The Effect Of H9N2 Virus Infection On The Pathogenicity Of The Virulent NDV And The Role Of Chicken SOCS3 Protein During NDV Infection

Avian influenza?AI?and Newcastle disease?ND?caused by avian influenza virus?AIV?and Newcastle disease virus?NDV?,respectively,are two major infectious diseases to poultry industries.Infection with highly pathogenic AIV or NDV can produce 100%mortality in chickens.They are listed as notifiable diseases and first kind of infectious disease by Office International des Epizooties?OIE?and the ministry of agriculture of the People's Republic of China,respectively.The Guangdong province located in the southeast coast of China,hosts warm and humid climate,a large number of wetlands and live poultry markets,which provide an ideal environment for virus transmission.Although intensive immunization programs are implemented to control AI and ND,the outbreaks of AI and ND also occurred sporadically in China.To investigate the prevalence of AIV and NDV endangered the poultry industries of Guangdong province,we collected the tissues of poultries that maybe infect AIV or NDV to perform virus isolation and identification.In the present study,13 H5N6 AIVs,6 H7N9 AIVs,8 virulent NDVs?5 isolates of genotype VIId,2 isolates of genotype VI,and 1 isolate of genotype XII?,and 2 H9N2 AIVs were identified.Notablly,we detect both genotype VIId NDV and H9N2 AIV in two chicken samples,indicating that co-infection of H9N2 AIV and virulent NDV was occurred in Guangdong province.Meanwhile,we performed animal experiment to evaluate the pathogenicity and transmission of the representative strains,a H5N6 HPAIV?QY01?and a H7N9 HPAIV?Z1?in chicken.Chickens were inoculated intranasally with 10⁶EID₅₀ of the indicated virus.Twelve hours after inoculation,three sentinel chickens were co-housed with the inoculated chickens in each group.The results showed that both viruses could efficiently infect,transmit and caused death in chickens,but the H5N6 HPIAV?QY01?replicated much more efficiently and is more lethal in chickens,when compared with the H7N9 HPAIV?Z1?.In order to detect the effect of H9N2 AIV infection on the pathogenicity of vNDV in chickens,we conducted animal experiment using a H9N2 virus and a genotype VIId NDV strain?rGM?.The results showed that chickens infected simultaneously with H9N2 AIV and vNDV did not affect the mortality,but delaying the replication of vNDV in chickens at the early stage of NDV infection;however,chicken infected with H9N2 AIV two days prior to being infected with vNDV increased the vNDV replication and the mortality.To further explore the possible mechanism of H9N2 AIV affects the pathogenicity of vNDV in chickens,we conducted the RNA-seq of the target tissue,lungs,of NDV and H9N2AIV.The results demonstrated that the differential expressed genes?DEGs?of the chicken lungs infected simultaneously with H9N2 AIV and vNDV,were enriched in cell growth and metabolism,compared with chicken infected with vNDV alone;however,the DEGs of the lungs of chickens infected H9N2 AIV two days prior to being infected with vNDV,were enriched in host immune response,with a down-regulation,when compared with chicken infected with vNDV alone.Sequentially,some genes were validated by qRT-PCR.These data indicated that H9N2 AIV could compete with vNDV for energy and other materials for virus replication,delaying the replication of vNDV,when chicken infected simultaneously with H9N2 AIV and vNDV;H9N2 AIV could suppress the host immune response,facilitating the replication of vNDV and aggravating the NDV infection.Our transcriptome analyses showed that the expression level of suppressor of cytokine signaling 3?SOCS3?was significantly increased in the lungs of chickens after infection the virulent NDV,rGM.SOCS3,a negative regulatory protein in host immune responses,plays an important role in the immune response of host by inhibiting the over expression of a large number of imunity molecules.Previous research has demonstrated that many pathogens could antagonize the antiviral response of host,by induced the expression of SOCS3.In view of the mechanism of NDV escape from immune response of host remained largely unknown,we explore the potential role of chicken SOCS3 during NDV infection by the methods of over-expression,siRNA interfere and specific inhibitor,according to the recent research.Our results displayed that the expression of SOCS3 was induced by NDV in chicken cells.However,the UV-inactive NDV couldn't induce the expression of SOCS3,indicating that virus replication was required for this process.The treatment of DF-1 cells with the specific NF-kB inhibitor BAY11-7082 prior to NDV infection can suppress the expression of SOCS3,suggesting that the expression of SOCS3 was induced by NDV in a NF-kB dependent manner.We also showed that the replication of NDV was affected by the expression of SOCS3 by using the means of over-expression or siRNA interference.The expressions of interferon stimulated genes?ISGs?,such as Mx1,Viperin and IFIT5,were significantly decreased in DF-1 cells,whose SOCS3-3 expression was knocked-down by siRNA in response to NDV infection,indicating that the expression of ISGs can be mediated by SOCS3 during NDV infection.In conclusion,the primary AIVs and NDVs was H5N6 and H7N9 HPAIVs,and genotype VII NDV,respectively,endangered the poultry industries in Guangdong province.We also found that co-infection of H9N2 virus and VII NDV in chickens.The animal expriments revealed that the virus antagonism was occurred because of competing with each other for the material of replication at the early stage of infection,when H9N2 AIV and vNDV simultaneous infected SPF chickens;H9N2 AIV could aggravate vNDV infection by suppressing the host immune response.In addition,we also explored the potential role of chicken SOCS3 during NDV infection,indicating that NDV is capable of inducing SOCS3expression in a NF-kB dependent manner to diminish the responsiveness of host to IFNs.