Isolation and full-genome sequence of two reticuloendotheliosis virus strains from mixed infections with Marek's disease virus in China.

Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China.

First isolation of reticuloendotheliosis virus from mallards in China.

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in many avian hosts worldwide. REV infection has never been reported in mallard ducks, however. To identify REV infection in mallards, we collected 40 mallard duck samples from Jilin Province of China. In this study, the REV strain, DBYR1102, was first isolated from a mallard in China and identified by PCR, indirect immunofluorescence assay and electron microscopy. The gp90 gene and complete LTR of DBYR1102 were amplified and sequenced. Phylogenetic analysis based on gp90 genes of REV indicated that the REV strain DBYR1102 is closely related to strain HLJR0901 from northeastern China, the prairie chicken isolate APC-566, and REV subtype III, represented by chick syncytial virus. This new strain is distantly related to two other subtypes of REV, 170A and SNV. Phylogenetic analysis based on the LTR yielded information similar to that obtained with the gp90 genes. The results of this study not only expand our epidemiological understanding of REV in the wild birds of China but also demonstrate the potential role of wild waterfowl in REV transmission.

Development and application of DETECTR-based rapid detection for pathogenic Bacillusanthracis.

Bacillus anthracis (B. anthracis) is a gram-positive bacterium responsible for the acute disease anthrax. Rapid and reliable identification of pathogenic B. anthracis is important in the detection of natural infectious disease cases or bio-threats. Herein, a DNA endonuclease targeted CRISPR trans reporter (DETECTR) detection platform based on recombinase polymerase amplification (RPA) was studied. The DETECTR system targeted three sequences from B. anthracis (the BA_5345 chromosomal specific marker, the protective antigen gene pag A from pXO1 plasmid and the capsule-biosynthesis-related gene cap A from pXO2 plasmid). We developed a rapid (<40 min), easy-to-implement and accurate identification method for of B. anthracis nucleic acid with near two-copies sensitivity. The combination of tripartite primer sets is effective for the reliable identification of B. anthracis but also for fast screening of pathogenic strains. More importantly, DETECTR correctly detected simulated clinical blood samples and firstly detected positive samples collected from the location of world War-II site, preserved at north-east China (45°36'55.940″ N, 126°38'33.738″ E) with high sensitivity and specificity. Our study provides insight into the DETECTR-based detection of B. anthracis. We present a novel screening and diagnostic option for pathogenic B. anthracis that can be performed on a user-friendly portable device. Based on its proven reliability, sensitivity, specificity and simplicity, our proposed method can be readily adapted to detect pathogenic B. anthracis, anthrax and biothreats.

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model.

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE(-)/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE(-)/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.

[Construction and identification of recombinant BHV-1 expressing foot and mouth disease virus VP1 gene].

OBJECTIVE: In order to construct the recombinant bovine hepervirus-1 (BHV-1) which expressed foot and mouth disease virus (FMDV) VP1 gene, we constructed a BHV-1 gE gene transfer vector by inserting the synthetic VP1 gene of FMDV (O/China/99) under the immediate-early promoter of cytomegalovirus. METHODS: The mixtures of parental virus (BHV-1/gE(-)/LacZ+) DNA and transfer vector was transfected into bovine turbinate cells using calcium phosphate-mediated transfection. Then the propagated viruses were harvested. The recombinant BHV-1 (designated BHV-1/gE(-)/VP1) was obtained by selection for white virus plaques. RESULTS: PCR results showed that VP1 gene was successfully inserted into the genome of BHV-1/gE(-). The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting. CONCLUSION: The research provided a basis for development of BHV-1 vector vaccines for FMD and other important bovine infectious diseases.

Molecular characteristics and evolutionary analysis of a very virulent infectious bursal disease virus.

