Screening interferon antagonists from accessory proteins encoded by P gene for immune escape of Caprine parainfluenza virus 3.

The Caprine parainfluenza virus 3 (CPIV3) is a novel Paramyxovirus that is isolated from goats suffering from respiratory diseases. Presently, the pathogenesis of CPIV3 infection has not yet been fully characterized. The Type I interferon (IFN) is a key mediator of innate antiviral responses, as many viruses have developed strategies to circumvent IFN response, whether or how CPIV3 antagonizes type I IFN antiviral effects have not yet been characterized. This study observed that CPIV3 was resistant to IFN-α treatment and antagonized IFN-α antiviral responses on MDBK and goat tracheal epithelial (GTE) cell models. Western blot analysis showed that CPIV3 infection reduced STAT1 expression and phosphorylation, which inhibited IFN-α signal transduction on GTE cells. By screening and utilizing specific monoclonal antibodies (mAbs), three CPIV3 accessory proteins C, V and D were identified during the virus infection process on the GTE cell models. Accessory proteins C and V, but not protein D, was identified to antagonize IFN-α antiviral signaling. Furthermore, accessory protein C, but not protein V, reduced the level of IFN-α driven phosphorylated STAT1 (pSTAT1), and then inhibit STAT1 signaling. Genetic variation analysis to the PIV3 accessory protein C has found two highly variable regions (VR), with VR2 (31-70th aa) being involved in for the CPIV3 accessory protein C to hijack the STAT1 signaling activation. The above data indicated that CPIV3 is capable of inhibiting IFN-α signal transduction by reducing STAT1 expression and activation, and that the accessory protein C, plays vital roles in the immune escape process.

Epidemiological investigation and genomic characterization of Caprine herpesvirus 1 from goats in China.

Caprine herpesvirus 1 (CpHV-1) is a member of the alpha subfamily of herpersviruses, and is responsible for genital lesions and latent infections in goat population worldwide. Here, we describe goats suffered severe respiratory diseases caused by alphaherpesvirus during 2013 to 2014 in Jiangsu province of China. CpHV-1 was detected out by PCR with a prevalence of 21.1% (40/190), among which three novel CpHV-1 strains were firstly identified and isolated in China. Phylogenetic analysis of glycoprotein B (gB) gene revealed that these new viruses were closely clustered with CpHV-1 strain E/CH. The isolate JSHA1405 was further studied by transmission electron microscopy, and displayed typical herpesvirus morphology. Then, for the first time, complete viral genome of JSHA1405 was sequenced by Illumina Hiseq and third-generation sequencing technology. The viral genome is 134,617 bp in length and the genome characteristics were deeply analyzed. 69 open reading frames were predicted and annotated, which was less than that of BoHV-1. Phylogenetic analysis of the complete genome revealed that JSHA1405 was classified into the same branch with previous CpHV-1 strains as well. Moreover, the pathogenicity test is further evidence that JSHA1405 strain induced obvious symptoms of high fever and nasal discharge in infected goats, consistent with clinical manifestations. This is the first report about isolation and identification of CpHV-1 in China and the first characterization of CpHV-1 genome structure. The research also provides a basis for understanding the characteristics, viral genome and pathogenicity of the virus.

The novel caprine parainfluenza virus type 3 showed pathogenicity in Guinea pigs.

Caprine parainfluenza virus type 3 (CPIV3) is one of the important viral respiratory tract agents in goats. The pathogenicity of CPIV3 has been examined in goats but it has not been explored in other laboratory animals. In the present study, an experimental infection of guinea pigs with CPIV3 was performed. The virus-inoculated guinea pigs displayed clinical signs related to the respiratory disease at 2-12 days post inoculation (dpi). Five infected guinea pigs died during 2 and 7 dpi. Apparent gross pneumonic lesions including consolidation and congestion in one or more lung lobes were observed in necropsied and dead animals. Histo-pathological changes in lungs including expansions of the alveolar interstitium, congestion, macrophage infiltration and compensatory emphysema were also observed. Virus was detectable at 2-10 dpi, 2-10 dpi and 2-7 dpi, as detected by virus isolation, real-time RT-PCR and immunohistochemistry staining, respectively. Viremia was also confirmed after CPIV3 infection during 3-7 dpi. The severe pathological lesions and highest viral load were observed before 7 dpi. Viral specific hemagglutination inhibition and neutralizing antibodies were produced from 7 dpi and 10 dpi, respectively, which related to the clearance of virus. The results present here indicated that guinea pig could be an ideal laboratory animal model for CPIV3 studies in the future.

