 1b isolates from different regions of China were confirmed by serotyping and biochemical characterization. Antimicrobial susceptibility testing showed that 85% of these isolates were multidrug-resistant strains and antibiotic susceptibility profiles varied between geographical locations. Strains from Yunnan were far more resistant than those from Xinjiang, while only one strain from Shanghai was resistant to ceftazidime and aztreonam. Fifteen cephalosporin resistant isolates were identified in this study. ESBL genes (blaSHV, blaTEM, blaOXA, and blaCTX-M and ampC genes (blaMOX, blaFOX, blaMIR(ACT-1, blaDHA, blaCIT and blaACC were subsequently detected among the 15 isolates. The results showed that these strains were positive only for blaTEM, blaOXA, blaCTX-M, intI1, and intI2. Furthermore, pulsed-field gel electrophoresis (PFGE analysis showed that the 40 isolates formed different profiles, and the PFGE patterns of Xinjiang isolates were distinct from Yunnan and Shanghai isolates by one obvious, large, missing band. In summary, similarities in resistance patterns were observed in strains with the same PFGE pattern. Overall, the results supported the need for more prudent selection and use of antibiotics in China. We suggest that antibiotic susceptibility testing should be performed at the start of an outbreak, and antibiotic use should be restricted to severe Shigella cases, based on resistance pattern variations observed in different regions. The data obtained in the current study might help to develop a strategy for the treatment of infections caused by S. flexneri 1b in China.
Jamal, Syed M; Ferrari, Giancarlo; Ahmed, Safia; Normann, Preben; Belsham, Graham J
Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate appropriate vaccine selection and tracing of outbreaks. The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998-2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1 have circulated in Pakistan during this time. These are Group-II, -VI and, recently, a novel Group (designated here as Group-VII). This new Group has not been detected in neighbouring Afghanistan during the study period but viruses from Groups I and -II are in circulation there. Using near complete genome sequences, from FMD viruses of serotypes Asia-1 and A that are currently circulating in Pakistan, we have identified an interserotypic recombinant virus, which has the VP2-VP3-VP1-2A coding sequences derived from a Group-VII Asia-1 virus and the remainder of the genome from a serotype A virus of the A-Iran05(AFG-07) sub-lineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses. Copyright © 2011 Elsevier B.V. All rights reserved.
Halsey, Eric S.; Marks, Morgan A.; Gotuzzo, Eduardo; Fiestas, Victor; Suarez, Luis; Vargas, Jorge; Aguayo, Nicolas; Madrid, Cesar; Vimos, Carlos; Kochel, Tadeusz J.; Laguna-Torres, V. Alberto
Background Disease caused by the dengue virus (DENV) is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype. Methodology and Principal Findings Between the years 2005–2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%), DENV-2 (4.3%), DENV-3 (41.5%), or DENV-4 (14.4%). When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations. Conclusions/Significance Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype. PMID:22563516
Eric S Halsey
Full Text Available Disease caused by the dengue virus (DENV is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4 with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype.Between the years 2005-2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%, DENV-2 (4.3%, DENV-3 (41.5%, or DENV-4 (14.4%. When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations.Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype.
P. G. Howell
Full Text Available Cohorts of yearlings were sampled over a period of 6 years in a retrospective serological survey to establish the annual prevalence of serotype specific antibody to equine encephalosis virus on Thoroughbred stud farms distributed within defined geographical regions of South Africa. Seasonal seroprevalence varied between 3.6% and 34.7%, revealing both single and multiple serotype infections in an individual yearling. During the course of this study serotypes 1 and 6 were most frequently and extensively identified while the remaining serotypes 2, 3, 4, 5 and 7 were all identified as sporadic and localized in fections affecting only individual horses. This study of the seasonal prevalence of equine encephalosis virus has a corollary and serves as a useful model in the seasonal incidence of the serotypes of African horse sickness and bluetongue in regions where the respective diseases are endemic.
Hu, H.-P.; Hsieh, S.-C.; King, C.-C.; Wang, W.-K.
In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates
Wekesa, S. N.; Muwanika, V. B.; Siegismund, H. R.
Foot‐and‐mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these virus...
Khanam, Saima; Rajendra, Pilankatta; Khanna, Navin; Swaminathan, Sathyamangalam
Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective
Full Text Available Abstract Background Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. Results This work stems from the emergence of (i the DEN virus envelope (E domain III (EDIII as the most important region of the molecule from a vaccine perspective and (ii the adenovirus (Ad as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Conclusion Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has
Yiltawe Simwal Wungak
Full Text Available Aim: This study aimed to determine the foot-and-mouth disease virus (FMDV serotypes circulating, the prevalence of FMDV serotypes, and the spatial distribution of FMDV among sedentary and pastoral cattle herds in the North-Central Nigeria. Materials and Methods: A cross-sectional study was undertaken, during which a total of 155 sera that tested positive for foot-and-mouth disease (FMD 3ABC non-structural protein antibodies were selected and screened for FMD structural protein serotypes, A, O, SAT 1, and SAT 2 using a solid-phase competitive enzyme-linked immunosorbent assay (ELISA. Epithelial tissue specimens were collected during outbreak investigations which were tested for FMD using an antigen capture ELISA for serotype A, O, SAT 1, and SAT 2. Results: An overall serotype-specific prevalence of 79.35 (95% confidence interval [CI]: 72.4-85.18 was recorded for serotype O, 65.2% (95% CI: 57.41-72.3 for serotype A, 52.9% (95% CI: 45.03-60.67 for SAT-2, and 33.55% (95% CI: 26.45-41.26 for SAT-1. Evidence of exposure to multiple FMDV serotypes showed that 12.26% of the sera samples had antibodies against four serotypes circulating, 30.97% had antibodies against three serotypes circulating, 22.58% had antibodies against two serotypes, and 17% showed exposure to only one serotype. Clinical specimens (epithelial tissue collected during outbreak investigations showed that serotype O has the highest proportion of 50% with serotype A - 25%; SAT 2 - 20.8%; and SAT 1 - 4.1%. Conclusion: The study detected diffuse and co-circulation of serotypes A, O, SAT1, and SAT2 within the study area, and hence the need for the appropriately matched multivalent vaccine is strongly advocated for FMD control in Nigeria.
Ashshi, Ahmed Mohamed
Transmission of dengue virus (DENV) through blood transfusion has been documented and hence screening for DENV during blood donation has been recently recommended by the American Association of Blood Banks and Centres of Disease Control and Prevention. DENV is endemic in the Western province of the Kingdom of Saudi Arabia (KSA) and serotypes 1, 2 and 3, but not 4, have been detected. However, little is known regarding the rates of DENV during blood donation in the kingdom. The aim of this study was therefore to measure the prevalence of dengue virus and its serotypes in eligible Saudi blood donors in the endemic Western region of KSA. This was a cross-sectional study and serum samples were collected from 910 eligible Saudi male blood donors. DENV IgM and IgG antibodies were measured serologically by ELISA while viral serotypes were detected by a single step IVD CE certified multiplex RT-PCR kit. The overall prevalence was 39 and 5.5% for IgG+ and IgM+, respectively. There were 12 (1.3%) with exclusively IgM+, 317 (34.8%) exclusively IgG+ and 38 (4.2%) with dual IgM+/IgG+ donors. The overall prevalence was 3.2% (n = 29) and 2.3% (n = 21) for primary and secondary infections. PCR was positive in 5.5% (n = 50) and, DENV-2 (n = 24; 48%) was the most frequent serotype and was significantly higher than DENV-1 (20%; P = 0.02) and DENV-3 (2%; P = 0.1 × 10 -5 ) but not DENV-4 (30%; P = 0.2). There was no significant difference between both DENV-4 and DENV-1 (P = 0.4). The combination of the PCR and serology findings showed that 22 (2.4%) and 28 (3.1%) donors had primary and secondary viremic infections, respectively. The detected rates of DENV by PCR suggest a potential high risk of viral transmission by blood transfusion. To the best of our knowledge, this study is the first to report the detection of DENV-4 serotype in Saudi Arabia. More studies are required to measure the precise prevalence of DENV serotypes and their potential
Doijad, Swapnil P.; Barbuddhe, Sukhadeo B.; Garg, Sandeep; Poharkar, Krupali V.; Kalorey, Dewanand R.; Kurkure, Nitin V.; Rawool, Deepak B.; Chakraborty, Trinad
A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids. PMID:26360831
Swapnil P Doijad
Full Text Available A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26% strains as weak, 27 (27.55% strains as moderate, and 9 (9.18% strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015 was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.
Reid, Scott M.; Mioulet, Valerie; Knowles, Nick J.
transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative...... by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world....
Maciel-de-Freitas, Rafael; Sylvestre, Gabriel; Gandini, Mariana; Koella, Jacob C.
BACKGROUND: Dengue virus (DENV) is transmitted by Aedes aegypti, a species that lives in close association with human dwellings. The behavior of DENV-infected mosquitoes needs further investigation, especially regarding the potential influence of DENV on mosquito biting motivation and avidity. METHODOLOGY/PRINCIPAL FINDINGS: We orally challenged 4-5 day-old Ae. aegypti females with a low passage DENV serotype -2 (DENV-2) to test whether the virus influences motivation to feed (the likelihood ...
Background: Culicoides sonorensis (Diptera: Ceratopogonidae) is a vector of epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD) and other wild ruminants. Although historically rare, reports of clinica...
Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D.
The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes. PMID:23770669
Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D
The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.
Ali, Amjad; Ahmad, Habib; Idrees, Muhammad; Zahir, Fazli; Ali, Ijaz
Dengue virus is circulating in Pakistan since 1994, which causes major and minor outbreaks in many areas of the country. The incidence of dengue in Pakistan in past years mainly restricted to parts of Sindh and Punjab provinces. As such, a severe dengue outbreak appeared in Pakistan in 2011, particularly in Punjab province with Lahore as the most hit city (290 deaths). In 2013, for the first time in the history of Pakistan, dengue outbreak erupted in Swat District, Khyber Pakhtunkhwa, which claimed more than 57 lives. Hence this study was conducted to document circulating serotypes of dengue virus in Pakistan in 2011 and 2013 dengue outbreaks in two different territories/areas of the country. In total, 1340 blood samples from people having dengue (ELISA positive) and/or dengue like symptoms from various cities/areas of Punjab and Swat, Khyber Pakhtunkhwa (KP) were collected and analyzed by reverse transcription polymerase chain reaction (RT-PCR) using serotype specific primers. The results indicated that all the four dengue virus serotypes were circulating in Punjab Province with highest frequency of DENV-2 (41.64 %) and DENV-3 (41.05 %). Similarly, DENV-2 (41.66 %) and DENV-3 (35.0 %) were dominant serotypes detected in KP-based people lived in Punjab. On the other hand only DENV-2 (40.0 %) and DENV-3 (60.0 %) were detected in Swat District. Furthermore an important observation noted in this study was mixed infection of DENV-2 and DENV-3 in Punjab in 2011 (3.81 %) and in people from KP infected in Punjab (8.33 %) which may account for the high mortality and morbidity rates as compared to previous outbreaks. Over all male population was mostly infected as compared to females and people in the age group between 15 to 45 was the highest infected group. The findings of this study indicate that all four serotypes of dengue virus are circulating in Punjab whereas serotypes 2 and 3 introduced for the first time into Swat, KP in 2013; about 600 km away from Lahore
Dolz, Roser; Pujols, Joan; Ordóñez, German; Porta, Ramon; Majó, Natàlia
As part of an epidemiological surveillance of infectious bronchitis virus (IBV) in Spain, four Spanish field isolates showed high S1 spike sequence similarities with an IBV sequence from the GenBank database named Italy 02. Given that little was known about this new emergent IBV strain we have characterized the four isolates by sequencing the entire S1 part of the spike protein gene and have compared them with many reference IBV serotypes. In addition, cross-virus neutralization assays were conducted with the main IBV serotypes present in Europe. The four Spanish field strains and the Italy 02 S1 sequence from the NCBI database were established as a new genotype that showed maximum amino acid identities with the 4/91 serotype (81.7% to 83.7%), the D274 group that included D207, D274 and D3896 strains (79.8% to 81.7%), and the B1648 serotype (79.3% to 80%). Furthermore, on the basis of these results, it was demonstrated that the Italy 02 genotype had been circulating in Spain since as early as 1997. Based on the average ratio of synonymous:non-synonymous (dS/dN) amino acid substitutions within Italy 02 sequences, no positive selection pressures were related with changes observed in the S1 gene. Moreover, phylogenetic analysis of the S1 gene suggested that the Italy 02 genotype has undergone a recombination event. Virus neutralization assays demonstrated that little antigenic relatedness (less than 35%) exists between Italy 02 and some of the reference IBV serotypes, and indicated that Italy 02 is likely to be a new serotype.
Thong, K L; Goh, Y L; Radu, S; Noorzaleha, S; Yasin, R; Koh, Y T; Lim, V K E; Rusul, G; Puthucheary, S D
The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.
Thong, K. L.; Goh, Y. L.; Radu, S.; Noorzaleha, S.; Yasin, R.; Koh, Y. T.; Lim, V. K. E.; Rusul, G.; Puthucheary, S. D.
The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia. PMID:12089269
El-Shehawy, Laila I; Abu-Elnaga, Hany I; Rizk, Sonia A; Abd El-Kreem, Ahmed S; Mohamed, A A; Fawzy, Hossam G
In February 2012, a massive new foot-and-mouth disease (FMD) outbreak struck Egypt. In this work, one-step RT-PCR assays were used for in-house detection and differentiation of foot-and-mouth disease virus (FMDV) in Egypt in this year using pan-serotypic and serotype-targeting sequence primers. FMDV SAT2 was the dominant virus in the examined isolates from the epidemic. The complete VP1 coding regions of two isolates were sequenced. The two isolates had 99.2 % sequence identity to most contemporary Egyptian SAT2 reference viruses, whereas they had 89.7-90.1 % identity to the SAT2/EGY/2/2012 isolate, which was collected from Alexandria, Egypt, and previously sequenced by WRLFMD. Phylogenetic analysis showed that Egypt had one topotype and two lineage of FMDV SAT2 in 2012. The Egyptian and the Palestinian 2012 strains were associated mainly with topotype VII, lineage SAT2/VII/Ghb-12, while the virus isolated from Alexandria Governorate belonged to the SAT2/VII/Alx-12 lineage. Topotype VII also comprised lineages that included strains isolated from Libya in 2012 and 2003. Furthermore, within the same topotype, the Egyptian SAT2/2012 isolates were related to strains from Saudi Arabia, Sudan, Eritrea, Cameroon and Nigeria. Nevertheless, more epidemiological work with neighboring countries is needed to prevent cross-border spread of disease and to reach a precise conclusion about the origin of the 2012 FMDV SAT2 emergency in the Middle East.
Strizki, J M; Repik, P M
We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M(r) (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The protein profiles of the 33 North American (NA)-serotype viruses examined were remarkably homogeneous, with variation detected only in the E1 protein of two isolates. In contrast, considerable heterogeneity was observed in the migration profiles of both the E1 and E2 glycoproteins of the 13 South American (SA)-type viruses examined. Peptide mapping of individual virion proteins using limited proteolysis with Staphylococcus aureus V8 protease confirmed that, in addition to the homogeneity evident among NA-type viruses and relative heterogeneity among SA-type viruses, the E1 and E2 proteins of NA- and SA-serotype viruses exhibited serotype-specific structural variation. The C protein was highly conserved among isolates of both virus serotypes. Endoglycosidase analyses of intact virions did not reveal substantial glycosylation differences between the glycoproteins of NA- and SA-serotype viruses. Both the HMW protein and the E2 protein (doublet) of EEE virus appeared to contain, at least in part, high-mannose type N-linked oligosaccharides. No evidence of O-linked glycans was found on either the E1 or the E2 glycoprotein. Despite the observed structural differences between proteins of NA- and SA-type viruses, Western blot analyses utilizing polyclonal antibodies indicated that immunoreactive epitopes appeared to be conserved.
Ullah, A; Jamal, S M; Romey, A; Gorna, K; Kakar, M A; Abbas, F; Ahmad, J; Zientara, S; Bakkali Kassimi, L
This study reports characterization of foot-and-mouth disease virus (FMDV) in samples collected from Balochistan, Pakistan. FMDV was detected by pan-FMDV real-time RT-PCR in 31 samples (epithelial and oral swabs) collected in 2011 from clinical suspect cases. Of these, 29 samples were serotyped by serotype-specific real-time RT-PCR assays and were confirmed by sequencing the VP1 coding region. Sixteen samples were found positive for serotype A and eight for serotype Asia-1, whereas five samples were found positive for both serotypes A and Asia-1. Two serotype A positive samples were found positive for two different strains of serotype A FMDV each. Phylogenetic analyses of serotype A FMDVs showed circulation of at least three different sublineages within the A-Iran05 lineage. These included two earlier reported sublineages, A-Iran05 HER -10 and A-Iran05 FAR -11 , and a new sublineage, designated here as A-Iran05 BAL -11 . This shows that viruses belonging to the A-Iran05 lineage are continuously evolving in the region. Viruses belonging to the A-Iran05 FAR -11 sublineage showed close identity with the viruses circulating in 2009 in Pakistan and Afghanistan. However, viruses belonging to the A-Iran05 HER -10 detected in Balochistan, Pakistan, showed close identity with the viruses circulating in Kyrgyzstan, Iran and Kazakhstan in 2011 and 2012, showing that viruses responsible for outbreak in these countries have a common origin. Serotype Asia-1 FMDVs reported in this study all belonged to the earlier reported Group-VII (Sindh-08), which is currently a dominant strain in the West Eurasian region. Detection of two different serotypes of FMDV or/and two different strains of the same serotype in one animal/sample shows complexity in occurrence of FMD in the region. © 2016 Blackwell Verlag GmbH.
Sangula, Abraham; Belsham, Graham; Muwanika, Vincent
Amongst the SAT serotypes of foot-and-mouth disease virus (FMDV), the SAT 2 serotype is the most widely distributed throughout sub-Saharan Africa. Kenyan serotype SAT 2 viruses have been reported to display the highest genetic diversity for the serotype globally. This complicates diagnosis...... and control, and it is essential that patterns of virus circulation are known in order to overcome these difficulties. This study was undertaken to establish patterns of evolution of FMDV serotype SAT 2 in Kenya using complete VP1 coding sequences in a dataset of 65 sequences from Africa, collected over...
Full Text Available Objective. To determine the serotype and antibiotic resistance profile of Salmonella spp. isolated from retail ground beef in Mexico City. Materials and methods. A total of 100 samples of ground beef were analyzed. The pathogen was isolated by conventional methods and confirmed by PCR (invA gene, 284 bp. The antibiotic resistance profile was determined by the Kirby-Bauer method while serotyping was performed according to the Kauffman-White scheme. Results. We isolated a total of 19 strains of Lomita (6, Derby (4, Senftenberg (2, Javiana and Cannsttat (1 and undeter- mined (5 serotypes. The strains showed a high resistance rate to ampicillin (18/19, carbenicillin (16/19, tetracyclin (13/19, and trimethoprim-sulfamethoxazole (13/19. Multidrug resistance was observed in 14 isolates. Conclusions. Several Salmonella spp. serotypes of public health significance are circulating in ground beef sold in the major Mexican city. Some of these strains are multi-drug resistance.
Lauderdale, T. L.; Aarestrup, Frank Møller; Chen, P. C.
(41%) and was highly prevalent in Salmonella enterica serotype Typhimurium (72.7%, 176/242) the most common serotype. Additional resistance to trimethoprim was present in 155 (19.4% overall) of the ACSSuT R-type isolates from several serotypes. Reduced susceptibility to fluoroquinolone (FQ...... multiresistant to other antimicrobials. Studies are needed to determine the sources of different multidrug-resistant serotypes. Continued national surveillance is underway to monitor changes in resistance trends and to detect further emergence of resistant Salmonella serotypes in Taiwan. (c) 2006 Elsevier Inc...
Kotaki, Tomohiro; Yamanaka, Atsushi; Mulyatno, Kris Cahyo; Churrotin, Siti; Sucipto, Teguh Hari; Labiqah, Amaliah; Ahwanah, Nur Laila Fitriati; Soegijanto, Soegeng; Kameoka, Masanori; Konishi, Eiji
Indonesia is one of the biggest dengue endemic countries, and, thus, is an important place to investigate the evolution of dengue virus (DENV). We have continuously isolated DENV in Surabaya, the second biggest city in Indonesia, since 2008. We previously reported sequential changes in the predominant serotype from DENV type 2 (DENV-2) to DENV type 1 (DENV-1) in November 2008 and from DENV-1 to DENV-2 in July 2013. The predominance of DENV-2 continued in 2014, but not in 2015. We herein phylogenetically investigated DENV-2 transitions in Surabaya between 2008 and 2014 to analyze the divergence and evolution of DENV-2 concomitant with serotype shifts. All DENV-2 isolated in Surabaya were classified into the Cosmopolitan genotype, and further divided into 6 clusters. Clusters 1-3, dominated by Surabaya strains, were defined as the "Surabaya lineage". Clusters 4-6, dominated by strains from Singapore, Malaysia, and many parts of Indonesia, were the "South East Asian lineage". The most recent common ancestor of these strains existed in 1988, coinciding with the time that an Indonesian dengue outbreak took place. Cluster 1 appeared to be unique because no other DENV-2 isolate was included in this cluster. The predominance of DENV-2 in 2008 and 2013-14 were caused by cluster 1, whereas clusters 2 and 3 sporadically emerged in 2011 and 2012. The characteristic amino acids of cluster 1, E-170V and E-282Y, may be responsible for its prevalence in Surabaya. No amino acid difference was observed in the envelope region between strains in 2008 and 2013-14, suggesting that the re-emergence of DENV-2 in Surabaya was due to the loss or decrease of herd immunity in the 5-year period when DENV-2 subsided. The South East Asian lineage primarily emerged in Surabaya in 2014, probably imported from other parts of Indonesia or foreign countries. Copyright © 2015 Elsevier B.V. All rights reserved.
Full Text Available The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal.We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses.Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.
Antibiogram of E. coli serotypes isolated from children aged under five with acute diarrhea in Bahir Dar town. Ayrikim Adugna1, Mulugeta Kibret1, Bayeh Abera2, Endalkachew Nibret1, Melaku Adal1. 1. Department of Biology, Science College, Bahir Dar University. 2. Department of Microbiology, Parasitology and ...
Das, S; Pingle, M R; Muñoz-Jordán, J; Rundell, M S; Rondini, S; Granger, K; Chang, G-J J; Kelly, E; Spier, E G; Larone, D; Spitzer, E; Barany, F; Golightly, L M
The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.
Soto Garita, Claudio
An immunopotentiation trial has used sera from dengue seropositive patients from Costa Rica's endemic areas. The detection and semi-quantification of immunopotentiating antibodies were optimized against dengue virus serotype 1. The cell line K562 (human erythromyeloblastoid leukemia cells) has been more efficient than the U937 (human histiocytic lymphoma cells). A more adequate detection of immunopotentiating antibodies was determined. The optimal infection and virus-antibody incubation parameters are demonstrated for the detection of immunopotentiating antibodies with the immunostaining technique. The immuno-optimized assay has allowed the detection and semi-quantification of immunopotentiating antibodies against serotype 1 of dengue virus. Samples of strong positive, weak positive and dengue negative sera are analyzed. The end has been to evaluate the usefulness in the detection and semi-quantification of immunopotentiating antibodies. The presence of immunopotentiating antibodies was demonstrated against dengue virus serotype 1 in endemic zones of Costa Rica, to complement with the evaluation of the other existing serotypes is recommended [es
Subramaniam, Saravanan; Mohapatra, Jajati K; Das, Biswajit; Sanyal, Aniket; Pattnaik, Bramhadev
In recent times, majority of the foot-and-mouth disease (FMD) outbreaks in India are caused by serotype O Ind2001 lineage. The lineage has diverged into four sub-lineages (Ind2001a, b, c and d). We report here the genetic and antigenic analyses of nine Ind2001d isolates that caused outbreaks during April 2013-March 2014 in India. The length of the genomes of outbreak viruses varied between 8153 and 8181 nucleotides without any insertion or deletion in the coding region. Of the nine isolates analyzed antigenically against the currently used Indian vaccine strain INDR2/1975, eight showed good cross serological match (>0.3) indicating optimal antigenic coverage by the vaccine strain. An unprecedented deletion of 22 nucleotides between position 57 and 78 was observed in the 3' untranslated region of one of the isolates without compromising the virus viability, which imply that partial distortion in SL2 of 3'UTR may not have influence on virus viability at least under in-vitro conditions. Recently the Ind2001 lineage has been reported from several countries including Libya and spread of this lineage across a wide geographical area needs to be monitored carefully to avoid any future pandemic. Copyright © 2014 Elsevier B.V. All rights reserved.
Zhang, Qingxun; Liu, Xinsheng; Fang, Yuzhen; Pan, Li; Lv, Jianliang; Zhang, Zhongwang; Zhou, Peng; Ding, Yaozhong; Chen, Haotai; Shao, Junjun; Zhao, Furong; Lin, Tong; Chang, Huiyun; Zhang, Jie; Wang, Yonglu; Zhang, Yongguang
Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I–VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10−3 substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown. PMID:25793223
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
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.
Wongsawan, Kanruethai; Gottschalk, Marcelo; Tharavichitkul, Prasit
In this present study, the serotype of 40 Streptococcus suis isolates from submaxillary glands of pig carcasses sold in wet markets in Chiang Mai Province, northern Thailand, was investigated. Eleven serotypes, including types 2, 3, 4, 5, 7, 8, 9, 17, 21, 22 and 31, were found in the isolates by a Multiplex PCR combined with serum agglutination. Of the eleven serotypes present, type 3 was the most prevalent, while types 2, 4, 5 and 21 were of primary interest due to their human isolate serotype. The mrp+/epf - /sly - genotype was found to be the most prevalent genotype. This study indicates the importance of effective control of human S. suis infection due to raw pork or pig carcass handling in northern Thailand.
Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E
Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.
Ruder, M G; Stallknecht, D E; Allison, A B; Mead, D G; Carter, D L; Howerth, E W
Epizootic hemorrhagic disease viruses (EHDVs) are orbiviruses transmitted by Culicoides biting midges to domestic and wild ruminants. EHDV-1 and EHDV-2 are endemic in the United States, where epizootic hemorrhagic disease is the most significant viral disease of white-tailed deer (WTD;Odocoileus virginianus) and reports of epizootic hemorrhagic disease in cattle are increasing. In 2006, a reassortant EHDV-6 was isolated from dead WTD in Indiana and has been detected each subsequent year over a wide geographic region. Since EHDV-6 is not a historically endemic serotype in the United States, it is important to understand infection outcome in potential hosts. Specifically, we aimed to evaluate the pathogenicity of the virus in 2 primary US ruminant hosts (WTD and cattle) and the susceptibility of a confirmed US vector (Culicoides sonorensis). Five WTD and 4 cattle were inoculated with >10(6)TCID50EHDV-6 by intradermal and subcutaneous injection. All 5 WTD exhibited moderate to severe disease, and 3 died. Viremia was first detected 3 to 5 days postinfection (dpi) with surviving animals seroconverting by 10 dpi. Two of 4 inoculated cattle had detectable viremia, 5 to 10 dpi and 7 to 24 dpi, respectively. No clinical, hematologic, or pathologic abnormalities were observed. Antibodies were detected by 10 dpi in 3 of 4 cows.C. sonorensis were fed on WTD blood spiked with EHDV-6 and held for 4 to 14 days postfeeding at 25°C. From 4 to 14 days postfeeding, 19 of 171 midges were virus isolation positive and 6 of 171 had ≥10(2.7)TCID50EHDV-6. Although outcomes varied, these studies demonstrate the susceptibility of ruminant and vector hosts in the United States for this recently emerged EHDV serotype. © The Author(s) 2015.
Manchala Nageswar Reddy
Full Text Available Background Dengue is a global human public health threat, causing severe morbidity and mortality. The occurrence of sequential infection by more than one serotype of dengue virus (DENV is a major contributing factor for the induction of Dengue Hemorrhagic Fever (DHF and Dengue Shock Syndrome (DSS, two major medical conditions caused by DENV infection. However, there is no specific drug or vaccine available against dengue infection. There are reports indicating the increased incidence of concurrent infection of dengue in several tropical and subtropical regions. Recently, increasing number of DHF and DSS cases were reported in India indicating potential enhancement of concurrent DENV infections. Therefore, accurate determination of the occurrence of DENV serotype co-infections needs to be conducted in various DENV prone parts of India. In this context, the present study was conducted to analyse the magnitude of concurrent infection in northern Kerala, a southwest state of India, during three consecutive years from 2013 to 2015. Methods A total of 120 serum samples were collected from the suspected dengue patients. The serum samples were diagnosed for the presence of dengue NS1 antigen followed by the isolation of dengue genome from NS1 positive samples. The isolated dengue genome was further subjected to RTPCR based molecular serotyping. The phylogenetic tree was constructed based on the sequence of PCR amplified products. Results Out of the total number of samples collected, 100 samples were positive for dengue specific antigen (NS1 and 26 of them contained the dengue genome. The RTPCR based molecular serotyping of the dengue genome revealed the presence of all four serotypes with different combinations. However, serotypes 1 and 3 were predominant combinations of concurrent infection. Interestingly, there were two samples with all four serotypes concurrently infected in 2013. Discussion All samples containing dengue genome showed the presence of
Full Text Available Abstract Backgroud Foot-and-mouth disease virus (FMDV serotype Asia1 generally infects cattle and sheep, while its infection of pigs is rarely reported. In 2005-2007, FMD outbreaks caused by Asia1 type occurred in many regions of China, as well as some parts of East Asia countries. During the outbreaks, there was not any report that pigs were found to be clinically infected. Results In this study, a strain of FMDV that isolated from pigs was identified as serotype Asia1, and designated as "Asia1/WHN/CHA/06". To investigate the genomic feature of the strain, complete genome of Asia1/WHN/CHA/06 was sequenced and compared with sequences of other FMDVs by phylogenetic and recombination analysis. The complete genome of Asia1/WHN/CHA/06 was 8161 nucleotides (nt in length, and was closer to JS/CHA/05 than to all other strains. Potential recombination events associated with Asia1/WHN/CHA/06 were found between JS/CHA/05 and HNK/CHA/05 strains with partial 3B and 3C fragments. Conclusion This is the first report of the isolation and identification of a strain of FMDV type Asia1 from naturally infected pigs. The Asia1/WHN/CHA/06 strain may evolve from the recombination of JS/CHA/05 and HNK/CHA/05 strains.
Gatti, B M; Ramirez Gronda, G A; Etchevarría, M; Vescina, C M; Varea, A M; González Ayala, S E
Haemophilus influenzae (Hi) is the causative agent of several human diseases such as sepsis, meningitis, celulitis, and osteoarthritis. We investigated the isolation of Hi serotypes from sterile sites in sick children. One hundred and seventy nine strains from 146 patients were studied, period 1996-2002, at the Microbiology Laboratory, Hospital de Niños Superiora Sor María Ludovica, Argentina. The serotype distribution was:1 a, 112 b,1 c,1 d, 4 e, 3 f y 24 no typable. Since the beginning of universal Hi b vaccination in 1998, we have observed the fast decrease of serotype b and a relative increase of other serotypes.
Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel
A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...
Paulo Roberto Santana de Melo
Full Text Available By 2002, dengue virus serotype 1 (DENV-1 and DENV-2 had circulated for more than a decade in Brazil. In 2002, the introduction of DENV-3 in the state of Bahia produced a massive epidemic and the first cases of dengue hemorrhagic fever. Based on the standardized frequency, timing and location of viral isolations by the state's Central Laboratory, DENV-3 probably entered Bahia through its capital, Salvador, and then rapidly disseminated to other cities, following the main roads. A linear regression model that included traffic flow, distance from the capital and DENV-1 circulation (r² = 0.24, p = 0.001 supported this hypothesis. This pattern was not seen for serotypes already in circulation and was not seen for DENV-3 in the following year. Human population density was another important factor in the intensity of viral circulation. Neither DENV-1 nor DENV-2 fit this model for 2001 or 2003. Since the vector has limited flight range and vector densities fail to correlate with intensity of viral circulation, this distribution represents the movement of infected people and to some extent mosquitoes. This pattern may mimic person-to-person spread of a new infection.
Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H
To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.
Yang, Baolin; Wang, Mingxia; Liu, Wenming; Xu, Zhiqiang; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li
Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8 TLLEDRILT 16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr 8 and Asp 12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.
Nielsen, R.; Andresen, Lars Ole; Plambeck, Tamara
Nine Danish Actinobacillus pleuropneumoniae biotype 1 isolates were shown by latex agglutination and indirect haemagglutination to possess capsular polysaccharide epitopes identical to those of serotype 2 strain 1536 (reference strain of serotype 2) and strain 4226 (Danish serotype 2 strain). Imm...... in the LPS of strains 1536 and 7317 were revealed. Since an antigenic determinant specific for the 9 isolates could not be demonstrated with the methods used, the strains are proposed to be designated K2:O7....
