Isolation And Characterization Of H5N6 Influenza Virus From Cat And Its Regulation Of Interferon ? Response

Cats,as a companion animal,are closely related to humans.Previous studies showed that cats can naturally infect a variety of influenza A viruses,including H5N1 highly pathogenic avian influenza virus,H3N2 low pathogenic canine influenza virus,H1N1 seasonal human influenza virus and H7N2 low pathogenic avian influenza virus.Therefore,cats as a potential 'gene pool' may produce new mammalian adaption influenza A viruses,and may provide an opportunity for avian influenza viruses to adapt to mammalian,then increasing the risk of pandemic in human.So,monitoring and research on influenza A viruses in cats is urgently and importantlyIn our study,an avian H5N6 feline influenza virus was isolated and the whole genome sequence of H5N6 virus from NCBI and GISAID database was selected to analyze its evolutionary characteristics.These results showed that:First,the HA gene of this H5N6 feline influenza virus belongs to the H5 branch of 2.3.4.4 and is closely related to avain Second,after analysis,mammalian(human,cat and pig)viruses were closely related to the avian influenza virus strains.Third,the HA gene of this virus has a highly pathogenic cleavage sites,the head of NA protein has 11 amino acids deletions,and the amino acid of NS1 has 5 amino acid deletions.These results suggest that this virus may be a highly pathogenic influenza virus,and the adaptability to host cells or the virulence of it has changedTo understand the pathogenicity of H5N6 feline influenza virus and the interreaction with host cells:first,we did animal experiments on chicken,mouse or cat by intranasal or intratracheal,the results showed that this virus had a high lethality in chicken and mice,and can transmission in chicken.However,its clinical symptoms of cats only showed fever and pathological changes in the lungs.Interesting,the positive serum was found in the cohabitation group,indicating that virus can transmission in cats.Then,we also did the iTRAQ for difference protein analysis after infection with CRFK(crandell-rees feline kidney,CRFK)cells.A total of 373 differential proteins were detected,of which 204 were up-regulated and 169 were down-regulated.The functions of these differential proteins are mainly enriched in immune-related pathways including P13K-Akt signaling pathway,ECM-receptor interaction,p53 signaling pathway,PPAR signaling pathway,and the NF-?B signaling pathway.Five of the differential proteins were selected and verified by Western-blot and q-PCR.This study was to understand the effect of H5N6 influenza virus on host innate immune response.By constructing a dual-fluorescent reporter system driven by feline and human IFN-? responsive promoters,it was found that NF-?B or IRF3 pathways can inhibit IFN-?response induced by infection of feline or human H5N6 influenza virus or NS1 protein expressed.We also found that overexpression of IFN-? could inhibit H5N6 influenza virus replication before virus infection,but overexpression of IFN-? had no significant effect on influenza virus replication after virus infection.In summary,first of all,an avain H5N6 feline influenza virus was isolated in our study.And the phylogenetic tree analysis showed that this virus belongs to 2.3.4.4 clade of H5.And the amino acid sequence of NS 1 of this virus is similar to that of avian influenza virus H5N6 NS1,and most of the strains have 5 amino acid deletions.The internal genes of the mammalian H5N6 IAVs were mainly derived from the low pathogenic H9N2/H7N9 virus,and this is better to know the evolution direction.Afterwards,the pathogenicity of this virus to chicken and mammals,it was found to have a high mortality rate in chickens,and a mild clinical response in cats.Then,Proteomics studies on infected cell samples showed that the differential proteins were significantly enriched in the immune-related pathways,indicating the viral infection caused a strong immune response in the host,and provided a theoretical basis for the subsequently explored of the virus pathogenicity and host immunity.Finally,we also found that the NS1 protein of this H5N6 virus can block the NF-?B or IRF3 pathways,then blocked the expression of IFN-? by inhibiting the IFN-? promoter.These results may provide a theoretical basis for the innate immune and antiviral research after host infection with H5N6 feline influenza virus.Simulaneously,it also may serve as a guidance for drug treatment of reasonable and effective time.