Transcriptome analysis reveals differential immune related genes expression in bovine viral diarrhea virus-2 infected goat peripheral blood mononuclear cells (PBMCs).

BACKGROUND: Bovine viral diarrhea virus (BVDV) is an economically important viral pathogen of domestic and wild ruminants. Apart from cattle, small ruminants (goats and sheep) are also the susceptible hosts for BVDV. BVDV infection could interfere both of the innate and adaptive immunity of the host, while the genes and mechanisms responsible for these effects have not yet been fully understood. Peripheral blood mononuclear cells (PBMCs) play a pivotal role in the immune responses to viral infection, and these cells were the target of BVDV infection. In the present study, the transcriptome of goat peripheral blood mononuclear cells (PBMCs) infected with BVDV-2 was explored by using RNA-Seq technology. RESULTS: Goat PBMCs were successfully infected by BVDV-2, as determined by RT-PCR and quantitative real-time RT-PCR (qRT-PCR). RNA-Seq analysis results at 12 h post-infection (hpi) revealed 499 differentially expressed genes (DEGs, fold-change ≥ ± 2, p < 0.05) between infected and mock-infected PBMCs. Of these genes, 97 were up-regulated and the remaining 352 genes were down-regulated. The identified DEGs were found to be significantly enriched for locomotion/ localization, immune response, inflammatory response, defense response, regulation of cytokine production, etc., under GO enrichment analysis. Cytokine-cytokine receptor interaction, TNF signaling pathway, chemokine signaling pathway, etc., were found to be significantly enriched in KEGG pathway database. Protein-protein interaction (PPI) network analysis indicated most of the DEGs related to innate or adaptive immune responses, inflammatory response, and cytokine/chemokine-mediated signaling pathway. TNF, IL-6, IL-10, IL-12B, GM-CSF, ICAM1, EDN1, CCL5, CCL20, CXCL10, CCL2, MAPK11, MAPK13, CSF1R and LRRK1 were located in the core of the network and highly connected with other DGEs. CONCLUSIONS: BVDV-2 infection of goat PBMCs causes the transcription changes of a series of DEGs related to host immune responses, including inflammation, defense response, cell locomotion, cytokine/chemokine-mediated signaling, etc. The results will be useful for exploring and further understanding the host responses to BVDV-2 infection in goats.

Epidemiological investigation and phylogenetic analysis of caprine parainfluenza virus type 3 in sheep of China.

Caprine parainfluenza virus type 3 (CPIV3) is a new member of the Respirovirus genus in the Paramyxivirudae family, mainly causing respiratory disease. Up to now, accumulating evidence has focused on CPIV3 infection in goats, with little understood about its epidemiology in sheep. To that end, we collected 1,163 sheep sera samples from nine provinces/autonomous regions in 2012 and 1,863 samples from six provinces/autonomous regions during 2016-2017, with serological prevalence of 50.3% (95% CI: 47.5, 53.3) and 64.9% (95% CI: 62.9, 67.2), respectively. Pathogenic detection by qRT-PCR was also performed on serum samples collected in 2016-2017, and the percentage of CPIV3 positive samples was 21.5% (95% CI: 19.7, 23.5). Sequence alignment and phylogenetic analyses revealed 11 novel CPIV3 strains based on the M gene sequences. The M gene and full-length sequences of CPIV3 strains derived from sheep shared high nucleotide similarity with goat-origin strains, indicating conserved genome characteristics between the viruses. Furthermore, sequence evolution and epidemiological analysis show that CPIV3 is widespread throughout China. This is the first report describing CPIV3 infection in sheep in China, showing a high sero-prevalence and contributes to the assessment of the epidemiology of CPIV3 in China.