Zanolari, P; Bruckner, L; Fricker, R; Kaufmann, C; Mudry, M; Griot, C; Meylan, M
Bluetongue virus serotype 8 (BTV-8) has caused disease in domestic ruminants in several countries of northern Europe since 2006. In 2008 a mass-vaccination program was launched in most affected countries using whole virus inactivated vaccines. To evaluate 2 inactivated vaccines (Bovilis BTV 8; BTVPUR AlSap8) for immunogenicity and safety against BTV-8 in South American camelids (SAC) in a field trial. Forty-two SAC (25 Alpacas, 17 Llamas) aged between 1 and 16 years. The animals were vaccinated twice at intervals of 21 days. They were observed clinically for adverse local, systemic, or both reactions throughout the trial. Blood samples collected on days 0, 14, 21, 43, and 156 after vaccination were tested for the presence of BTV-8 virus by real time-polymerase chain reaction and of specific antibodies by competitive ELISA and a serum neutralization test. All vaccinated animals developed antibodies to BTV-8 after the 2nd administration of the vaccine. No adverse effects were observed except for moderate local swellings at the injection site, which disappeared within 21 days. Slightly increased body temperatures were only observed in the first 2 days after vaccination. The BTV was not detected in any of the samples analyzed. The administration of the 2 inactivated commercial vaccines was safe and induced seroconversion against BTV-8 in all vaccinated animals. The results of this study suggest that 2 doses injected 3 weeks apart is a suitable vaccination regimen for SAC.
Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.; Afonso, Claudio L.
Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13.
Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.
Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13. PMID:27469958
Lorusso, Alessio; Sghaier, Soufien; Di Domenico, Marco; Barbria, Mohamed Elias; Zaccaria, Guendalina; Megdich, Aida; Portanti, Ottavio; Seliman, Imed Ben; Spedicato, Massimo; Pizzurro, Federica; Carmine, Irene; Teodori, Liana; Mahjoub, Mejdi; Mangone, Iolanda; Leone, Alessandra; Hammami, Salah; Marcacci, Maurilia; Savini, Giovanni
Bluetongue (BT), is one of the OIE-listed major diseases of ruminants. Following the official report of BT virus serotype 3 (BTV-3) in a sheep in Cap Bon (Tunisia), blood and serum samples of ruminants were collected from some areas of Tunisia to further investigate the presence of this virus in the country. A quantitative real time RT-PCR has been first developed for the detection and quantitation of BTV-3 RNA from field specimens. Out of 62 collected blood samples, 23 were shown to be positive for BTV-3 RNA. Isolation on cell cultures was also possible from six samples. Genome sequencing revealed the circulation of two unrelated western strains of BTV-3, one circulating in Cap Bon and neighboring areas, and the other circulating nearby the border with Libya. The presence of a putative novel BTV serotype (BTV-Y TUN2017) in sheep introduced from Libya to Tunisia, genomically related to the BTV strain contaminating a commercially-available sheep pox vaccine and to BTV-26, has been also demonstrated. This finding highlights the pressing need for a prompt production and release of a novel inactivated BTV-3 vaccine to be used in case of emergence or proactively in the areas of Southern Europe at major risk of BTV introduction. The assessment of a novel vaccine will certainly exalt the role and importance of surveillance activities and collaboration with Northern African countries. Copyright © 2018 Elsevier B.V. All rights reserved.
Chen, Ming; Wang, Rui; Luo, Fu-Guang; Huang, Yan; Liang, Wan-Wen; Huang, Ting; Lei, Ai-Ying; Gan, Xi; Li, Li-Ping
Recent studies have shown that group B streptococcus (GBS) may be infectious across hosts. The purpose of this study is to investigate the pathogenicity of clinical GBS isolates with serotypes Ia, III and V from human and cow to tilapia and the evolutionary relationship among these GBS strains of different sources. A total of 27 clinical GBS isolates from human (n=10), cow (n=2) and tilapia (n=15) were analyzed using serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Among them, 15 isolates were tested for their pathogenicity to tilapia. The results showed that five human GBS strains (2 serotype III, 2 serotype Ia and 1 serotype V) infected tilapia with mortality rate ranging from 56.67% to 100%, while the other five human GBS strains tested were unable to infect tilapia. In addition, two cow GBS strains C001 and C003 of serotype III infected tilapia. However, they had significantly lower pathogenicity than the five human strains. Furthermore, human GBS strains H005 and H008, which had very strong ability to infect tilapia, had the same PFGE pattern. MLST analysis showed that the five human and the two cow GBS strains that were able to infect tilapia belonged to clonal complexes CC19, CC23 and CC103. The study for the first time confirmed that human or cow GBS clonal complexes CC19, CC23 and CC103 containing strains with serotypes Ia, III and V could infect tilapia and induce clinical signs under experimental conditions. Copyright © 2015 Elsevier B.V. All rights reserved.
Manchala, Nageswar Reddy; Dungdung, Ranjeet; Pilankatta, Rajendra
Human serum protein profiling of the individual infected with multiple dengue virus serotypes for identifying the potential biomarkers and to investigate the cause for the severity of dengue virus infection. Dengue virus NS1-positive serum samples were pooled into two groups (S2 and S3) based on the molecular serotyping and number of heterotypic infections. The pooled serum samples were subjected to two-dimensional gel electrophoresis (2DGE) to identify the differentially expressed proteins. The peptide masses of upregulated protein were detected by matrix-assisted laser desorption-ionisation time-of-flight MALDI-TOF mass spectrometry and analysed by MASCOT search engine. The results were compared with the control group (S1). The commonly upregulated protein was validated by quantitative ELISA and compared with control as well as single serotypic infected samples. Based on 2DGE, total thirteen proteins were differentially upregulated in S2 and S3 groups as compared to control. Some of the upregulated proteins were involved in mediating the complement activation of immune response. The apolipoprotein A-1 (APO A-1) was upregulated in S2 and S3 groups. Upon validation, APO A-1 levels were increased in line with the number of heterotypic infection of dengue viruses. Heterotypic infection of dengue viruses upregulate the serum proteins involved in the complement pathway in the early phase of infection. There was a significant increase in the level of APO A-1 in three different serotypic infections of dengue virus as compared to control. Further, the role of APO-A1 can be explored in elucidating the mechanism of dengue pathogenesis. © 2017 John Wiley & Sons Ltd.
Full Text Available Dengue disease is currently a major health problem in Indonesia and affects all provinces in the country, including Semarang Municipality, Central Java province. While dengue is endemic in this region, only limited data on the disease epidemiology is available. To understand the dynamics of dengue in Semarang, we conducted clinical, virological, and demographical surveillance of dengue in Semarang and its surrounding regions in 2012. Dengue cases were detected in both urban and rural areas located in various geographical features, including the coastal and highland areas. During an eight months' study, a total of 120 febrile patients were recruited, of which 66 were serologically confirmed for dengue infection using IgG/IgM ELISA and/or NS1 tests. The cases occurred both in dry and wet seasons. Majority of patients were under 10 years old. Most patients were diagnosed as dengue hemorrhagic fever, followed by dengue shock syndrome and dengue fever. Serotyping was performed in 31 patients, and we observed the co-circulation of all four dengue virus (DENV serotypes. When the serotypes were correlated with the severity of the disease, no direct correlation was observed. Phylogenetic analysis of DENV based on Envelope gene sequence revealed the circulation of DENV-2 Cosmopolitan genotype and DENV-3 Genotype I. A striking finding was observed for DENV-1, in which we found the co-circulation of Genotype I with an old Genotype II. The Genotype II was represented by a virus strain that has a very slow mutation rate and is very closely related to the DENV strain from Thailand, isolated in 1964 and never reported in other countries in the last three decades. Moreover, this virus was discovered in a cool highland area with an elevation of 1,001 meters above the sea level. The discovery of this old DENV strain may suggest the silent circulation of old virus strains in Indonesia.
Samir K Saha
Full Text Available BACKGROUND: Streptococcus pneumoniae is a leading cause of meningitis in countries where pneumococcal conjugate vaccines (PCV targeting commonly occurring serotypes are not routinely used. However, effectiveness of PCV would be jeopardized by emergence of invasive pneumococcal diseases (IPD caused by serotypes which are not included in PCV. Systematic hospital based surveillance in Bangladesh was established and progressively improved to determine the pathogens causing childhood sepsis and meningitis. This also provided the foundation for determining the spectrum of serotypes causing IPD. This article reports an unprecedented upsurge of serotype 2, an uncommon pneumococcal serotype, without any known intervention. METHODS AND FINDINGS: Cases with suspected IPD had blood or cerebrospinal fluid (CSF collected from the beginning of 2001 till 2009. Pneumococcal serotypes were determined by capsular swelling of isolates or PCR of culture-negative CSF specimens. Multicenter national surveillance, expanded from 2004, identified 45,437 patients with suspected bacteremia who were blood cultured and 10,618 suspected meningitis cases who had a lumber puncture. Pneumococcus accounted for 230 culture positive cases of meningitis in children <5 years. Serotype-2 was the leading cause of pneumococcal meningitis, accounting for 20.4% (45/221; 95% CI 15%-26% of cases. Ninety eight percent (45/46 of these serotype-2 strains were isolated from meningitis cases, yielding the highest serotype-specific odds ratio for meningitis (29.6; 95% CI 3.4-256.3. The serotype-2 strains had three closely related pulsed field gel electrophoresis types. CONCLUSIONS: S. pneumoniae serotype-2 was found to possess an unusually high potential for causing meningitis and was the leading serotype-specific cause of childhood meningitis in Bangladesh over the past decade. Persisting disease occurrence or progressive spread would represent a major potential infection threat since serotype-2
Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.
Serotype and DNA fingerprint methods were used to study Pasteurella multocida isolated from 320 wild birds of North America. Isolates were collected during 1978-93. The HhaI profiles of 314 isolates matched the HhaI profile of somatic reference type 1, strain X-73; somatic type 1 antigen was expressed by 310 isolates, and the serotype of four isolates was undetected. Differentiation of the 314 isolates was observed by digestion of DNA with HpaII. None of the HpaII profiles matched the HpaII profile of X-73 (designated HhaI 001/HpaII 001). Three HpaII profiles were recognized among the somatic type 1 isolates: HpaII 002 (n = 18), HpaII 003 (n = 122), and HpaII 004 (n = 174). Profile HpaII 002 was found among isolates collected during 1979-83. Profile HpaII 003 was identified from isolates collected during 1979-89, with the exception of two isolates in 1992. The HpaII 004 profile was identified from isolates collected during 1983-93. Of the six remaining isolates, four expressed somatic type 4 and had HhaI profiles identical to the somatic type 4 reference strain P-1662 profile (designated HhaI 004); these isolates were differentiated by digestion of DNA with HpaII. One isolate was identified as serotype F:11, and another was serotype A:3,4. In the present study, 314 of 316 (99.4%) isolates from wild birds in the Central, Mississippi, and Pacific flyways during 1978-93, were P. multocida somatic type 1.
Full Text Available The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26 in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.
Hammond, R W; Crosslin, J M
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.
Maree, Francois; de Klerk-Lorist, Lin-Mari; Gubbins, Simon; Zhang, Fuquan; Seago, Julian; Pérez-Martín, Eva; Reid, Liz; Scott, Katherine; van Schalkwyk, Louis; Bengis, Roy; Charleston, Bryan; Juleff, Nicholas
Foot-and-mouth disease (FMD) virus (FMDV) circulates as multiple serotypes and strains in many regions of endemicity. In particular, the three Southern African Territories (SAT) serotypes are maintained effectively in their wildlife reservoir, the African buffalo, and individuals may harbor multiple SAT serotypes for extended periods in the pharyngeal region. However, the exact site and mechanism for persistence remain unclear. FMD in buffaloes offers a unique opportunity to study FMDV persistence, as transmission from carrier ruminants has convincingly been demonstrated for only this species. Following coinfection of naive African buffaloes with isolates of three SAT serotypes from field buffaloes, palatine tonsil swabs were the sample of choice for recovering infectious FMDV up to 400 days postinfection (dpi). Postmortem examination identified infectious virus for up to 185 dpi and viral genomes for up to 400 dpi in lymphoid tissues of the head and neck, focused mainly in germinal centers. Interestingly, viral persistence in vivo was not homogenous, and the SAT-1 isolate persisted longer than the SAT-2 and SAT-3 isolates. Coinfection and passage of these SAT isolates in goat and buffalo cell lines demonstrated a direct correlation between persistence and cell-killing capacity. These data suggest that FMDV persistence occurs in the germinal centers of lymphoid tissue but that the duration of persistence is related to virus replication and cell-killing capacity. Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in domestic livestock and wildlife species. African buffaloes (Syncerus caffer) are the primary carrier hosts of FMDV in African savannah ecosystems, where the disease is endemic. We have shown that the virus persists for up to 400 days in buffaloes and that there is competition between viruses during mixed infections. There was similar competition in cell culture: viruses that killed cells quickly persisted more
In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...
Tegar A. P. Siregar
conservation ofT and B cell epitope in NS3 protein among DENV-4 strains and four serotypes DENV of Indonesia strains.Methods: Research was held at the Department of Microbiology, Faculty of Medicine, UniversitasIndonesia, June 2013 to April 2014. NS3 amino acid sequence of DENV-4 081 strain was obtained afterNS3 gene of DENV-4 081 PCR products were sequenced. T and B cell epitopes of NS3 protein of DENV-4081 strain were analysed and compared to NS3 proteins of 124 DENV-4 strains around the world and fourserotypes of Indonesia strains. World strains were isolated from America (i.e. Venezuela, Colombia, etc.and Asia (i.e. China, Singapore, etc.. For the comparison, T and B cell epitope positions of NS3 proteinwere obtained from published report.Results: Eight positions of T cell epitopes and two positions of B cell epitopes of NS3 DENV-4 081 wereidentical and conserved to NS3 protein of 124 DENV-4 strains around the world. B cell epitope of NS3 DENV-4 081 protein at aa 537-544 was found identical and conserved to four serotypes DENV of Indonesia strains.Conclusion: This wide conservation of T and B epitopes in almost all DENV-4 strains around the worldand all serotypes of Indonesia strains. (Health Science Journal of Indonesia 2015;6:126-31Keywords: dengue virus, NS3 protein, T cell epitope, B cell epitope
Lotfollahi, Lida; Chaharbalesh, Ardalan; Ahangarzadeh Rezaee, Mohammad; Hasani, Alka
Listeria monocytogenes is a foodborne pathogen causing listeriosis, which potentially affects all individuals, especially pregnant women and immunocompromised persons. The present study investigated the prevalence, antimicrobial susceptibility and serotypes distribution of the isolated L. monocytogenes from Iran. Twenty two (4.97%) of 442 human, food and livestock samples were found to be positive for L. monocytogenes. L. monocytogenes was identified in 8.8% of 125 human samples, 2.99% of 267 food and 6% of 50 livestock samples. The standard disk diffusion method and minimum inhibitory concentration (MIC) assay were used for antimicrobial susceptibility testing and multiplex PCR for serotyping. Among the 22 isolates tested, 6 (27.2%) displayed resistance to penicillin G, with all of the isolates and 2 (9%) of them showing intermediate susceptibility to clindamycin and rifampicin, respectively. According to the MIC assay, the rate of resistance to penicillin G was the same as that of disk diffusion method, but 16 (72.7%) of isolates showed intermediate susceptibility to clindamycin using E-test. In the multiplex PCR, 19 (86.4%) of isolates belonged to serotype 1/2c or 3c and the remaining 3 isolates were identified as (4b, 4d or 4e) and (1/2a or 3a), respectively. The occurrence of resistance to penicillin G, which can be used in the treatment of listeriosis, is very alarming and more prevalence of 1/2c serotype, in comparison to 3 other important ones (1/2a, 1/2b and 4b), in Iran has been reported for the first time. To the best of our knowledge, this is the first study showing the distribution of various serogroups of L. monocytogenes from human and livestock in Iran. Copyright © 2017 Elsevier Ltd. All rights reserved.
Full Text Available Non-polio enteroviruses (NPEVs are among the most common viruses infecting humans worldwide. Most of these infections are asymptomatic but few can lead to systemic and neurological disorders like Acute Flaccid Paralysis (AFP. Acute Flaccid Paralysis is a clinical syndrome and NPEVs have been isolated frequently from the patients suffering from AFP but little is known about their causal relationship. The objective of this study was to identify and characterize the NPEV serotypes recovered from 184 stool samples collected from AFP patients in Federally Administered Tribal Areas (FATA in north-west of Pakistan. Overall, 44 (95.6 % isolates were successfully typed through microneutralization assay as a member of enterovirus B species including echovirus (E-2, E-3, E-4, E-6, E-7, E-11, E-13, E-14, E-21 and E-29 while two isolates (PAK NIH SP6545B and PAK NIH SP1202B remained untypeable. The VP1 and capsid regions analysis characterized these viruses as EV-B93 and EV-B106. Phylogenetic analysis confirmed that PAK NIH isolates had high genetic diversity and represent distinct genotypes circulating in the country. Our findings highlight the role of NPEVs in AFP cases to be thoroughly investigated especially in high disease risk areas, with limited surveillance activities and health resources.
Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H
VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.
Jamal, Syed Muhammad; Ferrari, Giancarlo; Ahmed, Safia
Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate...... appropriate vaccine selection and tracing of outbreaks.The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998–2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1...... of the A-Iran05AFG-07 sub-lineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses....
Kim, Boram; Hur, Jin; Lee, Ji Yeong; Choi, Yoonyoung; Lee, John Hwa
Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP). Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined. Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect β-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR. Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains). Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.
Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong
Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection. Copyright © 2015 Elsevier B.V. All rights reserved.
Folster, Jason P; Campbell, Davina; Grass, Julian; Brown, Allison C; Bicknese, Amelia; Tolar, Beth; Joseph, Lavin A; Plumblee, Jodie R; Walker, Carrie; Fedorka-Cray, Paula J; Whichard, Jean M
Salmonella enterica is one of the most common causes of bacterial foodborne illness in the United States. Although most Salmonella infections are self-limiting, antimicrobial treatment of invasive salmonellosis is critical. The primary antimicrobial treatment options include fluoroquinolones or extended-spectrum cephalosporins, and resistance to these antimicrobial drugs may complicate treatment. At present, S. enterica is composed of more than 2,600 unique serotypes, which vary greatly in geographic prevalence, ecological niche, and the ability to cause human disease, and it is important to understand and mitigate the source of human infection, particularly when antimicrobial resistance is found. In this study, we identified and characterized 19 S. enterica serotype Albert isolates collected from food animals, retail meat, and humans in the United States during 2005 to 2013. All five isolates from nonhuman sources were obtained from turkeys or ground turkey, and epidemiologic data suggest poultry consumption or live-poultry exposure as the probable source of infection. S. enterica serotype Albert also appears to be geographically localized to the midwestern United States. All 19 isolates displayed multidrug resistance, including decreased susceptibility to fluoroquinolones and resistance to extended-spectrum cephalosporins. Turkeys are a likely source of multidrug-resistant S. enterica serotype Albert, and circulation of resistance plasmids, as opposed to the expansion of a single resistant strain, is playing a role. More work is needed to understand why these resistance plasmids spread and how their presence and the serotype they reside in contribute to human disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Full Text Available Background/Aim. Pseudomonas aeruginosa (P. aeruginosa is devided into 20 serotypes on the base of the International Antigenic Typing Scheme. P. aeruginosa serotyping is important because of few reasons but epidemiological is the most important. The aim of the study was serotyping of P. aeruginosa clinical isolates, analysing of single clinical isolates P. aeruginosa present in the particular samples, and analysing of pyocianin and fluorescin production in different isolates of P. aeruginosa. Methods. A total of 223 isolates of P. aeruginosa, isolated in the microbiological laboratory of the Health Center “Aleksinac”, Aleksinac, were examinated. P. aeruginosa isolates were put on the pseudomonas isolation agar, pseudomonas agar base, acetamid agar, asparagin prolin broth, pseudomonas asparagin broth, Bushnnell-Haas agar, cetrimid agar base, King A and King B plates, plates for pyocianin production, plates for fluorescin production and tripticasa soya agar (Himedia. Polyvalent and monovalent serums were used in the agglutination (Biorad. Pigment production was analysed on the bases of growth on the plates for pyocianin and fluorescin production. Results. Serologically, we identificated the serovars as follows: O1, O3, O4, O5, O6, O7, O8, O10, O11 and O12. O1 (38% was the most often serovar, then O11 (19% and O6 (8.6%. A total of 18.6% (42 isolates did not agglutinate with any serum, whereas 21 isolates agglutinated only with polyvalent serum. The majority of P. aeruginosa isolates produced fluorescin, 129 (58.54%, 53 (22.94% produced pyocianin whereas 49 (21.21% isolates produced both pigments. Conclusion. P. aeruginosa was isolated most of the from urine, sputum and other materials. The majority often serovars were O1, O6 and O11. The most of isolates produced fluorescin (58.54%, while 22.94% producted pyocianin and 21.21% both pigments.
Kiely, R A
The screening of 2000 women of childbearing age in Cork between 2004 and 2006 produced 37 erythromycin-resistant group B streptococcus (GBS) isolates. PCR analysis was performed to determine the basis for erythromycin resistance. The ermTR gene was most frequently expressed (n = 19), followed by the ermB gene (n = 8). Four isolates harboured the mefA gene. Six isolates yielded no PCR products. Some phenotype-genotype correlation was observed. All isolates expressing the mefA gene displayed the M phenotype whilst all those expressing ermB displayed the constitutive macrolide resistance (cMLS(B)) phenotype. Of 19 isolates that expressed the ermTR gene, 16 displayed the inducible macrolide resistance (iMLS(B)) phenotype. Serotype analysis revealed that serotypes III and V predominated in these isolates. The identification of two erythromycin-resistant serotype VIII isolates among this collection represents the first reported finding of erythromycin resistance in this serotype. A single isolate was non-typable using two latex agglutination serotyping kits.
Clinical Survey of Dengue Virus Circulation in the Republic of Djibouti between 2011 and 2014 Identifies Serotype 3 Epidemic and Recommends Clinical Diagnosis Guidelines for Resource Limited Settings.
Erwan Le Gonidec
Full Text Available Dengue virus is endemic globally, throughout tropical and sub-tropical regions. While the number of epidemics due to the four DENV serotypes is pronounced in East Africa, the total number of cases reported in Africa (16 million infections remained at low levels compared to Asia (70 million infections. The French Armed forces Health Service provides epidemiological surveillance support in the Republic of Djibouti through the Bouffard Military hospital. Between 2011 and 2014, clinical and biological data of suspected dengue syndromes were collected at the Bouffard Military hospital and analyzed to improve Dengue clinical diagnosis and evaluate its circulation in East Africa. Examining samples from patients that presented one or more Dengue-like symptoms the study evidenced 128 Dengue cases among 354 suspected cases (36.2% of the non-malarial Dengue-like syndromes. It also demonstrated the circulation of serotypes 1 and 2 and reports the first epidemic of serotype 3 infections in Djibouti which was found in all of the hospitalized patients in this study. Based on these results we have determined that screening for Malaria and the presence of the arthralgia, gastro-intestinal symptoms and lymphopenia < 1,000cell/ mm3 allows for negative predictive value and specificity of diagnosis in isolated areas superior to 80% up to day 6. This study also provides evidence for an epidemic of Dengue virus serotype 3 previously not detected in Djibouti.
Sasmono, R Tedjo; Wahid, Isra; Trimarsanto, Hidayat; Yohan, Benediktus; Wahyuni, Sitti; Hertanto, Martin; Yusuf, Irawan; Mubin, Halim; Ganda, Idham J; Latief, Rachmat; Bifani, Pablo J; Shi, Pei-Yong; Schreiber, Mark J
Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007-2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the
Kannika, K; Pisuttharachai, D; Srisapoome, P; Wongtavatchai, J; Kondo, H; Hirono, I; Unajak, S; Areechon, N
This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems. Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas. Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand. We identified two major serotypes of S. agalactiae isolates associated with the outbreak in
To assess diversity of Salmonella enterica serotypes present in poultry and their environment from Southern Brazil, the Kauffman-White-LeMinor (KWL) scheme was used to serotype a total of 155 isolates. Isolates were then re-examined with nested PCR and sequencing of the dkgB-linked Intergenic Sequ...
Le Gall, O L; Lanneau, M; Candresse, T; Dunez, J
The RNA-2 of a carrot isolate from the English serotype of tomato black ring nepovirus (TBRV-ED) has been sequenced. It is 4618 nucleotides long and contains one open reading frame encoding a polypeptide of 1344 amino acids. The 5' non-coding region contains three repetitions of a stem-loop structure also conserved in TBRV-Scottish and grapevine chrome mosaic nepovirus (GCMV). The coat protein domain was mapped to the carboxy-terminal one-third of the polyprotein. Sequence comparisons indicate that TBRV-ED RNA-2 probably arose by an RNA recombination event that resulted in the exchange of the putative movement protein gene between TBRV and GCMV.
Thrane, Sandra Wingaard; Taylor, Véronique L.; Lund, Ole
aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web-service, and aptly facilitate high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability...... often associated with P. aeruginosa isolates from chronic infections, and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1649 genomes resulted in successful serogroup assignments in 99.27% of the cases...
Gaensbauer, James T; Asturias, Edwin J; Soto, Monica; Holt, Elizabeth; Olson, Daniel; Halsey, Neal A
To inform estimations of the potential impact of recently introduced pneumococcal conjugate vaccine (PCV), we report results of 11 years of pre-PCV surveillance for invasive pneumococcal disease (IPD) among children in Guatemala City. Cases of IPD in children younger than 5 years were identified by active surveillance at 3 referral hospitals in Guatemala City from October 1996 through 2007. Clinical and demographic data were obtained, and isolates of Streptococcus pneumoniae from normally sterile sites were serotyped using latex agglutination and confirmed by Quellung reaction. Four hundred fifty-two cases of IPD were identified with a case fatality rate of 21%. Meningitis was the most common cause of death (77% of all deaths) and occurred more often in infancy (median age 5 months) than other clinical syndromes. Of the 137 isolates serotyped, type 1 (26 cases, 17%), type 2 (25 cases, 16%) and type 5 (18 cases, 12%) were the most common. Serotype 2 was associated with a higher case fatality rate (28%), higher rate of meningitis (68%) and occurred in younger infants (median age, 3.5 months) than other common serotypes. Recently introduced PCV13 includes 73% of observed serotypes in the study. Infants with IPD presented at a young age. Serotype 2, rarely reported as a significant cause of IPD and not included in available PCVs, was a common cause of disease in this population. PCV13 introduction in Guatemala, begun in 2013, may not have as great an impact in disease reduction as has been observed in other countries.
Klein, Jörn; Parlak, Ü.; Özyörük, F.
the region encoding the immuno-dominant GH-loop. Also a close relationship to Foot-and-Mouth Disease virus (FMDV) serotype A isolates obtained from outbreaks in Iraq and Iran were detected and a clustering of isolates collected during the same period of time were found. The analysis of the deduced amino...... comparison reported elsewhere do not substantiate such a conclusion. There is evidence that IRN99 was introduced to Turkey, in all probability from Iran. Since, a member of the IRN96 lineage was included as a component of the FMDV vaccine produced since 2000, the outbreaks caused by IRN96 strains in 2004...
Nova Nayarit-Ballesteros; María Salud Rubio-Lozano; Enrique Delgado-Suárez; Danilo Méndez-Medina; Diego Braña-Varela; Oscar Rodas-Suárez
Objective. To determine the serotype and antibiotic resistance profile of Salmonella spp. isolated from retail ground beef in Mexico City. Materials and methods. A total of 100 samples of ground beef were analyzed. The pathogen was isolated by conventional methods and confirmed by PCR (invA gene, 284 bp). The antibiotic resistance profile was determined by the Kirby-Bauer method while serotyping was performed according to the Kauffman-White scheme. Results. We isolated a total of 19 strains o...
Cui, Chen; Huang, Ligang; Li, Jing; Zou, Xingqi; Zhu, Yuanyuan; Xie, Lei; Zhao, Qizu; Yang, Limin; Liu, Wenjun
Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.
Swaminathan Sathyamangalam; Khanna Navin; Rajendra Pilankatta; Khanam Saima
Abstract Background Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during expos...
Full Text Available A total of 150 purified isolates of Pasteurella multocida serotype B were used (Salmah, 2004 for plasmid DNA curing experiment to determine hyaluronidase activity, antibiotic resistance pattern (ARP and mice lethality test (LD50 for their role of pathogenicity. A plasmid curing experiment was carried out by using the intercalating agent; ethidium bromide and rifampicin, where it was found all the plasmids had been cured (plasmidless from Pasteurella multocida. All of these plasmidless isolates maintained their phenotypic characteristics. They showed the same antibiotic resistancepattern as before curing, produced hyaluronidase and possessed lethality activity in mice when injected intraperitoneally(i.p. Based on this observation, the antibiotic resistance, hyaluronidase activity and mice virulence could probably be chromosomal-mediated. Plasmids were detected 100% in all P. multocida isolates with identical profile of 2 plasmids size 3.0 and 5.5 kb. No large plasmids could be detected in all isolates. Since all the isolates appeared to have identicalplasmid profiles, they were subjected to restriction enzyme(RE analysis. From RE analysis results obtained, it can be concluded that the plasmid DNA in serotype B isolates are identical. Only 4 of 32 REs were found to cleave these plasmids with identical restriction fingerprints; BglII, HaeIII, RsaI and SspI. From RE analysis results, it can be concluded that the plasmid DNA isolates are identical. This plasmid might not played any role in pathogenicity of Pasteurella multocida serotype B, however this information is important for the construction of shuttle vectors in genetic studies of the pathogenicity of haemorrhagic septicaemia(HS.
Joanne, Sylvia; Vythilingam, Indra; Teoh, Boon-Teong; Leong, Cherng-Shii; Tan, Kim-Kee; Wong, Meng-Li; Yugavathy, Nava; AbuBakar, Sazaly
To determine the susceptibility status of Aedes albopictus with and without Wolbachia to the four dengue virus serotypes. Two newly colonised colonies of Ae. albopictus from the wild were used for the study. One colony was naturally infected with Wolbachia while in the other Wolbachia was removed by tetracycline treatment. Both colonies were orally infected with dengue virus-infected fresh blood meal. Dengue virus load was measured using quantitative RT-PCR at four-time intervals in the salivary glands, midguts and ovaries. Wolbachia did not significantly affect Malaysian Ae. albopictus dengue infection or the dissemination rate for all four dengue virus serotypes. Malaysian Ae. albopictus had the highest replication kinetics for DENV-1 and the highest salivary gland and midgut infection rate for DENV-4. Wolbachia, which naturally exists in Malaysian Ae. albopictus, does not significantly affect dengue virus replication. Malaysian Ae. albopictus is susceptible to dengue virus infections and capable of transmitting dengue virus, especially DENV-1 and DENV-4. Removal of Wolbachia from Malaysian Ae. albopictus would not reduce their susceptibility status. © 2017 John Wiley & Sons Ltd.
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; Silveira, Nelson José Freitas da; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil
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...
Grant, Clare F J; Carr, B Veronica; Kotecha, Abhay; van den Born, Erwin; Stuart, David I; Hammond, John A; Charleston, Bryan
Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype does not confer interserotype protection. However, some historical data have shown that interserotype protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study, we have investigated the kinetics of the FMDV-specific antibody-secreting cell (ASC) response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar for all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O, A, and Asia1 serotypes) a B cell response to FMDV SAT1 and serotype C was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific ASCs in the absence of circulating FMDV-specific ASCs, indicating the presence of short-lived ASCs, a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid. IMPORTANCE We have demonstrated the development of intraserotype response following a sequential vaccination regime of four different FMDV serotypes. We have found indication of short-lived ASCs in the local lymphoid tissue, further evidence of a TI-2 response to FMDV. Copyright © 2017 American Society for Microbiology.
Sivanandan, V; Halvorson, D A; Laudert, E; Senne, D A; Kumar, M C
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.
Culshaw, Abigail; Ladell, Kristin; Gras, Stephanie; McLaren, James E; Miners, Kelly L; Farenc, Carine; van den Heuvel, Heleen; Gostick, Emma; Dejnirattisai, Wanwisa; Wangteeraprasert, Apirath; Duangchinda, Thaneeya; Chotiyarnwong, Pojchong; Limpitikul, Wannee; Vasanawathana, Sirijitt; Malasit, Prida; Dong, Tao; Rossjohn, Jamie; Mongkolsapaya, Juthathip; Price, David A; Screaton, Gavin R
Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8 + T cell populations specific for variants of the nonstructural protein epitope NS3 133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3 133 -DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2 + TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2 + TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.
Gerbier, G.; Baldet, T.; Tran, A.; Hendrickx, G.; Guis, H.; Mintiens, K.; Elbers, A.R.W.; Staubach, C.
