Evolution Of H4 Subtype Avian Influenza Viruses And The Molecular Basis Of Pathogenesis In Mice

The H4 subtype avian influenza viruses(AIVs)have been widely circulating in the world and have such a wide host range from wild and domestic avian species to various mammalian species including pigs and seals.Live poultry markets(LPMs),where numerous birds are brought together,are the highly productive sources of AIVs in China,which leads to AIV reassortment,dissemination and interspecies transmission.Coexistence of multiple subtypes of avian influenza viruses enhances the risk of reassortments and mutations of H4 AIVs.However,so far,the evolution and pathogenic mechanism in poultry and mice of H4 AIVs remain largely unclear.In the present study,a total of eight H4 AIV strains isolated in duck farm and LPMs were characterized.The same amino acid sequence(PEKASR/GLF)at the cleavage site of HA were shown in all the eight H4 isolates,suggesting they are low pathogenic AIVs.None of the amino acid residues involved in the recognition of human-type receptors was found in these isolates,indicating that all the viruses prefer avian-like(?-2,3-sialic acid)receptor.The potential glycosylation sites of HA and NA proteins were conservative,and deletions in NA stem had not been found.No mutations in PB2 protein associated with mammailian adaptaiton were detected.None of other amantadine-resistant mutations in M2 protein was detected from other isolates except for V27 I mutation from viruses MH-2/H4N6,44-2/H4N6,46-2/H4N6 and 67-2/H4N6.The othor seven H4 AIVs possessed a PB1-F2 protein with 91 amino acids other than 29-1/H4N2 with 58 amino acids.Besides,N66 S mutation was found in PB1-F2 protein of 421-2/H4N6 virus.Phylogenetic analysis indicated that these strains are divided into two groups in the Eurasian lineage,and the Group1 of HA gene was prevalent in domestic.Eight genes of MH-2/H4N6 isolated from a duck farm were closely related to three H4N6 viruses from LPM,suggesting a potential AIV link between farms and LPMs.Additionally,the HA,NA,PB2,NP,and NS genes of two other H4N6 viruses isolated in LPM clustered with that of MH-2/H4N6.However,the remaining genes were more closely related to other sublineages,suggesting that MH-2/H4N6-originated viruses reassorted with other viruses in LPM.To determine the pathogenicity of the H4 isolates in different hosts,duck,chicken and mice were chosen as waterfowl,poultry and mammal models,respectively.Animal experiments showed none of the H4 AIVs induced clinical symptoms in animals.All or partial viruses replicated and transmitted in ducks and chickens,respectively.And systemic infections were observed in both ducks and chickens inoculated with certain viruses.Eight strains of the H4 AIVs could be detected in the lungs of mice,indicating the potential of waterfowl-origin H4 AIVs to infect mammals without prior adaptation.Based on above data,we chose DK29(short for 29-1/H4N2),which could hardly infect mice,as a parent virus to adapt in mouse lungs.Finally,a high-virulent mice-adapted virus(MA-50)was obtained,whose lethality in mice was up to 100%.Sequence and the pathogenicity analysis wereperformed in every five generations,and growth dynemics of DK29,MA-20 and MA-50 in DF1,MDCK and A549 cells at different temperatures were analyzed.Sequence analysis revealed multiple amino acid mutations in all eight genes were appeared during virus adaption.Increased replication ability in the fifth generation was observed,and no significant difference showed in the next few generations.The pathogenicity of each generation enhanced along with the virus passages.P25 began to obtain the ability to induce death in mice,and the lethality of MA-50 reached 100%.According to the growth curve of viruses in different cells,mice-adapted viruses had better replication abilities in both poultry-origin cells and mammalian cells.However,no difference of the replication ability of all three viruses was shown in MDCK in temperature of 37?,while poorer replication of DK29 was observed in temperature of 33? compared with MA-20 and MA-50,indicating MA-50 adapted to mammalian cells better,which enhanced its ability to infect mammals.To explore the molecular mechanism of interspecies infection of H4 AIV,reverse genetic systems of DK29 and MA-50 were eatablished first.Based on the reverse genetic system,we generated reassortant viruses containing HA and NA genes(HA,NA,M and NS genes)from DK29 in the MA-50 background,and vice versa in the DK29 background.According to the pathogenicity of these viruses in mice,both the surface and internal genes palyed roles in the virulence changes.We further generated reassortant viruses by replacing each single gene of MA-50 in the DK29 background,and the replication and pathogenicity of these viruses in mice were evaluated.Results showed that the viruses possessing the single gene of PB2,PA or HA from MA-50 replicated better in the lungs and nasal turbinates of mice.Besides,higher MCP-1 expression levels were shown in the lungs of mice inoculated with the viruses contained PB2 or PA gene from MA-50.On the other hand,reassortant viruses containing single gene of DK29 in the MA-50 background were rescued and evaluated in mice.The viruses contained PB2 or(HA+NA)gene from DK29 showed lower lethality compared with other recombinant viruses.The IL-6,IFN-? and IFN-? down-regulated significantly in the lungs of mice inoculated with the virus possessing(HA+NA)gene from DK29,and the expression level of IFN-? and IFN-?,in the group inoculated with the viruses contained PB2 or PB1 gene from DK29,were also significantly lower than that in MA-50-inoculated group,suggesting alleviatived inflammation reactions.Therefore,PB2 and HA genes played key roles in the virulence changes of H4 AIV during adaption in mice.In addition,HA played a role in the receptor binding specificity changes and PB2 took part in the polymerase activity changes during the virus adaption in mice.According to the amino acids differences in PB2 and HA genes between DK29 and MA-50,site mutation plasmids were constructed to rescue the single mutation viruses in the background of DK29 and MA-50,respectively.Mice infection results showed that mutation at the position of 701 or 680 in PB2 protein led to virulence changes in mice via affecting the polymerase activity of virus.We preliminarily elaborated the molecular basis of infectivity and lethality of H4 subtype avian influenza virus in mice.