Analysis of microRNAs Expression Profiles in Madin-Darby Bovine Kidney Cells Infected With Caprine Parainfluenza Virus Type 3.

Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.

Porcine Viperin protein inhibits the replication of classical swine fever virus (CSFV) in vitro.

BACKGROUND: Classical swine fever virus (CSFV) is the causative pathogen of Classical swine fever (CSF), a highly contagious disease of swine. Viperin is one of the hundreds of interferon-stimulated genes (ISGs), and possesses a wide range of antiviral activities. The aim of this study was to explore whether porcine Viperin has the anti-CSFV activity. METHOD: The influences of CSFV infection on Viperin expression and Newcastle disease virus (NDV)/Pseudorabies virus (PRV)-induced Viperin expression were examined in 3D4/21 cells and porcine peripheral blood mononuclear cells (PBMCs). Porcine Viperin gene was amplified to generate cell line PK-Vi over-expressing Viperin. CSFV was inoculated in the cell lines and viral load was detected by qRT-PCR, virus titration and Western blot. The influence of Viperin expression on CSFV binding, entry and release in the cells was also examined. The co-localization of Viperin with CSFV and its proteins (E2, NS5B) was determined by confocal laser scanning microscopy test. Co-IP assay was performed to check the interaction of Viperin with CSFV proteins. RESULTS: CSFV infection could not induce Viperin expression in vitro while significantly inhibiting NDV/PRV-induced Viperin expression at 12, 24 and 48 h post infection (hpi; P < 0.05). The proliferation of CSFV in PK-Vi was significantly inhibited at 24, 48 and 72 hpi (P < 0.05), comparing with control cells (PK-C1 expressing EGFP). Virus in both cell culture supernatants and cell pellets were reduced equally. CSFV binding and entry in the cells were not interfered by Viperin expression. These results indicated its anti-CSFV function occurred during the genome and/or protein synthesis step. Confocal laser scanning microscopy test showed the Viperin-EGFP protein co-localized with CSFV E2 protein in CSFV infected PK-Vi cells. Further experiments indicated that Viperin protein co-localized with E2 and NS5B proteins of CSFV in the transfected 293 T cells. Furthermore, Co-IP assay confirmed the interaction of Viperin with E2 protein, but not NS5B. CONCLUSION: Porcine Viperin effectively inhibited CSFV replication in vitro, potentially via the interaction of Viperin with CSFV E2 protein in cytoplasm. The results provided foundation for further studies of the interaction of Viperin with CSFV and other viruses.

Development of a blocking ELISA for Caprine parainfluenza virus type 3.

Caprine parainfluenza virus type 3 (CPIV3) is a novel pathogen mainly causing respiratory diseases in goats. At present, there are no high throughput and rapid testing methods available for epidemiological investigation. In this study, we designed a modified method for selection of hybridomas that secrete monoclonal antibodies (mAb) specific for CPIV3. The monoclonal antibodies were obtained by combination of indirect enzyme-linked immunosorbent assay (iELISA) and blocking ELISA (bELISA). The technique was efficient to determine each mAb with specificity and sensitivity. One bELISA was validated for the serological diagnosis of CPIV3. After optimization conditions were established, a total of 205 reference goat sera were tested in parallel by bELISA and by virus neutralization (VN) for their relative performances. The cut-off point was ultimately defined as 33.6% by ROC curve analysis. The bELISA specificity and sensitivity were 99.2% and 98.7%, respectively, and agreement with the VN test was >99.0%. Furthermore, testing another 2919 goat sera by bELISA demonstrated 39.3% prevalence in the goat population, more sensitive than HI detection. This new bELISA would offer higher throughput, sensitivity, and specific detection for CPIV3, and will be of great value not only for surveillance, but also for monitoring the efficiency of vaccination programs in the future.