The knowledge of the place where a disease is first introduced and from where it later spreads is a key element for the understanding of an epizootic. For a contagious disease, the main method is back tracing. For a vector-borne disease such as the Bluetongue virus serotype 8 epizootic that occurred
R. J. Zende,
Full Text Available Aim: Present study was undertaken to find out the frequency of few virulent genes and prevalence of related strains of Escherichia coli isolated from chicken meat obtained from chicken retail shops by Polymerase Chain Reaction (PCR.Materials and Methods: 66 samples of freshly slaughtered chicken meat were collected from 22 identified retail shops located at Mumbai city, randomly. Processed meat samples were cultured in EMB agar and presumptive colonies were confirmed by various biochemical tests. PCR method was accustomed for identification of the genes coding for heat-stable enterotoxin a (STa, heat labile enterotoxin (LT, shiga-like toxins 1 and 2 (SLT1 and SLT2. E. coli isolates were sent to National Salmonella and Escherichia Centre, CRI, Kasauli, HP, India for serotyping.Results: 11 (16.67% E. coli strains were isolated from 66 chicken meat samples. 3 (27.27% out of 11 harbored the gene for SLT2, and 2 (18.18% for STa. None of the strain contains SLT1 and LT genes. Serotypes detected were rough, O2, O20, O22, O102 each for one isolate and 6 isolates were untypable (UT.Conclusion: The results concluded that chicken meat samples analysed harbored genes for shiga like toxins and enterotoxins and different serotypes of E. coli. These findings indicating that regular monitoring of chicken meat is essential for this pathogen to prevent potential public health problems.
Full Text Available Background: Uropathogenic Escherichia coli (UPEC O-serogroups with their phylogenetic background are the most prevalent causes of urinary tract infections (UTIs. Objectives: The association of O types with phylogenetic background was assessed among E. coli isolates collected from patients with UTI. Patients and Methods: In this study, 186 patients with UTI, referred to two hospitals affiliated to Zabol University of Medical Sciences in southeast of Iran, were enrolled during January to July 2013. Phylogenetic groups and serotyping were performed using multiplex-PCR method. Results: A total of 100 E. coli strains were isolated from the urine samples. The most common types of O antigens were O2 (16.43%, O6 (16.43% and O18 (13.69%. The phylogenetic analysis showed that 63 O-antigen-positive isolates were mainly segregated from the phylogenetic group B2 (56% and the substantial prevalence (30% belonged to the phylogenetic group D. Conclusions: This was the first report of E. coli serotyping in patient with UTI from southeast of Iran as well as investigation of their relation with phylogenetic pattern by multiplex-PCR. Further studies from other parts of Iran and on other serotypes are recommended.
Jamal, Syed M; Belsham, Graham J
Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor
Syed M Jamal
Full Text Available Rapid and accurate diagnosis of foot-and-mouth disease (FMD and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08. In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples and specificity (100% each for serotypes O, A and Asia-1 positive samples for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for
Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.
Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution. Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes. Towards the determination of a structure for AAV type 6, this paper reports the high-yield production, purification, crystallization and preliminary diffraction studies of infectious AAV-6 virions. The crystals diffracted to 3.2 Å resolution using synchrotron radiation. The most promising crystal form belonged to space group R3 and appeared to be suitable for initial structure determination
Full Text Available Avian paramyxovirus (APMV serotypes 1-9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2-9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT assay in chicken eggs and intracerebral pathogenicity index (ICPI test in 1-day-old SPF chicks demonstrated that APMV types 2-9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2-9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2-9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1 and exhibited restricted viral replication of the APMVs (including APMV-1 to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1-9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes.
Mullens, B A; Tabachnick, W J; Holbrook, F R; Thompson, L H
Culicoides variipennis sonorensis females were fed bluetongue virus serotype 11 mixed in sheep blood and were held at constant temperatures of 32, 27, 21 and 15 degrees C. Virogenesis, as measured by enzyme-linked immunosorbent assay (ELISA), proceeded significantly faster at higher temperatures. Based on ELISA absorbance > or = 0.2, some flies first were categorized as infected after 1 day, 2 days and 4 days at 32, 27 and 21 degrees C, respectively. Peak levels of virus antigen were seen after 5-7, 7-13 and 18-22 days for flies held at 32, 27 and 21 degrees C, respectively. There was no significant virus replication in flies held at 15 degrees C for 22 days, but latent virus replicated and was detected easily (44% infection) 4-10 days after these flies were transferred to 27 degrees C. The implications for temperature effects on bluetongue epizootiology are discussed.
Full Text Available In a prospective field study conducted from July 2000 to June 2001, adult Aedes aegypti and Ae. albopictus mosquitoes were caught from the municipality of Nova Iguaçu, State of Rio de Janeiro, Brazil. Virus isolation in Ae. albopictus clone C6/36 cell line and a semi-nested reverse transcription-polymerase chain reaction detected only dengue virus type 3 in three pools of Ae. aegypti, despite the co-circulation of DEN-1, DEN-2 and DEN-3 serotypes in that area. No viruses were detected in Ae. albopictus mosquitoes. This virological surveillance consists in a sentinel system alerting for dengue outbreaks.
Lin, Jung-Chung; Koh, Tse Hsien; Lee, Nelson; Fung, Chang-Phone; Chang, Feng-Yee; Tsai, Yu-Kuo; Ip, Margaret; Siu, L Kristopher
In Klebsiella pneumoniae liver abscess (KP-LA), K. pneumoniae K2 is the most frequently isolated serotype after K1, but this serotype has been much less studied. In the present study, the molecular types sequences type (MLST) of serotype K2 isolates from three different regions in Asia were identified and the virulence of these isolates was investigated. Eight different MLSTs were found among 26 isolates (ST 65, 66, 86, 373, 374, 375, 380, and 434). There were two major MLST groups, ST-65-like (42%) and ST86-like (46%). No isolates contained allS while all isolates contained rmpA. The prevalence of aerobactin gene and kfu were 25/26 (96%) and 3/26 (11.5%) respectively. Although liver abscess isolates were generally more resistant (11/15 isolates) to serum killing, there was no specific distribution of serum killing resistant or susceptible ST types between stool carriage and liver abscess isolates. Neutrophil phagocytosis showed that the liver abscess and carriage isolates varied in their susceptibility to phagocytosis. Strains with resistance to both neutrophil phagocytosis and serum killing were generally hypervirulent with lethality at LD50 K2 isolates. Unlike serotype K1 KP-LA that mainly belong to ST-23, ST-65-like and -86-like are the two major MLST types among serotype K2 isolates from Singapore, Hong Kong and Taiwan.
Vaddadi, K; Gandikota, C; Jain, P K; Prasad, V S V; Venkataramana, M
The burden of dengue virus infections increased globally during recent years. Though India is considered as dengue hyper-endemic country, limited data are available on disease epidemiology. The present study includes molecular characterization of dengue virus strains occurred in Hyderabad, India, during the year 2014. A total of 120 febrile cases were recruited for this study, which includes only children and 41 were serologically confirmed for dengue positive infections using non-structural (NS1) and/or IgG/IgM ELISA tests. RT-PCR, nucleotide sequencing and evolutionary analyses were carried out to identify the circulating serotypes/genotypes. The data indicated a high percent of severe dengue (63%) in primary infections. Simultaneous circulation of all four serotypes and co-infections were observed for the first time in Hyderabad, India. In total, 15 patients were co-infected with more than one dengue serotype and 12 (80%) of them had severe dengue. One of the striking findings of the present study is the identification of serotype Den-1 as the first report from this region and this strain showed close relatedness to the Thailand 1980 strains but not to any of the strains reported from India until now. Phylogenetically, all four strains of the present study showed close relatedness to the strains, which are reported to be high virulent.
Carmo, Andreia Moreira Dos Santos; Suzuki, Rodrigo Buzinaro; Cabral, Aline Diniz; Costa, Renata Torres da; Massari, Gabriela Pena; Riquena, Michele Marcondes; Fracasso, Helio Augusto Alves; Eterovic, Andre; Marcili, Arlei; Sperança, Márcia Aparecida
Dengue virus, represented by four distinct, genetically diverse serotypes, is the etiologic agent of asymptomatic to severe hemorrhagic diseases. The spatiotemporal dynamics of dengue serotypes and its association to specific diseases vary among the different regions worldwide. By 2007, and in São Paulo State, Brazil, dengue-case concentration in urban centers had changed to increased incidence in small- and medium-sized towns, the case of Marília. The aim of this article was to distinguish dengue serotypes circulating during the 2007 Marília outbreak and define their association to demographic and hematological patient profiles, as well as the phylogenetic relationships among the different viruses. PCR amplicons corresponding to the junction of capsid and dengue pre-membrane encoding genes, obtained from dengue serologically positive patients, were sequenced. Hematological and demographic data of patients with different Dengue serotypes were evaluated by univariate and bivariate statistics. Dengue PCR sequences were used in phylogenetic relationships analyzed for maximum parsimony. Molecular typing confirmed co-circulation of the dengue serotypes 1 (DENV1) and 3 (DENV3), which presented divergent correlation patterns with regard to hematological descriptors. The increase in atypical lymphocytes, a likely indication of virus load, could be significantly associated to a decrease in leukocyte counts in the DENV3 group and platelet in the DENV1. Phylogenetic reconstitution revealed the introduction of DENV1 from northern Brazil and local divergence of DENV3 by either microevolution or viral introduction from other geographical regions or both. Dengue dynamics showed regional molecular-epidemiologic specificity, which has important implications for introduction of vaccines, disease management, and transmission control. Copyright © 2017 Elsevier B.V. All rights reserved.
Höglund Åberg, Carola; Antonoglou, Georgios; Haubek, Dorte; Kwamin, Francis; Claesson, Rolf; Johansson, Anders
The cytolethal distending toxin (Cdt) is a highly conserved exotoxin that are produced by a number of Gram negative bacteria, including Aggregatibacter actinomycetemcomitans, and affects mammalian cells by inhibiting cell division and causing apoptosis. A complete cdt-operon is present in the majority of A. actinomycetemcomitans, but the proportion of isolates that lack cdt-encoding genes (A, B and C) varies according to the population studied. The objectives of this study were to examine serotype, Cdt-genotype, and Cdt-activity in isolates of A. actinomycetemcomitans collected from an adolescent West African population and to examine the association between the carrier status of A. actinomycetemcomitans and the progression of attachment loss (AL). A total of 249 A. actinomycetemcomitans isolates from 200 Ghanaian adolescents were examined for serotype and cdt-genotype by PCR. The activity of the Cdt-toxin was examined by DNA-staining of exposed cultured cells and documented with flow cytometry. The periodontal status of the participants was examined at baseline and at a two-year follow-up. Presence of all three cdt-encoding genes was detected in 79% of the examined A. actinomycetemcomitans isolates. All these isolates showed a substantial Cdt-activity. The two different cdt-genotypes (with and without presence of all three cdt-encoding genes) showed a serotype-dependent distribution pattern. Presence of A. actinomycetemcomitans was significantly associated with progression of AL (OR = 5.126; 95% CI = [2.994-8.779], padolescents showed a distribution of serotype and cdt-genotype in line with results based on other previously studied populations. Presence of A. actinomycetemcomitans was significantly associated with disease progression, in particular the b serotype, whereas the association with disease progression was not particularly related to cdt-genotype, and Cdt-activity.
Asfor, Amin S.; Upadhyaya, Sasmita; Knowles, Nick J.; King, Donald P.; Paton, David J.
Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design. PMID:24584474
E de Sousa
Full Text Available In order to maintain the high production and export rates achieved by the Brazilian poultry industry, it is necessary to prevent and control certain disease agents, such as Salmonella spp. Using bacterial cultures, the aim of the present study was to investigate the prevalence of Salmonella spp. in specimens collected from broiler facilities. Local wild birds were also sampled, as well as the feces of swine housed on the poultry farm. After sample collection, the isolated serotypes were subsequently inoculated into broiler chicks to determine their effects. Positive samples were collected from the following locations in the poultry facilities: poultry litter (S. serotype 4,5,12:R:-; S. Heidelberg; S. Infantis, broiler feces (S. Heidelberg; S. serotype 6,7:R:-; S. serotype 4,5,12:R:-; S. Tennessee, water (S. Glostrup; S. serotype 6,8:d:-;, and lesser mealworms (Alphitobius diaperinus found in the litter (S. Tennessee. Among the 36 wild birds captured, S. Heidelberg was isolated from one bird's organs and intestinal contents (Colaptes campestris, and S. Enteritidis was isolated from another bird's intestinal contents (Zenaida auriculata. Salmonella Panama and Salmonella Typhimurium were isolated from swine feces. One-day-old chicks (150 were divided into 10 groups of 15 animals each. Each group was orally inoculated with a previously isolated serotype of Salmonella. Soft stools were observed on the cage floor and around the birds' cloaca between 3 and 12 days post-infection (dpi. The different serotypes of Salmonella used to inoculate the chicks were re-isolated from the spleen, liver, and cecal content samples of the infected birds on 15 and 21 dpi.
Gelanew, Tesfaye; Hunsperger, Elizabeth
Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to - 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1-4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1-4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1-3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.
Kopp, Sarah J; Ranaivo, Hantamalala R; Wilcox, Douglas R; Karaba, Andrew H; Wainwright, Mark S; Muller, William J
Outcomes of neonates with herpes simplex virus (HSV) encephalitis are worse after infection with HSV-2 when compared with HSV-1. The proteins herpes virus entry mediator (HVEM) and nectin-1 mediate HSV entry into susceptible cells. Prior studies have shown receptor-dependent differences in pathogenesis that depend on route of inoculation and host developmental age. We investigated serotype-related differences in HSV disease and their relationship to entry receptor availability in a mouse model of encephalitis. Mortality was attenuated in 7-d-old, wild-type (WT) mice inoculated with HSV-1(F) when compared with HSV-2(333). No serotype-specific differences were seen after inoculation of adult mice. HSV-1 pathogenesis was also attenuated relative to HSV-2 in newborn but not adult mice lacking HVEM or nectin-1. HSV-2 requires nectin-1 for encephalitis in adult but not newborn mice; in contrast, nectin-1 was important for HSV-1 pathogenesis in both age groups. Early viral replication was independent of age, viral serotype, or mouse genotype, suggesting host responses influence outcomes. In this regard, significantly greater amounts of inflammatory mediators were detected in brain homogenates from WT newborns 2 d after infection compared with adults and receptor-knockout newborns. Dysregulation of inflammatory responses induced by infection may influence the severity of HSV encephalitis.
Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard
Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174 PPAVP 179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.
Michiel M. Harmsen
Full Text Available Intact (146S foot-and-mouth disease virus (FMDVs can dissociate into specific (12S viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.
Ahmed, Z; Pauszek, S J; Ludi, A; LaRocco, M; Khan, E-U-H; Afzal, M; Arshed, M J; Farooq, U; Arzt, J; Bertram, M; Brito, B; Naeem, K; Abubakar, M; Rodriguez, L L
We report the laboratory analysis of 125 clinical samples from suspected cases of foot-and-mouth disease (FMD) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT-PCR, of which 88 were also found to contain infectious FMD virus (FMDV) by virus isolation (VI), with strong correlation between these tests (κ = 0.96). Samples that were VI-positive were serotyped by antigen detection ELISA (Ag-ELISA) and VP1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n = 13), O (n = 36) and Asia-1 (n = 41), including three samples from which both serotypes Asia-1 and O were detected. Serotype A viruses were classified within three different Iran-05 sublineages: HER-10, FAR-11 and ESF-10. All serotype Asia-1 were within Group VII (Sindh-08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia-2 within two different sublineages: ANT-10 and BAL-09. Using VP1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag-ELISA to correctly determine serotype was 74%, and serotype-specific sensitivity was 8% for serotype A, 88% for Asia-1 and 89% for O. Serotype-specific specificity was 100% for serotype A, 93% for Asia-1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag-ELISA. This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV. © 2017 Blackwell Verlag GmbH.
A fowl adenovirus serotype 9, a non-pathogenic large double stranded DNA virus, was developed as a viral vector to express influenza genes as a potential vaccine. Two separate constructs were developed that expressed either the hemagglutinin gene of A/Chicken/Jalisco/2012 (H7) or A/ Chicken/Iowa/20...
Modumo, Jacob; Venter, Estelle H
Recent outbreaks of bluetongue virus (BTV) serotypes 2 and 8 in many European countries provided an opportunity to investigate the possibility of improving the safety of the modified live vaccines administered mainly in South Africa. Modified live vaccines (MLV) released at a titre of 5 x 104 PFU/mL, raised concerns and prompted the need to determine the minimum titre which will still be protective and also safe. The BTV serotypes 2 and 8 vaccines were produced at the following titres: 102 PFU/mL, 103 PFU/mL and 104 PFU/mL, and were injected into 24 sheep which were then monitored. Blood was collected on days 0, 3, 6, 9, 12, 15, 18, 21, 25, 28 and 4 months post vaccination, for seroconversion and viraemia studies. These sheep were later challenged at 4 months post vaccination using BTV infected cell culture material, they were then observed and bled and again tested for viraemia. There was no viraemia post vaccination, however, a febrile reaction did occur and seroconversion was demonstrated at low titres for both BTV 2 and 8. Although viraemia was demonstrated post challenge, sheep vaccinated with the low titre BTV 2 vaccine showed more than a 90% protection index at a lower titre of 103 PFU/mL, compared with BTV 8 that showed a protection index above 90% at all the titres used. It is recommended that for BTV 2 vaccine, sheep should be vaccinated at a titre of 103 PFU/mL and at a titre of 102 PFU/mL with BTV 8 vaccine.
Bachanek-Bankowska, Katarzyna; Mero, Herieth R.; Wadsworth, Jemma
Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping...... methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within...... sequencing. Samples (n = 71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious...
Pacheco, Juan M.; Lee, Kwang-Nyeong; Eschbaumer, Michael; Bishop, Elizabeth A.; Hartwig, Ethan J.; Pauszek, Steven J.; Smoliga, George R.; Kim, Su-Mi; Park, Jong-Hyeon; Ko, Young-Joon; Lee, Hyang-Sim; Tark, Dongseob; Cho, In-Soo; Kim, Byounghan; Rodriguez, Luis L.; Arzt, Jonathan
Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic. PMID:26735130
Martinez-Torrecuadrada, J.L.; Langeveld, J.P.M.; Venteo, A.
African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological...... in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coil using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most....... Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques...
Gay, Noellie; Le Hello, Simon; Weill, François-Xavier; de Thoisy, Benoit; Berger, Franck
In French Guiana, a French overseas territory located in the South American northern coast, nearly 50% of Salmonella serotypes isolated from human infections belong to serotypes rarely encountered in metropolitan France. A reptilian source of contamination has been investigated. Between April and June 2011, in the area around Cayenne, 151 reptiles were collected: 38 lizards, 37 snakes, 32 turtles, 23 green iguanas and 21 caimans. Cloacal swab samples were collected and cultured. Isolated Salmonella strains were identified biochemically and serotyped. The overall carriage frequency of carriage was 23.2% (95% confidence interval: 16.7-30.4) with 23 serotyped strains. The frequency of Salmonella carriage was significantly higher for wild reptiles. Near two-thirds of the Salmonella serotypes isolated from reptiles were also isolated from patients in French Guiana. Our results highlight the risk associated with the handling and consumption of reptiles and their role in the spread of Salmonella in the environment. Copyright © 2014 Elsevier B.V. All rights reserved.
Siemer, B.L.; Harrington, C.S.; Nielsen, E.M.
health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters....... conclusions: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. Significance and Impct...... diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical...
Manjunatha N Belaganahalli
Full Text Available Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSDS. Eubenangee virus (EUBV, Tilligery virus (TILV, Pata virus (PATAV and Ngoupe virus (NGOV are currently all classified within the Eubenangee virus species of the genus Orbivirus, family Reoviridae. Full genome sequencing confirmed that EUBV and TILV (both of which are from Australia show high levels of aa sequence identity (>92% in the conserved polymerase VP1(Pol, sub-core VP3(T2 and outer core VP7(T13 proteins, and are therefore appropriately classified within the same virus species. However, they show much lower amino acid (aa identity levels in their larger outer-capsid protein VP2 (<53%, consistent with membership of two different serotypes - EUBV-1 and EUBV-2 (respectively. In contrast PATAV showed significantly lower levels of aa sequence identity with either EUBV or TILV (with <71% in VP1(Pol and VP3(T2, and <57% aa identity in VP7(T13 consistent with membership of a distinct virus species. A proposal has therefore been sent to the Reoviridae Study Group of ICTV to recognise 'Pata virus' as a new Orbivirus species, with the PATAV isolate as serotype 1 (PATAV-1. Amongst the other orbiviruses, PATAV shows closest relationships to Epizootic Haemorrhagic Disease virus (EHDV, with 80.7%, 72.4% and 66.9% aa identity in VP3(T2, VP1(Pol, and VP7(T13 respectively. Although Ngoupe virus was not available for these studies, like PATAV it was isolated in Central Africa, and therefore seems likely to also belong to the new species, possibly as a distinct 'type'. The data presented will facilitate diagnostic assay design and the identification of additional isolates of these viruses.
Dahlén, G; Gmür, R; Yoshino, T
This study was conducted to reveal phenotypic, serological subtypes and antibiotic susceptibility among fresh isolates of Porphyromonas gingivalis in a Swedish population with periodontitis and periodontal abscess. Fifty-five subgingival strains were isolated and tentatively designated as P. gingivalis from 55 consecutive paper-point samples taken from 51 patients with periodontitis (at least one site with >6-mm pocket depth) in Sweden and were sent in for microbiological evaluation. Eight P. gingivalis strains from periodontal abscesses were also included. Four P. gingivalis strains served as reference and another four type strains were included. The strains were characterized by colony morphology, biochemical tests, enzyme profile, gas-liquid chromatography and antibiotic susceptibility. The strains were further characterized for whole cell protein profiles using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were identified to serotype by specific monoclonal antibodies. Among the 55 P. gingivalis strains 35 had smooth (S), 13 rough (R) and seven semi-rough colony morphologies. All strains were phenotypically homogeneous in biochemical tests, enzyme profile and antibiotic susceptibility. All strains produced phenylacetic acid and alpha-fucosidase. Almost all (96%) of the subgingival strains, but relatively fewer (62%) of the abscess strains, belonged to serotype A. Two subgingival and three abscess strains were classified as serotype B. No specific SDS-PAGE protein profiles were recorded for the two serotypes. The P. gingivalis strains from Swedish periodontitis cases showed homogeneity in terms of biochemical phenotypes and antibiotic susceptibility patterns. The strains fell into two serotypes, of which serotype A predominated in the periodontitis cases and serotype B was overrepresented in periodontal abscesses.
Hammer, Anne Sofie; Pedersen, Karl; Andersen, Thomas Holmen
Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm out...
Ehizibolo, D O; Haegeman, A; De Vleeschauwer, A R; Umoh, J U; Kazeem, H M; Okolocha, E C; Van Borm, S; De Clercq, K
Control measures for foot and mouth disease (FMD) in Nigeria have not been implemented due to the absence of locally produced vaccines and risk-based analysis resulting from insufficient data on the circulating FMD virus (FMDV) serotypes/strains. In 2013-2015, blood and epithelial samples were collected from reported FMD outbreaks in four states (Kaduna, Kwara, Plateau and Bauchi) in northern Nigeria. FMDV non-structural protein (NSP) seroprevalence for the outbreaks was estimated at 80% (72 of 90) and 70% (131 of 188) post-outbreak. Antibodies against FMDV serotypes O, A, SAT1, SAT2 and SAT3 were detected across the states using solid-phase competitive ELISA. FMDV genome was detected in 99% (73 of 74) of the samples from FMD-affected animals using rRT-PCR, and cytopathic effect was found in cell culture by 59% (44 of 74) of these samples. Three FMDV serotypes O, A and SAT2 were isolated and characterized. The phylogenetic assessments of the virus isolates showed that two topotypes of FMDV serotype O, East Africa-3 (EA-3) and West Africa (WA) topotypes were circulating, as well as FMDV strains belonging to the Africa genotype (G-IV) of serotype A and FMDV SAT2 topotype VII strains. While the serotype O (EA-3) strains from Nigeria were most closely related to a 1999 virus strain from Sudan, the WA strain in Nigeria shares genetic relationship with three 1988 viruses in Niger. The FMDV serotype A strains were closely related to a known virus from Cameroon, and the SAT2 strains were most closely related to virus subtypes in Libya. This study provides evidence of co-occurrence of FMDV serotypes and topotypes in West, Central, East and North Africa, and this has implication for control. The findings help filling the knowledge gap of FMDV dynamics in Nigeria and West Africa subregion to support local and regional development of vaccination-based control plans and international risk assessment. © 2017 Blackwell Verlag GmbH.
Lerch, Thomas F.; Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.
Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning. Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication
Huhtamo, E; Korhonen, Em; Vapalahti, O
Imported dengue cases originating from the Madeiran outbreak are increasingly reported. In 2012 five Finnish travellers returning from Madeira were diagnosed with dengue fever. Viral sequence data was obtained from two patients. The partial C-preM sequences (399 and 396 bp respectively) were found similar to that of an autochthonous case from Madeira. The partial E-gene sequence (933 bp) which was identical among the two patients grouped phylogenetically with South American strains of dengue virus serotype 1.
Full Text Available Streptococcus suis infection is a severe zoonotic disease commonly found in Northern Thailand where people often consume raw pork and/or pig’s blood. The most frequent clinical presentations are meningitis, sepsis, and endocarditis with higher rate of mortality and hearing loss sequelae. To clarify the correlation between pulsed-field gel electrophoresis (PFGE groups and mrp/epf/sly genotypes of S. suis serotype 2, 62 patient and 4 healthy pig isolates from Northern Thailand were studied. By PFGE analysis, at 66% homology, most human isolates (69.4% and 1 pig isolate were in group A, whereas 14.5% of human isolates and 3 out of 4 pig isolates were in group D. According to mrp/epf/sly genotypes, 80.6% of human isolates were identified in mrp+epf−sly− and only 12.9% were in mrp−epf−sly+ genotypes; in contrast, 1 and 3 pig isolates were detected in these two genotypes, respectively. Interestingly, all isolates of S. suis serotype 2 classified in PFGE groups A, B, and E were set in mrp+epf−sly− genotypes. These data show a close correlation between PFGE groups and mrp/epf/sly genotypes of human S. suis serotype 2.
Tharavichitkul, Prasit; Wongsawan, Kanreuthai; Takenami, Naoki; Pruksakorn, Sumalee; Fongcom, Achara; Gottschalk, Marcelo; Khanthawa, Banyong; Supajatura, Volaluk; Takai, Shinji
Streptococcus suis infection is a severe zoonotic disease commonly found in Northern Thailand where people often consume raw pork and/or pig's blood. The most frequent clinical presentations are meningitis, sepsis, and endocarditis with higher rate of mortality and hearing loss sequelae. To clarify the correlation between pulsed-field gel electrophoresis (PFGE) groups and mrp/epf/sly genotypes of S. suis serotype 2, 62 patient and 4 healthy pig isolates from Northern Thailand were studied. By PFGE analysis, at 66% homology, most human isolates (69.4%) and 1 pig isolate were in group A, whereas 14.5% of human isolates and 3 out of 4 pig isolates were in group D. According to mrp/epf/sly genotypes, 80.6% of human isolates were identified in mrp (+) epf (-) sly (-) and only 12.9% were in mrp (-) epf (-) sly (+) genotypes; in contrast, 1 and 3 pig isolates were detected in these two genotypes, respectively. Interestingly, all isolates of S. suis serotype 2 classified in PFGE groups A, B, and E were set in mrp (+) epf (-) sly (-) genotypes. These data show a close correlation between PFGE groups and mrp/epf/sly genotypes of human S. suis serotype 2.
Full Text Available Recent outbreaks of bluetongue virus (BTV serotypes 2 and 8 in many European countries provided an opportunity to investigate the possibility of improving the safety of the modified live vaccines administered mainly in South Africa. Modified live vaccines (MLV released at a titre of 5 x 104 PFU/mL, raised concerns and prompted the need to determine the minimum titre which will still be protective and also safe. The BTV serotypes 2 and 8 vaccines were produced at the following titres: 102 PFU/mL, 103 PFU/mL and 104 PFU/mL, and were injected into 24 sheep which were then monitored. Blood was collected on days 0, 3, 6, 9, 12, 15, 18, 21, 25, 28 and 4 months post vaccination, for seroconversion and viraemia studies. These sheep were later challenged at 4 months post vaccination using BTV infected cell culture material, they were then observed and bled and again tested for viraemia. There was no viraemia post vaccination, however, a febrile reaction did occur and seroconversion was demonstrated at low titres for both BTV 2 and 8. Although viraemia was demonstrated post challenge, sheep vaccinated with the low titre BTV 2 vaccine showed more than a 90% protection index at a lower titre of 103 PFU/mL, compared with BTV 8 that showed a protection index above 90% at all the titres used. It is recommended that for BTV 2 vaccine, sheep should be vaccinated at a titre of 103 PFU/mL and at a titre of 102 PFU/mL with BTV 8 vaccine.
Petersen, L.; Nielsen, E.M.; Engberg, J.
(MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within...... serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:12 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide...
Bruns, M; Dietzschold, B; Schneider, L G; Cox, J H [Federal Research Inst. for Animal Virus Diseases, Tuebingen (Germany, F.R.)
Radioimmunoassay (RIA) provides a sensitive serological procedure for detecting rabies virus ribonucleoprotein (RNP) as well as its specific antibodies. RIA was carried out using highly purified RNPs labelled by the chloramine-T method. This paper describes optimal conditions for iodination of RNP with high specific activity. The optimal concentrations of /sup 125/I, RNP, chloramine-T, and reducing agent as well as the effect of pH on the reaction were investigated. RIA proved to be extremely sensitive for detection of homologous antibodies. In competition experiments the part-relationship of the group-specific RNPs of the three rabies virus serotypes (HEP, MOK, and LBV) was confirmed.
MacLachlan, N J; Nunamaker, R A; Katz, J B; Sawyer, M M; Akita, G Y; Osburn, B I; Tabachnick, W J
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.
Ozkaya, Etem; Uysal, Gülnar; Atak, Tunca; Alkan, Mehmet
Enteroviruses have major clinical and public health importance and are one of the leading causes of aseptic meningitis. There are many diseases with similar clinical symptoms and cerebrospinal fluid (CSF) findings of aseptic meningitis, thus virus isolation and identification is crucial for definitive diagnosis. Virological diagnosis is nonetheless important to distinguish between induced meningitis and other treatable causes of disease with a similar clinical picture. A total of 249 samples obtained from 246 cases (age range: 0-15 years), prediagnosed as aseptic meningitis, were sent to Virology Laboratory of Refik Saydam Hygiene Center. The patients were followed at Department of Pediatric Infectious Diseases in the Social Security Hospital, Ankara, Turkey, between 2001 and 2004. Stool (n: 180), CSF (n: 54) and throat swab (n: 15) samples have been inoculated to RD (rhabdomyosarcoma), Hep-2 (human epithelioma) and L20B (transgenic mice) cell lines, and followed up for the presence of cytopathic effects. A total of 95 enterovirus strains were isolated from 85 (34.6%) cases, and serotyped by using RIVM (National Institute of Public and the Environment, Nederlands) antisera with microneutralization method. As a result, the most frequently isolated types were found as echovirus type 30 (n: 24) and coxsackievirus type B (n: 19), which were most frequently isolated between July to October. This is the first report from Turkey for aseptic meningitis cases due to echovirus type 25 (n:3), 18 (n:2), 14 (n:1), 13 (n:4), 11 (n:6), 9 (n:1), 6 (n:9), 5 (n:1), 4 (n:1) and coxsackievirus type A9 (n:1).
Full Text Available The aim of this study was to determine the serotype and clonal distribution of pneumococci causing acute otitis media (AOM and their relationship with recurrences and mixed infections with other microorganisms under the influence of the 7-valent pneumococcal conjugate vaccine (PCV7. To do this, all pneumococcal isolates collected from the spontaneous middle-ear drainage of children <5 years old diagnosed of AOM by their pediatrician or their general practitioner from 1999 to 2010 were phenotypically characterized and the most frequent serotypes were genotyped. In the 12-year study, 818 episodes of pneumococcal AOM were detected, mostly (70.5% in children younger than 2 years old. In 262 episodes (32%, the pneumococci were isolated with another bacterium, mainly (n=214 Haemophilus influenzae. Mixed infections were similar in children under or over 2 years old. The most frequent serotypes were 19A (n=227, 27.8%, 3 (n=92, 11.2% and 19F (n=74, 9%. Serotypes included in the PCV7 sharply decreased from 62.4% in the pre-vaccination (1999-2001 to 2.2% in the late post-vaccination period (2008-2010. Serotype diversity steadily increased after the introduction of the PCV7 but decreased from 2008-2010 due to the predominant role of serotype 19A isolates, mostly ST276 and ST320. The prevalence of serotype 3 doubled from 6.1% (20/326 in 1999-2004 to 14.6% (72/492 in 2005-2010. Relapses mainly occurred in male infants infected with isolates with diminished antimicrobial susceptibility. Reinfections caused by isolates with the same serotype but different genotype were frequent, highlighting the need for genetic studies to differentiate among similar strains. In conclusion, the main change in pneumococcal AOM observed after the introduction of the PCV7 was the sharp decrease in vaccine serotypes. Also notable was the high burden of serotype 19A in total pneumococcal AOM before and especially after the introduction of the PCV7, as well as in relapses and
Full Text Available Background: Food-borne diseases pose serious health problems, affecting public health and economic development worldwide. Methods: Salmonella was isolated from samples of chicken parts, skin samples of whole chicken carcasses, raw egg yolks, eggshells and chicken faeces. Resulting isolates were characterised by serogrouping, serotyping, antimicrobial susceptibility testing and detection of extended-spectrum β-lactamase (ESBL production. Antibiotic resistance genes and integrons were identified by polymerase chain reaction (PCR. Results: The detection rates of Salmonella were 60%, 64% and 62% in chicken parts, skin, and faeces, respectively, whereas the egg yolks and eggshells were uniformly negative. Salmonella Kentucky and S. Enteritidis serotypes comprised 43.6% and 2.6% of the isolates, respectively, whilst S. Typhimurium was absent. Variable resistance rates were observed against 16 antibiotics; 97% were resistant to sulfamethoxazole, 96% to nalidixic acid and tetracycline and 76% to ampicillin. Multidrug resistance was detected in 82% (64/78 of the isolates and ESBL production was detected in 8% (6/78. The β-lactamase blaTEM-1 gene was detected in 57.6% and blaSHV-1 in 6.8% of the isolates, whilst the blaOXA gene was absent. The sul1gene was detected in 97.3% and the sul2 gene in 5.3% of the isolates. Sixty-four of the 78 isolates (82% were positive for the integrase gene (int I from class 1 integrons, whilst int II was absent. Conclusion: This study reveals the presence of an alarming number of multidrug-resistant Salmonella isolates in the local poultry markets in Cairo. The high levels of drug resistance suggest an emerging problem that could impact negatively on efforts to prevent and treat poultry and poultry-transmitted human diseases in Egypt.
