Studies On The Molecular Evolution And Pathogenicity Mechanism For Eurasian Avian-like H1N1 Swine Influenza Viruses

Pigs are known as "mixing vessel" for influenza A virus,which are susceptible to both avian-and human-influenza viruses.Eurasian avian-like H1N1 swine influenza viruses(EA H1N1 SIVs)were firstly detected in January 1979 in Europe.They were subsequently becoming predominant in pigs in both Europe and Asia.EA H1N1 SIVs were continuously reassorted with other co-circulated influenza viruses in swine,including 2009 pandemic H1N1(A(H1N1)pdm09),classical swine H1N1(CS H1N1)influenza viruses,and triple reassortment H1N2(TR H1N2)influenza viruses,and generated various genotypes.China identified the first EA H1N1 SIV in Hong Kong in 2001.After a long-term evolution,the majaority of EA H1N1 SIVs preferentially bind to human ?-2,6-linked sialic acids receptor.Some of them could effectively transmitted via respiratory droplets among ferrets.Therefore,EA H1N1 SIVs have recently been considered to possess the most potential risk for the next influenza pandemic.In this study,we firstly performed a systematic analysis on the molecular evolution of EA H1N1 SIVs in China.From 2001 to 2018,EA H1N1 SIVs were isolated from both pigs and human cases in nineteen provinces/municipalities of China.All EA H1N1 SIVs were classified into 11 distinct genotypes,from genotype 1 to genotype 11.Genotype 1 viruses accounted for 55.3%of the EA H1N1 SIVs.And the eight gene segments of this genotype viruses were exclusively originated from EA H1N1 SIVs.Genotypes 2-11 were the reassortment genotypes of EA H1N1,A(H1N1)pdm09,and TR H1N2 viruses respectively.The prevalence of genotypes illustrated that genotype 1 was widely predominant from 2001 to 2013.From 2009 to 2013,the genotypes 2 and 4 were co-circulated with genotype 1.Since 2013,genotypes 3 and 5 have gradually been predominant in pigs in China.Guangdong province had the largest number of genotypes,with a total of 7 genotypes.Molecular biomarkers analysis showed that the majority of EA H1N1 SIVs contained HA-190D(85.9%,H3 numbering)and 225E(84.1%,H3 numbering),which might aid the viruses to preferentially bind to human ?-2,6-linked sialic acids receptor.EA H1N1 SIVs could occasionally cross species barriers and infect humans.China reported the first human case with EA H1N1 SIV infection in Jiangsu province in 2011.The virus isolated from the patient was termed as A/Jiangsu/1/2011(JS1).Eight gene segments of this virus were exclusively originated from EA H1N1 SIVs,which was belonged to genotype 1.In 2015,another triple reassortment EA H1N1 SIV[A/Hunan/42443/2015(HuN)]was isolated from the human infection case in Hunan province.The HA,NA and M gene segments of HuN virus were derived from EA H1N1 SIVs.The PB2,PB1,PA and NP gene segments were origined from A(H1N1)pdm09.The NS gene segment was derived from TR H1N2 SIVs.It was belonged to genotype 3.We found that the pathogenicity of genotype 3(HuN-like)viruses in mice was significantly higher than those of genotype 1(JS1-like)viruses.However,the underlying pathogenicity mechanism was still unknown.To figure out which gene determined the pathogenicity of the two genotype viruses in mice,gene segments of HuN-like and JS1-like viruses were reassorted to produce the different recombination viruses.The results showed that the NP gene was the major determinant of the pathogenicity of EA H1N1 SIVs in mice.By comparing the NP sequences,three species specific substitutions including R305K,F313V,and Q357K in the NP protein,were observed between HuN-like and JS1-like viruses.In order to determine the effects of the three substitutions on the pathogenicity in mice,we introduced corresponding mutations in HuN virus.The further study revealed that the NP-K357Q mutation significantly reduced the replication and virulence of EA H1N1 virus in mice.In addition,the NP-K357Q mutation reduced the virus polymerase activity and replication titer compared to rgHuN-WT.The results suggested that NP-Q357K mutation determined the pathogenicity of EA H1N1 SIVs in mice.Previous studies showed PB2-E627K,D701N and T271A mutations were well recognized molecular biomarkers which could enhance the viral pathogenesis of H5N1 and H7N9 influenza viruses in mammals.Both JS1-like and HuN-like viruses possessed amino acids 627E in PB2 protein.They possessed PB2-701D and 701 N,PB2-271T and 271A respectively in JS1-like and HuN-like viruses.Previous study showed the PB2-D701N mutation could enhance the pathogenicity of EA H1N1 SIVs in mice.However,the effect of PB2-T271A mutation on the pathogenicity of EA H1N1 SIVs has not been reported.Our study demonstrated that the mutation PB2-T271A could significantly enhance the viral replication in A549,PK15,and DF-1 cell lines and increased the virulence of the EA H1N1 SIVs in mice.It suggested that PB2-T271A mutation was also an important molecular biomarker affecting the pathogenicity of EA H1N1 SIVs in mice.Taken together,we performed a systematic evolutionary analysis and key molecular markers on the EA H1N1 SIVs in China since 2001.Currently,genotype 3 and 5 have been predominant in pigs in China.NP gene determined the pathogenicity of EA H1N1 SIVs.In addition,a novel molecular biomaker of NP-Q357K acted as a virulence determinant of EA H1N1 SIVs in mice.Another substitution of PB2-T271A could increase the virulenceof EA H1N1 SIVs in mice as well.The study provided scientific evidence for the risk assessments and the target surveillance of the EA SIVs.