Isolation And Characterization Of H1N1 Influenza A Virus From Swine And Contribution Of The Hemagglutinin(HA) Protein To Pathogenicity

H1N1 swine influenza virus(SIV)was the significant causative agent of swine influenza(SI).It is a negative-sense,single-stranded RNA virus in the family of Orthomyx-oviridae.SIV genome contains eight segments encoding 11 proteins.H1N1 SIV was initially discovered in the United States in 1918.Outbreaks caused by a novel H1N1 influenza A virus occurred in North America in 2009,and the so-called "2009 pandemic H1N1 virus" has continued to be spread globally and leaded to severe prevalences.This virus is able to infect not only mammals,including humans,via human-to-human transmission,but also animals such as pig,dog and ferret by interspecies transmission.It is worthynote that the 2009 pandemic H1N1 virus could be transmited to pigs from humans or pigs.The evolution and pathogenicity of the virus in porcine are largely unknown.In this study we sought to investigate the evolution and pathogenicity of the virus in pigs.2300 samples of suspicious lung tissues were collected in pig farms in Nanning,Liuzhou,Hezhou,Yulin,Qinzhou,Guigang,Beihai and Guilin of Guangxi from 2009 to 2011.Nine isolates were identified as H1N1 subtype influenza viruses by RT-PCR and sequence analysis.Three of those isolates,which were named as A/Guangxi/l/2011(SwGX1/11),A/Guangxi/3/2011(SwGX3/11)and A/Guangxi/12/2011(SwGX12/11),respectively,were determined as 2009 pandemic H1N1 virus,as they exhibited as high as 99.1%to 100%nucleotide identities in HA,NP,M,NS,PA,PB1,PB2 and NA genes with the references.There were 23,16 and 16 amino acids in SwGX1/11,SWGX3/11 and SwGX12/11 strains were found to be alterated,compared with A/Califomia/04/2009.It should be noted that most alternations were present in HA or NA gene.Critical sites relating to property of the isolated viruses were at position of 225 and 226 in HA gene,119,295 and 386 in NA,375 in NP.Importantly,the amino acids at positions of 225 and 226 of HA gene of SwGX12/11 were glycine and glutamine,which were hallmarks of human influenza A virus.Subsequently,we determined the pathogenicity of the 3 viruses in vitro and in vivo.The result indicated that these three viruses grew well on MDCK cells,and the virus titers reached 107.7,107.5 and 108.0 TCID50/ml,respectively.Eight BALB/c mice were infected with 106TCID50 of each virus in a volume of 50 ?l by the intranasal route.All mice infected with the three isolated viruses showed typical symptoms of influenza on 3th day post-infection.All mice infected with these viruses exhibited consistent and significant weight loss to a level between 9.13%and 23.04%.Two mice infected with the SwGX12/11 virus eventually succumbed to the infection by day 7 and 9 post-infection.To further investigate the difference in pathogenesis among all viruses,we examined the viral loads in lung and turbinate tissues of the challenged mice,three mice were euthanized on day 3 postinoculation.The viral titers of SwGXl/11,SwGX3/11 and SwGX12/11 in lung were 105.53±0.4,105.67±0.29 and 106.2±0.35 TCID50/mL,while the titers in turbinate tissue were 103.83±0.42,102.73±0.44 and 105.73±0.64 TCID50/mL,respectively.These results illustrated that the 3 isolates were highly pathogenic to mice,whereas SWGX12/11 strain caused the highest level of pathogenicity.To ascertain the role of the HA protein of the isolated viruses in pathogenicity,we rescued SwGX 12/11 virus using an eight-plasmid reverse-genetics system.The rescue virus,which was designated RSwGX12/11,exhibited 100%identity at nucleotide level and identical level of virulence in terms of TCID50,EID50 and pathogenicity to mice,compared to the wild-type SwGX 12/11 virus.To define the residues in HA protein that are important for pathogenicity,using the reverse genetics technique,we made two mutations,Q226R and G225D in HA protein,and rescued two mutant viruses which were named HAmutQ226R and HAmutG225D?Q226R.The two mutant viruses were not different in their TCID50,EID50 and pathogenicity to mice.Interestingly,the mutation introduced to the SwGX 12/11 virus changed dramatically the pathogenicity of RSwGX12/11 virus to mice as well as the replication in MDCK cell.The values of TCID50 of HAmutQ226R and HAmutG225D&Q226R virues were 104.5 and 103.8,which were significantly lower than that of RSwGX12/11 of which was 107 5,Consistently,the body weight losses casued by the two mutant viruses were 2.46%and 2.35%,which were much lower than that of RSwGX12/11 virus of which was 24.33%.The results clearly showed that the change from glutamine(Q)to arginine(R)at position 226 of HA resulted in dramatically reduced pathogenicity to BALB/c mice and level of replication in MDCK cells.Consistent with our findings,neither D225G nor G225D mutation in HA leaded to the change of pathogenicity and capability of replication when arginine(R)at position 226 of HA remained unmodified.These data suggested that arginine(R)at position 226 of HA played a critical role in the pathogenicity of the isolated viruses to mice.In summary,we investigated the epidemiology and pathogenicity of 2009 pandemic A(H1N1)influenza virus isolated from pigs in Guangxi,China.Our results indicate that 2009 pandemic A(H1N1)influenza virus has been circulating in pigs in Guangxi.Point mutation at position 225 and 226 of HA gene resulted in alternation of pathogenicity in mice and replication in MDCK cells.Our findings provide important scientific basis for the study on the pathogenesis of SIV.