The Study Of PA Gene's Effects On The Pathogenicity And Airborne Transmission Of H9N2 Subtype Avian Influenza Virus

Avian influenza(AI)has always been a topic of concern with 130 years of history.Avian influenza virus(AIV)can infect a variety of poultry,waterfowl,wild birds or migratory birds,we found that some AIV even can directly infect people and other mammals without adaptation in recent years.In 1997,Hong Kong H5N1 avian influenza infected humans and lead to death,has caused public panic.The H5N1 AIV which are the most common concern,are high pathogenic avian influenza(HPAI)and classified as Class A animal epidemics by the-World Organisation for Animal Health(OIE).HPAI has the characteristics of rapid transmission,high harm range,high mortality rate,it not only can infect poultry but also can infect mammals,when human infected HPAI the mortality rate up to 33%.In fact,the most common avian flu among poultry populations is the H9N2 subtype AIV.It belongs to low pathogenic avian influenza(LPAI).When Poultry infected H9N2 subtype AIV,there generally no significant clinical symptoms,mortality is also very low.When H9N2 subtype AIV infected with other pathogens,the poultry showed obvious clinical symptoms,H9N2 subtype AIV can provide multiple internal gene fragments,to form a new recombinant virus.According to the study,six internal genes of the H7N9 subtype AIV were derived from the H9N2 subtype.Therefore,strengthening the research of H9N2 subtype AIV infection and pathogenesis,is important to evaluate the influenza pandemic trend,guarantee the poultry industry development and protect human health,and also has important guiding significance to develop effective prevention and control measures.AIV genome is composed of 8 independent single-stranded negative-strand RNA fragments,PA is one of them.PA gene mainly encodes the influenza virus PA protein,and the study showed that the protein is closely related to the virus host specificity and adaptability.The 2009 new H1N1 can be efficiently transmitted through the aerosols in the population.The PA gene is one of the determinants.In this study,the H9N2 subtype AIV SD01 parent strain was used as the backbone,and the PA gene was replaced by reverse genetic manipulation to study the PA gene's effect on the airborne transmission ability and cross-transmission ability of H9N2 subtype AIV SD01 strain.It is divided into the following parts:First,rescue and identification of recombinant rSD01-PA strain virus.Based on the principle of homologous recombination,we constructed the H9N2 subtype AIV SD01 parent strain's eight transcription / expression plasmids and H1N1 influenza virus SD07 strain PA plasmid,using the homologous recombination primers designed by us and the bidirectional transcription expression vector pHW2000 presented by Dr.Robert Webster.Then sequenced the plasmids.The 293 T cells were co-transfected with SD07 strain PA plasmid and other 7 plasmids of SD01 parent strain to rescue rSD01-PA recombinant virus strain.Detected and calculated EID50: SD01 parent strain EID50 was 9.31±0.27/mL,rSD01-PA strain EID50 was 8.27±0.20/mL,there is a difference of 1.04 EID50/m L.The antigenic property test showed that the replacement of the PA gene did not affect the antigenicity of the strain.Second,the virus in the SPF chicken pathogenicity and transmission experimentsThe SD01 parent strain and rSD01-PA recombinant strain were diluted to 106-108EID50,and each dilution inoculated 5 4-week-old SPF chickens.At 5 dpi each group was randomly assigned to take the lung tissue,and measured virus titer in the tissue.The results showed that the virus titer of the two viruses in SPF chicken lung tissue has not significantly different(P>0.05)after the same dose of virus inoculated.With the increase of the inoculation dose,the titer of the virus in the lung tissue was also increased.It can be seen that the replacement of the viral PA gene did not affect its replication within the lung tissue of the SPF chicken.Virus aerosol transmission experiments was carried out in the SPF chicken positive and negative pressure isolator,60 SPF chickens were randomly divided into three groups:vaccination group,direct contact group and aerosol infection group.Regular collected the oropharyngeal cloacal cotton swabs,serum samples and air samples for detection.The results showed that in SD01 parent strain aerosol transmission experiments,we can detect the virus and antibodies from the aerosol infection group.The airborne AIV was detected at 4 dpi,and reached the peak at 8dpi,and then gradually reduced.In rSD01-PA recombinant strain aerosol transmission experiments,the inoculation group and the direct contact group had detected the virus and antibodies,but the aerosol infection group had no detectable virus,antibody detection was negative;no airborne AIV particles were collected,either.Transmission experiments showed that the rSD01-PA recombinant strain lost its ability to transmit airborne between SPF chickens.The new virus,which is reorganized by the sevengenes of SD01 and new H1N1 PA gene,has lost the ability to form aerosols.Third,the virus in guinea pigs pathogenicity and transmission experimentsTo investigate the pathogenicity of two strains on guinea pigs,we collected 5 female250 g guinea pigs each,and inoculated 300 ?L 107EID50 virus solution.At 5dpi toke the brain,turbinate,trachea,lung and kidney tissues of 3 guinea pigs,and detected virus titer of each tissue.The results showed that SD01 parent strain could not be replicated in guinea pigs after inoculated so we couldn't detect the virus,but rSD01-PA recombinant strains could replicate in guinea pigs turbinate and trachea,the titer was 3.76±0.64/g and 4.32±0.48/g,respectively.The replacement of the PA gene resulted in rSD01-PA recombinant strain acquired the ability of infecting guinea pigs and replicating in their turbinate bone and trachea.The aerosol transmission experiments of rSD01-PA recombinant strain were taken in the mouse positive and negative pressure isolator,15 250 g clean guinea pigs were randomly divided into 3 groups: inoculate group,direct contact group and aerosol infection group.Guinea pig's nasal washing samples,serum samples and air samples were collected regularly for identification.The results showed that the virus was detected in the nasal washing samples of the inoculated group and the direct contact group,and the corresponding antibody was detected in the serum.However,the aerosol infection group had no detectable virus in the nasal wash solution,no antibodies were detected in serum either.Airborne AIV in the isolator is negative.The transmission experiments showed that the rescued rSD01-PA virus strain has acquired the ability of infect guinea pigs,and could infect the guinea pig group by direct contact,but could not transmit the virus to the guinea pig by aerosol.Indicating that the new H1N1 influenza virus PA gen can enhances the ability of the strain to infect mammals by crossing the interspecific barrier.