Yeh, Jih-Ching; Lo, Dan-Yuan; Chang, Shao-Kuang; Chou, Chi-Chung; Kuo, Hung-Chih
Pasteurella multocida (PM) can cause progressive atrophic rhinitis and suppurative bronchopneumonia in pigs. The present study performed antimicrobial susceptibility testing and serotype and genotype identification on the 62 PM strains isolated from the lungs of diseased pigs with respiratory symptoms. Antimicrobial susceptibility testing examined 13 antimicrobial agents (amoxicillin, cefazolin, doxycycline, flumequine, enrofloxacin, florfenicol, kanamycin, lincomycin, Linco-Spectin (lincomycin and spectinomycin), erythromycin, tylosin, tilmicosin and tiamulin). Antimicrobial resistance ratios were over 40% in all of the antimicrobial agents except for cefazolin. The highest levels of resistance (100%) were found for kanamycin, erythromycin and tylosin. The majority of isolated strains was serotype D:L6 (n=35) followed by A:L3 (n=17). Comparison of the antimicrobial resistance levels between the two serotypes showed that the antimicrobial resistance rates were higher in D:L6 than in A:L3 for all the tested antimicrobials except for tylosin and tilmicosin. For PM with erm (B), erm (T) or erm (42), the results showed no significant difference compared with non-resistance gene strains in phenotype. Pulsed-field gel electrophoresis genotyping using Apa I restriction digestion of the genomic DNA demonstrated that there were 17 distinct clusters with a similarity of 85% or more, and the genotyping result was similar to that of serotyping. The results of the present study demonstrated that the PM isolated from diseased pigs in Taiwan was resistant to multiple antimicrobials, and the distribution of antimicrobial resistance was associated with pulsotype and serotype. © British Veterinary Association (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
Chen, Hao-tai; Peng, Yun-hua; Zhang, Yong-guang; Liu, Xiang-tao
An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, and A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Furthermore, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suita...
Ularamu, H G; Ibu, J O; Wood, B A; Abenga, J N; Lazarus, D D; Wungak, Y S; Knowles, N J; Wadsworth, J; Mioulet, V; King, D P; Shamaki, D; Adah, M I
This study describes the molecular characterization of 47 foot-and-mouth disease (FMD) viruses recovered from field outbreaks in Nigeria between 2007 and 2014. Antigen ELISA of viral isolates was used to identify FMD virus serotypes O, A and SAT 2. Phylogenetic analyses of VP1 nucleotide sequences provide evidence for the presence of multiple sublineages of serotype SAT 2, and O/EAST AFRICA 3 (EA-3) and O/WEST AFRICA topotypes in the country. In contrast, for serotype A, a single monophyletic cluster of viruses has persisted within Nigeria (2009-2013). These results demonstrate the close genetic relatedness of viruses in Nigeria to those from other African countries, including the first formal characterization of serotype O/EA-3 viruses in Nigeria. The introductions and persistence of certain viral lineages in Nigeria may reflect transmission patterns via nomadic pastoralism and animal trade. Continuous monitoring of field outbreaks is necessary to dissect the complexity of FMD epidemiology in sub-Saharan Africa. © 2016 Blackwell Verlag GmbH.
Bruun, B; Gahrn-Hansen, B; Westh, H
Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61...... sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized....
Full Text Available Background: Dengue Virus Infektion is major public health problem in Indonesia. Aedesaegypti is widespread in both urban and rural areas, where multiple virus Serotype are circulating. On 2013 outbreak ofdengue virus infection occur in East Java. Therefore study seroepidemiology in Bangkalan and Lombok had been done. Aim:to find a mutated strain ofDengue Virus in 4 cities ofIndonesia. Method: On 2011 and 2012 seroepidemiology study had been done in Dr. Soetomo Surabaya and Soerya Sidoarjo Hospital; and on 2013 study had been done in Surabaya, Bangkalan and Lombok Hospital . Diagnosis ofDengue Virus Infection was based on Criteri WHO - 2009. Virus isolation in Surabaya, Sidoarjo, Bangkalan and Lombok had been done. Result:a total of349 isolate were obtained from dengue patients sera collected in Surabaya and Sidoarjo, 2011–2012 showed that Den V1 (182, Den V2 (20 Den V4 (1 were found in Surabaya on 2011 and Den V 1 (79 , Den V 2 (7 were found in Surabaya on 2012; Den V1 (40, Den V 2 (3 were found in Sidoarjo on 2011 and Den V 1 (17 were found in Sidoarjo on 2012; Virus isolation in Surabaya on 2013, January: 237 serum sample were collected, found Den V 1 (8, Den V 3 (2 and Den V 4 (5. And PCR stereotyping of isolated viruses in Madura found Den V 1 (1 and Den V 4 (23. In Lombok found Den V 4 (4.It is possible to shift predominant strain in Surabaya , Genotype or Serotype shift might increase the number ofdengue patients. Conclusion: there were shift predominant strain in Surabaya especially Den V 1. Therefore to continuous surveillance ofcirculating viruses is required to predict the risk ofDHF and DF
Schaake, Julia; Kronshage, Malte; Uliczka, Frank; Rohde, Manfred; Knuuti, Tobias; Strauch, Eckhard; Fruth, Angelika; Wos-Oxley, Melissa
Yersinia enterocolitica is a human pathogen that is ubiquitous in livestock, especially pigs. The bacteria are able to colonize the intestinal tract of a variety of mammalian hosts, but the severity of induced gut-associated diseases (yersiniosis) differs significantly between hosts. To gain more information about the individual virulence determinants that contribute to colonization and induction of immune responses in different hosts, we analyzed and compared the interactions of different human- and animal-derived isolates of serotypes O:3, O:5,27, O:8, and O:9 with murine, porcine, and human intestinal cells and macrophages. The examined strains exhibited significant serotype-specific cell binding and entry characteristics, but adhesion and uptake into different host cells were not host specific and were independent of the source of the isolate. In contrast, survival and replication within macrophages and the induced proinflammatory response differed between murine, porcine, and human macrophages, suggesting a host-specific immune response. In fact, similar levels of the proinflammatory cytokine macrophage inflammatory protein 2 (MIP-2) were secreted by murine bone marrow-derived macrophages with all tested isolates, but the equivalent interleukin-8 (IL-8) response of porcine bone marrow-derived macrophages was strongly serotype specific and considerably lower in O:3 than in O:8 strains. In addition, all tested Y. enterocolitica strains caused a considerably higher level of secretion of the anti-inflammatory cytokine IL-10 by porcine than by murine macrophages. This could contribute to limiting the severity of the infection (in particular of serotype O:3 strains) in pigs, which are the primary reservoir of Y. enterocolitica strains pathogenic to humans. PMID:23959720
Dilan Amila Satharasinghe
Full Text Available Newcastle disease virus (NDV is a prototype member of avian paramyxovirus serotype 1 (APMV-1, which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004-2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a; however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13 shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13 carried nucleocapsid protein consist of 55-1801 nucleotides (nts and near-complete phosphoprotein (1804-3254 nts genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I to IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains
Satharasinghe, Dilan A; Murulitharan, Kavitha; Tan, Sheau W; Yeap, Swee K; Munir, Muhammad; Ideris, Aini; Omar, Abdul R
Newcastle disease virus (NDV) is a prototype member of avian paramyxovirus serotype 1 (APMV-1), which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing (NGS) on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004-2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a); however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13) shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13) carried nucleocapsid protein consist of 55-1801 nucleotides (nts) and near-complete phosphoprotein (1804-3254 nts) genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase) and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I-IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains of NDV in
da Moura, Aires Januário Fernandes; de Melo Santos, Maria Alice Varjal; Oliveira, Claudia Maria Fontes; Guedes, Duschinka Ribeiro Duarte; de Carvalho-Leandro, Danilo; da Cruz Brito, Maria Lidia; Rocha, Hélio Daniel Ribeiro; Gómez, Lara Ferrero; Ayres, Constância Flávia Junqueira
Dengue is an arboviral disease caused by dengue virus (DENV), whose main vectors are the mosquitoes Aedes aegypti and Aedes albopictus. A. aegypti is the only DENV vector in Cape Verde, an African country that suffered its first outbreak of dengue in 2009. However, little is known about the variation in the level of vector competence of this mosquito population to the different DENV serotypes. This study aimed to evaluate the vector competence of A. aegypti from the island of Santiago, Cape Verde, to four DENV serotypes and to detect DENV vertical transmission. Mosquitoes were fed on blood containing DENV serotypes and were dissected at 7, 14 and 21 days post-infection (dpi) to detect the virus in the midgut, head and salivary glands (SG) using RT-PCR. Additionally, the number of copies of viral RNA present in the SG was determined by qRT-PCR. Furthermore, eggs were collected in the field and adult mosquitoes obtained were analyzed by RT-PCR and the platelia dengue NS1 antigen kit to detect transovarial transmission. High rates of SG infection were observed for DENV-2 and DENV-3 whereas for DENV-1, viral RNA was only detected in the midgut and head. DENV-4 did not spread to the head or SG, maintaining the infection only in the midgut. The number of viral RNA copies in the SG did not vary significantly between DENV-2 and DENV-3 or among the different periods of incubation and the various titers of DENV tested. With respect to DENV surveillance in mosquitoes obtained from the eggs collected in the field, no samples were positive. Although no DENV positive samples were collected from the field in 2014, it is important to highlight that the A. aegypti population from Santiago Islands exhibited different degrees of susceptibility to DENV serotypes. This population showed a high vector competence for DENV-2 and DENV-3 strains and a low susceptibility to DENV-1 and DENV-4. Viral RNA copies in the SG remained constant for at least 21 dpi, which may enhance the vector
Ludi, A; Ahmed, Z; Pomeroy, L W; Pauszek, S J; Smoliga, G R; Moritz, M; Dickmu, S; Abdoulkadiri, S; Arzt, J; Garabed, R; Rodriguez, L L
Little information is available about the natural cycle of foot-and-mouth disease (FMD) in the absence of control measures such as vaccination. Cameroon presents a unique opportunity for epidemiological studies because FMD vaccination is not practiced. We carried out a prospective study including serological, antigenic and genetic aspects of FMD virus (FMDV) infections among different livestock production systems in the Far North of Cameroon to gain insight into the natural ecology of the virus. We found serological evidence of FMDV infection in over 75% of the animals sampled with no significant differences of prevalence observed among the sampled groups (i.e. market, sedentary, transboundary trade and mobile). We also found antibodies reactive to five of the seven FMDV serotypes (A, O, SAT1, SAT2 and SAT3) among the animals sampled. Finally, we were able to genetically characterize viruses obtained from clinical and subclinical FMD infections in Cameroon. Serotype O viruses grouped into two topotypes (West and East Africa). SAT2 viruses grouped with viruses from Central and Northern Africa, notably within the sublineage causing the large epidemic in Northern Africa in 2012, suggesting a common origin for these viruses. This research will guide future interventions for the control of FMD such as improved diagnostics, guidance for vaccine formulation and epidemiological understanding in support of the progressive control of FMD in Cameroon. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Full Text Available Species C human adenovirus serotype 5 (HAdV-C5 is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR, HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5.
Sluijs, van der M.; Timmermans, M.; Moulin, V.; Vonk Noordegraaf, C.; Vrijenhoek, M.; Debyser, I.; Smit, de A.J.; Moormann, R.J.M.
The ability of Bluetongue virus serotype 8 (BTV-8) originating from the 2006 European outbreak to cross the ovine placenta during early and mid gestation was investigated in two separate experiments. In the first experiment, 16 ewes were infected with BTV-8 at 70–75 days gestation. The foetuses were
Syrogiannopoulos, G A; Doit, C; Grivea, I N; Geslin, P; Bingen, E
In January 1996 the emergence of penicillin-susceptible, multiresistant serotype 6B Streptococcus pneumoniae isolates resistant to chloramphenicol, tetracycline, erythromycin, clindamycin and trimethoprim-sulfamethoxazole was observed in young carriers in the city of Patras, located in the southwestern region of Greece. Later, a significant spread of pneumococci with this unusual phenotype was noted in carriers living in various other areas of the country. Using restriction fragment length polymorphism of the ribosomal RNA genes, clonal relationships were found between these Greek strains and serotype 6B penicillin-susceptible, multiresistant pneumococci isolated in France between January 1992 and September 1996. The French and Greek isolates appear to have a common ancestry.
Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan.
Siu, L Kristopher; Fung, Chang-Phone; Chang, Feng-Yee; Lee, Nelson; Yeh, Kuo-Ming; Koh, Tse Hsien; Ip, Margaret
Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.
S F Swedan
Full Text Available Aims: Streptococcus pneumoniae, an opportunistic pathogen commonly carried asymptomatically in the nasopharynx of children, is associated with increasing rates of treatment failures due to a worldwide increase in drug resistance. We investigated the carriage of S. pneumoniae in children 5 years or younger, the identity of prevalent serotypes, the rates of resistance to macrolides and other antimicrobial agents and the genotypes responsible for macrolide resistance. Materials and Methods: Nasopharyngeal swabs were collected from 157 children under 5 years for cultural isolation of S. pneumoniae. Antibiogram of isolates was determined using the disk diffusion test, and the minimal inhibitory concentration to macrolides was determined using the E-test. Isolate serotypes and macrolide resistance genes, erm(B and mef(E, were identified using multiplex polymerase chain reactions. Results: S. pneumoniae was recovered from 33.8% of children; 41.9% among males and 21.9% among females (P = 0.009. The highest carriage rate occurred among age groups 7-12 months and 49-60 months. Most frequent serotypes were 19F, 6A/B, 11A, 19A, 14 and 15B/C. Resistance to macrolides was 60.4%. Resistance to oxacillin, trimethoprim/sulfamethoxazole and clindamycin was present among 90.6%, 54.7% and 32.1% of isolates, respectively. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. Isolates resistant to one or more macrolide drugs were more likely to be multidrug resistant. Resistance to clindamycin or oxacillin coexisted with macrolide resistance. Among the erythromycin-resistant isolates, erm(B, mef(E and erm(B and mef(E genes were present at rates of 43.8%, 37.5% and 6.3%, respectively. Erm(B and mef(E were associated with very high level and moderate-to-high level resistance to macrolides, respectively. Conclusion: A significant proportion of children harboured macrolide and multidrug-resistant S. pneumoniae.
Tian, Huaiyu; Sun, Zhe; Faria, Nuno Rodrigues; Yang, Jing; Cazelles, Bernard; Huang, Shanqian; Xu, Bo; Yang, Qiqi; Pybus, Oliver G; Xu, Bing
The incidence of dengue has grown dramatically in recent decades worldwide, especially in Southeast Asia and the Americas with substantial transmission in 2014-2015. Yet the mechanisms underlying the spatio-temporal circulation of dengue virus (DENV) serotypes at large geographical scales remain elusive. Here we investigate the co-circulation in Asia of DENV serotypes 1-3 from 1956 to 2015, using a statistical framework that jointly estimates migration history and quantifies potential predictors of viral spatial diffusion, including socio-economic, air transportation and maritime mobility data. We find that the spread of DENV-1, -2 and -3 lineages in Asia is significantly associated with air traffic. Our analyses suggest the network centrality of air traffic hubs such as Thailand and India contribute to seeding dengue epidemics, whilst China, Cambodia, Indonesia, and Singapore may establish viral diffusion links with multiple countries in Asia. Phylogeographic reconstructions help to explain how growing air transportation networks could influence the dynamics of DENV circulation.
Full Text Available The incidence of dengue has grown dramatically in recent decades worldwide, especially in Southeast Asia and the Americas with substantial transmission in 2014-2015. Yet the mechanisms underlying the spatio-temporal circulation of dengue virus (DENV serotypes at large geographical scales remain elusive. Here we investigate the co-circulation in Asia of DENV serotypes 1-3 from 1956 to 2015, using a statistical framework that jointly estimates migration history and quantifies potential predictors of viral spatial diffusion, including socio-economic, air transportation and maritime mobility data. We find that the spread of DENV-1, -2 and -3 lineages in Asia is significantly associated with air traffic. Our analyses suggest the network centrality of air traffic hubs such as Thailand and India contribute to seeding dengue epidemics, whilst China, Cambodia, Indonesia, and Singapore may establish viral diffusion links with multiple countries in Asia. Phylogeographic reconstructions help to explain how growing air transportation networks could influence the dynamics of DENV circulation.
Gaye, Alioune; Faye, Oumar; Diagne, Cheikh T; Faye, Ousmane; Diallo, Diawo; Weaver, Scott C; Sall, Amadou A; Diallo, Mawlouth
To investigate the potential for domestic and wild populations of Aedes aegypti from Dakar and Kedougou to develop a disseminated infection after exposure to DENV-3 and DENV-1. We have exposed sylvatic and urban population of Ae. aegypti from Senegal to bloomeals containing dengue serotype 1 and 3. At different incubation period, individual mosquito legs/wings and bodies were tested for virus presence using real time RT-PCR to estimate the infection and dissemination rates. The data indicated low susceptibility to DENV-3 (infection: 2.4-15.2%, and dissemination rates: 0-8.3%) and higher susceptibility to DENV-1 (infection and dissemination rates up to 50%). Aedes aegypti from Senegal seem able to develop a disseminated infection of DENV-1 and DENV-3. Further studies are needed to test their ability to transmit the two serotypes. © 2014 John Wiley & Sons Ltd.
Hadjilouka, Agni; Andritsos, Nikolaos D; Paramithiotis, Spiros; Mataragas, Marios; Drosinos, Eleftherios H
The aim of this study was to assess serotype prevalence and biodiversity of Listeria monocytogenes strains isolated from diverse food products, i.e., minced pork, fruits, and vegetables. Three hundred twenty-six samples previously purchased from supermarkets and street markets within the Athens area were studied for L. monocytogenes prevalence. A total of 121 strains were isolated from the 36 samples that were positive for L. monocytogenes. Serotyping was performed with multiplex PCR, and biodiversity was assessed with random amplified polymorphic DNA (RAPD) PCR analysis using M13, UBC155, and HLWL85 as primers and with repetitive element palindromic (rep) PCR analysis using (GTG)5 as the primer. The majority (17 of 22) of the contaminated minced pork samples contained strains identified as serotype 1/2a, either alone or in combination with strains belonging to serotypes 1/2b, 4a, 4c, or 4ab. However, all L. monocytogenes isolates from fruits and vegetables belonged to serotype 4b. Rep-PCR provided better differentiation of the isolates than did RAPD PCR and resulted in discrimination of the isolates into a larger number of unique profiles. Complete differentiation was achieved only with the combination of these subtyping techniques.
Lefebvre, David J; De Vleeschauwer, Annebel R; Goris, Nesya; Van Borm, Steven; Chimirri, Alba; Monforte, Anna Maria; Valdazo-Gonzalez, Begona; King, Donald P; Neyts, Johan; De Clercq, Kris
The stamping-out policy for the control of foot-and-mouth disease virus (FMDV) in countries that are free from FMD without vaccination has a dramatic socio-economic impact, huge animal welfare issues and may result in the loss of farm animal genetic resources. As an alternative to pre-emptive culling or emergency vaccination we further explore the possibility to use antiviral drugs in the event of an FMD outbreak. In the present study, we tested the in vitro cytotoxicity and anti-FMDV activity of 1,2,4,5-tetrahydro-[1,4]thiazepino[4,5-a]benzimidazole. The molecule was shown to inhibit the replication of reference strains of the Eurasian FMDV serotypes O, A, C and Asia but not the FMDV serotypes from the South African Territories (SAT) neither a related picornavirus, i.e. swine vesicular disease virus. The molecule can be added until 2h post inoculation in a 'single replication cycle experiment' without losing its antiviral activity. The genetic characterization of progressively selected resistant FMD viruses shows that the molecule presumably interacts with the non-structural 2C protein of FMDV. Further studies are required on the use of this molecule in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.
Schulz, Claudia; Eschbaumer, Michael; Rudolf, Miriam; König, Patricia; Keller, Markus; Bauer, Christian; Gauly, Matthias; Grevelding, Christoph G; Beer, Martin; Hoffmann, Bernd
Bluetongue (BT) is an infectious, non-contagious disease of wild and domestic ruminants. It is caused by bluetongue virus (BTV) and transmitted by Culicoides biting midges. Since 1998, BT has been emerging throughout Europe, threatening not only the naïve ruminant population. Historically, South American camelids (SAC) were considered to be resistant to BT disease. However, recent fatalities related to BTV in captive SAC have raised questions about their role in BTV epidemiology. Data on the susceptibility of SAC to experimental infection with BTV serotype 8 (BTV-8) were collected in an animal experiment. Three alpacas (Vicugna pacos) and three llamas (Lama glama) were experimentally infected with BTV-8. They displayed very mild clinical signs. Seroconversion was first measured 6-8 days after infection (dpi) by ELISA, and neutralising antibodies appeared 10-13 dpi. BTV-8 RNA levels in blood were very low, and quickly cleared after seroconversion. However, spleens collected post-mortem were still positive for BTV RNA, over 71 days after the last detection in blood samples. Virus isolation was only possible from blood samples of two alpacas by inoculation of highly sensitive interferon alpha/beta receptor-deficient (IFNAR(-/-)) mice. An in vitro experiment demonstrated that significantly lower amounts of BTV-8 adsorb to SAC blood cells than to bovine blood cells. Although this experiment showed that SAC are generally susceptible to a BTV-8 infection, it indicates that these species play a negligible role in BTV epidemiology. Copyright © 2011 Elsevier B.V. All rights reserved.
Karasin, Alexander I.; Landgraf, John; Swenson, Sabrina; Erickson, Gene; Goyal, Sagar; Woodruff, Mary; Scherba, Gail; Anderson, Gary; Olsen, Christopher W.
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...
Jiang, Lei; Zhao, Wenjun; Han, Zongxi; Chen, Yuqiu; Zhao, Yan; Sun, Junfeng; Li, Huixin; Shao, Yuhao; Liu, Liangliang; Liu, Shengwang
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.
Ortiz, Alma; Capitan, Zeuz; Mendoza, Yaxelis; Cisneros, Julio; Moreno, Brechla; Zaldivar, Yamitzel; Garcia, Mariana; Smith, Rebecca E; Motta, Jorge; Pascale, Juan Miguel
A one-step RT-PCR and one-enzyme RFLP was used to detect and distinguish among flaviviruses, including the four serotypes of dengue and the St. Louis Encephalitis, West Nile and Yellow Fever viruses in cultured virus samples or acute-phase human serum. Using a previously described RT-PCR, but novel RFLP procedure, results are obtained in 24 h with basic PCR and electrophoresis equipment. There is 95% agreement between RT-PCR/RFLP results and those achieved by indirect immunofluorescence assays, and 100% agreement between RT-PCR/RFLP results and gene sequencing. This method is more rapid than tests of cytopathic effect based on virus isolation in tissue culture, and simpler than real-time PCR. It does not require specialized equipment, radioisotopes or computer analysis and is a method that can be applied widely in the developing world. It allows for prompt determination of whether a flavivirus is the cause of illness in a febrile patient, rapid identification of dengue serotypes in circulation, and improved patient management in cases where prior dengue exposure make dengue hemorrhagic fever or dengue shock syndrome a risk. Copyright © 2012 Elsevier B.V. All rights reserved.
Renström Lena HM
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.
Bálint, Adám; Metreveli, Giorgi; Widén, Frederik; Zohari, Siamak; Berg, Mikael; Isaksson, Mats; Renström, Lena Hm; Wallgren, Per; Belák, Sándor; Segall, Thomas; Kiss, István
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.
Chiapponi, Chiara; Zanni, Irene; Garbarino, Chiara; Barigazzi, Giuseppe; Foni, Emanuela
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).
Full Text Available Quinolones have been extensively used for treatment of a variety of invasive and systemic infections of salmonellosis. Widespread use of these agents has been associated with the emergence and dissemination of quinolone-resistant pathogens. The quinolone resistance and plasmid-mediated quinolone resistance determinants (qnrA, qnrB, qnrS and aac(6’-Ib-cr of 85 Salmonella isolates from chicken carcasses were investigated in this study. Isolates were serotyped according to the Kauffman-White-Le Minor scheme, and broth microdilution method was used to determine quinolone resistance. Plasmid-mediated quinolone resistance genes were investigated by real-time PCR and positive results were confirmed by sequencing. Among the Salmonella isolates, 30/85 (35% and 18/85 (21% were found to be resistant to enrofloxacin (MIC ≥ 2 mg/ml, and danofloxacin (MIC ≥ 2 mg/ml, respectively. All the isolates were negative for qnrA, qnrB and aac(6’-Ib-cr genes, nevertheless 2% (S. Brandenburg and S. Dabou were positive for qnrS (qnrS1 determinant. This study is the first and unique investigating the plasmid- mediated quinolone resistance determinants of Salmonella isolated from chicken carcasses in Turkey.
Mwiine, F.N.; Ayebazibwe, C.; Olaho-Mukani, W.
Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non-structural......Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non......-structural proteins and structural proteins. Three hundred and forty-nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non-structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified...... antibodies. High prevalences of antibodies against non-structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than...
Qiu, Fang-Zhou; Shen, Xin-Xin; Zhao, Meng-Chuan; Zhao, Li; Duan, Su-Xia; Chen, Chen; Qi, Ju-Ju; Li, Gui-Xia; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun
Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.
Zhang, Yong; Hong, Mei; Sun, Qiang; Zhu, Shuangli; Tsewang; Li, Xiaolei; Yan, Dongmei; Wang, Dongyan; Xu, Wenbo
Molecular methods, based on sequencing the region encoding the complete VP1 or P1 protein, have enabled the rapid identification of new enterovirus serotypes. In the present study, the complete genome of a newly discovered enterovirus serotype, strain Q0011/XZ/CHN/2000 (hereafter referred to as Q0011), was sequenced and analyzed. The virus, isolated from a stool sample from a patient with acute flaccid paralysis in the Tibet region of China in 2000, was characterized by amplicon sequencing and comparison to a GenBank database of enterovirus nucleotide sequences. The nucleotide sequence encoding the complete VP1 capsid protein is most closely related to the sequences of viruses within the species enterovirus B (EV-B), but is less than 72.1% identical to the homologous sequences of the recognized human enterovirus serotypes, with the greatest homology to EV-B101 and echovirus 32. Moreover, the deduced amino acid sequence of the complete VP1 region is less than 84.7% identical to those of the recognized serotypes, suggesting that the strain is a new serotype of enterovirus within EV-B. The virus was characterized as a new enterovirus type, named EV-B111, by the Picornaviridae Study Group of the International Committee on Taxonomy of Viruses. Low positive rate and titer of neutralizing antibody against EV-B111 were found in the Tibet region of China. Nearly 50% of children ≤5 years had no neutralizing antibody against EV-B111. So the extent of transmission and the exposure of the population to this new EV are very limited. This is the first identification of a new serotype of human enterovirus in China, and strain Q0011 was designated the prototype strain of EV-B111. Copyright © 2014 Elsevier B.V. All rights reserved.
Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.
Pasteurella multocida isolates from 21 raptors were examined by DNA fingerprint profile and serotyping methods. Isolates were obtained from noncaptive birds of prey found in 11 states from November 28, 1979, through February 10, 1993. Nine isolates were from bald eagles, and the remaining isolates were from hawks, falcons, and owls. Seven isolates were members of capsule group A, and 14 were nonencapsulated. One isolate was identified as somatic type 3, and another was type 3,4,7; both had unique HhaI DNA fingerprint profiles. Nineteen isolates expressed somatic type 1 antigen; HhaI profiles of all type 1 isolates were identical to each other and to the HhaI profile of the reference somatic type 1, strain X-73. The 19 type 1 isolates were differentiated by sequential digestion of DNA with HpaII; four HpaII fingerprint profiles were obtained. The HpaII profile of one isolate was identical to the HpaII profile of strain X-73. Incidence of P. multocida somatic type 1 in raptors suggests that this type may be prevalent in other wildlife or wildlife environments.
Lorca-Oró, Cristina; Pujols, Joan; García-Bocanegra, Ignacio; Mentaberre, Gregorio; Granados, José Enrique; Solanes, David; Fandos, Paulino; Galindo, Iván; Domingo, Mariano; Lavín, Santiago; López-Olvera, Jorge Ramón
Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication. PMID:22666321
Tijana Knezevic, PhD
Full Text Available Mutations in Bcl-2–associated athanogene 3 (BAG3 were associated with skeletal muscle dysfunction and dilated cardiomyopathy. Retro-orbital injection of an adeno-associated virus serotype 9 expressing BAG3 (rAAV9-BAG3 significantly (p < 0.0001 improved left ventricular ejection fraction, fractional shortening, and stroke volume 9 days post-injection in mice with cardiac dysfunction secondary to a myocardial infarction. Furthermore, myocytes isolated from mice 3 weeks after injection showed improved cell shortening, enhanced systolic [Ca2+]i and increased [Ca2+]i transient amplitudes, and increased maximal L-type Ca2+ current amplitude. These results suggest that BAG3 gene therapy may provide a novel therapeutic option for the treatment of heart failure.
Kristensen, Thea; Normann, Preben; Gullberg, Maria
. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results...
Fernandes, Sueli Aparecida; Camargo, Carlos Henrique; Francisco, Gabriela Rodrigues; Bueno, Maria Fernanda Campagnari; Garcia, Doroti Oliveira; Doi, Yohei; Casas, Monique Ribeiro Tiba
We characterized extended-spectrum β-lactamases (ESBL) enzymes among Salmonella strains isolated in Brazil from 2009 to 2014. Salmonella recovered from both clinical and nonhuman (food, poultry, and environment) sources were subjected to antimicrobial susceptibility testing. β-lactamases genes were detected by polymerase chain reaction/sequencing; plasmid profiles and transferability were assessed by S1-pulsed field gel electrophoresis (PFGE). Genetic diversity was evaluated by XbaI-PFGE. Out of 630 Salmonella strains screened, 46 displayed ESBL phenotype, distributed across 11 different serotypes. bla CTX-M-8 and bla CTX-M-2 genes were detected at frequencies of 47% and 41%, respectively. bla SHV-5 and bla SHV-2 were also detected but in lower frequencies (4%, 2%). bla TEM-1 gene was detected in 22% of the strains. Most of the ESBL genes were transferable by conjugation, and the respective bla ESBL gene was detected in the recipient strain, indicating the location of ESBL determinants on transferable plasmids. XbaI-PFGE revealed genomic diversity of Salmonella Typhimurium bearing bla CTX-M-2 , bla CTX-M-8 , bla TEM-1 , and bla SHV-2 genes. Salmonella Muenchen (harboring bla CTX-M-2 ) and Salmonella Corvallis (bla CTX-M-8 and bla SHV-5 ) showed clonal relatedness within respective serotypes. Our findings underscore the occurrence of diverse ESBL genes in several Salmonella serotypes, reinforcing the need for continuous surveillance of resistance genes circulating in human and nonhuman sources.
