| The avian influenza virus(AIV)could infect wild birds and domestic poultry,many kinds of animals and even people.Wild birds as the natural host of AIV,especially migratory birds play an important role in the transcontinental transmission of avian influenza virus.In this study,the diversity,prevalence and evolution of avian influenza viruses among migratory birds were investigated through continuous monitoring in eastern China.The public health risks of avian influenza viruses were evaluated by experimental of pathogenicity and transmission capacity of the viruses in animal model.The results are of great significance to understand the pathogenic ecology of AIVs in migratory birds and to prevent and control the emergency infectious disease.From 2019 to 2021,Surveillance AIV in migratory birds passing through eastern China was carried out for three years according to migratory bird migration periods.A total of 51766 migratory bird samples and environmental samples were collected,covering more than 10 bird species.The 126 low pathogenic avian influenza virus(LPAIV)strains and 84 high pathogenic avian influenza virus(HPAIV)strains were isolated,including 11 HA subtypes and 9 NA subtypes in total.The results showed that there was abundant subtype diversity of avian influenza viruses in migratory birds,and the highly pathogenic avian influenza H5 subtype,which had been silent for several years,was introduced into China from Europe and reassorted.Among the low pathogenic avian influenza viruses,H6 subtype had the highest detection rate and isolation rate,and was the dominant subtype among migratory birds in recent three years,and also showed that the prevalence of H7 subtype in wild birds increased year by year.It was found that the H5 HPAIV,which had been silent for many years,spread across the continent to various regions of China through migration of birds with evolution,recombination if the virus.This study focused on monitoring migratory birds in cross-border areas during migration period.We found that the HPAIV H5N8 entered China through whooper swans in October 2020.The H5N8 subtype was found in wild bird samples in Jiangsu,Shaanxi,Ningxia,Henan,Inner Mongolia and Shanghai from late 2020 to mid-2021,Involving wild bird species for whooper swans,tundra swans and black necked grebes.Phylogenetic analysis of the entire H5N8 gene sequence showed that: All H5 HPAIVs isolated from migratory birds in 2020～2021 belonged to the 2.3.4.4b reassorted strain,and HA gene analysis showed that all H5 subtype HPAIVs clustered in the same evolutionary branch with the H5 HPAIVs prevalent in Europe in2020.All NA genes analysis revealed that the N8 gene was clustered in the same evolutionary clade as the H5N8 HPAIV endemic in Europe.Internal genetic analysis revealed a rearrangement of a strain(SMX01)with LPAIV circulating in Europe.In this strain,the PA gene was reassorted,and the low pathogenic avian influenza viruses clustered in the same evolutionary clade.Therefore,it is highly likely that the virus was introduced to China directly by migratory birds from Europe.In 2021,a reassortative H5N6 strain was found in our surveillance program.Analysis showed that the HA gene had a high similarity with the H5N8 HPAIVs recently circulating in China,however,NA and H5N6 AIV are clustered in the same evolutionary branch as those prevalent in China.Internal gene analysis also showed that PA and M genes were derived from H5N8 HPAIV,while PB2,PB1,NP and NS genes were rematched with LPAIV in China.However,no rearrangement of the two strains was found in the follow-up investigation,confirming that the strain was not prevalent in wild birds.H5N8 HPAIV rearrangement is not accidental,but provides the conditions for H5 HPAIV to acquire host adaptation.Our investigation revealed that the H5N1,H5N8,rearranged with LPAIV in Europe,entered China in October 2021.Subsequently,the new H5N1 rearranged strain became the main epidemic strain and was prevalent in wild birds in Inner Mongolia,Hebei,Henan and Shanxi provinces.H5N8 and H5N1 HPAIV have caused outbreaks in wild birds and poultry in many countries,but have not caused widespread infection in poultry in China,in which the compulsory vaccination in China played an important role.The results showed that H5 Nx HPAIVs entered China several times by migratory birds,and reassorted with low pathogenic avian influenza virus after re-transmission in China.All results indicate that the avian influenza viruses of migratory birds have diversity of subtype,and the reemergencing highly pathogenic avian influenza virus H5 Nx continues to be prevalent in migratory birds In order to evaluate the infection,human-to-human transmission and public health risk of H5 Nx isolates,we carried out the infection and transmission ability tests of H5N8,H5N1,H5N6 isolates and H5N8 strains with PA recombination by ueing a guinea pig models.We found that all the infected guinea pigs were infected by the 4 virus strains and antibodies turning positive were found.No virus replication and antibody conversion were detected in the direct contact groups and indirect transmission groups for the 4 virus strains.the results indiacted that these viruses have not yet acquired the ability to spread among mammals.In LPAIVs,transcontinental gene exchange between north American lineages and Eurasian lineages was found,with the typical H6 subtype of low pathogenic avian influenza viruses.The virus is widespread in wild birds and is usually strictly regional,with the Eurasian lineage predominating in China.In this study,two H6N8 LPAIVs were found to be north American lineages and Eurasian lineages rearrangement strains.Analysis showed that: NA segments clustered in the same evolutionary clade with those of H3N8 subtype AIV isolated from Alaska,North America.HA and the other 6 internal gene segments all belonged to Eurasian evolutionary clade,and the two avian influenza viruses had different reassortments.PB2 and M of the two strains were in the same evolutionary clade.However,PB1,PA,NP and NS genes are in different evolutionary branches.The most recent expected common ancestor(t MRCA)of the two strains was deduced by Bayesian inference to 2018.In conclusion,the long-distance migration of migratory birds provides the main conditions for frequent reassorting of influenza strains between Eurasian and North American lineages,resulting in frequent gene fragment exchange or recombination of influenza strains between different continents.In our survey,we also found that the positive rate and isolation rate of H7 LPAIV in wild birds increased gradually.The genetic evolution analysis of H7N3 showed that H7N3 AIVs prevalent in migratory birds was very different from H7N3 AIVs previously prevalent in China.The analysis of H7N3 AIVs isolated showed that the genes of 8 AIVs isolates were all rearranged.The HA gene was derived from H7N7 and NA gene from H5N3.The H7N3 reassorted AIVs was found to be the main epidemic strain of wild birds in China since 2020.In order to evaluate the infection ability of H7N3 LPAIV to mammals,the infection ability test in mice was carried out in BALB/c mous model we found that the survival rate of experimental mice was 100%,and the weight loss was only slight on the third day after infection.Titration of virus in viscera showed that the virus could be detected in the lungs and turbinate bones of mice.All the results showed that H7N3 avian influenza virus from migratory birds was low pathogenicity to mice.Our studies indicated that the avian influenza viruses circulating in migratory birds with diversity.The migration of migratory birds is one of the main factors leading to the transcontinental spread and epidemic of avian influenza virus,which provides opportunities for frequent gene reassorting of influenza viruses.In turn,the influenza virus acquired host adaptation and became widespread in wild birds.Therefore,strengthening the monitoring of migratory bird avian influenza virus and biological evaluation of migratory bird avian influenza virus can provide theoretical support for proactive early warning of epidemic disease. |
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