Infectious bursal disease virus (IBDV) poses a significant threat to the poultry industry. Viral protein 2 (VP2), the major structural protein of IBDV, has been subjected to frequent mutations that have imparted tremendous genetic diversity to the virus. To determine how amino acid mutations may affect the virulence of IBDV, we built a structural model of VP2 of a very virulent strain of IBDV identified in China, vvIBDV Gx, and performed a molecular dynamics simulation of the interaction between virulence sites. The study showed that the amino acid substitutions that distinguish vvIBDV from attenuated IBDV (H253Q and T284A) favor a hydrophobic and flexible conformation of ß-barrel loops in VP2, which could promote interactions between the virus and potential IBDV-specific receptors. Population sequence analysis revealed that the IBDV strains prevalent in East Asia show a significant signal of positive selection at virulence sites 253 and 284. In addition, a signal of co-evolution between sites 253 and 284 was identified. These results suggest that changes in the virulence of IBDV may result from both the interaction and the co-evolution of multiple amino acid substitutions at virulence sites.

Binding chicken Anx2 is beneficial for infection with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. The identification of host cellular molecules that bind to IBDV will improve the understanding of the underlying pathogenic mechanisms. In this study, using a virus overlay protein-binding assay (VOPBA) and mass spectrometry (MS) analysis, IBDV was found to bind chicken Anx2, a membrane protein fraction from DF-1 cells. Its interactions were further confirmed by an overlay assay. The results of an immunofluorescence assay and flow cytometry showed that Anx2 could be expressed and colocalized with IBDV on the surface of infected cells. Moreover, either the soluble recombinant Anx2 or an anti-Anx2 antibody could inhibit IBDV binding to and infection of DF-1 cells in a dose-dependent manner. The knockdown of Anx2 of DF-1 cells by small interfering RNA clearly reduced the subsequent virus yield, and overexpression of Anx2 was capable of enhancing the virus yield. These results indicate, for the first time, that binding to Anx2 is beneficial for IBDV infection.

Chondroitin sulfate N-acetylgalactosaminyltransferase-2 contributes to the replication of infectious bursal disease virus via interaction with the capsid protein VP2.

Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2.

Pathogenic characteristics of Marek's disease virus field strains prevalent in China and the effectiveness of existing vaccines against them.

The virulence of Marek's disease virus (MDV) is continuously evolving, and more virulent MDV pathotypes are emerging, thereby reducing the effectiveness of the existing vaccines. In this study, feather pulps were collected from diseased chickens in commercial chicken flocks in China that presented significant MD visceral tumors in 2011 and were inoculated into a monolayer of duck embryo fibroblasts (DEFs). Three field isolates of MDV were obtained by plaque cloning and identified as MDV via PCR and designated strains LCC, LLY, and LTS. Unvaccinated and CVI988 vaccine-vaccinated specific pathogen-free chickens were challenged at 7 days post vaccination (dpv) with 1000 plaque forming units of each of the respective MDV isolates. These strains induced gross MD lesions in all (100%) of the unvaccinated chickens, and the mortality rates of the unvaccinated chickens were 42.9%, 46.7%, and 23.1% by 60 days post challenge (dpc), respectively. The CVI988 vaccine induced protective indices (PIs) of 85.7, 92.3, and 66.7, respectively. These results showed that the pathogenic characteristics of the Chinese isolates were diverse and that vaccine CVI988 provided different levels of protection against them. These data indicated that the existence of variant MDV strains was a possible reason of immunity failure in China.

A reassortment vaccine candidate as the improved formulation to induce protection against very virulent infectious bursal disease virus.

Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease affecting all major poultry producing areas of the world. Infectious bursal disease virus (IBDV) is genetically prone to mutation so that vaccines have to be changed accordingly. However, the traditional method of vaccine development with blind passage could not fit the style of the emergency prevention of IBDV. In this study, for the first time, a segment-reassortment attenuated IBDV rXATB, consisting of modified segment A of a prevalent strain and segment B of an attenuated strain, was designed and rescued; rXATB was stable and could induce good humoral and cellular immune responses which resulted in excellent protection against the lethal challenge of vvIBDV without obvious immunosuppression in chicken. This study revolutionarily provides a new formulation based on reverse genetics to develop new vaccine against prevalent IBDV.

A single amino acid V4I substitution in VP1 attenuates virulence of very virulent infectious bursal disease virus (vvIBDV) in SPF chickens and increases replication in CEF cells.

