Protective Efficacy Of A Recombinant Marek’s Disease Virus Against H9Subtype Of Avian Influenza And Marek’s Disease

H9subtype avian influenza virus (AIV) could cause the respiratory symptoms and a drop in egg production. The virus could cause high morbidity and lethality to poultry when co-infected with other pathogens, which bring heavy economic loss to our poultry industry. In recent years, the dominant lineage of H9subtype AIVs is Y280-like lineage in China, In this study, we devoted to construct a stable polyvalent vaccine by using Marek’s disease virus CVI988/Rispens strain as the vector to express the hemagglutinin of H9subtype AIV, and the protective efficacy of the virus as a vaccine against both AIV and MDV was investigated. The recombinant virus provides new vaccines for prevention and control of H9subtype AIV.The sorf1-sorf2of IRS-US junction region of MDV CVI988/Rispens genome was used as the insertion site for foreign genes. The recombinant transfer vector plasmid pMDV-HA/GFP contains the HA gene of A/Chicken/Anhui/1/2010belongs to Y280-like lineage and a loxp-flanked EGFP expression cassette under the control of the LTR promoter from REV. MDV-extracted DNA and the transfer vector plasmid pMDV-HA/GFP transfected chick embryo fibroblast cells (CEF) to obtain a recombinant virus rMDV-HA/GFP through homologous recombination. The EGFP gene was removed by Cre-mediated recombination, to abtain the recombinant virus rMDV-HA which expressed HA protein under the control of REV LTR. According to the result of PCR and western-blot, we can draw the conclusion that the HA gene was correctly inserted into the genome of MDV CVI988/Rispens and stably expressed in vitro.This study evaluate the protective efficacy of rMDV-HA vaccination against H9subtypes of AIV belongs to Y280-like lineage. One hundred,1-day-old SPF chickens were divided into5groups, including rMDV-HA-vaccineted group, rMDV-HA secondary-vaccineted group, rMDV-HA and H9subtypes of AIV inactivated-vaccineted group, H9subtypes of AIV inactivated-vaccineted group and CVI988/Rispens-vaccineted group. The first three groups and CVI988/Rispens-vaccineted group were inoculated subcutaneously on the back of the neck with5×103PFU of rMDV-HA or CVI988/Rispens; rMDV-HA secondary-vaccineted group strengthen immunization in the10-day-old; rMDV-HA and inactivated-vaccineted group, inactivated-vaccineted group intramuscular injection0.25mL H9subtypes of AIV inactivated vaccine in the14-day-old SPF chickens; Serum was collected weekly after vaccination, sera were used to detect HI antibody titer of H9subtype AIV.35days of age after immunization, chickens from each group were challenged by the ocular-nasal administration of2×106EID50of the H9subtype of AIV per chicken. On days3,5post-challenge, tracheal and cloaca swabs were collected and used for testing the shedding rate. The chickens from each group were euthanized and necropsied10days post-challenge for pathological examination. The results showed that, in the5groups, the rMDV-HA and H9subtypes of AIV inactivated-vaccineted group was first to produce the protective effect of antibody titers, almost7days earlier than that in chickens receiving inactivated vaccine, they were observed to have mild clinical signs of disease and a lowest virus-shedding rate. These results showed that the rMDV-HA and H9subtypes of AIV inactivated-vaccineted group induced a comprehensive and rapid immune response against AIV.To determine the protective efficacy of rMDV-HA against the virulent RB1B strain of MDV. A total of60SPF chickens were randomly divided into three groups:1-day-old SPF chickens were vaccinated with the rMDV-HA or the CVI988/Rispens as described above and challenged intraperitoneally after7days with MDV RBIB strain (500PFU/chick). Twenty unvaccinated SPF chickens were used as the challenge controls. The chickens were examined for clinical signs of disease and mortality for6weeks after challenge. The rMDV-HA conferred100%protection to chickens against the virulent RB1B strain of MDV, just as the CVI988/Rispens strain did, whereas90%of the challenge control chickens died of MD. These results showed that the rMDV-HA can provide complete protection against the virulent RBIB strain of MDV.