Analysis On Pathogenicity Of Coinfection Of Avian Reticuloendotheliosis Virus With Leukosis Virus Subgroup A

Reticuloendotheliosis virus (REV) and avian leukosis virus subgroup A (ALV-A) infections have been recognized as a common condition in chicken flocks in China in recent years, and meanwhile the two viruses co-infects clinically. The REV-contaminated vaccine is considered to be one of the main sources causing the REV infection and dissemination. In the present study, a strain of REV was isolated from Marek’s disease (MD) vaccine, and REV and ALV-A co-infection model was established in SPF chickens, and the pathogenicity of REVand ALV-A co-infection for chickens was investigated in SPF chickens and vaccinated chickens. In addition, an indirect immunofluorescence assay (IFA) for detecting REV in contaminated MD vaccine was established and an infectious clone of REV was constructed.A strain of REV was isolated from Marek’s disease (MD) HVT live vaccine, which was named MD-2. Sequencing and comparative analysis of the MD-2genome showed that the virus was closely related to American pheasant/APC-566, Taiwan goose/3410/06, chicken/HLJR0901, with above99%nucleotide sequence identity, only96.7%nucleotide sequence identity to Chinese strain HA9901, and93.5%identity to American duck/SNV.One-day old SPF chickens were infected with REV or ALV-A alone or dural in order to analyze the pathogenicity of REV and ALV-A co-infection in chickens. The results indicated that the co-infection of these two viruses could induce obviously synergistic effect in pathogenicity for chickens, with higher mortality, lower body weight and significantly decrease of CD4+/CD8+ratio, bursa index and thymus index of the infected chickens, compared with single infection. Under routine immunization, live vaccines or inactivated vaccines immunization conditions,1-day old and10-day old chickens displayed weight loss and certain extend mortality when infected with REV and ALV-A. The co-infected chickens showed higher mortality, significantly decreased bursa index and thymus index than single infection, indicating an obviously synergistic effect in pathogenicity for the vaccinated chickens.Following immunization with inactivated vaccine, the HI titers of NDV, H5AIV and H9AIV in the co-infected chickens and single REV-infected chickens decreased significantly, with a sharp decline of HI titers in the co-infected group. Single REV-infected group and the co-infected group showed an inhibitory antibody response against IBD vaccination. The seroconversion rates of IBD antibody in single REV-infected group and co-infected group were obviously lower than control group under immunization with inactivated vaccine, while there were no obvious differences under the immunization with live vaccine.The results also showed that ALV-A infection could induce severe vertical transmission, while REV did not. At131day old, antibody positive rate of single ALV-A infection and co-infection were31%and48%respectively. Isolation rates of single ALV-A infection and co-infection from9-day old chicken embryos were57.9%(11/19) and20%(3/15), and66.7%(6/9) and36.4%(4/11) in7-day old chickens. Antibody positive rate of single ALV-A-infection and co-infection at10-day old was over80%. No obvious differences in mRNA transcription level of cytokines and Toll-like receptors were found among all groups.A recombinant plasmid of the REV MD-2full-length provirus genome was constructed with an enzyme site as genetic markers for identification. Rescued virus displayed consistent growth characteristics with the parental virus, with a stable inherited genetic markers. Five mutations in env gene were determined separately, showing that the1433th residue T mutant into G was a lethal mutation for REV.A IFA was established to detect REV contaminated in MD vaccine. The results showed that the minimum detection limit for REV contaminated in I subtype MD live vaccine, HVT live vaccine and bivalent live MD vaccine was5,10and15TCID50per500dose of vaccine with this method.As a whole, our results indicated the co-infection of REV and ALV-A exhibited a synergistic effect in pathogenicity for chickens, providing scientific evidence for control of the co-infection in poultry production.