| In 2014,human infection with H5N6 subtype of avian influenza virus has been found in Sichuan,Anhui and Guangdong areas.And the mortality rate is extremely high,which has resulted great concern for public health in the world.Apart from H5N1 and H7N9,H5N6 has become the third highly virulent virus that can infect people.Although it has not been found that the avian influenza virus can be transmitted among human beings,studies have shown that H5N1 virus can be transmitted among ferrets by air if a few site mutations or proper reassortment are found.If people are infected by H7N9 virus,the virus can be directly transmitted through the air among the ferrets without mutation.It has been reported that H5N6 virus can be transmitted by direct contact among the ferrets.Some scholars predict that H5,H7 or H9 subtype avian influenza viruses might be the next generation of prevalent strains.At the same time,the isolation rate of H5N6 virus among migratory birds and poultry is extremely high,which has become the main subtype among the poultry instead of H5N1 virus.Therefore,it is necessary to have a better understanding of the biological characteristics of the H5N6 subtype avian influenza virus transmitted among the migratory birds and poultry and clarify the risk of pathogenicity and transmission among the mammals,which have great significance for the scientific cognition,risk assessment,comprehensive prevention and control of H5N6 virus.In this study,H5N6 subtype avian influenza virus was first isolated from the migratory birds and poultry for the whole genome sequencing.Then,its genetic characteristics were interpreted through the analysis on its genetic evolution.Subsequently,the analysis on the biological characteristics was carried out to clarify whether the H5N6 subtype avian influenza virus from migratory birds and poultry could cross-infect mammals and cause disease.Besides,its risk of transmission among mammals was also studied.Then,the adaptation passage was adopted to simulate the adaptive process of H5N6 avian influenza virus in the mammals.Besides,the paper has also discussed whether the interspecies infection,pathogenicity and transmission risk of strains adapted to the mammals have been enhanced,looked for the key mutation sites of amino acids related to the pathogenicity and transmission capacity and preliminarily described the molecular mechanism.At last,we have also analyzed the air biological mechanism of the influenza virus that can be transmitted in perspective of the aerosology.The paper has provided the theoretical and technical support for the infection and transmission of H5N6 avian influenza virus in our country.The findings and conclusions obtained are as follows:1.In this study,we have successfully separated three strains of H5N6 avian influenza virus in one poultry market of Hubei Province(Duck Source A/duck/Hubei/01/2016(H5N6),DK-01 for short;chicken source A/chicken/Hubei/918/2016(H5N6),CK-165 for short;chicken source A/chicken/Hubei/918/2016(H5N6),CK-918 for short).Apart from that,professor Chai Hongliang from Northeast Forestry University also donated a strain of H5N6 avian influenza virus from chicken source to us(Anas clypeata source A/Northern Shoveler/Ningxia/488-53/2015(H5N6),NX-53 for short).In this paper,the whole genome sequencing has been performed on four strains.Besides,the sequence alignment and phylogenetic analysis have also been conducted.The results have shown that DK-01 and NX-53 are the recombinant virus of H5N6 avian influenza virus in which NA gene originates from H6N6 and the others are from H5N1.CK-165 and CK-918 are the recombinant virus of H5N6 and H9N2 avian influenza virus in which HA and NA genes originate from H6N6 and the others are from H9N2.The surface antigen genes,HA and NA of the four strains are highly homologous to strains of human-infected H5N6 virus.And the sequence similarity ranged from 96% to 98.5%.As a result,the separated strains in this study and strains of human infected H5N6 virus might originate from the same source.2.The binding specificity of poultry ?-2,3 receptors and the binding characteristic of unmanned ?