Pathogenicity and virus shedding ability of fowl adenovirus serotype 4 to ducks.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogen causing hepatitis-hydropericardium syndrome (HHS) in broilers. Since June 2015, it has emerged as one of the leading causes of economic losses in the poultry industry in China. Although most studies on FAdV-4 have focused on its pathogenicity to broilers, limited studies have been performed on other natural hosts such as ducks and geese. In this study, we assessed the pathogenicity of FAdV-4 to ducks of different ages through intramuscular injection and found that infected ducks showed severe growth depression. The infected ducks also suffered from extensive organ damage and had histopathological changes in the liver, spleen, and kidney. Although the virus infection caused lymphocyte necrosis of immune organs and the development of the bursa of Fabricius (bursa) was inhibited, the humoral immune response of infected ducks to FAdV-4 remained strong. The infected ducks also had high viral load in tissues and shed virus after the challenge. Overall, our research demonstrates that FAdV-4 can infect ducks and adversely affect the productivity of animals. And the viruses shed by infected ducks can pose a potential risk to the same or other poultry flocks.

Cellular hnRNPAB interacts with avian influenza viral protein PB2 and inhibits virus replication potentially by restricting PB2 mRNA nuclear export and PB2 protein level.

The PB2 protein of avian influenza virus (AIV) is essential for transcription and replication of virus genome. In this study, we reported that chicken heterogenous nuclear riboncleoprotein AB (hnRNPAB) cooperated with avian influenza viral protein PB2 and inhibited the polymerase activity and virus replication. We found that hnRNPAB was associated with PB2 mRNA and overexpression of hnRNPAB reduced PB2 mRNA nuclear export and PB2 protein level, but had no influence on PB2 mRNA level. At the same time, overexpression of hnRNPAB also reduced protein levels rather than mRNA levels of PA, PB1 and NP. In addition, overexpression of hnRNPAB restricted the polymerase activity and virus replication, while knockdown of hnRNPAB resulted in enhanced polymerase activity and virus replication. Lastly, virus infection induced the nuclear accumulation of hnRNPAB, but did not cause the change of expression level of endogenous hnRNPAB in DF-1 cells. Collectively, these findings suggested that hnRNPAB played a restrictive role in polymerase activity and virus replication potentially through inhibiting PB2 mRNA nuclear export and PB2 protein level.

Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus.

BACKGROUND: H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. RESULTS: The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2- 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. CONCLUSIONS: In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

SRSF10 inhibits the polymerase activity and replication of avian influenza virus by regulating the alternative splicing of chicken ANP32A.

Compared with mammalian ANP32A, most avian-coded ANP32A contains a 33 amino acids insertion (ch-ANP32A-33) or a 29 amino acids insertion (ch-ANP32A-29), which can rescue the mammalian-restricted avian influenza virus polymerase activity, with ch-ANP32A-33 exhibiting a more potent phenotype. The alternative splicing of 3' splice sites (SSs) of chicken ANP32A intron 4 generates full-length ch-ANP32A-33 and truncated ch-ANP32A-29. In this study, we found a splicing regulatory cis-element that affected the alternative splicing of 3' SSs by block-scanning mutagenesis. RNA affinity purification and mass spectrometry showed that the SRSF10 bound to the splicing cis-element and the binding was further identified and confirmed by RIP experiment. Overexpression of SRSF10 changed the ratio of the two chicken ANP32A transcripts with the increased ch-ANP32A-29 and the decreased ch-ANP32A-33. The knockdown of both of the ch-ANP32A-33 and ch-ANP32A-29 was harmful to avian influenza virus polymerase activity in DF-1 cells, but the restoration and increasement of only ch-ANP32A-29 could not completely rescue the activity of avian influenza virus polymerase. Overexpression of SRSF10 negatively affected the polymerase activity and replication of avian influenza virus, and the expression of ch-ANP32A-33 could partially recover the decrease of polymerase activity of avian influenza virus. By contrast, SRSF10 had weak inhibition on the polymerase activity of mammalian adapted influenza virus and had no effect on the replication of mammalian adapted influenza virus. Taken together, we demonstrated that SRSF10 acts as a negative regulator in polymerase activity and replication of avian influenza virus by binding to the splicing cis-element to regulate the alternative splicing of chicken ANP32A intron 4 for the reduced ch-ANP32A-33 and increased ch-ANP32A-29.

Insights into species-specific regulation of ANP32A on the mammalian-restricted influenza virus polymerase activity.

The ANP32A is responsible for mammalian-restricted influenza virus polymerase activity. However, the mechanism of ANP32A modulation of polymerase activity remains poorly understood. Here, we report that chicken ANP32A (chANP32A) -X1 and -X2 stimulated mammalian-restricted PB2 627E polymerase activity in a dose-dependent manner. Distinct effects of ANP32A constructs suggested that the 180VK181 residues within chANP32A-X1 are necessary but not sufficient to stimulate PB2 627E polymerase activity. The PB2 N567D, T598V, A613V or F636L mutations promoted PB2 627E polymerase activity and chANP32A-X1 showed additive effects, providing further support that species-specific regulation of ANP32A might be only relevant with the PB2 E627K mutation. Rescue of cycloheximide-mediated inhibition showed that ANP32A is species-specific for modulation of vRNA but not mRNA and cRNA, demonstrating chANP32A-X1 compensated for defective cRNPs produced by PB2 627E virus in mammalian cells. The promoter mutations of cRNA enhanced the restriction of PB2 627E polymerase in mammalian cells, which could be restored by chANP32A-X1, indicating that ANP32A is likely to regulate the interaction of viral polymerase with RNA promoter. Coimmunoprecipitation showed that ANP32A did not affect the primary cRNPs assembly. We propose a model that chANP32A-X1 regulates PB2 627E polymerase for suitable interaction with cRNA promoter for vRNA replication.