Primary surveys on molecular epidemiology of bovine viral diarrhea virus 1 infecting goats in Jiangsu province, China.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a pathogen of domestic and wildlife animals worldwide and is associated with several diseases. In China, there are many reports about genotyping of BVDV strains originated from cattle and pigs, and some of them focused on the geographical distributions of BVDV. Currently, the goat industry in Jiangsu province of China is under going a rapid expansion. Most of these goat farms are backyard enterprises and in close proximity to pig and cattle farms. However, there was very limited information about BVDV infections in goats. The objective of this study was to assess the frequency of BVDV infections of goats, the relationship of these infections to clinical signs and determine what BVDV genotypes are circulating in Jiangsu province. RESULTS: From 236 goat sera collected from six regions in Jiangsu province between 2011 and 2013, BVDV-1 was identified in 29 samples from the five regions by RT-PCR. The BVDV-1 infections occurred with/without clinical signs. Eight different BVDV-1 strains were identified from these positive samples based on the 5'-untranslated region (5'-UTR) sequences, and further clustered into four BVDV-1 subtypes on the phylogenetic analysis. Three were BVDV-1b, two BVDV-1m, two BVDV-1o, and one BVDV-1p, respectively. CONCLUSIONS: To our knowledge, this is the first report to investigate the occurrence of BVDV and the genotypes of BVDV infecting goats in China. The results indicated that BVDV-1 infections were indeed present and the viruses were with genetic variations in Chinese goat herds. The information would be very useful for prevention and control of BVDV-1 infections in China.

Rapid detection of novel caprine parainfluenza virus type 3 (CPIV3) using a TaqMan-based RT-qPCR.

Parainfluenza virus type 3 (PIV3) is one of the most important respiratory pathogens for humans and many animals. A novel caprine PIV3 (CPIV3) was recently identified and isolated from Chinese goat flocks with respiratory disease. In order to develop rapid and sensitive methods for CPIV3 detection in infected goats, a TaqMan RT-qPCR was established in this study based on the primers and probe designed to amplify a 150 nucleotide-long region located within the M gene of the virus. The method was able to detect about 1.0×10(1) DNA copies/µL with an efficiency of 99.6% and a R(2) value of 0.997. There were no cross-reaction observed using this technique against peste des petits ruminants virus (PPRV), border disease virus (BDV), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). One hundred and fourteen samples, including nasal swabs, feces swabs, sera, hearts, livers, spleens, lungs, kidneys, tracheas and hilar lymph nodes (HLNs) from six challenged goats, were evaluated by this technique. Using TaqMan RT-qPCR, CPIV3 was positively detected in 51 of 114 samples (44.74%), which was higher than RT-PCR (27.19%, 31/114) and virus isolation (14.9%, 17/114), respectively. The method also gave higher positive detection rate (35%, 42/120) than RT-PCR (28.33%, 34/120) from clinical samples. These data indicated that this method could be used for faster and more accurate monitoring of viral load, disease progression and vaccination efficacy of CPIV3 in goat flocks.

Pathogenicity and horizontal transmission studies of caprine parainfluenza virus type 3 JS2013 strain in goats.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals, but goat infection has been rarely reported. In 2014, one novel PIV3 strain was first isolated from goats suffered respiratory diseases in Jiangsu and Anhui provinces of eastern China and named as caprine PIV3 (CPIV3) JS2013. In order to systematically evaluate the pathogenicity and horizontal transmission ability of this new virus, experimental infection of goats with the CPIV3 strain was done. The virus-inoculated goats (challenge control (CC) group) displayed coughing and nasal discharges from 3days post infection (dpi) and lasted for about 2 weeks. Two goats in group CC showed fever between 7 and 12dpi. As detected by a TaqMan real time quantitative RT-PCR (qRT-PCR), viremia was detected during 3-11dpi, peaked at 6dpi; and virus shedding from nasal discharge and faeces were confirmed during 3-21dpi and 4-21dpi, respectively. Virus-specific HI antibodies and neutralizing antibodies (NAs) became positive since 7dpi and 14dpi; peaked at 14dpi and 28dpi, respectively; and lasted at least 70days. Pathological lesions were mainly found on the lungs and tracheas. In addition, viruses were also detected in part of the tracheal secretion and lung samples, and the viral load in tracheal secretion was higher than that in lungs. Goats in horizontal infected group (hCC, kept in different cages in the same house with CC group) showed to be horizontally infected, with slightly milder clinical signs and pathological changes; and slightly shorter period of viremia and virus shedding. This was the first report of the detailed pathogenicity characterization of the novel CPIV3 and demonstrated its horizontal transmission ability. The results would be helpful for further studies on the preventive and control strategies for CPIV3 infections.

