Biological Characteristics Of A Type H1N1 Subtype Influenza Virusin Guangdong Province In 2016?2017

Influenza which belongs to orthomyxoviridae virus can cause an acute,thermal respiratory infectious diseases.According to different genetic and antigenic characteristic of the virus particles nucleoprotein protein and matrix protein,influenza viruses can be divided into four type A,B,C,D,of which A influenza viruses(AIV)are the most.Pigs are considered as"mixing vessel"for AIV because of common their respiratory tracts contain both?-2,3 and?-2,6 sialic acid receptors.Avian,swine and human influenza viruses can recombine in pigs to produce novel viruses that can spread to humans and cause a pandemic,posing a serious threat to public health.Hemagglutinin(HA)and Neuraminidase(NA)are antigenic proteins on the surface of AIV particles,which can be classified into different subtypes basing on the differences between HA and NA proteins.Currently,the H1N1 AIV is one of the main subtypes circulating in humans and pigs.Two of the four human influenza pandemics in history were caused by the H1N1 subtype of influenza virus,including the Spanish flu in 1918 and the pandemic in 2009(pdm09 H1N1).Guangdong province is a large densely populated province in China with developed import and export trade,and it is a unique ecosphere of influenza virus.Therefore,the study on the biological characteristics of H1N1 AIV circulating in Guangdong population and the pigs is of great significance for the public health.Therefore,this study selected H1N1 subtype swine influenza virus(SIV)and human influenza virus in Guangdong as the research object,growth ability in vitro and pathogenicity characteristics of the virus through the analysis of genetic characteristics,genetic evolution to analyze the biological characteristics of the H1N1 AIV in Guangdong.In this study,a total of 319 nasal swabs from pig were collected in Guangdong in 2017.Five positive samples were screened out through M gene testing,with a positive rate of 1.58%.A total of 4 strains of SIV were isolated from 5 positive swabs through infected in MDCK cells,with a total virus isolation rate of 1.25%.Four SIV were identified as H1N1 subtype,and 33 human influenza virus were obtained from CDC of Guangzhou.The genetic evolution analysis of 37 influenza viruses showed that 4 SIV belonged to the same genotype,HA,NA and M genes belonged to the Eurasian avian-like clade(EA H1N1),internal genes PB2,PB1,PA and NP belonged to the pdm09 H1N1 clade,and NS genes belonged to the classical H1N1 clade(CS H1N1).33 human influenza virus belonged to genotype,and 8 gene belonged to the pdm09 H1N1 clade.Analysis of the receptor binding sites(RBS)showed that 4 strains of SIV were adaptive to mammals,and amino acid mutations occurred in the RBS of some human influenza virus,which may have shown adaptability to avian.Analysis at the antigen sites showed that there were no amino acid mutations in the four strains of SIV,but amino acid mutations took place in some human influenza virus.The replication ability of 37 AIV in different cells was detected,and the results showed that the virus titer of the SIV and human influenza virus in MDCK cells were 2.50?8.50lg TCID₅₀/m L and 3.30?7.67 lg TCID₅₀/m L,respectively.The virus titer of the SIV anf human influenza virus in PK15 cells was 3.33?6.33 lg TCID₅₀/m L and 1.67?3.90lg TCID₅₀/m L,respectively.The virus titer of the SIV and human influenza virus in ST cells was 4.50?6.50 lg TCID₅₀/m L,and 0?3.33 lg TCID₅₀/m L,respectively.The virus titer of the SIV and human influenza virus in 3D4/21 cells was 3.50?6.50 lg TCID₅₀/m L and 1.50?6.50 lg TCID₅₀/m L,respectively.The virus titer of the SIV and human influenza virus in PED cells was 1.67?6.83 lg TCID₅₀/m L,and 2.67?5.50 lg TCID₅₀/m L,respectively.The virus titer of the SIV and human influenza virus in IPEC-1 cells was 0.67?2.67 lg TCID₅₀/m L,and 0?1.50 lg TCID₅₀/m L,respectively.The pathogenicity of S182 and H752 were tested in vivo.The results showed that S182could cause clinical symptoms in pig after infecting.The virus titer of the nasal wash was1.70?2.37 lg TCID₅₀/m L in the infection group,and 1.37?3.37 lg TCID₅₀/m L in the contact group.The titer of virus in the organs of the infection group was 1.37?2.37 lg TCID₅₀/g/m L,while that in the contact group was 1.37?2.20 lg TCID₅₀/g/m L.The antibody titer of the infection group ranged from 1:10 to 1:20,while that of the contact group ranged from 1:10to 1:160.The H752 did not cause clinical symptoms in pig after infecting.The titer of the nasal washing in infection group was 1.20?2.37 lg TCID₅₀/m L,while that in the contact group was 1.20?2.20 lg TCID₅₀/m L.The virus titer in the organs of the infection group was1.37?2.20 lg TCID₅₀/g/m L,while that in the contact group was 1.37?2.20 lg TCID₅₀/g/m L.The antibody titer of the infection group were 1:10?1:20,while that of the contact group were 1:10?1:20.To sum up,the H1N1 SIV circulating in Guangdong province has been recombinated with other clade.Although H1N1 subtype of human influenza virus has not been recombinated with other clade,the amino acid on the antigen site,receptor binding site and internal genes related to the pathogenicity of the site in HA gene has been mutated.In this study,the two groups of viruses had similar growth capacity and titer in MDCK,3D4/21 and PED.Beside MDCK cells,the remaining two cell lines could be used as candidate cell lines for isolating SIV.The growth ability of SIV in PK15,ST and IPEC-1 was stronger than that of human influenza virus.Both groups of viruses were able to successfully infect pigs and caught transmission.And no significant differences in pathogenicity to pig were found.These findings will provide theoretical basis and data support for the surveillance and control of H1N1 subtype swine influenza virus,public health,the screening of swine-original cells suitable for replication of swine influenza virus,and the pathogenicity study of H1N1influenza virus circulating in swine in recent years.