| In recent years,avian influenza has not only caused great losses to the poultry industry in China,but also affected the population structure of wild birds and seriously threatened the public health and safety of the world.Migration of wild birds is a natural phenomenon which could cause the spread of influenza virus.Many scholars have the idea that wild birds are the natural hosts of avian influenza virus,especially such as echelons,closely related to the spread of the virus.Some researches have documented that several subtypes of influenza,including H5 and H7,have been isolated from 241 wild bird hosts of 27 orders.Since the spring of 2013,human infection with H7N9 subtype avian influenza virus has appeared in many areas of China.By the end of March 2019,615 of the 1567 confirmed cases had died,with a mortality rate of 39.25%,reminding people to pay attention to the potential threat of H7 subtype avian influenza virus to public health and safety.Two wild bird influenza viruses,a/Baer’s pochard/Hunan/414/2010(H7N1)(hereinafter referred to as 1T-H7N1)and a/lesser white fronted goose/Hunan/412/2010(H7N7)(hereinafter referred to as WT-H7N7),were isolated from the body of blue headed diving duck and small white geese respectively in our laboratory.The preliminary study shows that WT-H7N1 and WT-H7N7 have low pathogenicity to mice.In order to further study their adaptability in mammals,we used mice as mammalian model.passed the two viruses in mice continuously,preparation of mouse adapted strains and evaluation of pathogenicity,so as to provide theoretical reference for evaluating the impact ofwild bird H7subtypeinfluenza viruses on public health safety.1.Preparation of two H7 subtype wild bird avian influenza virus mouse adaptive strainsWT-H7N1 and WT-H7N7 strains were passaged in mice respectively,and the mice were dead at 2 days post infection(d.p.i.)in the fifth and seventhpassage.The virus was isolated from the lung of dead mice,named MA-P1P5,MA-P3P5,MA-H1P7 and MA-H2P7 respectively.The MLD50(lgEID50)of MA-P1P5 and MA-P3P5 were 2.25 and 1.75 respectively,which were 10000 times and 31623 times higher than WT-H7N1 virus;the MLD50(lgEID50)of MA-H1P7 and MA-H2P7 were 3.5 and 2.75 respectively,which were 1000 and 5000 times higher than WT-H7N7 virus.The results of whole gene sequencing showed that there were five amino acid mutations in MA-P1P5,including PB2-I615T、PB2-E627K、HA-E122K、HA-G214E、HA-G227E and NA-S350N and six amino acid mutations in MA-P3P5,including PB2-I615T、PB2-E627K、HA-E122K、HA-G214E、HA-G227E 和 NA-S350N.Four amino acid mutations(PB2-E627K、PB1-R118I、PA-L550M and HA-G214R)were found in MA-H1P7,while MA-H2P7 possessed five amino acid mutations,such as PB2-E627K、PB1-R118I、PA-L550M、HA-G214R and NA-S372N.2.In vitro and in vivo replication ability and tissue tropism of two mouse adaptive strainsIn order to study the replication ability of the mouse adaptive strain in vitro,the virus was diluted to 0.01 MOI and inoculated into MDCK cells.After inoculation,the virus titer was detected every 12hours until 72 hours.Compared with the parent virus,the virus titers(lgTCID50/ml)of the mouse-lung adapted strain was significantly improved at 48 hours,and MA-P1P5,MA-P3P5,MA-H1P7 and MA-H2P7 were 5.75,6.05,6.02 and 6.08,respectively.In order to further study the replication ability of the mouse adaptive strain in vivo,the virus titers in the lungs of mice inoculated by 104 EID50 MA-P1P5 virus were 108.5±0.1 EID50/g and 108.6±0.2 EID50/g at 3d.p.i and 5d.p.i,which were 40 times and 8 time higher than that of WT-H7N1 respectively,while the virus titers of MA-P3P5 virus were 107.4±0.2EID50/g 和107.9±0.1 EID50/g at 3d.p.i and 5d.p.i,which were 3 times and 1.6 time higher than that of WT-H7N1.The virus titers of MA-H1P7 were 107.9±0.9 EID50/g and 108.4±0.5 EID50/g at 3d.p.i and 5d.p.i respectively,which were 398 times and 126 times higher than that of WT-H7N7;And the virus titers of MA-H2P7 virus were 108.2±0.7 EID50/g and 107.1±0.1 EID50/g at 3d.p.i and 5d.p.i,which were 7943 times and 6 time higher than that of WT-H7N1.In order to study the histotropism of the mouse adaptive strain,mice were challenged with 106 EID50 virus,and the lungs,brain,liver,spleen,kidney and intestines of mice were collected in orders at 3 d.p.i,The WT-H7N1 virus could be detected in the lungs and the intestines of the mouse,while WT-H7N7 virus could be only detected the virus in the lungs.while the lung adapted strain of mice could detect the virus in the lungs,brain and liver Viruses were detected in spleen,kidney and intestines,which indicated that the two wild bird virus strains had increased histotropism in the process of adaptation.It is worth noting that mouse adaptive strains could be detected in lung,brain,liver,spleen,kidney and intestine,indicating that the tissue tropism of the virus is expansive during the process of adaptation.3.Analysis of amino acid mutation and pathogenicity of H7N1 and H7N7 subtype avian influenza virus during adaptation in miceIn order to study the relationship between gene mutation process and pathogenicity of WT-H7N1 and WT-H7N7 during the adaptation in mice,we sequenced the whole genome of mouse adaptive strains in different passage and evaluated the pathogenicity of the virus in mice.PB2-E627K appeared in the 5th generation of WT-H7N1 virus strain in the process of adaptation with MLD50(lgEID50)of 1.75 and pulmonary interstitial vascular congestion and vascular wall edema were observed in the pathological sections of lung in mice by 106EID50 virusat 3 d.p.i.PB2-E627K appeared in the 3th generation of WT-H7N7 virus strain in the process of adaptation with MLD50(lgEID50)of 4.5 and the edema,infiltration of inflammatory cells around pulmonary small vessels and bronchioles were observed in the pathological sections-of lung in mice infected by 106 EID50 dose of virus at 3 d.pi..In conclusion,we conclude that WT-H7N1 and WT-H7N7 subtype virus could adapt in mice and mouse adapted strains were established successfully.In the process of adaptation,wild virus strains significantly enhance the pathogenicity and tissue tropism of mice,and increase the ability of replication in vitro and vivo.At the same time,PB2-E627K mutation was present in all four mouse adaptive strains and may be the key factor to enhance the virulence.The above results show that wild bird H7 influenza virus has a strong adaptability to mammals(mice),and has a potential threat topublic health and safety,which needs more attention. |