Analysis on the complete genome of a novel caprine parainfluenza virus 3.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals. One unique caprine PIV3 (CPIV3) strain named JS2013 was isolated in Chinese goat flocks with respiratory diseases in 2013. Now, the complete genome sequence of the strain JS2013 had been determined. A total of 15 overlapping DNA clones, covering the entire genome of the virus, were obtained by primer walking RT-PCR. The sequences of the 3' and 5' termini of the viral genome were amplified by 3' and 5' RACE. The viral genome was 15,618 nucleotides (nt) in length, which was consisted of six genes in the order 5'-leader-N-P/C/V-M-F-HN-L-tailer-3'. The junction sequences between two genes were highly conserved gene start and stop signal sequences, and trinucleotide intergenic regions (IGR) similar to those of other reported PIV3 strains. Phylogenetic analysis based on the complete genomes of JS2013 with other strains of genus Respirovirus demonstrated that the JS2013 obviously differed from HPIV1, Sendai virus, HPIV3 and other reported BPIV3 genotypes. Further analysis of HN genes of JS2013 along with two more CPIV3 strains isolated later indicated that CPIV3 strains formed a separate cluster. The results presented here suggested that CPIV3 is a new member of the genus Respirovirus.

The swine CD81 enhances E2-based DNA vaccination against classical swine fever.

Classical swine fever (CSF) is a highly contagious and economically important viral disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV can induce neutralizing antibodies and protective immunity, and is widely used for novel vaccine development. The objective of this study was to explore whether a tetraspanin molecule CD81 could improve the immune responses of an E2-based DNA vaccine. Plasmids pVAX-CD81, pVAX-E2 and pVAX-CD81-E2 were constructed and the expression of target proteins was confirmed in BHK-21 cells by indirect immunofluorescence assay. BALB/c mice were divided into 5 groups and immunized with different plasmids (pVAX-E2, pVAX-CD81-E2, pVAX-E2+pVAX-CD81, pVAX-CD81 and PBS) three times with two weeks interval. The results showed that the introduction of CD81 promoted higher humoral and cellular immune responses than E2 expression alone (P<0.05). In addition, immunization with pVAX-CD81-E2 induced stronger immune responses than pVAX-E2+pVAX-CD81. Furthermore, four groups of pigs were immunized with pVAX-E2, pVAX-CD81-E2, pVAX-CD81 and PBS, respectively. Humoral and cellular immune responses detection showed similar results with those in mice. Compared to pVAX-E2, pVAX-CD81-E2 induced higher titers of neutralizing antibodies after viral challenge and conferred stronger protection. These results confirmed the capacity of swine CD81 enhancing the humoral and cellular responses with an adjuvant effect on CSFV DNA vaccine. This is the first report demonstrating the adjuvant effect of CD81 to enhance the DNA vaccination for swine pathogen.

Chinese border disease virus strain JSLS12-01 infects piglets and down-regulates the antibody responses of classical swine fever virus C strain vaccination.

During 2012 and 2013, several border disease virus (BDV) strains were identified from Chinese goat and sheep herds. At the same time, pigs from the same areas were found to be seropositive to BDV by ELISA, without showing clinical signs (unpublished data). To examine the susceptibility of pigs to the Chinese BDV strains, BDV isolate JSLS12-01, isolated from naturally infected sheep, was used to infect pigs. Antibody responses, viremia, clinical signs and pathological changes of the infected animals were examined. It confirmed that the current BDV strain could infect the domestic pigs, the animals showed viremia during 4 to 14 days post infection (dpi) and sero-conversion from 14dpi; no clinical and pathological changes were observed. In addition, CSFV maternal antibody did not influence BDV infection. Subsequently, pigs were infected with the BDV isolate and vaccinated with Hog cholera lapinized virus (HCLV) 21 days later to determine the effect of BDV infection on antibody induction of CSFV vaccination. The specific CSFV antibody and neutralizing antibody titers of the BDV infected group remained negative after the primary vaccination. Even after the boost vaccination, they were still significantly lower than those of the uninfected groups (p<0.05). These results indicated that BDV infection could down-regulate the antibody responses of CSFV C-strain vaccination. It should be paid attention that BDV prevalence in pig herds and in live vaccines might hamper the vaccination of CSF.