Full Text Available Serotype, mating type and ploidy of 84 strains of Cryptococcus neoformans isolated from 61 AIDS and 23 non-AIDS patients admitted in a tertiary teaching hospital in São Paulo, Brazil were examined. Among 61 strains isolated from AIDS patients, 60 strains were var. grubii (serotype A. Only one strain was var. gattii (serotype B. No var. neoformans (serotype D was found. Among 23 strains isolated from non-AIDS patients, 15 were var. grubii (serotype A and the remaining 8 were var. gattii, all of which were serotype B. Seventy-three of the 75 serotype A strains were the heterothallic alpha type (MATalpha and the remaining 2 were untypable (asexual. Most of the MATalpha strains (69/73 were haploid and the remaining 4 strains were diploid. Similarly, both of the 2 asexual strains among the 75 serotype A strains were haploid. There were no alpha-mating type (MATalpha strains among the 84 isolates. All of the 8 var. gattii strains were serotype B and haploid. Among a total of 84 strains tested, neither serotype AD nor serotype D were found. Neither triploid nor tetraploid were found. These results suggest that the serological, sexual and ploidy characteristics in C. neoformans strains isolated from AIDS patients in São Paulo were rather simple, whereas strains isolated from non-AIDS patients presented serotype A and B with predominance of serotype A.Foram estudados os sorotipos, "mating type" e ploidia de 84 amostras de C. neoformans isoladas de 61 pacientes com AIDS e 23 não-AIDS em São Paulo. Das amostras isoladas de pacientes com AIDS, 60 foram identificadas como var. grubii (sorotipo A e 1 como var. gattii (sorotipo B. Não houve isolamento do sorotipo D. Entre as amostras isoladas, de pacientes não-AIDS, 15 foram de var. grubii (sorotipo A e as 8 restantes de var. gattii, todos do sorotipo B. Setenta e três dos 75 sorotipos A foram identificadas como cepas heterotálicas do fenótipo alfa (MATalfa e as 2 remanescentes n
Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.
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.
Glass, S E; Naqi, S A; Grumbles, L C
An avian influenza virus with surface antigens similar to those of fowl plague virus (Hav 1 Nav 2) was isolated in 1979 from 2 commercial turkey flocks in Central Texas. Two flocks in contact with these infected flocks developed clinical signs, gross lesions, and seroconversion but yielded no virus. This was the first recorded incidence of clinical avian influenza in Texas turkeys and only the second time that an agent with these surface antigens was isolated from turkeys in U.S.
Full Text Available Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997 for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.
Full Text Available Streptococcus suis (S. suis infection is considered to be a major problem in the swine industry worldwide. Based on the capsular type, 33 serotypes of S. suis have been described, with serotype 2 (SS2 being the most frequently isolated from diseased piglets. Little is known, however, about the pathogenesis and virulence factors of S. suis. Research on bacteriophages highlights a new area in S. suis research. A S. suis serotype 2 bacteriophage, designated SMP, has been previously isolated in our laboratory. Here, we selected a lysogenic isolate in which the SMP phage was integrated into the chromosome of strain SS2-4. Compared to the wild-type isolate, the lysogenic strain showed increased mortality in zebra fish. Moreover the sensitivity of the lysogenic strain to lysozyme was seven times higher than that of the wild-type.
Wekesa, Sabenzia Nabalayo; Sangula, Abraham Kiprotich; Belsham, Graham J; Tjornehoj, Kirsten; Muwanika, Vincent B; Gakuya, Francis; Mijele, Dominic; Siegismund, Hans Redlef
Understanding the epidemiology of foot-and-mouth disease (FMD), including roles played by different hosts, is essential for improving disease control. The African buffalo (Syncerus caffer) is a reservoir for the SAT serotypes of FMD virus (FMDV). Large buffalo populations commonly intermingle with livestock in Kenya, yet earlier studies have focused on FMD in the domestic livestock, hence the contribution of buffalo to disease in livestock is largely unknown. This study analysed 47 epithelia collected from FMD outbreaks in Kenyan cattle between 2008 and 2012, and 102 probang and serum samples collected from buffalo in three different Kenyan ecosystems; Maasai-Mara (MME) (n = 40), Tsavo (TSE) (n = 33), and Meru (ME) (n = 29). Antibodies against FMDV non-structural proteins were found in 65 of 102 (64%) sera from buffalo with 44/102 and 53/102 also having neutralising antibodies directed against FMDV SAT 1 and SAT 2, respectively. FMDV RNA was detected in 42% of the buffalo probang samples by RT-qPCR (Cycle Threshold (Ct) ≤32). Two buffalo probang samples were positive by VI and were identified as FMDV SAT 1 and SAT 2 by Ag-ELISA, while the latter assay detected serotypes O (1), A (20), SAT 1 (7) and SAT 2 (19) in the 47 cattle epithelia. VP1 coding sequences were generated for two buffalo and 21 cattle samples. Phylogenetic analyses revealed SAT 1 and SAT 2 virus lineages within buffalo that were distinct from those detected in cattle. We found that FMDV serotypes O, A, SAT 1 and SAT 2 were circulating among cattle in Kenya and cause disease, but only SAT 1 and SAT 2 viruses were successfully isolated from clinically normal buffalo. The buffalo isolates were genetically distinct from isolates obtained from cattle. Control efforts should focus primarily on reducing FMDV circulation among livestock and limiting interaction with buffalo. Comprehensive studies incorporating additional buffalo viruses are recommended.
Sparding, Nadja; Dayie, Nicholas Tete Kwaku Dzifa; Mills, Richael O.
Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococcal strains are classified according to their capsular polysaccharide and more than 90 different serotypes are currently known. In this project, three distinct groups of pneumococcal carriage isolates from Gh...... in Ghana in that many new clones were identified. This supports the importance of continued monitoring of pneumococcal carriage in Ghana and elsewhere when vaccines, e.g., PCV-13, have been introduced to monitor the possible future spread of antimicrobial resistant clones....
Dobbs, Fred C; Goodrich, Amanda L; Thomson, Frank K; Hynes, Wayne
There is concern that ships' ballasting operations may disseminate Vibrio cholerae to ports throughout the world. Given evidence that the bacterium is indeed transported by ships, we isolated pandemic serotypes O1 and O139 from ballast tanks and characterized them with respect to antibiotic resistance and virulence genes ctxA and tcpA. We carried out concurrent studies with V. cholerae isolated from coastal waters. Of 284 isolates, 30 were serotype O1 and 59 were serotype O139. These serotypes were overrepresented in ballast tanks relative to the coastal waters sampled. All locations, whether coastal waters or ballast tanks, yielded samples from which serotype O1, O139, or both were isolated. There were three groups among the 62 isolates for which antibiotic characterization was conclusive: those exhibiting β-lactamase activity and resistance to at least one of the 12 antibiotics tested; those negative for β-lactamase but having antibiotic resistance; those negative for β-lactamase and registering no antibiotic resistance. When present, antibiotic resistance in nearly all cases was to ampicillin; resistance to multiple antibiotics was uncommon. PCR assays revealed that none of the isolates contained the ctxA gene and only two isolates, one O139 and one O1, contained the tcpA gene; both isolates originated from ballast water. These results support the bacteriological regulations proposed by the International Maritime Association for discharged ballast water.
The 7-valent pneumococcal conjugate vaccine (PCV7) was included in the routine infant immunization schedule in Ireland in September 2008. We determined the serotype of 977 S. pneumoniae isolates causing invasive disease between 2000-2002 and 2007-2008, assessed for the presence of the recently described serotype 6C and determined the susceptibility of isolates during 2007-2008 to penicillin and cefotaxime. Serotype 14 was the most common serotype during both periods and 7·7% of isolates previously typed as serotype 6A were serotype 6C. During 2000-2002 and 2007-2008, PCV7 could potentially have prevented 85% and 74% of invasive pneumococcal disease in the target population (i.e. children aged <2 years), respectively. The level of penicillin non-susceptibility was 17% in 2007-2008. Ongoing surveillance of serotypes is required to determine the impact of PCV7 in the Irish population and to assess the potential of new vaccines with expanded valency.
Full Text Available We developed a lateral flow strip using monoclonal antibodies (MAbs which allows for rapid antigen detection and serotyping of foot-and-mouth disease virus (FMDV. This FMDV serotyping strip was able to detect all 7 serotypes and distinguish serotypes O, A, C and Asia1. Its sensitivities ranged from 10(3 to 10(4 of a 50% tissue culture infectious dose of each FMDV stain; this is equal to those of the commercial product Svanodip (Boehringer Ingelheim Svanova, Uppsala, Sweden, which can detect all seven serotypes of FMDV, but does not distinguish them. Our evaluation of the FMDV serotyping strip using a total of 118 clinical samples (vesicular fluids, vesicular epithelial emulsions and oral and/or nasal swabs showed highly sensitive antigen detection and accuracy in serotyping in accordance with ELISA or RT-PCR. To the best of our knowledge, this is the first report on any FMDV serotyping strip that provides both rapid antigen detection and serotyping of FMDV at the same time on one strip without extra devices. This method will be useful in both FMD-free countries and FMD-infected countries, especially where laboratory diagnosis cannot be carried out.
Dolz, Roser; Pujols, Joan; Ordóñez, German; Porta, Ramon; Majó, Natàlia
An in-depth molecular study of infectious bronchitis viruses (IBV) with particular interest in evolutionary aspects of IBV in Spain was carried out in the present study based on the S1 gene molecular characterization of twenty-six Spanish strains isolated over a fourteen-year period. Four genotypes were identified based on S1 gene sequence analyses and phylogenetic studies. A drastic virus population shift was demonstrated along time and the novel Italy 02 serotype was shown to have displaced the previous predominant serotype 4/91 in the field. Detailed analyses of synonymous to non-synonymous ratio of the S1 gene sequences of this new serotype Italy 02 suggested positive selection pressures might have contributed to the successful establishment of Italy 02 serotype in our country. In addition, differences on the fitness abilities of new emergent genotypes were indicated. Furthermore, intergenic sequences (IGs)-like motifs within S1 gene sequences of IBV isolates were suggested to enhance the recombination abilities of certain serotypes.
dos Santos, Silvia R; Passadore, Lilian F; Takagi, Elizabeth H; Fujii, Cristiane M; Yoshioka, Cristina R M; Gilio, Alfredo E; Martinez, Marina B
The ten-pneumococcal conjugate vaccine (PCV10) was introduced into the national immunization program for childhood vaccination schedules by the Brazilian Health Public Service in March 2010. The aim of this study was to compare Streptococcus pneumoniae serotype distribution, antibiotic resistance patterns, and potential coverage before (January 2006-June 2010) and after (July 2010-September 2012) PCV10 introduction. The incidence of invasive pneumococcal disease (IPD), patient demographics, and disease characteristics were recorded. This study was conducted at the University Hospital of Sao Paulo University in Brazil from January 2006 to September 2012. Serotyping was performed using multiplex PCR typing, and antimicrobial sensitivity by Clinical and Laboratory Standards Institute (CLSI). A total of 259 S. pneumoniae strains were isolated from patients with IPD. The ages of the patients ranged from 3 months to 95 years old. The strains were isolated from cerebrospinal fluid, pleural fluid, and blood. The incidence of IPD among patients at HU-USP changed after the introduction of PCV10. The overall incidence of IPD was 3.42 cases per 1000 admissions in the vaccine pre- implementation period and of 2.99 cases per 1000 admissions in the vaccine post-implementation period. The incidence of IPD among children<2 y.o. attended at HU-USP changed significantly after the introduction of PCV10, from 20.30 to 3.97 of incidence. The incidence of PCV10- serotypes decrease from 16.47 to 0.44 in the same age, before and after PC10 implementation, respectively. Moreover, it was possible to realize the sensitivity to penicillin among isolates increased significantly in the post-vaccine period. Data from this study suggest that PCV10 contributed to decrease with PID rate among children less than 2 y.o. The resistance rate among pneumococcal isolates also could be observed since serotypes with greater resistance to beta lactam antibiotics were not easily isolated after vaccination
Das, Biswajit; Mohapatra, Jajati K; Pande, Veena; Subramaniam, Saravanan; Sanyal, Aniket
Three decades-long (1977-2013) evolutionary trend of the capsid coding (P1) region of foot-and-mouth disease virus (FMDV) serotype A isolated in India was analysed. The exclusive presence of genotype 18 since 2001 and the dominance of the VP3(59)-deletion group of genotype 18 was evident in the recent years. Clade 18c was found to be currently the only active one among the three clades (18a, 18b and 18c) identified in the deletion group. The rate of evolution of the Indian isolates at the capsid region was found to be 4.96×10(-3)substitutions/site/year. The timescale analysis predicted the most recent common ancestor to have existed during 1962 for Indian FMDV serotype A and around 1998 for the deletion group. The evolutionary pattern of serotype A in India appears to be homogeneous as no spatial or temporal structure was observed. Bayesian skyline plots indicate a sharp decline in the effective number of infections after 2008, which might be a result of mass vaccination or inherent loss of virus fitness. Analyses of variability at 38 known antigenically critical positions in a countrywide longitudinal data set suggested that the substitutions neither followed any specific trend nor remained fixed for a long period since frequent reversions and convergence was noticed. A maximum of 6 different amino acid residues was seen in the gene pool at any antigenically critical site over the decades, suggesting a limited combination of residues being responsible for the observed antigenic variation. Evidence of positive selection at some of the antigenically critical residues and the structurally proximal positions suggest a possible role of pre-existing immunity in the host population in driving evolution. The VP1 C-terminus neither revealed variability nor positive selection, suggesting the possibility that this stretch does not contribute to the antigenic variation and adaptation under immune selection. Copyright © 2016 Elsevier B.V. All rights reserved.
Backx, A.; Heutink, C.G.; Rooij, van E.M.A.; Rijn, van P.A.
Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven
Mwiine, Frank Norbert; Ayebazibwe, Chrisostom; Alexandersen, Søren
Foot-and-mouth disease (FMD) outbreaks in cattle occur annually in Uganda. In this study the authors investigated antibodies against FMD virus (FMDV) in cattle in surrounding areas of Lake Mburo National Park in South-western Uganda. Two hundred and eleven serum samples from 23 cattle herds were...... examined for the presence of antibodies against FMDV non-structural proteins and structural proteins using Ceditest® FMDV-NS and Ceditest® FMDV type O (Cedi Diagnostics BV, Lelystad, The Netherlands). Furthermore, serotype-specific antibodies against the seven serotypes of FMDV were determined using in......-house serotype-specific Solid Phase Blocking ELISAs (SPBE). Of the sera tested, 42.7% (90/211) were positive in the ELISA for antibodies against non-structural proteins, while 75.4% (159/211) had antibodies against the structural proteins of FMDV serotype O. Titres of ≥ 1:160 of serotype-specific antibodies...
Michele de Souza Bastos
Full Text Available INTRODUCTION: Manaus, the capital city of the state of Amazon with nearly 2 million inhabitants, is located in the middle of the Amazon rain forest and has suffered dengue outbreaks since 1998. METHODS: In this study, blood samples were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR, aimed at identifying dengue virus serotypes. RESULTS: Acute phase sera from 432 patients were tested for the presence of dengue virus. Out of the 432 patients, 137 (31.3% were found to be positive. All the four dengue virus serotypes were observed. CONCLUSIONS: The simultaneous circulation of the four dengue serotypes is described for the first time in Manaus and in Brazil.
Hood, M A; Ness, G E; Rodrick, G E
Two strains of Vibrio cholerae serotype O1 Inaba were isolated from eastern oysters, Crassostrea virginica, collected from estuarine waters in Florida during April 1980. The oyster meats and waters from which the oysters were collected had low fecal coliform counts, and the area had no prior evidence of sewage contamination. PMID:7235700
Full Text Available Abstract Background Shigella flexneri is the major pathogen causing bacillary dysentery. Fifteen serotypes have been recognized up to now. The genesis of new S. flexneri serotypes is commonly mediated by serotype-converting bacteriophages. Untypeable or novel serotypes from natural infections had been reported worldwide but have not been generated in laboratory. Results A new S. flexneri serotype-serotype 1 d was generated when a S. flexneri serotype Y strain (native LPS was sequentially infected with 2 serotype-converting bacteriophages, SfX first and then SfI. The new serotype 1 d strain agglutinated with both serotype X-specific anti-7;8 grouping serum and serotype 1a-specific anti- I typing serum, and differed from subserotypes 1a, 1b and 1c. Twenty four S. flexneri clinical isolates of serotype X were all converted to serotype 1 d by infection with phage SfI. PCR and sequencing revealed that SfI and SfX were integrated in tandem into the proA-yaiC region of the host chromosome. Conclusions These findings suggest a new S. flexneri serotype could be created in nature. Such a conversion may be constrained by susceptibility of a strain to infection by a given serotype-converting bacteriophage. This finding has significant implications in the emergence of new S. flexneri serotypes in nature.
Pires Neto, R J; Lima, D M; de Paula, S O; Lima, C M; Rocco, I M; Fonseca, B A L
Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.
Pires Neto R.J.
Full Text Available Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.
Diallo, Mawlouth; Ba, Yamar; Sall, Amadou A; Diop, Ousmane M; Ndione, Jacques A; Mondo, Mireille; Girault, Lang; Mathiot, Christian
After 8 years of silence, dengue virus serotype 2 (DENV-2) reemerged in southeastern Senegal in 1999. Sixty-four DENV-2 strains were isolated in 1999 and 9 strains in 2000 from mosquitoes captured in the forest gallery and surrounding villages. Isolates were obtained from previously described vectors, Aedes furcifer, Ae. taylori, Ae. luteocephalus, and--for the first time in Senegal--from Ae. aegypti and Ae. vittatus. A retrospective analysis of sylvatic DENV-2 outbreaks in Senegal during the last 28 years of entomologic investigations shows that amplifications are periodic, with intervening, silent intervals of 5-8 years. No correlation was found between sylvatic DENV-2 emergence and rainfall amount. For sylvatic DENV-2 vectors, rainfall seems to particularly affect virus amplification that occurs at the end of the rainy season, from October to November. Data obtained from investigation of preimaginal (i.e., nonadult) mosquitoes suggest a secondary transmission cycle involving mosquitoes other than those identified previously as vectors.
Ma, Yuling; Lu, Chengping; Fan, Hongjie
A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes (SS1, SS7, SS9) and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high homology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The induced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.
Joensen, Katrine Grimstrup; Tetzschner, Anna M. M.; Iguchi, Atsushi
typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-based in silico serotyping of E. coli applicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing...... tool. SerotypeFinder was evaluated on 682 E. coli genomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen genes wzx, wzy, wzm, and wzt and the flagellin...
Yuan Zong Hui
Full Text Available Salmonella spp. can indirectly infect humans via transfer from animals and animal-derived food products, and thereby cause potentially fatal diseases. Therefore, gaining an understanding of Salmonella infection in farm animals is increasingly important. The aim of this study was to identify the distribution of serotypes in Salmonella samples isolated from chickens (n = 837, pigs (n = 930, and dairy cows (n = 418 in central China (Henan, Hubei, and Hunan provinces in 2010–2011, and investigate the susceptibility of strains to antimicrobial agents. Salmonella isolates were identified by PCR amplification of the invA gene, serotypes were determined by using a slide agglutination test for O and H antigens, and susceptibility to 24 antimicrobials was tested using the agar dilution method. In total, 248 Salmonella strains were identified: 105, 105, and 38 from chickens, dairy cows, and pigs, respectively. Additionally, 209 strains were identified in unhealthy pigs from the Huazhong Agricultural University veterinary hospital. Among these 457 strains, the dominant serotypes were Typhimurium in serogroup B, IIIb in serogroup C, and Enteritidis in serogroup D. In antimicrobial susceptibility tests, 41.14% of Salmonella spp. were susceptible to all antimicrobial agents, 48.14% were resistant to at least one, and 34.72% were resistant to more than three classes. Strains were highly resistant to sulfamethoxazole-trimethoprim (39.61%, nalidixic acid (39.17%, doxycycline (28.22%, and tetracycline (27.58%. Resistance to cephalosporins and fluoroquinolones ranged from 5.25% to 7.44% and 19.04% to 24.51%, respectively. Among penicillin-resistant and cephalosporin-resistant strains, 25 isolates produced extended-spectrum β-lactamases (ESBLs. The multidrug-resistant and ESBL-producing Salmonella strains identified in healthy animals here will present a challenge for veterinary medicine and farm animal husbandry, and could also pose a threat to public health
Nan Nwe Win
Full Text Available Determination of hepatitis C virus (HCV genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4 region and 5′-untranslated region (5′-UTR, respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5′-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.
Opperman, Pamela A.; Rotherham, Lia S.; Esterhuysen, Jan; Charleston, Bryan; Juleff, Nicholas; Capozzo, Alejandra V.; Theron, Jacques
ABSTRACT Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been
Tan, Kim-Kee; Zulkifle, Nurul-Izzani; Abd-Jamil, Juraina; Sulaiman, Syuhaida; Yaacob, Che Norainon; Azizan, Noor Syahida; Che Mat Seri, Nurul Asma Anati; Samsudin, Nur Izyan; Mahfodz, Nur Hidayana; AbuBakar, Sazaly
Dengue is hyperendemic in most of Southeast Asia. In this region, all four dengue virus serotypes are persistently present. Major dengue outbreak cycle occurs in a cyclical pattern involving the different dengue virus serotypes. In Malaysia, since the 1980s, the major outbreak cycles have involved dengue virus type 3 (DENV3), dengue virus type 1 (DENV1) and dengue virus type 2 (DENV2), occurring in that order (DENV3/DENV1/DENV2). Only limited information on the DENV3 cycles, however, have been described. In the current study, we examined the major outbreak cycle involving DENV3 using data from 1985 to 2016. We examined the genetic diversity of DENV3 isolates obtained during the period when DENV3 was the dominant serotype and during the inter-dominant transmission period. Results obtained suggest that the typical DENV3/DENV1/DENV2 cyclical outbreak cycle in Malaysia has recently been disrupted. The last recorded major outbreak cycle involving DENV3 occurred in 2002, and the expected major outbreak cycle involving DENV3 in 2006-2012 did not materialize. DENV genome analyses revealed that DENV3 genotype II (DENV3/II) was the predominant DENV3 genotype (67%-100%) recovered between 1987 and 2002. DENV3 genotype I (DENV3/I) emerged in 2002 followed by the introduction of DENV3 genotype III (DENV3/III) in 2008. These newly emerged DENV3 genotypes replaced DENV3/II, but there was no major upsurge of DENV3 cases that accompanied the emergence of these viruses. DENV3 remained in the background of DENV1 and DENV2 until now. Virus genome sequence analysis suggested that intrinsic differences within the different dengue virus genotypes could have influenced the transmission efficiency of DENV3. Further studies and continuous monitoring of the virus are needed for better understanding of the DENV transmission dynamics in hyperendemic regions. Copyright © 2017 Elsevier B.V. All rights reserved.
Bello Gonzalez, Teresita; Rivera-Olivero, Ismar Alejandra; Sisco, María Carolina; Spadola, Enza; Hermans, Peter W; de Waard, Jacobus H
Serotype surveillance of Streptococcus pneumoniae is indispensable for evaluating the potential impact of pneumococcal conjugate vaccines. Serotyping by the standard Quellung reaction is technically demanding, time consuming, and expensive. A simple and economical strategy is multiplex PCR-based serotyping. We evaluated the cost effectiveness of a modified serial multiplex PCR (mPCR), resolving 24 serotypes in four PCR reactions and optimally targeting the most prevalent invasive and colonizing pneumococcal serotypes found in Venezuela. A total of 223 pneumococcal isolates, 140 invasive and 83 carriage isolates, previously serotyped by the Quellung reaction and representing the 18 most common serotypes/groups identified in Venezuela, were serotyped with the adapted mPCR. The mPCR serotyped 76% of all the strains in the first two PCR reactions and 91% after four reactions, correctly identifying 17 serotypes/groups. An isolate could be serotyped with mPCR in less than 2 minutes versus 15 minutes for the Quellung reaction, considerably lowering labor costs. A restrictive weakness of mPCR was found for the detection of 19F strains. Most Venezuelan 19F strains were not typeable using the mPCR, and two 19F cps serotype variants were identified. The mPCR assay is an accurate, rapid, and economical method for the identification of the vast majority of the serotypes from Venezuela and can be used in place of the standard Quellung reaction. An exception is the identification of serotype 19F. In this setting, most 19F strains were not detectable with mPCR, demonstrating a need of serology-based quality control for PCR-based serotyping.
Ba, Yamar; Sall, Amadou A.; Diop, Ousmane M.; Ndione, Jacques A.; Mondo, Mireille; Girault, Lang; Mathiot, Christian
After 8 years of silence, dengue virus serotype 2 (DENV-2) reemerged in southeastern Senegal in 1999. Sixty-four DENV-2 strains were isolated in 1999 and 9 strains in 2000 from mosquitoes captured in the forest gallery and surrounding villages. Isolates were obtained from previously described vectors, Aedes furcifer, Ae. taylori, Ae. luteocephalus, and—for the first time in Senegal—from Ae. aegypti and Ae. vittatus. A retrospective analysis of sylvatic DENV-2 outbreaks in Senegal during the last 28 years of entomologic investigations shows that amplifications are periodic, with intervening, silent intervals of 5–8 years. No correlation was found between sylvatic DENV-2 emergence and rainfall amount. For sylvatic DENV-2 vectors, rainfall seems to particularly affect virus amplification that occurs at the end of the rainy season, from October to November. Data obtained from investigation of preimaginal (i.e., nonadult) mosquitoes suggest a secondary transmission cycle involving mosquitoes other than those identified previously as vectors. PMID:12643833
Full Text Available Objective: To determine the antiviral activity of ethanol extracts derived from Cassia grandis leaves and Tabernaemontana cymosa bark against two dengue virus (DENV serotype 2 strains DENV-2/NG and DENV-2/1 6681 in two cell lines susceptible to infection, VERO and U937. Methods: The cytotoxic concentration 50 (CC50 was assessed using the MTT method, and the effective concentration 50 (EC50 was determined using the technique of inhibiting the production of infectious viral particles by the plating method. Further testing of dose-response inhibition was performed, and three experimental approaches were evaluated (pre-, trans- and posttreatment to determine the effect of the extracts according to the time of administration. Finally, a preliminary phytochemical analysis for both extracts was performed. Results: The cytotoxicity of the extracts was low (CC50>300 µg/mL, and the U937 cell line was more sensitive to the antiproliferative effect of both extracts. When the virus strain-dependent selectivities of the extracts were compared, it was found that both extracts were more selective in cultures infected with the DENV-2/NG strain than in those infected with the DENV-2/16681 strain. A dose-dependent inhibitory effect of the extracts was not observed in any of the evaluations. Finally, the highest inhibition was detected with the post-treatment approach with the Tabernaemontana cymosa extract (99.9% in both cell lines. Conclusions: A therapy with compounds derived from these extracts would inhibit viral replication and affect steps after viral internalization.
Park, Saeyoung; Gerber, Sabina; Lee, Jean C.
Most Staphylococcus aureus isolates produce either a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide, and the CP antigens are targets for vaccine development. Since CP5 and CP8 have similar trisaccharide repeating units, it is important to identify an epitope shared by both CP5 and CP8. To characterize cross-reactivity between CP5 and CP8, the immunogenicity of CP5 and CP8 conjugate vaccines in mice and rabbits was evaluated by serological assays. Immune sera were also tested for function...
Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei
The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.
Full Text Available Abstract A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations.
Thi Hoa Ngo
Full Text Available Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST and pulse field gel electrophoresis (PFGE. Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542 of pigs carried S. suis of one or multiple serotypes. 8% (45/542 carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%. 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.
William B Messer
Full Text Available Dengue viruses (DENV are enveloped single-stranded positive-sense RNA viruses transmitted by Aedes spp. mosquitoes. There are four genetically distinct serotypes designated DENV-1 through DENV-4, each further subdivided into distinct genotypes. The dengue scientific community has long contended that infection with one serotype confers lifelong protection against subsequent infection with the same serotype, irrespective of virus genotype. However this hypothesis is under increased scrutiny and the role of DENV genotypic variation in protection from repeated infection is less certain. As dengue vaccine trials move increasingly into field-testing, there is an urgent need to develop tools to better define the role of genotypic variation in DENV infection and immunity. To better understand genotypic variation in DENV-3 neutralization and protection, we designed and constructed a panel of isogenic, recombinant DENV-3 infectious clones, each expressing an envelope glycoprotein from a different DENV-3 genotype; Philippines 1982 (genotype I, Thailand 1995 (genotype II, Sri Lanka 1989 and Cuba 2002 (genotype III and Puerto Rico 1977 (genotype IV. We used the panel to explore how natural envelope variation influences DENV-polyclonal serum interactions. When the recombinant viruses were tested in neutralization assays using immune sera from primary DENV infections, neutralization titers varied by as much as ∼19-fold, depending on the expressed envelope glycoprotein. The observed variability in neutralization titers suggests that relatively few residue changes in the E glycoprotein may have significant effects on DENV specific humoral immunity and influence antibody mediated protection or disease enhancement in the setting of both natural infection and vaccination. These genotypic differences are also likely to be important in temporal and spatial microevolution of DENV-3 in the background of heterotypic neutralization. The recombinant and synthetic tools
Duintjer Tebbens, Radboud J; Hampton, Lee M; Thompson, Kimberly M
The endgame for polio eradication involves coordinated global cessation of oral poliovirus vaccine (OPV) with cessation of serotype 2 OPV (OPV2 cessation) implemented in late April and early May 2016 and cessation of serotypes 1 and 3 OPV (OPV13 cessation) currently planned for after 2018. The logistics associated with globally switching all use of trivalent OPV (tOPV) to bivalent OPV (bOPV) represent a significant undertaking, which may cause some complications, including delays that lead to different timing of the switch across shared borders. Building on an integrated global model for long-term poliovirus risk management, we consider the expected vulnerability of different populations to transmission of OPV2-related polioviruses as a function of time following the switch. We explore the relationship between the net reproduction number (Rn) of OPV2 at the time of the switch and the time until OPV2-related viruses imported from countries still using OPV2 can establish transmission. We also analyze some specific situations modeled after populations at high potential risk of circulating serotype 2 vaccine-derived poliovirus (cVDPV2) outbreaks in the event of a non-synchronous switch. Well-implemented tOPV immunization activities prior to the tOPV to bOPV switch (i.e., tOPV intensification sufficient to prevent the creation of indigenous cVDPV2 outbreaks) lead to sufficient population immunity to transmission to cause die-out of any imported OPV2-related viruses for over 6 months after the switch in all populations in the global model. Higher Rn of OPV2 at the time of the switch reduces the time until imported OPV2-related viruses can establish transmission and increases the time during which indigenous OPV2-related viruses circulate. Modeling specific connected populations suggests a relatively low vulnerability to importations of OPV2-related viruses that could establish transmission in the context of a non-synchronous switch from tOPV to bOPV, unless the gap
Full Text Available Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad, serotype 2 adeno-associated virus vectors (rAAV2, or self-complementary (sc AAV2 vectors carrying green fluorescent protein (GFP. Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.
Angen, Øystein; Jessing, Stine Graakjær; Ahrens, Peter
. However, a number of isolates show cross-reaction between serotypes and this has urged the development of quick, serotype specific DNA-based methods necessary. Serotype specific tests have until now been described for the serotypes 2, 5, and 6 (Jessing et al, 2003) and serotypes 1, 2 and 8 (Schuchert et...
Spanjaard, L.; Bol, P.; de Marie, S.; Zanen, H. C.
Case histories of 692 patients with meningococcal disease due to serogroup B, C, or W (W-135) were reviewed to study the association of the serotypes 2a and 2b with the course of disease. The case-fatality rate in group B disease was significantly associated with serotype 2b (B:2b) strains (P =
Vaishali P. Waman
Full Text Available Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae. There are four serotypes of Dengue Virus (DENV-1 to DENV-4, each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis
Ramirez-Carvajal, Lisbeth; Pauszek, Steven J; Ahmed, Zaheer; Farooq, Umer; Naeem, Khalid; Shabman, Reed S; Stockwell, Timothy B; Rodriguez, Luis L
Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008-2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008-2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.
Full Text Available Foot-and-mouth disease (FMD is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008-2009. Furthermore, signature amino acid (aa substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008-2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.
Qiu, S; Xu, X; Yang, C; Wang, J; Liang, B; Li, P; Li, H; Yi, S; Liu, H; Cui, X; Wu, Z; Xie, J; Jia, L; Wang, L; Hao, R; Jin, H; Wang, Y; Sun, Y; Song, H
We identified 2912 Shigella isolates from diarrhoeal patients in China during 2003-2013. The most common species was Shigella flexneri (55.3%), followed by Shigella sonnei (44.1%); however, S. sonnei is becoming increasingly prevalent. Among the S. flexneri isolates, serotypes 2a and X variant (-:7,8, E1037) were the two most prevalent serotypes, and serologically atypical isolates were also commonly identified. Overall, S. sonnei, S. flexneri 2a and S. flexneri X variant (-:7,8, E1037) accounted for 76.1% of all Shigella isolates, and their prevalence increased from 54.0% during 2003-2004 to 84.1% during 2011-2013. A change was observed in the serotype distribution of Shigella in China during this period, and we propose an ideal strategy to inform the development of a broadly effective Shigella vaccine candidate. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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
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.
Johansson, T.; Einer-Jensen, K.; Batts, W.; Ahrens, P.; Bjorkblom, C.; Kurath, G.; Bjorklund, H.; Lorenzen, N.
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.
Kiely, R A
This study determined the carriage rate and serotype distribution of group B Streptococcus (GBS) in women of child-bearing age in the southern region of Ireland. A total of 2000 vaginal swabs collected in two periods in 2004 and 2006 were examined and revealed a GBS carriage rate of 16·1%. Serotyping of isolates showed that serotypes Ia, II, III, IV, and V were the most prevalent. A high prevalence of serotype IV was found, increasing from 7·6% to 15·2% between 2004 and 2006. Random amplified polymorphic DNA analysis demonstrated considerable genetic heterogeneity in the serotype IV isolates. This serotype should be considered for inclusion in potential vaccines for use in Ireland.