Infectious bursal disease virus (IBDV) is a birnavirus that causes immunosuppressive disease in chickens. The emergence of very virulent IBDV (vvIBDV) has brought new challenges for this disease. The molecular determinants for the high pathogenicity of vvIBDV are not fully understood. Previous studies focused mostly on the VP2 protein on segment A, but recent evidence suggests that segment B also plays an important role. Previously we identified eight amino acid changes in the VP1 protein of vvIBDV. In this study, we investigated effect of amino acids substitutions in VP1 on viral replication and pathogenicity. We identified a Valine to Isoleucine substitution at amino acid position 4 (V4I) of VP1 that attenuates viral pathogenicity and reduces viral replication in SPF chickens but increases viral replication in CEF cells. This study confirms that VP1 of segment B play an important role in viral replication and pathogenicity of vvIBDV.

Mutations of residues 249 and 256 in VP2 are involved in the replication and virulence of infectious Bursal disease virus.

Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. Although the PDE and PFG domains of the capsid protein VP2 contribute significantly to virulence and fitness, the detailed molecular basis for the pathogenicity of IBDV is still not fully understood. Because residues 253 and 284 of VP2 are not the sole determinants of virulence, we hypothesized that other residues involved in virulence and fitness might exist in the PDE and PFG domains of VP2. To test this, five amino acid changes selected by sequence comparison of the PDE and PFG domains of VP2 were introduced individually using a reverse genetics system into the virulent strain (rGx-F9VP2). Then reverse mutations of the selected residues 249 and 256 were introduced individually into the attenuated strain (rGt). Seven modified viruses were generated and evaluated in vitro (CEF cells) and in vivo (SPF chicken). For residue 249, Q249R could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while R249Q could reduce in vitro and elevate in vivo the replication of rGt; meanwhile Q249R reduced the virulence of rGx-F9VP2 while R249Q increased the virulence of rGt, which indicated that residue 249 significantly contributed to the replication and virulence of IBDV. For residue 256, I256V could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while V256I could reduce in vitro but didn't change in vivo the replication of rGt; although V256I didn't increase the virulence of rGt, I256V obviously reduced the virulence of virulent IBDV. The present results demonstrate for the first time, to different extent, residues 249 and 256 of VP2 are involved in the replication efficiency and virulence of IBDV; this is not only beneficial to further understanding of pathogenic mechanism but also to the design of newly tailored vaccines against IBDV.

Isolation and genetic characterization of bovine parainfluenza virus type 3 from cattle in China.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important of the known viral respiratory pathogens of both young and adult cattle. However BPIV3 has not been detected or isolated in China prior to this study. In 2008, four BPIV3 strains were isolated with MDBK cells from cattle in China and characterized by RT-PCR, nucleotide sequence analysis, transmission electron microscope observation, hemadsorption and hemagglutination tests. Nucleotide phylogenetic analysis of partial hemagglutinin-neuraminidase (HN) gene for four isolates and the complete genome for the SD0835 isolate implicated that the four Chinese BPIV3 strains were distinct from the previously reported genotype A (BPIV3a) and genotype B (BPIV3b) and might be a potentially new genotype, which was tentatively classified as genotype C (BPIV3c). This is the first study to report the isolation and genetic characterization of BPIV3 from cattle in China.

Genotyping of bovine viral diarrhea viruses from cattle in China between 2005 and 2008.

Eighteen bovine viral diarrhea viruses (BVDV) from cattle in China between 2005 and 2008 were genetically typed by sequencing of the 5'-untranslated region (5'-UTR) of the viral genome and for selected isolates the N(pro) region. Phylogenetic reconstructions indicated that all of the 18 BVDV positive samples examined in this work clustered within the BVDV type 1 genotype. Of the 15 previously described subgenotypes of BVDV1 (1a-1o), 12 of the samples examined in this work clustered with the Chinese BVDV ZM-95 strain of pig origin, which was the prototype of BVDV1m, while 2 samples clustered with the BVDV1b. But 4 samples formed a separate group appearing to be a potentially new subgenotype, which was tentatively typed as "BVDV1p". Based on these results there appears to be highly genetic variation within the Chinese BVDV field isolates. As well, the phylogenetic reconstructions indicate that the clustering of the Chinese BVDV1m subgenotype in the phylogenetic tree is a result of geographic isolation. The information obtained from this work will be useful when carrying out epidemiological surveys of BVDV detected in China, especially for the BVDV1m detection in Chinese cattle.