Development and Application of an MRT-qPCR Assay for Detecting Coinfection of Six Vertically Transmitted or Immunosuppressive Avian Viruses.

Marek's disease virus (MDV), reticuloendotheliosis virus (REV), avian reovirus (ARV), chicken infectious anemia virus (CIAV), infectious bursal disease virus (IBDV), and fowl adenovirus (FAdV) are important causes of disease in poultry. To investigate the infection status of the above six viruses in chickens in China, 1,187 samples from chicken flocks were collected and tested using a newly developed multiplex reverse-transcription quantitative real-time PCR (MRT-qPCR) assay in the study. A series of validation tests confirmed that the MRT-qPCR assay has high specificity, sensitivity, and repeatability. As for six detected pathogens, CIAV had the highest detection ratio, while ARV was not detected in any samples. In the spleen samples, the coinfection rate for MDV and CIAV was 1.6%, and that for REV and CIAV was 0.4%. In the bursa samples, the coinfection rate for FAdV and CIAV was 0.3%, and that for IBDV and CIAV was 1%. In the thymus samples, the coinfection rates for MDV and CIAV and for REV and CIAV were both 0.8%. Our study indicates that the coinfection of these viruses was existing in chickens in China. Through the detection of clinical samples, this study provides data on the coinfections of the above six pathogens and provides a basis for the further study of viral coinfection in chickens.

Phylogenetic analyses and pathogenicity of a variant infectious bursal disease virus strain isolated in China.

Infectious bursal disease is an acute, highly contagious, immunosuppressive disease of young chickens caused by infectious bursal disease virus (IBDV). In recent years, there has been a notable increase in the isolation rates of variant IBDV strains in China; however, the pathogenicity of these variants is unclear. In the current study, we characterized variant IBDV strain ZD-2018-1 and assessed its pathogenicity in specific-pathogen-free chickens. Phylogenetic analysis revealed that ZD-2018-1 belonged to the variant IBDV strain, which showed several unique amino acid mutations compared with other previously-isolated variant IBDV strains. Pathogenicity assays showed that ZD-2018-1 was less virulent than very virulent IBDV strain SD-2013-1, and did not cause an obvious symptoms or death. In comparison, strain SD-2013-1 had a high mortality rate and caused severe lesions in various tissues. However, both of the strains induced obvious pathological lesions on the bursa of Fabricius, resulting in severe immunosuppression in the infected chickens. The results of this study present a systematic evaluation of the genetic characteristics, pathogenicity, and immunosuppressive properties of a new variant IBDV strain, and may help in the development of strategies for the prevention and control of IBDV in poultry.

A subunit vaccine based on fiber-2 protein provides full protection against fowl adenovirus serotype 4 and induces quicker and stronger immune responses than an inactivated oil-emulsion vaccine.

As the number of hepatitis hydropericardium syndrome (HHS) cases has increased in recent years in China, development of a safe and effective vaccine is now urgent. To address this problem a subunit vaccine is a good option, we here systematically investigated the minimum immune dose of a subunit vaccine against HHS based on recombinant fowl adenovirus serotype 4 (FAdV-4) fiber-2 protein and compared the effects between this subunit vaccine and an inactivated oil-emulsion FAdV-4 vaccine in a vaccination trial. The results revealed that the lowest dose of recombinant fiber-2 protein that could provide 100% protection against challenge with virulent FAdV-4 strain HB1501 as well as elicit protective immunity was 2.5 µg/bird. Neither clinical signs nor gross lesions were observed in chickens. In addition, immunization of specific-pathogen-free (SPF) chickens with recombinant fiber-2 protein (≥2.5 µg/bird) could induce quicker and stronger immune responses than the inactivated oil-emulsion FAdV-4 vaccine. These findings provide important information about the development of subunit vaccines for the control of HHS.

Recombinant Fiber-2 Protein-Based Indirect ELISA for Antibody Detection of Fowl Adenovirus Serotype 4.

Clinical cases of hepatitis-hydropericardium syndrome (HHS) from fowl adenovirus serotype 4 (FAdV-4) have increased in China since 2013. Therefore, the development of a new serologic method for HHS detection is now urgent. Here, the FAdV-4 strain JSJ13 was used to construct a plasmid for prokaryotic expression of the JSJ13 fiber-2 protein. The protein, purified by affinity chromatography, was refolded by gradient dialysis. After coating a 96-well plate with the purified fiber-2 protein (1.5 µg/ml), standard serum and secondary antibodies (1:200 and 1:6000 dilutions, respectively) were used to develop an indirect ELISA (I-ELISA). Nine field-collected serum samples and JSJ13-positive serum were tested by I-ELISA and the results corresponded with those of the serum neutralization test. The I-ELISA was used to test 450 clinical serum samples from different parts of China. Chickens from nonvaccinated flocks had low antibody titers and low virus positivity rates. In contrast, FAdV-4 vaccinated chickens were strongly positive, and the positivity rates of all the flocks exceeded 73.3%. The newly developed I-ELISA with the recombinant JSJ13 fiber-2 protein as the antigen detected antibodies to FAdV-4 accurately and sensitively.

Phylogenetic Analyses of Fowl Adenoviruses (FAdV) Isolated in China and Pathogenicity of a FAdV-8 Isolate.

Fowl adenoviruses (FAdVs) have a worldwide distribution and are associated with a variety of diseases, causing considerable economic losses to the poultry industry. We characterized 10 FAdVs isolated from China in 2015-2016 and assessed the pathogenicity of a FAdV-8 strain in specific-pathogen-free (SPF) chickens. Phylogenetic analysis of a hexon gene revealed that only 1 of the 10 isolates belonged to FAdV-8, whereas others belonged to FAdV-4, indicating that Chinese FAdVs were mainly FAdV-4 in recent years. The pathogenicity experiment of the FAdV-8 strain CH/SD/2015/09 showed that no clinical signs were observed in infected chickens. Necropsy displayed mild necrotic foci and petechial hemorrhage of livers collected at 5 days postinfection (dpi). Histopathologic examination identified the presence of intranuclear inclusion bodies in hepatocytes. No virus was detected in oral and cloacal swabs at 5 dpi, and only viral DNA could be measured in kidneys collected at the same time. The results revealed that CH/SD/2015/09 had no obvious pathogenicity in 5-wk-old SPF chickens, which could provide a better understanding about the pathogenicity of the FAdV-8 serotype.