Zhang, Yan; Wang, Nan; Cao, Jiyue; Chen, Huanchun; Jin, Meilin; Zhou, Hongbo
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.
Full Text Available Avian paramyxovirus serotype 4 (APMV-4 is found sporadically in wild birds worldwide, and it is an economically important poultry pathogen. Despite the existence of several published strains, very little is known about the distribution, host species, and transmission of APMV-4 strains. To better understand the relationships among these factors, we conducted an APMV-4 surveillance of wild birds and domestic poultry in six provinces of China suspected of being intercontinental flyways and sites of interspecies transmission. APMV-4 surveillance was conducted in 9,160 wild birds representing seven species, and 1,461 domestic poultry in live bird markets (LMBs from December 2013 to June 2016. The rate of APMV-4 isolation was 0.10% (11/10,621, and viruses were isolated from swan geese, bean geese, cormorants, mallards, and chickens. Sequencing and phylogenetic analyses of the 11 isolated viruses indicated that all the isolates belonging to genotype I were epidemiologically connected with wild bird-origin viruses from the Ukraine and Italy. Moreover, chicken-origin APMV-4 strains isolated from the LBMs were highly similar to wild bird-origin viruses from nearby lakes with free-living wild birds. In additional, a hemagglutination-negative APMV-4 virus was identified. These findings, together with recent APMV-4 studies, suggest potential virus interspecies transmission between wild birds and domestic poultry, and reveal possible epidemiological intercontinental connections between APMV-4 transmission by wild birds.
Green, Kelsie J; Chikh-Ali, Mohamad; Hamasaki, Randall T; Melzer, Michael J; Karasev, Alexander V
Poha, or cape gooseberry (Physalis peruviana L.), is a plant species cultivated in Hawaii for fresh fruit production. In 2015, an outbreak of virus symptoms occurred on poha farms in the South Kohala District of the island of Hawaii. The plants displayed mosaic, stunting, and leaf deformation, and produced poor fruit. Initial testing found the problem associated with Potato virus Y (PVY) infection. Six individual PVY isolates, named Poha1 to Poha6, were collected from field-grown poha plants and subjected to biological and molecular characterization. All six isolates induced mosaic and vein clearing in tobacco, and three of them exhibited O-serotype while the other three reacted only with polyclonal antibodies and had no identifiable serotype. Until now, PVY isolates have been broadly divided into pepper or potato adapted; however, these six PVY isolates from poha were unable to establish systemic infection in pepper and in four tested potato cultivars. Whole-genome sequences for the six isolates were determined, and no evidence of recombination was found in any of them. Phylogenetic analysis placed poha PVY isolates in a distinct, monophyletic "Poha" clade within the PVY C lineage, suggesting that they represented a novel, biologically and evolutionarily unique group. The genetic diversity within this poha PVY C clade was unusually high, suggesting a long association of PVY C with this solanaceous host or a prolonged geographical separation of PVY C in poha in Hawaii.
Moury, B; Cardin, L; Onesto, J P; Candresse, T; Poupet, A
ABSTRACT A survey for viruses in rose propagated in Europe resulted in detection of only Prunus necrotic ringspot virus (PNRSV) among seven viruses screened. Four percent of cut-flower roses from different sources were infected with PNRSV. Progression of the disease under greenhouse conditions was very slow, which should make this virus easy to eradicate through sanitary selection. Comparison of the partial coat protein gene sequences for three representative rose isolates indicated that they do not form a distinct phylogenetic group and show close relations to Prunus spp. isolates. However, a comparison of the reactivity of monoclonal antibodies raised against these isolates showed that the most prevalent PNRSV serotype in rose was different from the most prevalent serotype in Prunus spp. All of the 27 rose isolates tested infected P. persica seedlings, whereas three of the four PNRSV isolates tested from Prunus spp. were poorly infectious in Rosa indica plants. These data suggest adaptation of PNRSV isolates from Prunus spp., but not from rose, to their host plants. The test methodologies developed here to evaluate PNRSV pathogenicity in Prunus spp. and rose could also help to screen for resistant genotypes.
Full Text Available Abstract Foot-and-mouth disease virus (FMDV is a highly infectious member of the Picornaviridae inducing an acute disease of cloven-hoofed species. Vaccine-induced immune protection correlates with the presence of high levels of neutralizing antibodies but also opsonising antibodies have been proposed as an important mechanism of the immune response contributing to virus clearance by macrophages and leading to the production of type-I interferon (IFN by plasmacytoid dendritic cells (pDC. The present study demonstrates that the opsonising antibody titres mediating enhanced IFN-α responses in pDC were similar to neutralizing titres, when antigenically related viruses from the same serotype were employed. However, sera cross-reacted also with non-neutralized isolates of multiple serotypes, when tested in this assay. Both uncomplexed virus and immune complexed virus stimulated pDC via Toll-like receptor 7. An additional finding of potential importance for strain-specific differences in virulence and/or immunogenicity was that pDC activation by FMDV strongly differed between viral isolates. Altogether, our results indicate that opsonising antibodies can have a broader reactivity than neutralizing antibodies and may contribute to antiviral responses induced against antigenically distant viruses.
McNulty, M S; Allan, G M; McCracken, R M; McParland, P J
An influenza virus was isolated from turkeys with an acute disease causing 30% mortality. The virus was subtyped as H5 N8. The nomenclature A/turkey/Ireland/83 (H5 N8) is proposed for this isolate. The virus had an ICPI of 1.80 to 1.85 for 1-day-old chicks and an IVPI of 2.74 for 6-week-old chickens. Following oronasal inoculation of juvenile and adult turkeys, chickens and ducks with the isolate, 100% mortality occurred in turkeys and chickens. No clinical signs were observed in inoculated ducks, but all developed serum antibody titres against the virus.
Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J
The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.
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.
Kumar, Ravi; Rajak, Kaushal Kishor; Chandra, Tribhuwan; Muthuchelvan, Dhanavelu; Saxena, Arpit; Chaudhary, Dheeraj; Kumar, Ajay; Pandey, Awadh Bihari
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.
Walter H B Demczuk
Full Text Available Since implementation of the 13-valent polyvalent conjugate vaccine (PCV13 in Canada during 2010, the proportion of PCV13 serotypes causing invasive pneumococcal disease (IPD has declined from 55% (n = 1492 in 2010 to 31% (n = 764 in 2014. A concurrent increase of non-PCV13 serotypes has occurred and 22F has become the most prevalent serotype in Canada increasing from 7% (n = 183 to 11% (n = 283. Core single nucleotide variant phylogenetic analysis was performed on 137 Streptococcus pneumoniae serotype 22F isolates collected across Canada from 2005-2015. Six phylogenetic lineages (n = 117 were identified among a serotype 22F/ST433 clonal complex (CC, including a recently expanding erythromycin-resistant clone. Erythromycin-resistance was observed in 25 isolates possessing ermB, mef or a 23S rRNA A2061G point mutation; 2 penicillin-resistant isolates had recombinant pbp1a, pbp2a and/or pbp2x; 3 tetracycline-resistant isolates contained tetM; and 1 isolate was multidrug-resistant. Virulence factor analysis indicated a high level of homogeneity among the 22F/ST433 clonal complex strains. A group of 6 phylogenetic outlier strains had differing MLST, antimicrobial resistance and molecular profiles suggestive of capsule switching events. While capsule switch events among S. pneumoniae serotype 22F has been observed, increasing prevalence of S. pneumoniae serotype 22F can be attributed to an evolving homogenous clone expanding nationally through local transmission events.
Franco F. Calderaro
Full Text Available Abstract: Streptococcus suis is one of most important pathogens in the swine industry worldwide. Despite its importance, studies of S. suis characterization in South America are still rare. This study evaluates S. suis isolates from distinct Brazilian states, from 1999 to 2004, and its molecular and serological characterization. A total of 174 isolates were studied. S. suis identification was confirmed by PCR and isolates were further serotyped and genotyped by SE-AFLP and amplification of virulence markers. Serotype 1, 2, 3, 4, 7, 18, 22 and 32 were identified among the studied isolates, and only 4% were characterized as non-typeable. The mrp+/epf+/sly+ genotype was the most frequent. The SE-AFLP analysis resulted in 29 patterns distributed in three main clusters with over 65% of genetic similarity. Isolates presented a slight tendency to cluster according to serotype and origin; however, no further correlation with virulence genotypes was observed.
Dayie, Nicholas T. K. D.; Arhin, Reuben E.; Newman, Mercy J.
Background: The objective of this study was to determine the prevalence of nasopharyngeal carriage, serotype distribution, and penicillin resistance of Streptococcus pneumoniae in children 2 mu g/ml and were classified as fully penicillin resistant with 45% of the isolates having intermediate...... serotypes detected. The two penicillin resistant isolates (MIC 32 mu g/ml) were serotypes included in both PCV-13 and PPV-23. A nationwide monitoring system of penicillin susceptibility patterns and pneumococcal serotypes is recommended....
Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita
In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... probes. The limits of detection ware found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid...
Zhang, Rong; Wang, Xuan; Lü, Jianxin
To study the virulence and mechanism of carbapenem resistance of a clinical isolate of carbapenem-resistant K1 serotype Klebsiella pneumonia strain. Identification of isolate was carried out with VITEK-2 compact system. Antimicrobial susceptibility was determined by E-test; Metallo β-lactamases and carbapenemases screening were conducted by imipenem-EDTA double disc synergy test and modified Hodge test, respectively.Specific polymerehse chain reaction (PCR) and DNA sequencing were preformed to detect the virulence genes including K1, K2, K5, K20, K54, K57, magA, rmpA, wcaG and a series of β-lactamase resistence genes. Conjunction experiment was also performed. The plasmids of transconjugants were submitted to PCR-based replicon typing (PBRT) method. Molecular typing was performed by multilocus sequence typing (MLST). Antimicrobial susceptibility testing revealed that the Klebsiella pneumonia strain was resistant to most of the antibiotics used in clinic. Phynotype confirmary rest revealed the production of carbapanemases, while Metallo β-lactamases were negative; PCR and DNA sequencing confirmed the isolate was positive for blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-1 and virulence genes K1, magA, rmpA, wcaG simultaneously; blaKPC-2 was transferred from donor to Escherichia EC600 by conjunction experiment, while no virulence genes were found in the transconjugants. PBRT revealed that Frep plasmid was found in transconjugants. MLST analysis revealed that this strain belonged to ST23. K1 serotype Klebsiella pneumonia strain carries virulence genes and carbapenem resistance gene blaKPC-2, noteworthily the carbapenem resistance genes can be transferred through horizontal transmission on plasmids.
Irfan Ahmad Mir
Conclusions: Occurrence of high proportion of serovars in our study which can cause serious gastroenteritis in humans is a matter of concern. Salmonella Altona has been detected for the first time in India from poultry. This serotype is known to cause serious outbreaks of gastroenteritis in humans. Multidrug resistant isolates were recovered at high percentage which can be attributed to non-judicious use of antibiotics both in prophylaxis and treatment regimen. This observation draws serious attention as poultry serves as an important source of transmission of these multidrug resistant Salmonella serovars to humans.
Kawamura, Ryusuke; Shimura, Hanako; Mochizuki, Tomofumi; Ohki, Satoshi T; Masuta, Chikara
Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.
Christopher J. Kasanga
Full Text Available This study was conducted to investigate the presence of foot-and-mouth disease virus (FMDV in different geographic locations of Tanzania. Epithelial tissues and fluids (n = 364 were collected from cattle exhibiting oral and foot vesicular lesions suggestive of FMD and submitted for routine FMD diagnosis. The analysis of these samples collected during the period of 2002 and 2010 was performed by serotype-specific antigen capture ELISA to determine the presence of FMDV. The results of this study indicated that 167 out of 364 (46.1% of the samples contained FMDV antigen. Of the 167 positive samples, 37 (28.4% were type O, 7 (4.1% type A, 45 (21.9% SAT 1 and 79 (45.6% SAT 2. Two FMDV serotypes (O and SAT 2 were widely distributed throughout Tanzania whilst SAT 1 and A types were only found in the Eastern zone. Our findings suggest that serotypes A, O, SAT 1 and SAT 2 prevail in Tanzania and are associated with the recent FMD outbreaks. The lack of comprehensive animal movement records and inconsistent vaccination programmes make it difficult to determine the exact source of FMD outbreaks or to trace the transmission of the disease over time. Therefore, further collection and analysis of samples from domestic and wild animals are being undertaken to investigate the genetic and antigenic characteristics of the circulating strains, so that a rational method to control FMD in Tanzania and the neighbouring countries can be recommended.
Dolz, Roser; Vergara-Alert, Júlia; Pérez, Mónica; Pujols, Joan; Majó, Natàlia
Infectious bronchitis (IB) is a worldwide disease affecting chickens of all ages and causing important economic losses in poultry industry. Despite being one of the predominant IB virus (IBV) serotype in several European countries, slightly is known about pathogenesis and pathogenicity of Italy 02 serotype. In this study chicks and old hens were infected by oculo-nasal route with Italy 02 serotype. Clinical signs, gross and microscopic findings were evaluated, viral nucleic acid detection was assessed by in situ hybridization (ISH) in several tissues and viral RNA was detected by RT-PCR in trachea, kidney and nasal and cloacal swabs. Italy 02 serotype was demonstrated to cause severe respiratory and renal damage in one-day old chicks but not in adult hens in which only respiratory disease and drop in egg production was observed. The use of ISH technique demonstrated the presence of viral RNA in nasal turbinates prior to trachea, but more consistent and longer replication periods in enterocytes of lower gastrointestinal tract. The detection of viral nucleic acid in gut by RT-PCR was consistent and more persistent viral shedding was detected in faeces than in nasal exudates. We describe a complete update of IBV distribution in tissues by the use of molecular techniques and we also provide and in-depth pathological characterization of the new Italy 02 IBV serotype. Furthermore, new data about IBV pathogenesis essential in field control is afforded. Copyright Â© 2011 Elsevier B.V. All rights reserved.
Sucipto, Teguh Hari; Kotaki, Tomohiro; Mulyatno, Kris Cahyo; Churrotin, Siti; Labiqah, Amaliah; Soegijanto, Soegeng; Kameoka, Masanori
Dengue virus (DENV) infection is a major health issue in tropical and subtropical areas. Indonesia is one of the biggest dengue endemic countries in the world. In the present study, the phylogenetic analysis of DENV in Bangkalan, Madura Island, Indonesia, was performed in order to obtain a clearer understanding of its dynamics in this country. A total of 359 blood samples from dengue-suspected patients were collected between 2012 and 2014. Serotyping was conducted using a multiplex Reverse Transcriptase-Polymerase Chain Reaction and a phylogenetic analysis of E gene sequences was performed using the Bayesian Markov chain Monte Carlo (MCMC) method. 17 out of 359 blood samples (4.7%) were positive for the isolation of DENV. Serotyping and the phylogenetic analysis revealed the predominance of DENV-1 genotype I (9/17, 52.9%), followed by DENV-2 Cosmopolitan type (7/17, 41.2%) and DENV-3 genotype I (1/17, 5.9%) . DENV-4 was not isolated. The Madura Island isolates showed high nucleotide similarity to other Indonesian isolates, indicating frequent virus circulation in Indonesia. The results of the present study highlight the importance of continuous viral surveillance in dengue endemic areas in order to obtain a clearer understanding of the dynamics of DENV in Indonesia.
Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao
A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.
Nakai, Hiroyuki; Fuess, Sally; Storm, Theresa A.; Muramatsu, Shin-ichi; Nara, Yuko; Kay, Mark A.
Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 1012 vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression. PMID:15596817
Velthuis, A.G.J.; Mourits, M.C.M.; Saatkamp, H.W.; Koeijer, de A.; Elbers, A.R.W.
Background: Bluetongue (BT) is a vector-borne disease of ruminants caused by bluetongue virus that is transmitted by biting midges (Culicoides spp.). In 2006, the introduction of BTV serotype 8 (BTV-8) caused a severe epidemic in Western and Central Europe. The principal effective veterinary measure
Chotiwan, Nunya; Roehrig, John T.; Schlesinger, Jacob J.; Blair, Carol D.; Huang, Claire Y.-H.
Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection
Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: firstname.lastname@example.org [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)
Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.
Jamal, Syed Muhammad; Ferrari, Giancarlo; Ahmed, Safia
Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan; serotypes O, A and Asia-1 of the virus are responsible for the outbreaks in these countries with FMDV type O usually being the most common. In the present study, the nucleotide sequences encoding the FMDV capsid protein VP1 from...... in the region were found to be 6.65×10−3 (95% CI=5.49–7.80×10−3) and 7.80×10−3 (95% CI=6.72–8.89×10−3) substitutions per nucleotide per year, respectively. The present study reveals the presence of multiple (sub-)lineages of FMDV serotype O co-circulating in the region and that significant new variants...
Chiávetta, L; Chávez, E; Ruzic, A; Mollerach, M; Regueira, M
Neisseria meningitidis is an important cause of meningitis, bacteremia and septic shock syndrome. We herein present the distribution of serogroups, serotypes and serosubtypes of 2244 isolates of N. meningitidis from patients with meningitis or meningococcemia, received within the period 1993-2005, in the National Reference Laboratory, INEI-ANLIS "Dr. Carlos G. Malbrán", from 33 Argentine hospitals that are included in a National Network devoted to for the study of bacterial meningitis. Between 1993-1995, serogroup B was prevalent (66%) whereas in the period from 1995-2001, serogroup C prevailed (65%). However, following but after that period, the prevalence of serogroup B was recovered. In the last 5 years of the studied period, the serogroups Y and W135 represented as a whole a 15.6% as a whole whereas up to the year 2000 during the first 6 years they accounted for it was of 4.7%. Higher diversity in the distribution of serotypes and serosubtypes was observed within serogroup B. The nonsubtypable isolates throughout the period of study represented the 52.8%, this high percentage demonstrates the limited capacity of the serotyping for the determination of meningococcal/meningococcus subtypes. of meningococco.
Choudhary, Manish Chandra; Gupta, Ekta; Sharma, Shvetank; Hasnain, Nadeem; Agarwala, Pragya
In 2015, New Delhi witnessed a massive outbreak of Dengue virus (DENV) resulting in high morbidity and mortality. We report the molecular characterisation of the dominant circulating DENV strain to understand its evolution and dispersal. DENV infections were diagnosed by detection of IgM/NS1 antigen, and serotyping was performed by C-PrM PCR. Envelope gene was amplified, and variation(s) in envelope gene were analysed. Phylogenetic tree construction, time-based phylogeny and origin of DENV were analysed. Site-specific selection pressure of envelope gene variants was analysed. Confirmed DENV infection was observed in 11.34% (32 of 282) cases, while PCR positivity for C-PrM region was observed in 54.16% (13 of 24) of NS1 antigen-positive cases. All samples belonged to serotype 2 and cosmopolitan genotype. Phylogenetic analysis using envelope gene revealed segregation of cosmopolitan genotype strains into specific lineages. The Indian strains clustered separately forming a distinct monophyletic lineage (lineage III) with a signature amino acid substitution viz., I162V and R288K. Selection pressure analysis revealed that 215D, 288R and 304K were positively selected sites. The rate of nucleotide substitution was 6.93 × 10 -4 substitutions site-1 year-1 with time to most common ancestor was around 10 years with JX475906 (Hyderabad strain) and JN030345 (Singapore strain) as its most probable ancestor. We observed evolution of a distinct lineage of DENV-2 strains on the Indian subcontinent with possible changes in endemic circulating dengue strains that might give rise to more pathogenic strains. © 2017 John Wiley & Sons Ltd.
Desingu, Perumal Arumugam; Singh, Shambhu Dayal; Dhama, Kuldeep; Vinodhkumar, Obli Rajendran; Barathidasan, Rajamani; Malik, Yashpal Singh; Singh, Rajendra; Singh, Raj Kumar
Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.
Kobayashi, Miho; Takayama, Ikuyo; Kageyama, Tsutomu; Tsukagoshi, Hiroyuki; Saitoh, Mika; Ishioka, Taisei; Yokota, Yoko; Kimura, Hirokazu; Tashiro, Masato; Kozawa, Kunihisa
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.
Jamal, Syed Muhammad; Ferrari, Giancarlo; Ahmed, Safia
Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. Three different serotypes of the virus, namely O, A and Asia-1, are responsible for the outbreaks of this disease in these countries. In the present study, the nucleotide-coding sequences for the VP1 capsid protein (69 samples) ...
Drolet, Barbara S.; Reister-Hendricks, Lindsey M.; Podell, Brendan K.; Breitenbach, Jonathan E.; Mcvey, D.S.; Rijn, van Piet A.; Bowen, Richard A.
Bluetongue virus (BTV) is an orbivirus transmitted by biting midges (Culicoides spp.) that can result in moderate to high morbidity and mortality primarily in sheep and white-tailed deer. Although only 5 serotypes of BTV are considered endemic to the United States, as many as 11 incursive
Full Text Available Abstract Bluetongue virus serotype 8 (BTV-8, which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.
Jamal, Syed M.; Belsham, Graham
Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV....... Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection...... and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnosticassays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of OPan...
Lecouturier, Valerie; Berry, Catherine; Saulnier, Aure; Naville, Sophie; Manin, Catherine; Girerd-Chambaz, Yves; Crowe, James E; Jackson, Nicholas; Guy, Bruno
The recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1-4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1-4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these
World-wide there are at least 24 serotypes of bluetongue virus (BTV), a complex non-enveloped virus in the family Reoviridae, genus Orbivirus. Bluetongue (BT) is an arthropod-borne disease of cattle, sheep, goats, and deer and is transmitted by Culicoides biting midges. In 2006, bluetongue serotype ...
Full Text Available BACKGROUND: Dengue virus (DENV is transmitted by Aedes aegypti, a species that lives in close association with human dwellings. The behavior of DENV-infected mosquitoes needs further investigation, especially regarding the potential influence of DENV on mosquito biting motivation and avidity. METHODOLOGY/PRINCIPAL FINDINGS: We orally challenged 4-5 day-old Ae. aegypti females with a low passage DENV serotype -2 (DENV-2 to test whether the virus influences motivation to feed (the likelihood that a mosquito obtains a blood-meal and the size of its blood meal and avidity (the likelihood to re-feed after an interrupted first blood-meal. To assay motivation, we offered mosquitoes an anesthetized mouse for 2, 3, 4 or 5 minutes 7 or 14 days after the initial blood meals and measured the time they started feeding. 60.5% of the unexposed mosquitoes fed on the mouse, but only 40.5% of the positive ones did. Exposed but negative mosquitoes behaved similarly to unexposed ones (55.0% feeding. Thus DENV-2 infection decreased the mosquitoes' motivation to feed. To assay avidity, we offered the same mosquitoes a mouse two hours after the first round of feeding, and we measured the time at which they started probing. The exposed (positive or negative mosquitoes were more likely to re-feed than the unexposed ones and, in particular, the size of the previous blood-meal that kept mosquitoes from re-feeding was larger in the exposed than in the unexposed mosquitoes. Thus, DENV-2 infection increased mosquito avidity. CONCLUSIONS/SIGNIFICANCE: DENV-2 significantly decreased the mosquitoes' motivation to feed, but increased their avidity (even after taking account the amount of blood previously imbibed. As these are important components of transmission, we expect that the changes of the blood-feeding behaviour impact the vectorial capacity Ae. aegypti for dengue.
Maciel-de-Freitas, Rafael; Sylvestre, Gabriel; Gandini, Mariana; Koella, Jacob C
Dengue virus (DENV) is transmitted by Aedes aegypti, a species that lives in close association with human dwellings. The behavior of DENV-infected mosquitoes needs further investigation, especially regarding the potential influence of DENV on mosquito biting motivation and avidity. We orally challenged 4-5 day-old Ae. aegypti females with a low passage DENV serotype -2 (DENV-2) to test whether the virus influences motivation to feed (the likelihood that a mosquito obtains a blood-meal and the size of its blood meal) and avidity (the likelihood to re-feed after an interrupted first blood-meal). To assay motivation, we offered mosquitoes an anesthetized mouse for 2, 3, 4 or 5 minutes 7 or 14 days after the initial blood meals and measured the time they started feeding. 60.5% of the unexposed mosquitoes fed on the mouse, but only 40.5% of the positive ones did. Exposed but negative mosquitoes behaved similarly to unexposed ones (55.0% feeding). Thus DENV-2 infection decreased the mosquitoes' motivation to feed. To assay avidity, we offered the same mosquitoes a mouse two hours after the first round of feeding, and we measured the time at which they started probing. The exposed (positive or negative) mosquitoes were more likely to re-feed than the unexposed ones and, in particular, the size of the previous blood-meal that kept mosquitoes from re-feeding was larger in the exposed than in the unexposed mosquitoes. Thus, DENV-2 infection increased mosquito avidity. DENV-2 significantly decreased the mosquitoes' motivation to feed, but increased their avidity (even after taking account the amount of blood previously imbibed). As these are important components of transmission, we expect that the changes of the blood-feeding behaviour impact the vectorial capacity Ae. aegypti for dengue.
Yang, Ming; Caterer, Nigel R; Xu, Wanhong; Goolia, Melissa
Foot-and-mouth disease (FMD) is one of the world's most highly contagious animal diseases with tremendous economic consequences. A rapid and specific test for FMD diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. This project developed a multiplex lateral flow immunochromatographic strip test (multiplex-LFI) for the rapid detection and serotyping of FMD viruses. The monoclonal antibodies (mAbs) against serotypes O, A, and Asia 1 were used as capture mAbs. The mAbs were conjugated with fluorescein, rhodamine or biotin for serotype O, A and Asia 1, respectively. The detection mAbs which consisted of a serotype-independent mAb in combination with one serotype A-specific mAb and one Asia 1-specific mAb, were each colloidal gold-conjugated. The strips used in this study contained one control line and three test lines, which corresponded to one of the three serotypes, O, A or Asia 1. The newly developed multiplex-LFI strip test specifically identified serotype O (n=46), A (n=45) and Asia 1 (n=17) in all tested field isolates. The sensitivity of this strip test was comparable to the double antibody sandwich ELISA for serotypes O and A, but lower than the ELISA for serotype Asia 1. The multiplex-LFI strip test identified all tissue suspensions from animals that were experimentally inoculated with serotypes O, A or Asia 1. FMD viruses were detected in 38% and 50% of the swab samples from the lesion areas of experimentally inoculated sheep for serotypes O and A, respectively. The capability of the multiplex-LFI strip tests to produce rapid results with high specificity for FMD viruses of multiple serotypes makes this test a valuable tool to detect FMD viruses at outbreak sites. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.
Kasanga, C J; Wadsworth, J; Mpelumbe-Ngeleja, C A R; Sallu, R; Kivaria, F; Wambura, P N; Yongolo, M G S; Rweyemamu, M M; Knowles, N J; King, D P
This paper describes the molecular characterization of foot-and-mouth disease viruses (FMDV) recovered from outbreaks in Tanzania that occurred between 1967 and 2009. A total of 44 FMDV isolates, containing representatives of serotypes O, A, SAT 1 and SAT 2 from 13 regions of Tanzania, were selected from the FAO World Reference Laboratory for FMD (WRLFMD) virus collection. VP1 nucleotide sequences were determined for RT-PCR amplicons, and phylogenetic reconstructions were determined by maximum likelihood and neighbour-joining methods. These analyses showed that Tanzanian type O viruses fell into the EAST AFRICA 2 (EA-2) topotype, type A viruses fell into the AFRICA topotype (genotype I), type SAT 1 viruses into topotype I and type SAT 2 viruses into topotype IV. Taken together, these findings reveal that serotypes O, A, SAT 1 and SAT 2 that caused FMD outbreaks in Tanzania were genetically related to lineages and topotypes occurring in the East African region. The close genetic relationship of viruses in Tanzania to those from other countries suggests that animal movements can contribute to virus dispersal in sub-Saharan Africa. This is the first molecular description of viruses circulating in Tanzania and highlights the need for further sampling of representative viruses from the region so as to elucidate the complex epidemiology of FMD in Tanzania and sub-Saharan Africa. © 2014 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.
Nishikawa, F; Sugiyama, T
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.
Flores-Ocelotl, María R; Rosas-Murrieta, Nora H; Moreno, Diego A; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Domínguez, Fabiola; Santos-López, Gerardo
Urtica dioica, Taraxacum officinale, Calea integrifolia and Caesalpinia pulcherrima are widely used all over the world for treatment of different illnesses. In Mexico, these plants are traditionally used to alleviate or counteract rheumatism and inflammatory muscle diseases. In the present study we evaluated the activity of aqueous and methanolic extracts of these four plants, on the replication of dengue virus serotype 2 (DENV2). Extraction process was carried out in a Soxtherm® system at 60, 85 and 120 °C; a chemical fractionation in silica gel chromatography was performed and compounds present in the active fractions were identified by HPLC-DAD-ESI/MSn. The cytotoxic concentration and the inhibitory effect of extracts or fractions on the DENV2 replication were analyzed in the BHK-21 cell line (plaque forming assay). The half maximal inhibitory concentration (IC 50 ) and the selectivity index (SI) were calculated for the extracts and fractions. The methanolic extracts at 60 °C of T. officinale and U. dioica showed the higher inhibitory effects on DENV2 replication. After the chemical fractionation, the higher activity fraction was found for U. dioica and T. officinale, presenting IC 50 values of 165.7 ± 3.85 and 126.1 ± 2.80 μg/ml, respectively; SI values were 5.59 and 6.01 for each fraction. The compounds present in T. officinale, were luteolin and caffeoylquinic acids derivatives and quercertin diclycosides. The compounds in the active fraction of U. dioica, were, chlorogenic acid, quercertin derivatives and flavonol glycosides (quercetin and kaempferol). Two fractions from U. dioica and T. officinale methanolic extracts with anti-dengue activity were found. The compounds present in both fractions were identified, several recognized molecules have demonstrated activity against other viral species. Subsequent biological analysis of the molecules, alone or in combination, contained in the extracts will be carried out to develop therapeutics
Kimberly A. Dowd
Full Text Available Recent epidemics of Zika virus (ZIKV have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.
Setchanova, Lena Petrova; Alexandrova, Alexandra; Dacheva, Daniela; Mitov, Ivan; Kaneva, Radka; Mitev, Vanio
A pneumococcal conjugate vaccine (PCV10) was introduced in Bulgarian national immunization program since April 2010. Clonal composition based on pulsed-field gel electrophoresis and multilocus sequence typing genotyping of 52 serotype 19A Streptococcus pneumoniae isolates was analyzed. These were invasive and respiratory isolates collected between 1992 and 2013 from both children (78.8% clone. The most frequent sequence type (ST) was ST230 (48.1%) and together with four other closely related STs (15.4%), belonging to ST1611, ST276, ST7466, and ST2013, which were single- and double-locus variants; they were included in the main CC230. The disappearance of highly drug-resistant ST663 clone and emergence of new clones as CC320 and CC199 was also observed among the rest 19A isolates. A comparison of clonal composition between invasive and noninvasive isolates did not show a great genetic diversity among both kinds of isolates. Continuous surveillance of serotype 19A population following the introduction of PCV10 is essential to evaluate the impact of the vaccine on the epidemiology of this serotype.
Wang, S; Liu, X; Xu, X; Zhao, Y; Yang, D; Han, X; Tian, M; Ding, C; Peng, D; Yu, S
Pathogens utilize type III secretion systems to deliver effector proteins, which facilitate bacterial infections. The Escherichia coli type III secretion system 2 (ETT2) which plays a crucial role in bacterial virulence, is present in the majority of E. coli strains, although ETT2 has undergone widespread mutational attrition. We investigated the distribution and characteristics of ETT2 in avian pathogenic E. coli (APEC) isolates and identified five different ETT2 isoforms, including intact ETT2, in 57·6% (141/245) of the isolates. The ETT2 locus was present in the predominant APEC serotypes O78, O2 and O1. All of the ETT2 loci in the serotype O78 isolates were degenerate, whereas an intact ETT2 locus was mostly present in O1 and O2 serotype strains, which belong to phylogenetic groups B2 and D, respectively. Interestingly, a putative second type III secretion-associated locus (eip locus) was present only in the isolates with an intact ETT2. Moreover, ETT2 was more widely distributed in APEC isolates and exhibited more isoforms compared to ETT2 in human extraintestinal pathogenic E. coli, suggesting that APEC might be a potential risk to human health. However, there was no distinct correlation between ETT2 and other virulence factors in APEC.
Wheeler, Richard W.; Davies, Robert L.; Dalsgaard, Inger
the restriction enzyme NotI. Serotype O1 isolates responsible for ERM in rainbow trout in both the US and Europe, and including biotype 2 isolates, represented a distinct subgroup of similar pulsotypes. Biotype 2 isolates, responsible for outbreaks of the disease in rainbow trout in the UK, Denmark and Spain, had....... In contrast, US biotype 2 isolate YRNC10 had an identical pulsotype and OMP profile to UK biotype 2 isolates, suggesting that there had been exchange of these isolates between the UK and the US in the past. UK Atlantic salmon isolates were genetically and serologically diverse, with 12 distinct pulsotypes...... their likely origins and relationships, a geographically and temporally diverse collection of isolates were characterised by serotyping, biotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) profiling. A total of 44 pulsotypes were identified from 160 isolates by PFGE, using...
