Genomic sequence of an avian paramyxovirus type 1 strain isolated from Muscovy duck (Cairina moschata) in China

The complete sequence of an avian paramyxovirus type 1 (APMV-1) strain, FP1/02, isolated from Muscovy duck in China, was determined. Sequence analysis indicated that the complete genome of strain FP1/02 contained 15,192 nucleotides (nt), following the rule of six. The genome contained an extra 6-nt insertion in the non-coding region of the NP gene when compared with other APMV-1 strains, such as strains La Sota and Beaudette C. The cleavage site of the F protein was (112)R-R-Q-K-R↓F(117), indicating that the FP1/02 strain was virulent, but the morbidity and mortality varied with the species of duck. Genotypic analysis based on the F gene revealed that APMV-1 FP1/02 was a member of genotype VII. Phylogenetic analysis showed that the FP1/02 strain shared high identity with other APMV-1 strains such as ZJ1, SF02 and NA-1 isolated from geese.     

A rapid molecular strategy for early detection and characterization of Vietnamese foot-and-mouth disease virus serotypes O, A, and Asia 1

A one-step RT-PCR method using newly designed primers VN-VP1F/VN-VP1R targeting the full VP1 capsid protein-coding gene, combined with direct sequencing of its PCR product, has been developed successfully for universal detection and characterization of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The one-step RT-PCR amplified 821-bp dsDNA products covering the entire VP1 gene of FMDV serotypes O, A, and Asia 1. The obtained dsDNA products were suitable for direct sequencing, cloning, and other molecular epidemiology studies of Vietnamese FMDV strains, which eliminated the need for cell culture and virus purification. This one-step RT-PCR system was applied to detect and characterize 55 field FMDV strains, including 34 serotype O, 17 serotype A, and 4 serotype Asia 1 isolates collected from endemic outbreaks in Vietnam from 2005 to 2010. Interestingly, the PCR products obtained from the present PCR method could be used as DNA templates for the second PCR typing method using serotypes O, A, and Asia 1-specific primers (Le et al., 2011). The use of the second PCR amplification increased markedly the sensitivity of the test for FMDV detection. The present RT-PCR method promises to be an effective tool for molecular epidemiological studies of FMD in Vietnam.     

Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread

Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.     

Molecular epidemiology of enzootic rabies in California.

BACKGROUND: Molecular characterization of rabies virus has been used to trace spillover transmission from reservoir species to non-reservoir animals and humans (molecular epidemiology), and to monitor emergence of specific strains of the virus into new species and geographic areas (molecular surveillance). OBJECTIVES: To characterize the enzootic strains of rabies virus in California wildlife for epidemiological investigation of transmission to non-reservoir animals and humans. STUDY DESIGN: Molecular characterization was performed on rabies strains from 213 bats, 276 terrestrial animals and one human case, by RT-PCR amplification of the viral nucleocapsid (N) gene followed by Dde I digestion and restriction endonuclease analysis (REA). Brain material from 88 terrestrial animals and 74 bats was stained with a panel of 20 monoclonal antibodies (MABs) directed against the N protein. In order to characterize rabies from very small quantities of brain tissue a nested RT-PCR was developed and evaluated for sensitivity of rabies detection. RESULTS AND CONCLUSIONS: Enzootic terrestrial rabies in California is confined to the Central Valley, the western slope of the Sierra Nevada, and the Central and Northern Pacific Coast Ranges. No terrestrial reservoirs were identified south of the Tehachapi Mountains or east of the Sierra Nevada. Bat strains accounted for rabies in terrestrial animals in these regions. Among terrestrial rabies strains REA identified ten genotypes that segregated geographically and were associated with skunk and fox populations from distinct locations. Co-circulation of three genotypes occurred in Placer County, which had the highest incidence of rabies in skunks. In regions with terrestrial enzootic rabies, the strain from that region accounted for 73% of spillover cases. Bat strains accounted for the remaining 27%. Among terrestrial animals MABs identified three predominant patterns. In rabies strains from bats REA identified ten major and two minor patterns primarily associated with genus and species of bat. Sharing of strains between species was observed. An additional sixteen unclassified REA bat patterns were observed in only one or two individuals of various species. MABs identified four major patterns in bats associated with genus and species of bat with considerable variability. The bat strains most frequently detected in spillover cases throughout California were from the California myotis (Myotis californicus) and Mexican free-tailed bat (Tadarida brasiliensis). Nested RT-PCR increased the detection level of rabies virus 100,000-fold, to 0.03 TCID50.     

Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America

Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.     

Real-time RT-PCR assay for rapid and specific detection of classical swine fever virus: comparison of SYBR Green and TaqMan MGB detection methods using novel MGB probes

Real-time PCR is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. The basic principle is the recurring measurement of a fluorescent signal, which is proportional to the amount of amplification product. In our trial two detection systems were tested for classical swine fever virus (CSFV) detection and for its discrimination from other pestiviruses; non-specific dsDNA-binding dye SYBR Green and specific fluorogenic TaqMan MGB probes. Real-time RT-PCR assays were evaluated for diagnostic sensitivity and specificity by different pestiviral reference and field strains. With both approaches, SYBR Green and TaqMan probes, respectively, all of the CSFV strains isolated on cell culture were detected and also clearly distinguished from other pestiviruses. However, the established one-step real-time TaqMan RT-PCR assay was shown to be more appropriate for pestivirus quantitation, it reduces the risk of contamination and is less time consuming.     

Development of one-step multiplex RT-PCR method for simultaneous detection and differentiation of foot-and-mouth disease virus serotypes O, A, and Asia 1 circulating in Vietnam

A one-step multiplex RT-PCR method using new primers was developed for the simultaneous detection and differentiation of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The RT-PCR method used a cocktail of one universal minus-sense primer designed in the 2B gene and three serotype-specific plus-sense primers designed in the hypervariable regions of the capsid VP1 coding gene of FMDV. These serotype-specific primer pairs amplified 658, 535, and 427 bp PCR products corresponding to FMDV serotypes O, Asia 1, and A, respectively. In this study, six well-characterized FMDV strains belonging to serotypes O, A, and Asia 1 were used as reference strains for validation tests. Among these six FMDV strains were three vaccine strains for type O (O1/Manisa), A (A22/Iraq), and Asia 1 (As1/Shamir/89). The other reference strains included one pandemic strain of FMDV serotype Asia 1 (Asia1/MOG/05) and two pandemic strains of FMDV serotype O (O/UKG/34/2001 and O/SKR/2000). For field application, 37 positive-clinical samples and 18 cell culture-adapted viruses belonging to serotypes O, A, and Asia 1, as confirmed previously by antigen ELISA for FMDV detection, were used. The present method showed high sensitivity and specificity and can be adapted for detection and typing of FMDV serotypes O, A, and Asia 1 circulating in Vietnam.     

Comparison of strain typing results for Clostridium difficile isolates from North America

Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.     

Genomic characterization of two avian paramyxovirus type 2 isolates from chickens in China

The complete genome sequences were determined for avian paramyxovirus type 2 (APMV-2) strains F8 and NK isolated from chickens in China. Both strains had a genome of 14,904 nucleotides (nt) in length, which followed the “rule of six”. Each genome consisted of six genes in the order 3′-N-P-M-F-HN-L-5′, with a 55-nt leader at the 3′ end and a 154-nt trailer at the 5′ end. Sequence alignment and phylogenetic analysis showed that APMV-2 strains F8 and NK shared the highest sequence identity with APMV-2 prototype strain Yucaipa, being classified in the same subgroup as strains Yucaipa, England and Kenya, while strain Bangor represented another subgroup of APMV-2. Among the APMVs, APMV-2 strains F8 and NK exhibited a closer evolutionary relationship with APMV-7 and APMV-8 representative strains.     

Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

Rapid and reliable detection and classification of infectious bursal disease viruses (IBDVs) is of crucial importance for disease surveillance and control. This study presents the development and validation of a real-time RT-PCR assay to detect and discriminate very virulent (vv) from non-vv (classic and variant) IBDV strains. The assay uses two fluorogenic, minor groove-binding (MGB) TaqMan probes targeted to a single nucleotide polymorphism (SNP) embedded in a highly conserved genomic region. The analytical sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 10(2) and 10(8) standard RNA copies per reaction. Good reproducibility was also detected, with intra- and inter-assay coefficients of variation ranging from 0.13% to 2.23% and 0.26% to 1.92%, respectively. The assay detected successfully all the assessed vv, classical, and variant field and vaccine strains and correctly discriminated all vvIBDV strains from non-vvIBDV strains. Other common avian RNA viruses tested negative, indicating high specificity of the assay. The high sensitivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation.