Pathogenicity Of Goose-Origin H5N6 Subtype AIVs In Chickens

Avian influenza viruses?AIVs?belong to influenza viruses A,which are known to infect a variety of species and cause disease.Avian influenza has broken out in poultry over70 countries since 2003.Highly pathogenic avian influenza viruses?HPAIVs?,including H5 and H7 subtypes,can cause systemic infections in poultry with a fatality rate up to100%.It can also infect people and cause clinical symptoms and death occasionally.Since1996,The H5 HPAIV subtype was firstly isolated from China in geese.After the first human case infected with H5N1 AIV and caused death in Hong Kong in 1997,the subtype began to be widespread in poultry in China.Since 2015,the H5N6 AIV become a predominant epidemic strain in China and caused a series of outbreaks.Therefore,we should pay more attention to the source,pathogenicity and the transmissionbility of H5N6subtype AIVs.To understand the genetic properties of H5N6 subtype AIVs in South China in2015²016,the genetic evolution analysis showed that the HA genes of the 10 H5N6 AIVs belonged to clade 2.3.4.4.The HA,PA,PB1,M,NP and NS genes of the 10 strains all located in the Mix-like 1 group,consisting of isolates from countries such as China,Vietnam and Laos.Their PB2 genes clustered in the Mix-like 2 group,who came from countries such as China,Vietnam and Japan.In addition,the NA genes of them were classified into Eurasian Lineage.Therefore,the 10 strains were recombinant between different genotypes.The molecular characteristics analysis showed that the cleavage sites of these 10 viruses in HA protein all had multiple basic amino acids?RERRRK/GLF?sequences,which consistent with the molecular characteristics of HPAIVs.The amino acid was Q and G in position 226 and 228 of the 10 strains in HA,respectively,indicating that they were more likely to bind the 2,3-NeuAcGal of the AIVs receptor.There was some potential glycosylation sites in HA and NA proteins of the 10 strains.They all had 12 amino acid deleted at positions 59⁷0 in NA protein,5 amino acid deleted at positions 80⁸4 in NS1 protein.Only the GS116 virus had the V27A mutation in M2 protein related to amantadine resistance in the 10 viruses.To study the pathogenicity of H5N6 AIVs from waterfowls,we selected GS144 and GS148 viruses to inoculate specific pathogen free?SPF?chickens at a dose of 10⁴ EID₅₀/0.1mL.The results exhibited that they showed different pathogenicity and transmissibility in chickens.The lethality of GS144 inoculated chickens was 100%,The virus titers in their lung,kidney,liver,trachea,spleen,and brain between 6.83 lg EID₅₀⁸.25 lg EID₅₀,showing high pathogenicity?HP?;the lethality of GS148 inoculated chickens was 0%,the virus titers in the 6 organs between 2.17 lg EID₅₀³.83 lg EID₅₀,showing low pathogenicity?LP?;The lethality of GS144 contact chickens was 87.5%,the virus titers in the 6 organs between 7.17 lg EID₅₀⁹.17 lg EID₅₀;The lethality of GS148 contact chickens was 0%,the virus titers in the 6 organs between 1.83 lg EID₅₀⁴.17 lg EID₅₀.This showed that the pathogenicity and transmissibility of GS144 virus is more stronger than GS148.In order to determine the antiviral immune response and related antiviral immune factors produced by H5N6 strains in inoculated chickens,we used quantitative Real-Time PCR?qRT-PCR?to detect the mRNA expression levels of genes such as MDA5,MAVS,TRAF3,IFN-?,Mx1,IL-6,and TLR3 in the lungs of inoculated chickens.The results showed that in the GS144 inoculated chickens,the mRMA expression of MDA5,MAVS,TRAF3,IFN-?,Mx1,IL-6 and TLR3 was up-regulated by 2.66 fold,5.35 fold,4.01 fold,5.84 fold,3.53 fold,3.32 fold and 3.50 fold,respectively;In the GS148 inoculated chickens,the mRMA expression of MDA5,MAVS,TRAF3,IFN-?,Mx1,IL-6 and TLR3 was up-regulated by 1.35 fold,1.42 fold,1.04 fold,1.24 fold,1.13 fold,1.15 fold,and 1.13 fold,respectively.Therefore,both GS144 and GS148 strains could activate the natural antiviral immune response in chickens,and the effect of GS144 virus was stronger than GS148virus.In this study,we used the cDNA of chicken embryo fibro?CEF?as a template to clone The CDS sequence of chicken TRAF3?chTRAF3?by RT-PCR technique.Sequence analysis showed that the CDS of chTRAF3 gene is 1395 bp long and encodes 465 amino acids.The homology comparison results showed that the homology of the chTRAF3protein with the mallard duck TRAF3 was 79%,the homology of the chTRAF3 protein with the human TRAF3 was 66.6%,and the homology of the chTRAF3 protein with the murine TRAF3 was 65.9%.Structural predictions indicate that the CDS region of chTRAF3 mainly contains two domains,namely the ZN RING domain?AA52-87?and the C-terminal transmembrane domain?MATH??AA316-439?.In order to study the role of chTRAF3 in the antiviral innate immune signaling pathway of chickens,we connected chTRAF3 into pCAGGS vector to obtain a eukaryotic expression plasmid which the target protein was successfully expressed in the 293T cell line.chTRAF3 was over-expressed in DF-1 and CEF cells,and the relative mRNA expression levels of cytokines such as OAS,Mx1,IFN-?,and IL-6 were detected by qRT-PCR.The results showed that the mRNA expressions of OAS,Mx1,IFN-?and IL-6were up-regulated by 6.72 fold,7.78 fold,7.33 fold and 9.04 fold in DF-1 cells,and the expression of IRF3 and IFN-?were increased by 1.59 fold and 1.69 fold in CEF,respectively.Therefore,overexpression of chTRAF3 gene can effectively activate the cytokines downstream TRAF3 in the RLRs antiviral innate immune signaling pathway.