Characterization of one sheep border disease virus in China.

BACKGROUND: Border disease virus (BDV) causes border disease (BD) affecting mainly sheep and goats worldwide. BDV in goat herds suffering diarrhea was recently reported in China, however, infection in sheep was undetermined. Here, BDV infections of sheep herds in Jiangsu, China were screened; a BDV strain was isolated and identified from the sheep flocks in China. The genomic characteristics and pathogenesis of this new isolate were studied. RESULTS: In 2012, samples from 160 animals in 5 regions of Jiangsu province of China were screened for the presence of BDV genomic RNA and antibody by RT-PCR and ELISA, respectively. 44.4% of the sera were detected positively, and one slowly grown sheep was analyzed to be pestivirus RNA positive and antibody-negative. The sheep kept virus positive and antibody negative in the next 6 months of whole fattening period, and was defined as persistent infection (PI). The virus was isolated in MDBK cells without cytopathic effect (CPE) and named as JSLS12-01. Near-full-length genome sequenced was 12,227 nucleotides (nt). Phylogenetic analysis based on 5'-UTR and N(pro) fragments showed that the strain belonged to genotype 3, and shared varied homology with the other 3 BDV strains previously isolated from Chinese goats. The genome sequence of JSLS12-01 also had the highest homology with genotype BDV-3 (the strain Gifhorn). Experimental infections of sheep had mild clinical signs as depression and short-period mild fever (5 days). Viremia was detected in 1-7 days post-infection (dpi), and seroconversion began after 14 dpi. CONCLUSIONS: This study reported the genomic and pathogenesis characterizations of one sheep BDV strain, which confirmed the occurrence of BDV infection in Chinese sheep. This sheep derived BDV strain was classified as BDV-3, together with the goat derived strains in China. These results might be helpful for further understanding of BDV infection in China and useful for prevention and control of BDV infections in the future.

A novel parainfluenza virus type 3 (PIV3) identified from goat herds with respiratory diseases in eastern China.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals, but goat infection has been rarely reported. Starting in Aug 2013, goats in the Jiangsu and Anhui provinces of eastern China suffered severe respiratory diseases. In order to identify the causative agent, numerous related pathogens were tested with RT-PCR or PCR. A unique PIV3 strain was detected in most of the clinical nasal swabs or serum samples. The virus was isolated on MDBK cells and characterized by RT-PCR, nucleotide sequence analysis and hemagglutination test. The entire M and F gene coding regions, HN, 5'-UTR-N and L gene fragments were amplified using pairs of degenerate primers. Nucleotide, amino acid sequence alignments and phylogenetic analyses based on these genes indicated that the goat-derived PIV3 strain was distinct from previously reported BPIV3 genotypes and HPIV3 strains. The novel isolate, named JS2013, might be a potentially new member of the respirovirus genus. Goats were experimentally infected with JS2013 culture. The virus-inoculated goats displayed coughing and nasal discharges that were related to respiratory diseases. Viremia and virus shedding were detected during 4-10 days post-inoculation (dpi). Virus-specific HI antibodies became positive from 14 dpi. This is the first report of the detection of PIV3 from Chinese goat herds and genetic and pathogenetic characterization of the novel goat-derived PIV3.

Genome Sequence of Border Disease Virus Strain JSLS12-01, Isolated from Sheep in China.

Border disease virus (BDV) is a recognized virus in the genus Pestivirus and causes border disease (BD) in sheep and goats. Here, a novel BDV strain, JSLS12-01, was identified from sheep in Jiangsu Province, China. The complete coding sequence (CDS) was finished, which provides a better understanding of the molecular evolution of BDV isolates.

Detection of border disease virus (BDV) in goat herds suffering diarrhea in eastern China.

