Yihong Xiao

Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015.

There have been many outbreaks of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, in Chinese chicken farms since June 2015. HPS was mainly found in miscellaneous meat-type chickens, Ma chickens, layer chicks and Three-yellow chickens, while it was occasionally found in white broilers. To determine the specific causative pathogen and pathogenicity of HPS in chickens, we collected 25 suspected cases and performed clinical pathology and aetiology analyses. The results showed that the 25 cases exhibited multifocal hepatitis with intra-nuclear inclusion bodies and 70 nm-latticed viral particles in the cell nuclei. All samples were positive for fowl adenovirus (FAdV), and sequencing results showed that the hexon gene shared the highest nucleotide similarities with the hexon gene of group 1 serotype 4 (FAdV-4). FAdV-4 was highly pathogenic to embryos and specific pathogen-free chickens, causing 100 and 70 % mortality rates, respectively. Thus, FAdV-4 is associated with HPS outbreaks in China.

MYH9 is an Essential Factor for Porcine Reproductive and Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is an important swine disease worldwide. PRRSV has a limited tropism for certain cells, which may at least in part be attributed to the expression of the necessary cellular molecules serving as the virus receptors or factors on host cells for virus binding or entry. However, these molecules conferring PRRSV infection have not been fully characterized. Here we show the identification of non-muscle myosin heavy chain 9 (MYH9) as an essential factor for PRRSV infection using the anti-idiotypic antibody specific to the PRRSV glycoprotein GP5. MYH9 physically interacts with the PRRSV GP5 protein via its C-terminal domain and confers susceptibility of cells to PRRSV infection. These findings indicate that MYH9 is an essential factor for PRRSV infection and provide new insights into PRRSV-host interactions and viral entry, potentially facilitating development of control strategies for this important swine disease.

Identification of atypical porcine pestivirus infection in swine herds in China

Atypical porcine pestivirus (APPV) have been detected in swine herds from the USA, Germany, the Netherlands, Spain and most recently in Austria, suggesting a wide geographic distribution of this novel virus. Here, for the first time, we reported APPV infection in swine herds in China. Newborn piglets from two separate swine herds in Guangdong province were found showing typical congenital tremors in July and August 2016. RT-PCR, sequencing and phylogenetic analysis showed APPV infection occurred. Phylogenetic analysis showed that Chinese APPV strains, GD1 and GD2, formed independent branch from the USA, Germany and the Netherlands. Nucleotide identities between members of the APPV ranged between 83.1% and 83.5%, and this showed APPV is highly diverse. It is apparent that this provides the first molecular evidence of APPV infection in swine herds in China.

HAEMANGIOMAS, LEIOMYOSARCOMA AND MYELOMA CAUSED BY SUBGROUP J AVIAN LEUKOSIS VIRUS IN A COMMERCIAL LAYER FLOCK

An outbreak of simultaneously occurring haemangiomas, leiomyosarcoma and myeloma was observed in a commercial layer flock in China. The sick chickens were extremely thin and dehydrated. Scattered haemangiomas were found on the claws, breast and wings. At necropsy, haemangiomas and some other nodular tumours were also found in the internal organs. In addition, diffuse enlargement of the liver and spleen appeared in some birds. Histopathologically, haemangiomas were typically cavernous haemangiomas and haemangioendothelioma. In the diffusely swollen liver and spleen, multifocal or widespread marrow tumour cells filled with ball-like acidophilic particles in cytosol were observed, which are the characteristic pathological changes of avian myelocytomatosis. The nodular tumour cells formed by muscle bundles were of variable size, irregular shape, poorly differentiated and malaligned. Immunohistochemistry for vimentin, cytokeratin, actin (smooth muscle) and actin (sarcomeric) and Massons staining confirmed the different cell lineage of the nodular tumour, thus leading to the diagnosis of leiomyosarcoma. The seroprevalence of avian leukosis subgroup J (ALV-J) antibodies was 13.46% (7/52), while ALV-A/B and reticuloendotheliosis virus (REV) antibodies were not detectable. The DF-1 cells inoculated by virus extracted from liver samples from 24 infected chickens were cultured and the group-specific antigen (GSA) was identified by ELISA. All samples were positive for ALV, which was further identified as ALV-J by immunofluorescence assay (IFA). PCR analysis revealed that three isolates of ALV-J proviral sequence were close to the HPRS-103 prototype strain and other Chinese field strains isolated in recent years, while one isolate (DP01) had a lower homology with them. This is the first report that ALV-J infection caused the simultaneous occurrence of haemangiomas, leiomyosarcoma and myeloma in a commercial layer flock.

Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination.

Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China.

Distribution of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in different stages of gestation sows: HP-PRRSV distribution in gestation sows

Highly pathogenic PRRSV (HP-PRRSV) emerged in China in 2006 and caused severe reproductive losses, particularly in late-term sows. To determine whether these reproductive failures were related to the susceptibility of late-term sows to HP-PRRV, 60- and 90-days of gestation sows were infected with HP-PRRSV isolate TA-12 (GenBank accession HQ417620). A monoclonal antibody specific to the C-terminal of the nucleocapsid protein was used to evaluate viral distribution by IHC. This showed that HP-PRRSV had a similar distribution in both sets of sows. However, HP-PRRSV infection led to dramatically decreased serum levels of luteinizing hormone (LH) and 17-β-estradiol (E2) in late-term sows, while only E2 was decreased in the 60-day sows. These results indicate that HP-PRRSV-induced reproductive failure is more likely due to reproductive hormone level imbalances rather than tissue tropism differences.

Anti-idiotypic antibodies reduce efficacy of the attenuated vaccine against highly pathogenic PRRSV challenge

BackgroundThe inability of current vaccines to provide effective protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection is not fully understood. One of the reasons might be the presence of anti-idiotypic antibodies (Ab2s) to the envelope glycoprotein GP5 induced by PRRSV infection since our previous studies demonstrated the presence of auto-Ab2s (aAb2s) in pigs infected with PRRSV. To test this hypothesis, PRRSV negative piglets were injected with a monoclonal Ab2 (Mab2-5G2) and aAb2s that are specific for anti-GP5 antibody, vaccinated with the attenuated PRRSV vaccine CH-1R and then challenged with the highly pathogenic PRRSV HuN4 strain. The animals were evaluated for clinical signs, pathological changes of the thymus and lungs, viremia, levels of serum antibodies and cytokines.ResultsThe piglets injected with Mab2-5G2 or aAb2, and who received the attenuated PRRSV vaccine CH-1R before challenge, produced high levels of anti-N antibodies, IL-2 and IL-4, but low levels of neutralizing antibodies. After PRRSV HuN4 challenge, the animals showed obvious clinical signs, including lung lesions, severe thymus atrophy and decreased production of IL-4 and higher level of viremia.ConclusionWhen anti-GP5 Ab2s are present, the use of attenuated PRRSV vaccine CH-1R against HP-PRRSV infection is not recommended. It can result in poor health status with pneumonia and thymus atrophy.

Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.

Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection.

Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14–3–3 Proteins

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins.

Florfenicol-induced Mitochondrial Dysfunction Suppresses Cell Proliferation and Autophagy in Fibroblasts

Florfenicol (FLO) is one of the most popular antibiotics used in veterinary clinic and aquaculture. FLO can inhibit both bacterial and mitochondrial protein synthesis. However, the effects of FLO on mitochondrial function and cellular homeostasis remain unclear. Here we show that FLO inhibits expression of mitochondrial DNA-encoded proteins, decreases mitochondrial membrane potential, and promotes generation of reactive oxygen species (ROS) in vitro. As a result, activities of mitochondrial respiratory chain complex I and IV and the cellular ATP level are decreased and mitochondrial morphology is damaged. FLO represses cell growth and proliferation by suppression of phosphorylation of p70S6K through AMPK/mTOR/p70S6K pathway. Furthermore, FLO also induces G0/G1 cell cycle arrest via increase of p21 levels through activating ROS/p53/p21 pathway. Moreover, the clearance of damaged mitochondria by autophagy is impaired, leading to cell proliferation inhibition and promotes cell senescence. In addition, FLO-induced upregulation of cytosolic p53 may contribute to mitophagy deficiency via regulation of Parkin recruitment. In summary, our data suggest that florfenicol is an inhibitor of mitochondrial protein synthesis that can induce noticeable cytotoxicity. Thus, these findings can be useful for guiding the proper use of FLO and the development of safe drugs.