-2,6 receptors have been found in NX-53 virus from the migratory bird source.The weight of infected guinea pig reduced slightly at first and then was back to normal.No death was found.Virus could always be detected in the lungs of infected guinea pig and pathological injury was also found.After the guinea pig was infected,the virus was detected in its nasal lavage fluid.However,the virus could not be transmitted among the guinea pigs through direct contact and air.Sequently,in this study,we have created the guinea pig-adapted strain in its body through the continuous passage and found that the pathogenicity of the guinea pig was enhanced.The tenth-generation virus could cause 100% death rate,which can also cross the blood brain barrier and be detected in the brain.However,it still can't be transmitted among the guinea pigs through the direct contact and air.From the sequence analysis,eight key amino acid sites related to the enhancement of pathogenicity have been found,which are HA-I67 V,HA-R239 H,NP-H52 Y,NP-A430 T,PA-T97 I,PA-S388 C,PB2-T23 I and PB2-E627 K respectively.From the above analysis,it has shown that bird source H5N6 avian influenza virus can be transmitted to the mammals,which can also be replicated and proliferated in the mammals.However,the pathogenicity is relatively low.When the virus was adapted to the mammals,the pathogenicity was obviously enhanced.However,it can't be transmitted and the epidemic risk among human beings is relatively low.3.The pathogenicity and transmission capacity of three different poultry source H5N6 avian influenza virus are different among the mammals.DK-01 virus has the binding specificity of ?-2,3 receptors and very weak ?-2,6 receptors.The binding specificity of poultry ?-2,3 receptors and the binding characteristic of manned ?-2,6 receptors have been found in CK-918 and CK-165 virus.The weight of the guinea pig infected by DK-01 and CK-165 reduced obviously.Death has been found and the death rate was 60%.However,the pathogenicity of CK-918 virus was extremely low.The weight of infected guinea pig reduced slightly at first and then was back to normal.No death was found.Virus could be always detected in the lungs of guinea pig infected by the three strains and pathological injury was also found.From the experiment of transmission,CK-165 virus can be transmitted among the guinea pigs through direct contact.However,the airborne transmission was not found.The above results show that H5N6 influenza virus from the poultry source can infect the mammals.The binding specificity of human receptor can be found in certain strains and the pathogenicity is extremely high.As a result,the virus can be transmitted among the mammals through direct contact,which has potential public health risks and poses a significant threat to human health.4.Adaptive passage of CK-918 virus with low pathogenicity was performed in the guinea pig.It has been found that the pathogenicity of the virus can be significantly enhanced.The second-generation virus can result in 100% death rate.The study show ed that mutation at NS1-G183 R site played an important role in the enhancement of the pathogenicity(first found in the world).The mutation has significantly improved the replication capacity of H5N6 avian influenza virus in the mammals and broadened the tissue distribution in the guinea pig,which can suppress the interferon IFN-? passage.In addition,we have performed the adaptive passage of CK-165 virus originating from the poultry and being transmitted through the direct contact in the guinea pig to acquire the adapted virus strains which can be transmitted through the air.Then,we can find the amino acid sites related to the airborne transmission.However,after 15-generation adaptation,the pathogenicity of the virus has been enhanced,but it still can not be transmitted through the air.As a result,it has been found that H5N6 influenza virus from the poultry source can be transmitted to human beings through the direct contact and can result in high pathogenicity.But its epidemic risk among human beings is extremely low.5.In this study,we have analyzed the aerobiological mechanism of the transmission of the influenza virus among guinea pigs from the perspective of the aerobiology.Results of the study are as following.After the guinea pig was infected by the untransmissible influenza virus,it still can breathe out the viral aerosol.However,the exhaled aerosol dose(48±31 copies/L)was much lower than the exhaled dose of guinea pigs infected with airborne influenza virus(11432±3892 copies/L),which was 238 times different.At the same time,size of aerosol particles exhaled by the former(3.89±0.07 ?m)was significantly higher than that exhaled by the latter(2.43±0.18 ?m);No significant difference has been found in the sedimentation and decay law of the untransmissible virus and airborne virus.The half-infected dose of untransmissible viral aerosols for guinea pigs was not significantly different from the that of airborne viral aerosols for guinea pigs.The above study has shown that the small dose of exhaled virus aerosol and large particle size may be the main reason for its inability to transmit when the untransmissible virus infected the guinea pigs. |
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