Identification Of Virulence Markers And Pathogenesis Of PB2 Protein Of H5N8 Avian Influenza Viruses With Enhanced Pathogenicity In Mice

H5N1 subtype highly pathogenic avian influenza virus(AIV)A/Goose/Guangdong/1/1996(Gs/GD/1996)was first detected from goose in 1996 in Guangdong,China,after that the viruses of H5N1 subtype always exist in our country and infect both wild birds and poultry.H5 subtype highly pathogenic AIVs not only cause high morbidity and mortality in poultry,but also occasionally cause high mortality in humans by cross-species transmission.Recently,the H5N1 viruses evolved into novel reassortant H5Nx viruses with different neuraminidase genes,including H5N2,H5N6 and H5N8,in which the novel H5N8 viruses distribute widely.Therefore,it is crucial to understand the viral pathogenesis and identify virulence markers of the novel H5N8 viruses.In this study,we characterized two H5N8 AIVs,which shared high similarity of their genetic background and exhibited remarkably different pathogenicity in mice,and analyzed the molecular basis and host mechanism for different pathogenicity.1.Selection and characterization of H5N8 AIVs with different pathogenicity im mice The pathogenicity in mice of 10 H5N8 AIVs which from different hosts isolated from 2012 to2014 were determined.Two isolates,A/goose/Eastern China/CZ/2013(CZ)and A/duck/Eastern China/JY/2014(JY),which exhibited significantly different virulence in mice,were selected as model viruses.Phylogenetic analysis of the HA gene indicated that the two viruses belonged to a newly evolved clade 2.3.4.4 and comparison analysis of the whole-genome sequences showed that there were only 25 amino-acid differences throughout eight genes.Hemagglutination(HA)assays using ?-2,3-sialidase-treated goose red blood cells demonstrated that both viruses exhibited a dual-receptor-binding preference.Viral growth kinetics in vizo indicated that both viruses replicated capably in CEF cells to a high titer of about 8 log10 TCID5O/mL.In MDCK cells,however,CZ replicated efficiently(7 log 10TCID50/mL),while JY grew to peak titers below 4 log10TCID50/mL.Animal studies indicated that although both viruses were highly virulent in chickens,they exhibited significantly different virulence in mice.CZ was highly virulent(MLD50= 101.6EID50),whereas JY had low virulence(MLD50>106.5 EID50).2.Synergistic effect of PB2 283M and 526R contributes to enhanced virulence of H5N8 influenza viruses in miceA set of reassortant viruses were generated by exchanging a single gene between r-CZ and r-JY viruses and virulence in mice of each reassortant virus was determined in contrast to their parental viruses.The results showed that when compared to the parental r-CZ virus,the reassortants having PB2,PB1 and PA from the JY virus displayed attenuation.However,the JY based reassortant carrying only PB2 from CZ virus(JY-CZPB2)showed an increased virulence in mice.Through site-directed mutation of amino acid in PB2 gene between r-CZ and r-JY viruses,we demonstrated that 283M and 526R in PB2 of the H5N8 viruses contributed to viral polymerase activity,viral replication in mammalian cells(MDCK)and virulence in mice.Further study confirmed that the 283M and 526R in PB2 had similar impacts on a H5N1 AIV.Therefore,283M in PB2 is a new mammalian virulence marker and synergistic effect of 283M and 526R is a critical factor for viral high pathogenicity in mice.3,Host factors for enhanced virulence in mice infected by H5N8 AIV with PB2 I283M and K526R mutationsThe lungs of mice infected with parental virus r-JY and mutant virus JYPB2-I283M-K526R at 3 days post infection(dpi)were collected and subjected to a high-throughput RNA-seq analysis?The results showed that both sequenced numbers of transcripts and expression levels of host differentially expressed genes(DEGs)were higher in JYPB2-I283M-K526R-infected mice than that in r-JY-infected mice.Gene Ontology(GO)enrichment analysis showed that DEGs in r-JY-infected mice were mainly associated with functions in response to stress,defense response and immune response,while DEGs in JYPB2-I283M-K526R-infected mice were associated with functions in response to stress,immune system process and protein binding.The numbers of host immune-related genes induced by the mutant virus were about 4 fold higher than that induced by r-JY.Further,heat map profiles revealed that expression levels of immune response-related genes in JYPB2-I283M-K526R-infected mice were higher than that in the parental virus-infected mice,suggesting that mutant virus induced an excessive innate immune response in mouse lungs,In addition,JYPB2-I283M-K526R virus induced stronger apoptosis in mouse lungs than r-JY and mock-infected control at 3 dpi.Dual sequencing of viral transcripts and qRT-PCR results revealed that expression levels of the mutant virus genes were higher than that of parental virus at 3 dpi,which is consistent with higher viral titers in lungs of the mutant virus-infected mice.In conclusion,the synergistic effect of 283M and 526R in PB2 is responsible for high virulence of the H5N8 viruses in mice.Moreover,the induced abnormal host innate immune response is correlated with the high virulence of CZ virus in mice.