BACKGROUND: Border disease virus (BDV) is an important pathogen in sheep and goat production. Neither epidemiological investigation nor any reports of BDV infection was available in China. During Jan to Apr, 2012, several herd goats in Anhui and Jiangsu provinces in eastern China suffered unremitting diarrhea, with morbidity and mortality of about 28-37% and 10-15%, respectively. In the present study, sera and tissue samples from diseased goats of four farms were taken for BDV detection, isolation and identification. RESULTS: Panpesti generic primers and border disease virus (BDV)-specific primers targeting the 5'-UTR region produced RT-PCR positive bands for sera (24/28) and tissue samples (7/30). Twenty positive sera and tissue samples were inoculated onto Madin-Darby bovine kidney (MDBK) cells for virus isolation. Finally, three different strains of BDV, named AH12-01, AH12-02 and JS12/04, were successfully isolated as identified by RT-PCR using 5'-UTR and N(pro) gene primers, sequencing and electron microscopy. Sequences of 5'-UTR and N(pro) genes of them were used for phylogenetic analysis and comparison to other reference sequences available in GenBank. The results indicated AH12-01, AH12-02 and JS12/04 possess high relationship with the BDV 3 group viruses and differed with each other. CONCLUSION: This is the first detection of BDV from goats with diarrhea and confirmation of BDV infection in China.

Development and partial validation of a recombinant E2-based indirect ELISA for detection of specific IgM antibody responses against classical swine fever virus.

Detecting classical swine fever virus specific antibody responses is critical for prevention and control of CSF. In this study, a ΔE2-based indirect ELISA was developed to detect specific IgM antibodies against CSFV. The optimized conditions that were determined experimentally are: a ΔE2 antigen concentration of 0.5 µg/ml, a serum sample dilution of 1/100 incubated at 37°C for 1.5 h, and a HRP conjugated rabbit anti-pig IgM dilution of 1/50,000 incubated at 37°C for 1 h. Three hundred clinical sera were tested with ΔE2-IgM-ELISA and IDEXX ELISA and the positive rates were 77.3% (232/300) and 71.7% (215/300), respectively. Concordance rate between them was 80.3% (241/300). The 59 inconsistent sera were tested further: among the 21 IDEXX ELISA +/ΔE2-IgM-ELISA - and 38 IDEXX ELISA +/ΔE2-IgM-ELISA - samples, 17 and 24 were determined positive by virus neutralization test; 15 and 25 were tested positive by ΔE2-IgG-ELISA, respectively. In addition, the E2-specific IgM antibody response in 15 vaccinated piglets could be detected 2 weeks post-vaccination and earlier than specific IgG antibody. It increased regularly and reached high levels by 6 weeks post-vaccination. The ΔE2-IgM-ELISA could be used for clinical detection and for exploring the kinetics of IgM antibody response.

Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter.

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease in young chickens and results in considerable economic losses for the poultry industry. To suppress the replication of IBDV, two short hairpin RNAs (shRNAs) were designed for targeting the VP1 and VP2 genes of IBDV. Recombinant plasmids carrying each shRNA or two shRNAs were constructed based on vector pSilencer2.1-U6 in which the human U6 promoter was replaced with chicken U6 promoter. In chicken embryo fibroblasts, transfection with these shRNA plasmids 24h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID(50)) from 10(8.75) TCID(50)/0.1 mL to 10(3.75)-10(1.0) TCID(50)/0.1 mL. In 10-day old specific pathogen-free (SPF) chicken embryos, incubation with a mixture of IBDV B87 and a shRNA plasmid via the allantoic cavity resulted in 100% mortality and high IBDV virus titer in the control group but 25-0% mortality and near normal embryo development in the specific shRNA groups; additionally, IBDV VP1 and VP2 mRNA levels were reduced by 72-95% in the shRNA groups as compared with the control groups. When challenged with a virulent strain IBDV GX8/99, 14-day-old chickens pre-treated with the single shRNA plasmids or the dual shRNA plasmid showed approximately 70% or 90% survival at 5 days post-challenge while those pre-treated with control plasmid or saline had less than 5% survival. The current study suggests that two IBDV shRNAs expressed by a plasmid under chicken U6 promoter could effectively and synergistically reduce IBDV replication in vitro and in vivo.