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.
Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio
Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.
Turki, Yousra; Mehr, Ines; Ouzari, Hadda; Khessairi, Amel; Hassen, Abdennaceur
Salmonella enterica isolates representing commonly isolated serotypes in Tunisia were analyzed using genotyping and phenotyping methods. ERIC and ITS-PCR applied to 48 Salmonella spp. isolates revealed the presence of 12 and 10 different profiles, respectively. The distribution of profiles among serotypes demonstrated the presence of strains showing an identical fingerprinting pattern. All Salmonella strains used in this study were positive for the sdiA gene. Three Salmonella isolates belonging to serotypes Anatum, Enteritidis and Amsterdam were negative for the invA gene. The spvC gene was detected in thirteen isolates belonging to serotypes Anatum, Typhimurium, Enteritidis, Gallinarum and Montevideo. Antibiotic resistance was frequent among the recovered Salmonella isolates belonging to serotypes Anatum, Typhimurium, Enteritidis, Zanzibar and Derby. The majority of these isolates exhibited resistance to at least two antibiotic families. Four multidrug-resistant isolates were recovered from food animals and poultry products. These isolates exhibited not only resistance to tetracycline, sulphonamides, and ampicillin, but also have shown resistance to fluoroquinolones. Common resistance to nalidixic acid, ciprofloxacin and ofloxacin in two S. Anatum and S. Zanzibar strains isolated from raw meat and poultry was also obtained. Furthermore, wastewater and human isolates exhibited frequent resistance to nalidixic acid and tetracycline. Of all isolates, 33.5% were able to form biofilm.
Ortrud Monika Barth
Full Text Available One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2. Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.
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.
Circulación de un linaje diferente del virus dengue 2 genotipo América / Asia en la región amazónica de Perú, 2010 Circulation of a different lineage of dengue virus serotype 2 American / Asian genotype in the Peruvian amazon, 2010
Full Text Available El objetivo del estudio fue determinar el genotipo del virus dengue tipo 2 (DENV-2 que circuló en la región Amazónica de Perú entre noviembre de 2010 y enero de 2011. Se analizaron ocho muestras de pacientes captados durante la vigilancia para dengue en las ciudades de Iquitos, Yurimaguas, Trujillo, Tarapoto y Lima entre noviembre de 2010 y enero de 2011 que fueron remitidas al Instituto Nacional de Salud. Se realizó el aislamiento viral en la línea C6/36 HT y la extracción del ARN viral. Se aplicaron técnicas de biología molecular para establecer el serotipo (RT - PCR múltiple y genotipo (RT-Nested PCR de la región E/NS1 seguidas de secuenciación y análisis filogenético. El análisis filogenético reveló la introducción de un linaje diferente que ingresó a Perú a finales del 2010. Estos aislamientos encontrados en Iquitos y otras ciudades de Perú están muy relacionados con aislamientos de DENV-2 que circularon en Brasil durante el 2007 y 2008 asociados con casos de dengue grave y muertes. En conclusión se detectó la introducción de un linaje diferente del DENV-2 genotipo América/Asia en Perú que podría estar asociado con la presencia de casos más graves de dengue.Our objective was to determine the genotype of the dengue virus type 2 (DENV-2 that circulated in the Amazon region of Peru between November 2010 and January 2011. We analyzed eight samples collected during dengue surveillance activities in the cities of Iquitos, Yurimaguas, Trujillo, Tarapoto and Lima between November 2010 and January 2011 that were sent to Insitituto Nacional de Salud. The viruses were isolated in C6/36 HT cell line. Viral RNA was extracted and the serotype (RT - PCR multiplex and genotype (RT-Nested PCR of the region E/NS1 were determined. Finally, the E/ NS1 amplicons were sequenced and analyzed by phylogeny. The phylogenetic analysis revealed the introduction of a different lineage which entered in Peru by the end of 2010. These isolates
Cong, Wei; Jin, Hong; Jiang, Chengda; Yan, Weiyao; Liu, Mingqiu; Chen, Jiulian; Zuo, Xiaoping; Zheng, Zhaoxin
In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 109 CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID50 of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 109 CFU of p3D-NT56/S. cho were protected in 9 days. PMID:20167192
Gelbíčová, T; Pantůček, R; Karpíšková, R
The aim of this study was to assess the potential risk posed to the human population by the presence of Listeria monocytogenes serotype 1/2c in food based on the characterization of virulence factors of Listeria involved in the invasion of host cells and sensitivity to antimicrobial agents. In addition to sequencing of the inlA and inlB genes, the presence of genes lapB, aut, fbpA, ami, vip and llsX was tested. A premature stop codon (PMSC) in the inlA gene was detected in all tested strains of serotype 1/2c and, concurrently, two novel PMSC mutation types were identified. However, neither PMSC in the inlB gene nor deletion of the lapB, aut, fbpA, ami and vip genes were found in any of the strains. The presence of the llsX gene was not confirmed. Even though all L. monocytogenes strains showed sensitivity to the tested antimicrobials on the basis of their phenotype, sequencing revealed the presence of IS1542 insertion in the inlA gene, indicating the possibility of sharing of mobile genetic elements associated with antimicrobial resistance among strains. Other than the presence of PMSCs in the inlA gene, no PMSC in inlB or deletion of other factors linked to the invasiveness of listeria were detected. Tested strains showed sensitivity to antibiotics used in the therapy of listeriosis. Strains of L. monocytogenes serotype 1/2c typically carry a PMSC in the inlA gene, but these strains still represent a potential threat to public health. The possibility of transfer of IS1542, associated with resistance to vancomycin, between enterococci and Listeria spp. was revealed. © 2016 The Society for Applied Microbiology.
Full Text Available In the present study, the relationships between serotype, pathogenic profile and in vitro biofilm formation of 106 Listeria monocytogenes strains, having no epidemiological correlation and isolated from different environmental and food sources, were analyzed. The quantitative assessment of the in vitro biofilm formation was carried out by using a microtiter plate assay with spectrophotometric reading (OD620. The isolates were also submitted to serogrouping using the target genes lmo0737, lmo1118, ORF2819, ORF2110, prs, and to the evaluation of the presence of the following virulence genes: prfA, hlyA, rrn, inlA, inlB, iap, plcA, plcB, actA and mpl, by multiplex PCRs. The 62% of the strains showed weak or moderate in vitro ability in biofilm formation, in particular serotypes 1/2b and 4b, frequently associated with sporadic or epidemic listeriosis cases. The 25% of these isolates showed polymorphism for the actA gene, producing a fragment of 268-bp instead of the expected 385-bp. The deletion of nucleotides in this gene seems to be related to enhanced virulence properties among these strains. Strains belonging to serotypes associated with human infections and characterized by pathogenic potential are capable to persist within the processing plants forming biofilm.
Renström Lena HM; Isaksson Mats; Berg Mikael; Zohari Siamak; Widén Frederik; Metreveli Giorgi; Bálint Ádám; Wallgren Per; Belák Sándor; Segall Thomas; Kiss István
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...
Borah, B; Deka, P; Sharma, K; Baro, S; Hazarika, A K; Das, C; Garam, G B; Boro, P; Ltu, K
Foot-and-mouth disease (FMD) is a contagious disease of cloven-hoofed animals that causes substantial and perpetual economic loss. Apart from the contagious nature of the disease, the FMD virus can establish in a "carrier state" among all cloven-hoofed animals. The Mithun (Bos frontalis), popularly called the "Cattle of Mountain," is found in the geographically isolated, hilly region of north-east India: Arunachal Pradesh, Nagaland, Manipur and Mizoram. Despite the geographical inaccessibility, infection by FMD virus has emerged as the single most devastating disease among Mithun after the eradication of rinderpest from this region. Samples from outbreaks of FMD in Mithun were analysed by sandwich ELISA, multiplex RT-PCR (MRT-PCR) and liquid-phase blocking enzyme-linked immunosorbent assay and isolated in the BHK-21 cell line. The results indicate the presence of FMDV serotype "O." The sequencing and molecular phylogenies have revealed close relationships in the lineage of type "O" isolates from Bangladesh. The findings will provide useful information for further research and development of a sustainable programme for the progressive control of FMD in the Mithun population. © 2017 Blackwell Verlag GmbH.
Fales, W H; Morehouse, L G; Mittal, K R; Bean-Knudsen, C; Nelson, S L; Kintner, L D; Turk, J R; Turk, M A; Brown, T P; Shaw, D P
The antimicrobial susceptibility of 73 Actinobacillus (Haemophilus) pleuropneumoniae isolates from swine in Missouri was determined with a microdilution minimal inhibitory concentration test system. Serotyping was accomplished by means of co-agglutination. Serotype 1 (39/73) and serotype 5 (30/73) were commonly found, whereas serotype 7 (4/73) was infrequently encountered. Most isolates (MIC90) were found susceptible to ampicillin (amoxicillin), cephalothin, penicillin, erythromycin, gentamicin, and kanamycin. Marked resistance was found with oxytetracycline, tylosin, and sulfadimethoxine. The data indicate that use of ampicillin (amoxicillin) or penicillin may correlate well with the favorable outcome of treatment.
Thrane, Sandra Wingaard; Taylor, Véronique L.; Freschi, Luca
that serotype switching in combination with an antibiotic resistance determinant contributed to the dissemination of the O12 serotype in the clinic. This selective advantage coincides with the introduction of fluoroquinolones in the clinic. With the PAst program isolates can be serotyped using WGS data......Introduction: Since the 1980’s the serotype O12 of Pseudomonas aeruginosa has emerged as the predominant serotype in clinical settings and in epidemic outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics.Methods: In this study, we explore how......).Results: While most serotypes were closely linked to the core genome phylogeny we observed horizontal exchange of LPS genes among distinct P. aeruginosa strains. Specifically, we identified a ‘serotype island’ containing the P. aeruginosa O12 LPS gene cluster and an antibiotic resistance determinant (gyrAC248T...
Albariño, César G; Guerrero, Lisa Wiggleton; Chakrabarti, Ayan K; Kainulainen, Markus H; Whitmer, Shannon L M; Welch, Stephen R; Nichol, Stuart T
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.
Boscher, Evelyne; Houard, Emmanuelle; Denis, Martine
This study was undertaken to acquire new data on the prevalence of Listeria monocytogenes in sows and fattening pigs in farrow-to-finish pig farms, and to analyze distribution of serotypes and genotypes of the bacterium within farms. Detection of L. monocytogenes was carried out on 730 pooled feces samples from sows in 73 pig farms and on 172 pooled feces samples from fattening pigs in 43 of these farms. Isolates were serotyped and typed by pulsed-field gel electrophoresis. For sows, 46% of the farms and 11% of the samples were positive for L. monocytogenes. A total of 124 isolates were collected and distributed in four serotypes: 1/2a (41%), 1/2b (36%), 4b (21%), and 1/2c (2%). Positive farms harbored one to three serotypes. The genetic diversity was high; 51 genetic profiles were obtained with 25, 16, 9, and 1 for the serotypes 1/2a, 1/2b, 4b, and 1/2c, respectively. Positive farms harbored 1 to 6 genetic profiles. Isolates showing similar genotypes occurred in several farms. For fattening pigs, 25% of the farms and 14.5% of the samples were positive for L. monocytogenes. The 34 isolates belonged to four serotypes: 1/2a (32%), 1/2b (41%), 4b (24%), and 1/2c (3%). They were distributed in 20 genotypes: 6 for 1/2a; 8 for 1/2b, 5 for 4b, and 1 for 1/2c. Similar serotypes and pulsotypes were recovered in sows and fattening pigs from the same farms, suggesting common sources of contamination.
Ahamed, Syed Fazil; Vivek, Rosario; Kotabagi, Shalini; Nayak, Kaustuv; Chandele, Anmol; Kaja, Murali-Krishna; Shet, Anita
Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially. Copyright © 2017 Elsevier B.V. All rights reserved.
Virgolino Helaine A
Full Text Available Abstract Background Hepatitis B virus (HBV isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil. Results Genotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%, and most of these isolates were classified as subgenotype A1 (138/153; 90.2%. Genotype D was the most common genotype in the South (84.2% and Central (47.6% regions. The prevalence of genotype F was low (13% countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5% belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127. Conclusion The presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F
Lee, Seo-Yong; Lee, Yeo-Joo; Kim, Rae-Hyung; Park, Jeong-Nam; Park, Min-Eun; Ko, Mi-Kyeong; Choi, Joo-Hyung; Chu, Jia-Qi; Lee, Kwang-Nyeong; Kim, Su-Mi; Tark, Dongseob; Lee, Hyang-Sim; Ko, Young-Joon; Seo, Min-Goo; Park, Jung-Won; Kim, Byounghan; Lee, Myoung-Heon; Lee, Jong-Soo; Park, Jong-Hyeon
There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV. IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries
Ying Xiu eToh
Full Text Available Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of one year. We found that serotype cross-reactive antibodies peaked three weeks after infection and subsided within one year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.
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.
Thong, K. L.; Goh, Y. L.; Radu, S.; Noorzaleha, S.; Yasin, R.; Koh, Y. T.; Lim, V. K. E.; Rusul, G.; Puthucheary, S. D.
The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.2...
Background Even though dengue has been recognized as one of the major public health threats in Pakistan, the understanding of its molecular epidemiology is still limited. The genotypic diversity of Dengue virus (DENV) serotypes involved in dengue outbreaks since 2005 in Pakistan is not well studied. Here, we investigated the origin, diversity, genetic relationships and geographic distribution of DENV to understand virus evolution during the recent expansion of dengue in Pakistan. Methods The study included 200 sera obtained from dengue-suspected patients from 2006 to 2011. DENV infection was confirmed in 94 (47%) sera by a polymerase chain reaction assay. These included 36 (38.3%) DENV-2, 57 DENV-3 (60.6%) and 1 DENV-4 (1.1%) cases. Sequences of 13 whole genomes (6 DENV-2, 6 DENV-3 and 1 DENV-4) and 49 envelope genes (26 DENV-2, 22 DENV-3 and 1 DENV-4) were analysed to determine the origin, phylogeny, diversity and selection pressure during virus evolution. Results DENV-2, DENV-3 and DENV-4 in Pakistan from 2006 to 2011 shared 98.5-99.6% nucleotide and 99.3-99.9% amino acid similarity with those circulated in the Indian subcontinent during the last decade. Nevertheless, Pakistan DENV-2 and DENV-3 strains formed distinct clades characterized by amino acid signatures of NS2A-I116T + NS5-K861R and NS3-K590R + NS5-S895L respectively. Each clade consisted of a heterogenous virus population that circulated in Southern (2006–2009) and Northern Pakistan (2011). Conclusions DENV-2, DENV-3 and DENV-4 that circulated during 2006–2011 are likely to have first introduced via the southern route of Pakistan. Both DENV-2 and DENV-3 have undergone in-situ evolution to generate heterogenous populations, possibly driven by sustained local DENV transmission during 2006–2011 periods. While both DENV-2 and DENV-3 continued to circulate in Southern Pakistan until 2009, DENV-2 has spread in a Northern direction to establish in Punjab Province, which experienced a massive dengue
First page Back Continue Last page Overview Graphics. Virus isolation: Specimen type and probable transmission. Over 500 CHIK virus isolations were made. 4 from male Ae. Aegypti (?TOT). 6 from CSF (neurological involvement). 1 from a 4-day old child (transplacental transmission.
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
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.
Berhane, Y; Joseph, T; Kehler, H; Hisanaga, T; Embury-Hyatt, C; Diederich, S; McGreevy, K Hooper; Handel, K; Cottam-Birt, C; Pasick, J
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.
Ayebazibwe, C.; Mwiine, F. N.; Balinda, S. N.
In East Africa, the foot-and-mouth disease (FMD) virus (FMDV) isolates have over time included serotypes O, A, C, Southern African Territories (SAT) 1 and SAT 2, mainly from livestock. SAT 3 has only been isolated in a few cases and only in African buffalos (Syncerus caffer). To investigate...... the presence of antibodies against FMDV serotypes in wildlife in Uganda, serological studies were performed on buffalo serum samples collected between 2001 and 2003. Thirty-eight samples from African buffalos collected from Lake Mburo, Kidepo Valley, Murchison Falls and Queen Elizabeth National Parks were...... screened using Ceditest® FMDV NS to detect antibodies against FMDV non-structural proteins (NSP). The seroprevalence of antibodies against non-structural proteins was 74%. To characterize FMDV antibodies, samples were selected and titrated using serotype-specific solid phase blocking enzyme linked...
Gutiérrez, C B; Rodríguez Barbosa, J I; Tascón, R I; Costa, L; Riera, P; Rodríguez Ferri, E F
Seventy-one isolates of Actinobacillus pleuropneumoniae isolated from the lungs of pigs in outbreaks of pleuropneumonia in Spain were serotyped by indirect haemagglutination. Serotype 4 (42.2 per cent), serotype 7 (22.5 per cent) and serotype 2 (12.8 per cent) were predominant, whereas serotypes 1, 3, 6, 8, 9, 12 and untypable isolates were present only in small numbers. Serotypes 1, 2, 4 and 7 originated mainly from cases of acute pleuropneumonia, whereas serotypes 3, 6, 8, 9 and 12 were associated with chronically infected herds. The susceptibility of the isolates to 20 antimicrobial agents was determined by agar disc diffusion. Most were susceptible to cefuroxime, cefaclor, cefazolin, kanamycin, tobramycin, gentamicin, oxolinic acid, ciprofloxacin, enoxacin, thiamphenicol, colistin and trimethoprim/sulphamethoxazole. Marked resistance was found with amoxicillin, ticarcillin, oxytetracycline, doxycycline and metronidazole. Rifampicin, fosfomycin and tiamulin were the agents most effective against the isolates tested.
Dr DADIE Thomas
Feb 17, 2014 ... The virulence, serotype and phylogenetic traits of diarrhoeagenic Escherichia coli were detected in 502 strains isolated during digestive infections. Molecular detection of the target virulence genes, rfb gene of operon O and phylogenetic grouping genes Chua, yjaA and TSPE4.C2 was performed.
Hu, Beixia; Huang, Yanyan; He, Yefeng; Xu, Chuantian; Lu, Xishan; Zhang, Wei; Meng, Bin; Yan, Shigan; Zhang, Xiumei
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
Tereza Cristina A. Leal
Full Text Available Sixteen strains of Yersinia enterocolitica serotype O:3, isolated from apparently healthy pigs collected in Rio de Janeiro, and four human strains of serotypes O:4, O:5, O:6 and O:13 were analyzed by RAPD-PCR. The strains were grouped into five genotypic profiles according to the amplification patterns obtained with three random primers. Fifteen of the 16 pig strains had identical amplification patterns, which was named genotypic profile 1. The one different profile was named genotypic profile 2. Genotypic profile 1 was also exhibited by the O:6 human serotype strain. The O:4 and O:13 human serotype strains showed similar amplification profiles with two primers. However, the third primer induced a distinct profile in each strain. Therefore, these two strains were placed into genotypic profile 3 and 4, respectively. Each primer produced a completely different amplification profile in the O:5 human serotype strain; therefore, it was named genotypic profile 5. The presence or absence of plasmids in the strains studied did not affect the amplification results. These results show that genetic variations can exist within a serotype, and strains of different serotypes can exhibit the same amplification profile when compared using other primers.Foram utilizados três "primers" aleatórios para caracterizar pela técnica RAPD-PCR 16 cepas de Yersinia enterocolitica do sorotipo O:3, isoladas de suínos sadios do Rio de Janeiro. Pelos resultados dos padrões de amplificação, as 16 cepas dos suínos e as 4 cepas humanas usadas como referência (sorotipos O:4, O:5, O:6 e O:13 foram agrupadas em 5 perfis genotípicos. Quinze cepas de suínos apresentaram um padrão de amplificação idêntico (perfil genotípico 1 e somente uma apresentou um perfil de amplificação diferente (perfil genotípico 2. O mesmo padrão de amplificação do perfil genotípico 1 foi também observado em uma cepa humana do sorotipo O:6. As cepas humanas dos sorotipos O:4 e O:13
Soda, Kosuke; Cheng, Ming-Chu; Yoshida, Hiromi; Endo, Mayumi; Lee, Shu-Hwae; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Wang, Ching-Ho; Kida, Hiroshi
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...
Teoh, Boon-Teong; Sam, Sing-Sin; Abd-Jamil, Juraina
Ancestral sylvatic dengue virus type 1, which was isolated from a monkey in 1972, was isolated from a patient with dengue fever in Malaysia. The virus is neutralized by serum of patients with endemic DENV-1 infection. Rare isolation of this virus suggests a limited spillover infection from an otherwise restricted sylvatic cycle. PMID:21029545
Full Text Available Abstract Background Dengue fever (DF and dengue hemorrhagic fever (DHF represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus that comprises four distinct serotypes (DENV-1 to DENV-4. Fluorescence in situ hybridization (FISH has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae. The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use
Full Text Available Little is known about the natural history of dengue in Papua New Guinea (PNG. We assessed dengue virus (DENV-specific neutralizing antibody profiles in serum samples collected from northern and southern coastal areas and the highland region of New Guinea between 1959 and 1963. Neutralizing antibodies were demonstrated in sera from the northern coast of New Guinea: from Sabron in Dutch New Guinea (now known as West Papua and from four villages in East Sepik in what is now PNG. Previous monotypic infection with DENV-1, DENV-2, and DENV-4 was identified, with a predominance of anti-DENV-2 neutralizing antibody. The majority of positive sera demonstrated evidence of multiple previous DENV infections and neutralizing activity against all four serotypes was detected, with anti-DENV-2 responses being most frequent and of greatest magnitude. No evidence of previous DENV infection was identified in the Asmat villages of the southern coast and a single anti-DENV-positive sample was identified in the Eastern Highlands of PNG. These findings indicate that multiple DENV serotypes circulated along the northern coast of New Guinea at different times in the decades prior to 1963 and support the notion that dengue has been a significant yet neglected tropical infection in PNG for many decades.
Full Text Available Salmonella is frequently found in poultry and represent an important source for human gastrointestinal infections worldwide. The aim of this study was to investigate the prevalence, genotypes and antimicrobial resistance of Salmonella serotypes in broilers from Ecuador. Caeca content from 388 at random selected broiler batches were collected in 6 slaughterhouses during 1 year and analyzed by the ISO 6579/Amd1 protocol for the isolation for Salmonella. Isolates were serotyped and genotypic variation was acceded by pulsed field gel electrophoresis. MIC values for sulfamethoxazole, gentamicin, ciprofloxacin, ampicillin, cefotaxime, ceftazidime, tetracycline, streptomycin, trimethropim, chloramphenicol, colistin, florfenicol, kanamycin and nalidixic acid were obtained. Presence of blaCTX-M, blaTEM, blaSHV and blaCMY; and mcr-1 plasmid genes was investigated in resistant strains to cefotaxime and colistin respectively. Prevalence at batch level was 16.0%. The most common serotype was S. Infantis (83.9% followed by S. Enteritidis (14.5% and S. Corvallis (1.6%. The pulsed field gel electrophoresis analysis showed that S. Corvallis, S. Enteritidis and S. Infantis isolates belonged to 1, 2 and 12 genotypes respectively. S. Infantis isolates showed high resistance rates to 12 antibiotics ranging from 57.7% (kanamycin up to 98.1% (nalidixic acid and sulfamethoxazole. All S. Enteritidis isolates showed resistance to colistin. High multiresistant patterns were found for all the serotypes. The blaCTX-M gene was present in 33 S. Infantis isolates while mcr-1 was negative in 10 colistin resistant isolates. This study provides the first set of scientific data on prevalence and multidrug-resistant Salmonella coming from commercial poultry in Ecuador.
Anil Babu Korrapati
Full Text Available BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs. This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5 vector to deliver a short-hairpin RNA (shRNA targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.
Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying
Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.
Full Text Available The pathogenesis of dengue virus (DV infection has not been completely defined and change of redox status mediated by depletion of glutathione (GSH in host cell is a common result of viral infection. Our previous study has demonstrated that DV serotype 2 (DV2 infection alters host intracellular GSH levels, and exogenous GSH inhibits viral production by modulating the activity of NF-κB in HepG2 cells. GSH is the most powerful intracellular antioxidant and involved in viral infections. Thus, this study was to investigate whether DV2 infection can induce alteration in redox balance and effect of GSH on the disease in HepG2 xenografts SCID mice. Our results revealed that mice infected with DV2 showed alterations in oxidative stress by increasing the level of malondialdehyde (MDA, an end product of lipid peroxidation, and GSSG/GSH ratio. DV2-infected mice also showed a decrease in the activity of catalase (CAT and total superoxide dismutase (T-SOD in the serum and/or observed organs, especially the liver. Moreover, DV2 infection resulted in elevated serum levels of the cytokines tumor necrosis factor-α and interlukin-6 and obvious histopathological changes in the liver. The administration of exogenous GSH significantly reversed all of the aforementioned pathological changes and prevented significant liver damage. Furthermore, in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells. These results suggest that GSH prevents DV2-induced oxidative stress and liver injury in mice by inhibiting proinflammatory cytokine production, and GSH and may be a promising therapeutic agent for prevention of oxidative liver damage during DV infection.
McEgan, Rachel; Chandler, Jeffrey C; Goodridge, Lawrence D; Danyluk, Michelle D
Identification of Salmonella serotypes is important for understanding the environmental diversity of the genus Salmonella. This study evaluates the diversity of Salmonella isolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmental Salmonella isolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608-3615, 2006, http://dx.doi.org/10.1128/JCM.00701-06). Multiplex PCR was performed on 562 Salmonella isolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the Salmonella-positive water samples, all Salmonella isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three Salmonella enterica serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:-. A majority (141/223) of Salmonella isolates clustered into one genotypic group. Salmonella isolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the Salmonella
Full Text Available Escherichia coli sequence type (ST131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5 and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E. Fourteen out of the 23 ST131 isolates tested (61% killed 90 to 100% of mice challenged, and 18 of 23 (78% at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h to low final lethality (death at 7 days but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.
Prevalencia de anticuerpos neutralizantes contra los serotipos del virus dengue en universitarios de Tabasco, México Prevalence of neutralizing antibodies to dengue virus serotypes in university students from Tabasco, Mexico
Gilma Guadalupe Sánchez-Burgos
Full Text Available OBJETIVO: Determinar la seroprevalencia de anticuerpos neutralizantes de los serotipos del virus dengue en estudiantes universitarios de Tabasco, México, durante los meses de septiembre a noviembre del año 2005. MATERIAL Y MÉTODOS: Se determinó la presencia de IgG contra el virus en el suero de estudiantes que acudieron al centro clínico de la universidad; en los sueros positivos se determinaron los anticuerpos neutralizantes mediante el ensayo de reducción de placa lítica. RESULTADOS: La prevalencia de IgG contra el dengue fue de 9.1%; de esta proporción, los anticuerpos neutralizantes fueron DENV-1 (20%, DENV-2 (100%, DENV-3 (4% y DENV-4 (68%. CONCLUSIONES: Este estudio muestra que el serotipo transmitido con mayor frecuencia en el estado de Tabasco es el DENV-2, aunque no ha sido el aislado con más frecuencia. La elevada prevalencia de anticuerpos neutralizantes contra el DENV-4, al parecer de reacción cruzada, podría explicar la baja circulación de este serotipo en Tabasco.OBJECTIVE: Determine the seroprevalence of neutralizing antibodies to dengue virus in students from the state university of Tabasco, Mexico. MATERIAL AND METHODS: A transversal study was conducted of serum collected from students between September and November, 2005. The sera were screened for anti-dengue IgG and those that had evidence of dengue antibodies were analyzed by a plaque reduction neutralization test. RESULTS: Prevalence of anti-dengue IgG was 9.1%. The frequency of neutralizing antibodies was 100% for DENV-2, 68% for DENV-4, 20% for DENV-1, and 4 % for DENV-3. CONCLUSIONS: We found that in this population, DENV-2 circulates more than DENV-3 despite the fact that DENV-3 is more frequently isolated. Unexpectedly, neutralizing antibodies against DENV-4 were frequently found even though this serotype is almost extinct; thus, it is probable that cross-immunity could suppress DEN-4 transmission, as has been suggested.
Xu, Min; Wang, Shujie; Li, Linxi; Lei, Liancheng; Liu, Yonggang; Shi, Wenda; Wu, Jiabin; Li, Liqin; Rong, Fulong; Xu, Mingming; Sun, Guangli; Xiang, Hua; Cai, Xuehui
Porcine reproductive and respiratory syndrome virus (PRRSV) and Streptococcus suis are common pathogens in pigs. In samples collected during the porcine high fever syndrome (PHFS) outbreak in many parts of China, PRRSV and S. suis serotype 7 (SS7) have always been isolated together. To determine whether PRRSV-SS7 coinfection was the cause of the PHFS outbreak, we evaluated the pathogenicity of PRRSV and/or SS7 in a pig model of single and mixed infection. Respiratory disease, diarrhea, and anorexia were observed in all infected pigs. Signs of central nervous system (CNS) disease were observed in the highly pathogenic PRRSV (HP-PRRSV)-infected pigs (4/12) and the coinfected pigs (8/10); however, the symptoms of the coinfected pigs were clearly more severe than those of the HP-PRRSV-infected pigs. The mortality rate was significantly higher in the coinfected pigs (8/10) than in the HP-PRRSV- (2/12) and SS7-infected pigs (0/10). The deceased pigs of the coinfected group had symptoms typical of PHFS, such as high fever, anorexia, and red coloration of the ears and the body. The isolation rates of HP-PRRSV and SS7 were higher and the lesion severity was greater in the coinfected pigs than in monoinfected pigs. HP-PRRSV infection increased susceptibility to SS7 infection, and coinfection of HP-PRRSV with SS7 significantly increased the pathogenicity of SS7 to pigs.
Full Text Available Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV and Streptococcus suis are common pathogens in pigs. In samples collected during the porcine high fever syndrome (PHFS outbreak in many parts of China, PRRSV and S. suis serotype 7 (SS7 have always been isolated together. To determine whether PRRSV-SS7 coinfection was the cause of the PHFS outbreak, we evaluated the pathogenicity of PRRSV and/or SS7 in a pig model of single and mixed infection. Results Respiratory disease, diarrhea, and anorexia were observed in all infected pigs. Signs of central nervous system (CNS disease were observed in the highly pathogenic PRRSV (HP-PRRSV-infected pigs (4/12 and the coinfected pigs (8/10; however, the symptoms of the coinfected pigs were clearly more severe than those of the HP-PRRSV-infected pigs. The mortality rate was significantly higher in the coinfected pigs (8/10 than in the HP-PRRSV- (2/12 and SS7-infected pigs (0/10. The deceased pigs of the coinfected group had symptoms typical of PHFS, such as high fever, anorexia, and red coloration of the ears and the body. The isolation rates of HP-PRRSV and SS7 were higher and the lesion severity was greater in the coinfected pigs than in monoinfected pigs. Conclusion HP-PRRSV infection increased susceptibility to SS7 infection, and coinfection of HP-PRRSV with SS7 significantly increased the pathogenicity of SS7 to pigs.
Cham, Tat-Chuan; Chang, Yen-Chen; Tsai, Pei-Shiue; Wu, Ching-Ho; Chen, Hui-Wen; Jeng, Chian-Ren; Pang, Victor Fei; Chang, Hui-Wen
Unlike for serotype II feline coronaviruses (FCoV II), the cellular receptor for serotype I FCoV (FCoV I), the most prevalent FCoV serotype, is unknown. To provide a platform for assessing the pattern by which FCoV I attaches to its host receptor(s), HEK293 cell lines that stably express the ectodomains of the spike (S) proteins derived from a FCoV I feline enteric coronavirus strain UU7 (FECV UU7) and a feline infectious peritonitis virus strain UU4 (FIPV UU4) were established. Using the recombinant S proteins as probes to perform S protein affinity histochemistry in paraffin-embedded tissues, although no tissue or enteric binding of FECV UU7 S protein was detected, it was found that by immunohistochemistry that the tissue distribution of FIPV UU4 S protein-bound cells correlated with that of FIPV antigen-positive cells and lesions associated with FIP and that the affinity binding of FIPV UU4 S protein on macrophages was not affected by enzymatic removal of host cell-surface sialic acid with neuraminidase. These findings suggest that a factor(s) other than sialic acid contribute(s) to the macrophage tropism of FIPV strain UU4. This approach allowed obtaining more information about both virus-host cell interactions and the biological characteristics of the unidentified cellular receptor for FCoV I. © 2017 The Societies and John Wiley & Sons Australia, Ltd.
Saw, Wuan Geok; Tria, Giancarlo; Grüber, Ardina; Subramanian Manimekalai, Malathy Sony; Zhao, Yongqian; Chandramohan, Arun; Srinivasan Anand, Ganesh; Matsui, Tsutomu; Weiss, Thomas M.; Vasudevan, Subhash G.; Grüber, Gerhard
Infection by the four serotypes of
Olesen, Niels Jørgen; Skall, Helle Frank
This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus.......This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus....
Ingle, Danielle J; Valcanis, Mary; Kuzevski, Alex; Tauschek, Marija; Inouye, Michael; Stinear, Tim; Levine, Myron M; Robins-Browne, Roy M; Holt, Kathryn E
The lipopolysaccharide (O) and flagellar (H) surface antigens of Escherichia coli are targets for serotyping that have traditionally been used to identify pathogenic lineages. These surface antigens are important for the survival of E. coli within mammalian hosts. However, traditional serotyping has several limitations, and public health reference laboratories are increasingly moving towards whole genome sequencing (WGS) to characterize bacterial isolates. Here we present a method to rapidly and accurately serotype E. coli isolates from raw, short read WGS data. Our approach bypasses the need for de novo genome assembly by directly screening WGS reads against a curated database of alleles linked to known and novel E. coli O-groups and H-types (the EcOH database) using the software package srst2. We validated the approach by comparing in silico results for 197 enteropathogenic E. coli isolates with those obtained by serological phenotyping in an independent laboratory. We then demonstrated the utility of our method to characterize isolates in public health and clinical settings, and to explore the genetic diversity of >1500 E. coli genomes from multiple sources. Importantly, we showed that transfer of O- and H-antigen loci between E. coli chromosomal backbones is common, with little evidence of constraints by host or pathotype, suggesting that E. coli ' strain space' may be virtually unlimited, even within specific pathotypes. Our findings show that serotyping is most useful when used in combination with strain genotyping to characterize microevolution events within an inferred population structure.
Kostolansky, F.; Styk, B.; Russ, G.
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)
Mendez Jairo A
Full Text Available Abstract Background Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70's when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1, there are not studies about its origin, genetic diversity and distribution. Results We used 224 bp corresponding to the carboxyl terminus of envelope (E gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found. Conclusion DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages.
Takayama, Koichi; Reynolds, Paul N.; Short, Joshua J.; Kawakami, Yosuke; Adachi, Yasuo; Glasgow, Joel N.; Rots, Marianne G.; Krasnykh, Victor; Douglas, Joanne T.; Curiel, David T.
The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion
Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.
Michael T Karwacki
Full Text Available Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications.
Yoshioka, Cristina R M; Martinez, Marina B; Brandileone, Maria C C; Ragazzi, Selma B; Guerra, Maria L L S; Santos, Silvia R; Shieh, Huei H; Gilio, Alfredo E
To identify the most common pneumococcal serotypes in children hospitalized with invasive pneumonia, correlate isolated serotypes with those included in conjugate vaccines, and ascertain the sensitivity of the isolated pneumococcal strains to penicillin and other antibiotics. From January 2003 to October 2008, a retrospective study of hospitalized children with a diagnosis of Streptococcus pneumoniae pneumonia was conducted at the university hospital of Universidade de São Paulo. Criteria for inclusion were: age greater than 29 days and less than 15 years, radiological and clinical diagnosis of pneumonia, and isolation of Streptococcus pneumoniae in blood cultures and/or pleural effusion. The study included 107 children. The most common serotypes were 14 (36.5%), 1 (16%), 5 (14.6%), 6B (6.3%) and 3 (4.2%). The proportion of identified serotypes contained in the heptavalent, 10-valent and 13-valent conjugate vaccines was 53.1, 86.5, and 96.9%, respectively. Pneumococcal strains were sensitive to penicillin (minimum inhibitory concentration, MIC ≤ 2 µg/mL) in 100 cases (93.5%) and displayed intermediate resistance (MIC = 4 µg/mL) in 7 cases (6.5%). No strains were penicillin-resistant (MIC ≥ 8 µg/mL) according to the Clinical and Laboratory Standards Institute 2008 standards. Tested isolates were highly sensitive to vancomycin, rifampicin, ceftriaxone, clindamycin, erythromycin, and chloramphenicol. Our results confirm a significant potential impact of conjugate vaccines, mainly 10-valent and 13-valent, on invasive pneumonia. Furthermore, susceptibility testing results show that penicillin is still the treatment of choice for invasive pneumonia in our setting.
Douglas, Kirk O; Lavoie, Marc C; Kim, L Mia; Afonso, Claudio L; Suarez, David L
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.
Oncino, C; Hemmer, O; Fritsch, C
The satellite RNAs (sat-RNAs) associated with some isolates of tomato black ring virus (TBRV) consist of single-stranded molecules of about 1375 nucleotides, encoding a nonstructural protein of 48K which has been shown to be involved in the replication of the sat-RNA. The TBRV sat-RNAs are also dependent for their replication and for their encapsidation on the helper virus. To characterize the nature of the association between sat-RNA and helper virus, transcripts of sat-RNA from TBRV isolates C and L (respectively, of serotypes G and S) have been prepared and inoculated onto Chenopodium quinoa leaves or protoplasts. Transcript of the TBRV sat-RNA C is efficiently multiplied when coinoculated with the genomic RNAs of TBRV isolate G (used instead of TBRV isolate C, because isolate G was depleted of sat-RNA), but does not multiply with TBRV isolate L. On the other hand, transcript of the sat-RNA L is able to multiply with the cognate helper virus and, less efficiently, with grapevine chrome mosaic virus (another nepovirus, 80% similar to TBRV), but does not multiply with TBRV G. The specificity of the association resides at the level of sat-RNA replication. Analysis of the multiplication of chimeric sat-RNAs, obtained by exchanging different regions between the two sat-RNAs C and L, showed that the 5' and the 3' noncoding regions of the sat-RNA, although important for replication, are not implicated in specificity. The results suggest that the determinants of the specificity are contained in the 48K sat-RNA-encoded protein.
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.
Hatakeyama, Kaoru; Sadamasu, Kenji; Kai, Akemi
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.
Wang, Shaohui; Meng, Qingmei; Dai, Jianjun; Han, Xiangan; Han, Yue; Ding, Chan; Liu, Haiwen; Yu, Shengqing
Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.
Full Text Available Systemic infections by avian pathogenic Escherichia coli (APEC are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.
[Genetic characterisation of Powassan virus (POWV) isolated from Haemophysalis longicornis ticks in Primorye and two strains of Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus): Alma-Arasan virus (AAV) isolated from Ixodes persulcatus ticks in Kazakhstan and Malyshevo virus isolated from Aedes vexans nipponii mosquitoes in Khabarovsk kray].
L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Deriabin, P G; Gitel'man, A K; Botikov, A G; Aristova, V A
The complete genomes of the three tick-borne flaviviruses (genus Flavivirus, fam. Bunyaviridae) were sequenced: Povassan virus (POWV, strain LEIV-3070Prm, isolated from Haemophysalis logicornis in Primorsky Krai, Russia in 1977), Alma-Arasan virus (AAV, strain LEIV-1380Kaz, isolated from Ixodes persulcatus ticks in Kazakhstan in 1977) and Malyshevo virus (isolated from a pool of Aedes vexans nipponii mosquitoes, in the Khabarovsk Krai, Russia in 1978). It is shown that AAV and Malyshevo virus are the strains of Tick-borne encephalitis virus (TBEV) and belong to Sibirian and Far-Eastern genotypes, respectively (GenBank ID: AAV KJ744033; strain Malyshevo KJ744034). Phylogenetically AAV is closest related (94,6% nt and 98,3% aa identity) to TBEV strains, isolated in Sibiria (Vasilchenko, Aino, Chita-653, Irkutsk-12). Malyshevo virus is closest related (96,4% nt and 98,3% nt identity) to strains of TBEV, isolated in Far Eastern part of Russia (1230, Spassk-72, Primorye-89). POWV LEIV-3070Prm has 99.7% identity with the prototype strain POWV LB, isolated in Canada and 99.5% of isolates with Far-Eastern strains of POWV (Spassk-9 and Nadezdinsk-1991).
Full Text Available Zika virus (Flavivirus genus is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments.We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015. Here, we describe the patient's clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3' un-translated region, to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains.We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas.
Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J
A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward...
Muggeridge, Martin I.; Grantham, Michael L.; Johnson, F. Brent
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
Slotved, Hans-Christian; Dayie, Nicholas T K D; Banini, Josephine A N
was performed on a single subcultured colony. Gram staining was performed, and isolates were evaluated for beta-haemolytic reactions. Furthermore, the isolates were serotyped using the GBS latex serotyping kit. RESULTS: The carriage rates were found to be 25.5% (95% CI: 19.6-32.1) to 28.0% (95% CI: 21...... of this study revealed that prevalence of GBS colonization in pregnant women in Greater Accra region is high and comparable to rates observed in South Africa and Western countries. The most prevalent serotypes were serotypes VII and IX, which have not been observed before in West Africa....
Field, H J; Darby, G; Wildy, P
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.
Full Text Available In this paper we report the results of n.105 E. coli strains serotyping, isolated during the period 2000-2004 in Lombardia and Emilia Romagna (North Italy from avian species (poultry and turkeys, starting from cloacal swabs. The most frequently identified serogroup was O78 both in poultry and turkeys, with a large prevalence over the other detected serogroups. Remarkable was the non typeable percentage among the examined strains, datum which is in accordance with our and other authors’ previous studies.
Full Text Available Lung abscess has been considered to be a rare complication of pneumococcal infection, and most cases are reported to be Streptococcus pneumoniae serotype 3. A 67-year-old man presented with fever and was diagnosed to have lung abscess caused by S. pneumoniae serotype 6B. The minimal inhibitory concentration (MIC of penicillin for the isolate was 1 μg/mL. He was treated with high-dose intravenous sulbactam/ampicillin as definitive therapy based on susceptibility testing for S. pneumoniae and recovered successfully without surgical intervention. S. pneumoniae serotype 6B can cause lung abscess. Keywords: Streptococcus pneumoniae, Lung abscess, Serotype 6B, Penicillin-resistant Streptococcus pneumoniae
Hammond, R W; Crosslin, J M; Pasini, R; Howell, W E; Mink, G I
Prunus necrotic ringspot ilarvirus (PNRSV) exists as a number of biologically distinct variants which differ in host specificity, serology, and pathology. Previous nucleotide sequence alignment and phylogenetic analysis of cloned reverse transcription-polymerase chain reaction (RT-PCR) products of several biologically distinct sweet cherry isolates revealed correlations between symptom type and the nucleotide and amino acid sequences of the 3a (putative movement protein) and 3b (coat protein) open reading frames. Based upon this analysis, RT-PCR assays have been developed that can identify isolates displaying different symptoms and serotypes. The incorporation of primers in a multiplex PCR protocol permits rapid detection and discrimination among the strains. The results of PCR amplification using type-specific primers that amplify a portion of the coat protein gene demonstrate that the primer-selection procedure developed for PNRSV constitutes a reliable method of viral strain discrimination in cherry for disease control and will also be useful for examining biological diversity within the PNRSV virus group.
Spencer, Brady L; Shenoy, Anukul T; Orihuela, Carlos J; Nahm, Moon H
As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ The coding sequence of wciZ contains eight consecutive TA repeats [(TA) 8 ]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA) 7 serotype 15C is ∼0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA) 7 serotype 15C, and 15BΔ wciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C. Copyright © 2017 American Society for Microbiology.
Hatrongjit, Rujirat; Kerdsin, Anusak; Gottschalk, Marcelo; Takeuchi, Dan; Hamada, Shigeyuki; Oishi, Kazunori; Akeda, Yukihiro
Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. It has been reported that S. suis infection in humans is mostly caused by serotype 2. However, human cases caused by other serotypes have rarely been reported. This is the first report of a human case of infection with S. suis serotype 31 in Thailand. A 55-year-old male alcohol misuser with liver cirrhosis was admitted with sepsis to a hospital in the Central Region of Thailand. He had consumed a homemade, raw pork product prior to the onset of illness. He was alive after treatment with ceftriaxone and no complication occurred. An isolate from blood culture at the hospital was suspected as viridans group Streptococcus. It was confirmed at a reference laboratory as S. suis serotype 31 by biochemical tests, 16S rDNA sequencing, and multiplex polymerase chain reaction for serotyping, but it was untypable by the co-agglutination test with antisera against recognized S. suis serotypes, suggesting loss of capsular material. The absence of a capsule was confirmed by transmission electron microscopy. The isolate was confirmed to be sequence type 221, with 13 putative virulence genes that are usually found in serotype 2 strains. We should be aware of the emergence of S. suis infections caused by uncommon serotypes in patients with predisposing conditions. Laboratory capacity to identify S. suis in the hospital is needed in developing countries, which can contribute to enhanced surveillance, epidemiological control, and prevention strategies in the prevalent area.
Liu, Yan-Xia; Li, Fen-Xiang; Liu, Zhuan-Zhuan; Jia, Zhi-Rong; Zhou, Yan-He; Zhang, Hao; Yan, Hui; Zhou, Xian-Qiang; Chen, Xiao-Guang
Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut. © 2016 Institute of Zoology, Chinese Academy of Sciences.
Full Text Available Abstract Background Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes. Results In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH. Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF. Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP and EF (MRP-EF-, suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7. Conclusions In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.
Chen, Yixiang; Meng, Xueqiong; Liu, Qi; Huang, Xia; Huang, Shengbin; Liu, Cuiquan; Shi, Kaichuang; Guo, Jiangang; Chen, Fangfang; Hu, Liping
Our aim in this study was to determine the genetic characterization and probable origin of the H1N2 swine influenza virus (A/Swine/Guangxi/13/2006) (Sw/GX/13/06) from lung tissue of a pig in Guangxi province, China. Eight genes of Sw/GX/13/06 were cloned and genetically analyzed. The hemagglutinin (HA), nucleoprotein (NP), matrix (M) and non-structural (NS) genes of Sw/GX/13/06 were most closely related to genes from the classical swine H1N1 influenza virus lineage. The neuraminidase (NA) and PB1 genes were most closely related to the corresponding genes from the human influenza H3N2 virus lineage. The remaining two genes PA and PB2 polymerase genes were most closely related to the genes from avian influenza virus lineage. Phylogenetic analyses revealed that Sw/GX/13/06 was a human/swine/avian H1N2 virus, and closely related to H1N2 viruses isolated from pigs in United States (1999-2001) and Korea (2002). To our knowledge, Sw/GX/13/06 was the first triple-reassortant H1N2 influenza A virus isolated from a pig in China. Whether the Sw/GX/13/06 has a potential threat to breeding farm and human health remains to be further investigated.
FIGUEIREDO Luiz Tadeu Moraes
Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination
Alexander, D J; Spackman, D
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.
Schmidt, Candice; Cibulski, Samuel Paulo; Muterle Varela, Ana Paula; Mengue Scheffer, Camila; Wendlant, Adrieli; Quoos Mayer, Fabiana; Lopes de Almeida, Laura; Franco, Ana Cláudia; Roehe, Paulo Michel
In this study, the full-genome sequence of a reassortant H1N2 swine influenza virus is reported. The isolate has the hemagglutinin (HA) and neuraminidase (NA) genes from human lineage (H1-δ cluster and N2), and the internal genes (polymerase basic 1 [PB1], polymerase basic 2 [PB2], polymerase acidic [PA], nucleoprotein [NP], matrix [M], and nonstructural [NS]) are derived from human 2009 pandemic H1N1 (H1N1pdm09) virus. Copyright © 2014 Schmidt et al.
Deniz Akgun Karapinar
Full Text Available Aim: In this study, the distribution of serogroup/serotype and antibiotic susceptibility testing of Streptococcus pneumoniae strains, recovered from pediatric and adult patients were evaluated. Material and Method: A total of 80 clinical isolates recovered from 19 pediatric and 61 adult patients were performed by latex aglutination method and antibiotic susceptibility tests in Istanbul University, Istanbul Faculty of Medicine, Medical Microbiology Laboratories. Results: Sixty-two strains (76 %, were serogroup/serotyped and 18 (23 % strains couldn%u2019t serogroup/serotyped. The most frequent identified serogroups were 19, 14, 23, 6, 4 in pediatrics, and 3, 19, 23 and 9 in adults. In adults, serogroups 3, 9, 5, 8, 18, 1, 15 were determined, but these serogroups weren%u2019t found in pediatrics. Vaccine serotypes rates were found as 53 % in pediatric and as 85 % in adults. The serogroups 2, 7, 10, 11, 12, 17, 20, 22, 33 were not detected, which are available in vaccine serotypes. Only 1 (1 % strain was found to exhibit low level resistance to penicillin and high level resistance wasn%u2019t found in any strain. Resistant results for trimethoprim-sulfamethoxazole, erythromycin, chloramphenicol and ofloxacin were found as 45 (56 %, 22 (27.5 %, 7 (9 %, 2 (2.5 %, respectively. All strains were found susceptible to vancomycin, linezolid and levofloxacin. The most resistant serogroups were 19, 23, 9 and 14 in the tested antibiotics. Multidrug resistance was found in 9 (11 % strains and these strains were found as serogroups 19, 23, 9, 6 and 14. Discussion: The epidemiological studies are important that the distribution of serotype and antibiotic resistance vary depending on many factors like age, and geographic region.
Full Text Available Abstract Background Group B Streptococcus (GBS serotype (Ia, Ib, II-IX correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan, the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations. Methods To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps genes associated with pathogen virulence. Results Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively. A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p Conclusion This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.
Luciano Matsumiya Thomazelli
Full Text Available A novel avian paramyxovirus (APMV isolated from a migratory bird cloacal swab obtained during active surveillance in April 2012 in the Lagoa do Peixe National Park, Rio Grande do Sul state, South of Brazil was biologically and genetically characterized. The nucleotide sequence of the full viral genome was completed using a next-generation sequencing approach. The genome was 14,952 nucleotides (nt long, with six genes (3'-NP-P-M-F-HN-L-5' encoding 7 different proteins, typical of APMV. The fusion (F protein gene of isolate RS-1177 contained 1,707 nucleotides in a single open reading frame encoding a protein of 569 amino acids. The F protein cleavage site contained two basic amino acids (VPKER↓L, typical of avirulent strains. Phylogenetic analysis of the whole genome indicated that the virus is related to APMV-10, -2 and -8, with 60.1% nucleotide sequence identity to the closest APMV-10 virus, 58.7% and 58.5% identity to the closest APMV-8 and APMV-2 genome, respectively, and less than 52% identity to representatives of the other APMVs groups. Such distances are comparable to the distances observed among other previously identified APMVs serotypes. These results suggest that unclassified/calidris_fuscicollis/Brazil/RS-1177/2012 is the prototype strain of a new APMV serotype, APMV-15.
Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay
Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.
Muthukrishnan, Madhanmohan; Singanallur, Nagendrakumar B; Ralla, Kumar; Villuppanoor, Srinivasan A
Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.
Clinical Survey of Dengue Virus Circulation in the Republic of Djibouti between 2011 and 2014 Identifies Serotype 3 Epidemic and Recommends Clinical Diagnosis Guidelines for Resource Limited Settings.
Le Gonidec, Erwan; Maquart, Marianne; Duron, Sandrine; Savini, Hélène; Cazajous, Geraldine; Vidal, Pierre-Olivier; Chenilleau, Marie-Caroline; Roseau, Jean-Baptiste; Benois, Alain; Dehan, Céline; Kugelman, Jeffrey; Leparc-Goffart, Isabelle; Védy, Serge
Dengue virus is endemic globally, throughout tropical and sub-tropical regions. While the number of epidemics due to the four DENV serotypes is pronounced in East Africa, the total number of cases reported in Africa (16 million infections) remained at low levels compared to Asia (70 million infections). The French Armed forces Health Service provides epidemiological surveillance support in the Republic of Djibouti through the Bouffard Military hospital. Between 2011 and 2014, clinical and biological data of suspected dengue syndromes were collected at the Bouffard Military hospital and analyzed to improve Dengue clinical diagnosis and evaluate its circulation in East Africa. Examining samples from patients that presented one or more Dengue-like symptoms the study evidenced 128 Dengue cases among 354 suspected cases (36.2% of the non-malarial Dengue-like syndromes). It also demonstrated the circulation of serotypes 1 and 2 and reports the first epidemic of serotype 3 infections in Djibouti which was found in all of the hospitalized patients in this study. Based on these results we have determined that screening for Malaria and the presence of the arthralgia, gastro-intestinal symptoms and lymphopenia Djibouti.
Costa, Natália S; Pinto, Tatiana C A; Merquior, Vânia L C; Castro, Luciana F S; da Rocha, Filomena S P; Morais, Jaqueline M; Peralta, José M; Teixeira, Lúcia M
Streptococcus pneumoniae remains as an important cause of community-acquired bacterial infections, and the nasopharynx of asymptomatic carriers is the major reservoir of this microorganism. Pneumococcal strains of serotype 14 and serogroup 9 are among the most frequently isolated from both asymptomatic carriers and patients with invasive disease living in Brazil. Internationally disseminated clones belonging to such serotypes have been associated with the emergence and spread of antimicrobial resistance in our setting, highlighting the need for epidemiological tracking of these isolates. In this scenario, Multiple Loci VNTR Analysis (MLVA) has emerged as an alternative tool for the molecular characterization of pneumococci, in addition to more traditional techniques such as Multi-Locus Sequence Typing (MLST) and Pulsed-Field Gel Electrophoresis (PFGE). In the present study, 18 VNTR loci, as well as other previously described reduced MLVA panels (7 VNTR loci), were evaluated as tools to characterize pneumococcal strains of serotypes 14, 9N and 9V belonging to international and regional clones isolated in Brazil. The 18 VNTR loci panel was highly congruent with MLST and PFGE, being also useful for indicating the genetic relationship with international clones and for discriminating among strains with indistinguishable STs and PFGE profiles. Analysis of the results also allowed deducing a novel shorter 7 VNTR loci panel, keeping a high discriminatory power for isolates of the serotypes investigated and a high congruence level with MLST and PFGE. The newly proposed simplified panel was then evaluated for typing pneumococcal strains of other commonly isolated serotypes. The results indicate that MLVA is a faster and easier to perform, reliable approach for the molecular characterization of S. pneumoniae isolates, with potential for cost-effective application, especially in resource-limited countries.
Full Text Available Dengue virus (DENV infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.
Body, Mohammad H; Alrarawahi, Abdulmajeed H; Alhubsy, Saif S; Saravanan, Nirmala; Rajmony, Sunil; Mansoor, Muhammad Khalid
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.
Zhan, Bujie; Angen, Øystein; Hedegaard, Jakob
Actinobacillus pleuropneumoniae is a bacterial pathogen that causes highly contagious respiratory infection in pigs and has a serious impact on the production economy and animal welfare. As clear differences in virulence between serotypes have been observed, the genetic basis should be investigat...... at the genomic level. Here, we present the draft genome sequences of the A. pleuropneumoniae serotypes 2 (strain 4226) and 6 (strain Femo)....
Full Text Available Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for β-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with MLSB phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2.
Nusa, Roy; Prasetyowati, Heni; Meutiawati, Febrina; Yohan, Benediktus; Trimarsanto, Hidayat; Setianingsih, Tri Yuli; Sasmono, R Tedjo
Dengue is endemic and affects people in all Indonesian provinces. Increasing dengue cases have been observed every year in Sukabumi in West Java province. Despite the endemicity, limited data is available on the genetic of dengue viruses (DENV) circulating in the country. To understand the dynamics of dengue disease, we performed molecular and serological surveillance of dengue in Sukabumi. A total of 113 patients were recruited for this study. Serological data were obtained using anti-dengue IgM and IgG tests plus dengue NS1 antigen detection. Dengue detection and serotyping were performed using real-time RT-PCR. Viruses were isolated and the envelope genes were sequenced. Phylogenetic and evolutionary analyses were performed to determine the genotype of the viruses and their evolutionary rates. Real-time RT-PCR detected DENV in 25 (22%) of 113 samples. Serotyping revealed the predominance of DENV-2 (16 isolates, 64%), followed by DENV-1 (5 isolates, 20%), and DENV-4 (4 isolates, 16%). No DENV-3 was detected in the samples. Co-circulation of genotype I and IV of DENV-1 was observed. The DENV-2 isolates all belonged to the Cosmopolitan genotype, while DENV-4 isolates were grouped into genotype II. Overall, their evolutionary rates were similar to DENV from other countries. We revealed the distribution of DENV serotypes and genotypes in Sukabumi. Compared to data obtained from other cities in Indonesia, we observed the differing predominance of DENV serotypes but similar genotype distribution, where the infecting viruses were closely related with Indonesian endemic viruses isolated previously.
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.
Full Text Available Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA and matrix (M genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.
Full Text Available Starting in late November 2010, the H5N1 highly pathogenic avian influenza (HPAI virus was isolated from many types of wild ducks and raptors and was subsequently isolated from poultry in Korea. We assessed the genetic and pathogenic properties of the HPAI viruses isolated from a fecal sample from a mandarin duck and a dead Eurasian eagle owl, the most affected wild bird species during the 2010/2011 HPAI outbreak in Korea. These viruses have similar genetic backgrounds and exhibited the highest genetic similarity with recent Eurasian clade 188.8.131.52 HPAI viruses. In animal inoculation experiments, regardless of their originating hosts, the two Korean isolates produced highly pathogenic characteristics in chickens, ducks and mice without pre-adaptation. These results raise concerns about veterinary and public health. Surveillance of wild birds could provide a good early warning signal for possible HPAI infection in poultry as well as in humans.
Pedersen, Kerri; Marks, David R; Arsnoe, Dustin M; Afonso, Claudio L; Bevins, Sarah N; Miller, Patti J; Randall, Adam R; DeLiberto, Thomas J
Since their introduction to the United States in the late 19th century, mute swans (Cygnus olor) have become a nuisance species by causing damage to aquatic habitats, acting aggressively toward humans, competing with native waterfowl, and potentially transmitting or serving as a reservoir of infectious diseases to humans and poultry. In an effort to investigate their potential role as a disease reservoir and to establish avian health baselines for pathogens that threaten agricultural species or human health, we collected samples from 858 mute swans and tested them for avian paramyxovirus serotype 1 (APMV-1), avian influenza virus (AIV), and Salmonella spp. when possible. Our results indicate that exposure to APMV-1 and AIV is common (60%, n = 771, and 45%, n = 344, antibody prevalence, respectively) in mute swans, but detection of active viral shedding is less common (8.7%, n = 414, and 0.8%, n = 390, respectively). Salmonella was isolated from three mute swans (0.6%, n = 459), and although the serovars identified have been implicated in previous human outbreaks, it does not appear that Salmonella is commonly carried by mute swans.
Bauer, Katja; Richter, Martina; Wutzler, Peter; Schmidtke, Michaela
In the flu season 2005/2006 amantadine-resistant human influenza A viruses (FLUAV) of subtype H3N2 circulated in Germany. This raises questions on the neuraminidase inhibitor (NAI) susceptibility of FLUAV. To get an answer, chemiluminescence-based neuraminidase inhibition assays were performed with 51 H1N1, H1N2, and H3N2 FLUAV isolated in Germany from 2001 to 2005/2006. According to the mean IC(50) values (0.38-0.91 nM for oseltamivir and 0.76-1.13 nM for zanamivir) most H1N1 and H3N2 FLUAV were NAI-susceptible. But, about four times higher zanamivir concentrations were necessary to inhibit neuraminidase activity of H1N2 viruses. Two H1N1 isolates were less susceptible to both drugs in NA inhibition as well as virus yield reduction assays. Results from sequence analysis of viral hemagglutinin and neuraminidase genes and evolutionary analysis of N2 gene revealed (i) different subclades for N2 in H1N2 and H3N2 FLUAV that could explain the differences in zanamivir susceptibility among these viruses and (ii) specific amino acid substitutions in the neuraminidase segment of the two less NAI-susceptible H1N1 isolates. One H3N2 was isolate proved to be a mixture of a NA deletion mutant and full-length NA viruses.
Cho, Ying-Chun; Chiu, Nan-Chang; Lu, Chun-Yi; Huang, Daniel Tsung-Ning; Huang, Fu-Yuan; Chang, Luan-Yin; Huang, Li-Min; Chi, Hsin
After the introduction of 13-valent pneumococcal conjugate vaccine (PCV13) against Streptococcus pneumoniae, public health officials in Taiwan monitored a decline in circulating vaccine serotypes and the emergence of nonvaccine serotypes in children with invasive pneumococcal disease. A gradually expanded PCV13 national immunization program was launched in 2013 in Taiwan. Here, we evaluate the changes in the distribution of pneumococcal serotypes and antimicrobial nonsusceptibility in children during the evolution of vaccination policy. S. pneumoniae isolates from children with pneumococcal disease were collected and serotyped from 2010 to 2015 in northern Taiwan. PCVs were administered at the recipients' expense between 2010 and 2012, and then PCV13 was partially reimbursed by the government beginning in 2013. The distribution and diversity of serotypes were analyzed along with their antimicrobial susceptibilities. Among a total of 498 isolates, the proportion of invasive pneumococcal disease isolates declined (47.1%-10.6%) during the study period, and serotype diversity increased after 2011. Between 2010 and 2012, the dominant serotypes were 19A, 19F, 3, 6B and 14, and serotype 19A rose from 44.1% to 57.5%. Serotypes 19A, 15A, 19F and 15B were more prevalent from 2013 to 2015, and serotype 19A decreased from 42.1% to 4.5%. Serotypes 19F and 15A became the most commonly detected serotypes in 2015. Overall, PCV13 additional serotypes were reduced by 80% (P program is effective against pneumococcal disease in Taiwanese children, mainly by reducing PCV13 additional serotypes.
A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.
Full Text Available Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs. Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII, which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs. These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent
Full Text Available Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR and TDR-related hemolysin (TRH genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST analysis, and 48 sequence types (STs were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17 and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these environmental pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.
Budziszewska, Marta; Obrepalska-Steplowska, Aleksandra; Wieczorek, Przemysław; Pospieszny, Henryk
A new virus was isolated from greenhouse tomato plants showing symptoms of leaf and apex necrosis in Wielkopolska province in Poland in 2003. The observed symptoms and the virus morphology resembled viruses previously reported in Spain called Tomato torrado virus (ToTV) and that in Mexico called Tomato marchitez virus (ToMarV). The complete genome of a Polish isolate Wal'03 was determined using RT-PCR amplification using oligonucleotide primers developed against the ToTV sequences deposited in Genbank, followed by cloning, sequencing, and comparison with the sequence of the type isolate. Phylogenetic analyses, performed on the basis of fragments of polyproteins sequences, established the relationship of Polish isolate Wal'03 with Spanish ToTV and Mexican ToMarV, as well as with other viruses from Sequivirus, Sadwavirus, and Cheravirus genera, reported to be the most similar to the new tomato viruses. Wal'03 genome strands has the same organization and very high homology with the ToTV type isolate, showing only some nucleotide and deduced amino acid changes, in contrast to ToMarV, which was significantly different. The phylogenetic tree clustered aforementioned viruses to the same group, indicating that they have a common origin.
Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li
The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.
Wedderkopp, A.; Nielsen, E.M.; Pedersen, Karl
During the period January 1998-December 2001, all Danish broiler flocks were monitored bacteriologically for thermophilic campylobacters and isolates were stored at -80 degreesC. Six neighbouring broiler farms in a small community were selected for detailed examination of all Campylobacter jejuni...... isolated (n = 180) from these farms during 1998-2000 using Penner serotyping and pulsed-field gel electrophoresis (PFGE). The area and the farms were selected according to their prevalence of campylobacter so that both farms with low and high frequencies of campylobacter positive flocks were included...... in the study. The frequency of campylobacter positive flocks on the six farms ranged from 24.5 to 72.7%. One hundred and eighty of the isolates were C. jejuni (included in this study), 14 isolates were C. coli whereas 7 isolates belonged to other species but were not further identified. By serotyping of all C...
Kong, Wei Li; Huang, Liang Zong; Qi, Hai Tao; Cao, Nan; Zhang, Liang Quan; Wang, Heng; Guan, Shang Song; Qi, Wen Bao; Jiao, Pei Rong; Liao, Ming; Zhang, Gui Hong
In China H3N2 and H1N1 swine influenza viruses have been circulating for many years. In January 2010, before swine were infected with foot and mouth disease in Guangdong, some pigs have shown flu-like symptoms: cough, sneeze, runny nose and fever. We collected the nasopharyngeal swab of all sick pigs as much as possible. One subtype H1N2 influenza viruses were isolated from the pig population. The complete genome of one isolate, designated A/swine/Guangdong/1/2010(H1N2), was sequenced and compared with sequences available in GenBank. The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. HA, NP, M and NS were shown to be closely to swine origin. PB2 and PA were close to avian origin, but NA and PB1were close to human origin. It is a result of a multiple reassortment event. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially before the emergence of highly pathogenic FMDs in pigs in Guangdong.
Full Text Available Abstract In China H3N2 and H1N1 swine influenza viruses have been circulating for many years. In January 2010, before swine were infected with foot and mouth disease in Guangdong, some pigs have shown flu-like symptoms: cough, sneeze, runny nose and fever. We collected the nasopharyngeal swab of all sick pigs as much as possible. One subtype H1N2 influenza viruses were isolated from the pig population. The complete genome of one isolate, designated A/swine/Guangdong/1/2010(H1N2, was sequenced and compared with sequences available in GenBank. The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. HA, NP, M and NS were shown to be closely to swine origin. PB2 and PA were close to avian origin, but NA and PB1were close to human origin. It is a result of a multiple reassortment event. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV surveillance, especially before the emergence of highly pathogenic FMDs in pigs in Guangdong.