Duck-originated H6N6 Subtype Avian Influenza Virus Infects Mice And Its Replication In Human Lung Tissues

Objective:The H6N6 subtype avian influenza virus is widespread in wild birds and poultry animals.The virus continues to evolve,and its host range gradually expands from poultry to mammals,some viruses even have the ability to recognize and bind to human-like receptors SAα-2,6Gal.Worryingly,the serum of some exposed people is positive for the specific antibody of H6 subtype avian influenza virus.Therefore,the H6N6 subtype avian influenza virus could cross the species barrier to infect mammals and even humans potential.Studies have confirmed that quail and chickens were the intermediate hosts for avian influenza viruses crossing species to humans.Waterfowl ducks always contribute to the transmission of avian influenza virus to land birds,but whether duck-originated H6N6 subtype avian influenza virus could cross the species barrier to mammals and humans is still unclear.In this study,the H6N6 subtype avian influenza virus isolated from aquatic duck was used.Gene sequencing and evolutionary analysis were used to ascertain the origin of the H6N6 subtype avian influenza virus and the changes in the key amino acid sites of 8 gene segments.Then,the viruses were inoculated into mice in vivo and human respiratory tissues in vitro to observe the pathogenicity of the virus to mice and its replicate in human lung tissues,preliminarily explore whether the H6N6 subtype avian influenza virus derived from aquatic duck has the potential to infect mice and humans.And it would lay the foundation for further exploration of the cross-species transmission of the H6N6 subtype avian influenza virus.Methods:1.Multiplication of avian influenza virus and determination of HA titer,EID₅₀ and TCID₅₀:Three strains of duck-originated H6N6 subtype avian influenza virus A/DK/KM/1135/2019（KM1135）,A/DK/GY/1774/2019（GY1774）,A/DK/GY/6952/2019（GY6952）were replificated in chicken embryos.The chicken embryo allantoic fluid was harvested,and HA titer,EID₅₀ and TCID₅₀were detected on the allantoic fluid to determine the virus concentration used in infection experiment in mice and in human respiratory tissues.2.Genetic and phylogenetic analyses:Viral RNA was extracted with the RNeasy kit（Qiagen,Valencia,CA）and was reverse transcribed.PCR reaction amplification was performed by using segment-specific primers,and the products were purified.The whole virus gene was sequenced by the Illumina Solexa system.Sequence comparison and homology analysis were carried out with Bioedit to analyze the changes of amino acid sites of eight gene segments of three strains of H6N6 viruses.Did model test first and then phylogenic trees were generated by the Maximum-likelihood method using the MEGA-X software with 1000 boot-strap replications.3.Avian influenza virus infected BALB/c mice:The mice were inoculated with virus through nose and eye.After inoculation,the weight,body temperature and mental state of the mice were recorded every day.The throat swabs and blood of each group of mice were collected on 3 d,5 d,7 d,and 14 d post-inoculation.Three mice in each group were euthanized on days 3,5,and 7 post-inoculation,respectively.Turbinate,tracheal and lung tissues were collected and divided into two parts.One part was ground and centrifuged to obtain supernatant and then isolated and cultured in 10-day-old embryonated chicken eggs and MDCK.The other part was fixed in 10%formalin solution at room temperature for 24 hours for pathological examination and virus protein detection.4.Avian influenza virus replicated in human lung tissue in vitro:The patient’s respiratory tract surgical resection specimens were collected cut into small tissue pieces,and then inoculate with the viruses in vitro.Tissue culture medium and 2tissue blocks were collected on 12 h,24 h,36 h,48 h,72 h post-inoculation,one tissue block was homogenized and then the virus was isolated and cultured;the other was fixed in 10%formalin,used for HE staining and immunohistochemical staining.Results:1.HA titer and EID₅₀ and TCID₅₀ of avian influenza virus post-replication:Among of the three strains,the HA titer,EID₅₀ value,and TCID₅₀ value of the KM1135 virus strain were the highest.Therefore,the infection ability and pathogenicity of km1135 strain were the strongest.The HA titer,EID₅₀ value,and TCID₅₀ value of the GY6952 virus strain are the lowest,and the virus has the weakest infectivity and pathogenicity.2.Gene sequencing and evolutionary analysis results:The HA cleavage site sequence of the three viruses is PQIETR/GL,with only a single basic amino acid belonging to the molecular characteristics of low-pathogenic AIV.H156K mutation of HA were found in three viruses,but no mutations in key amino acid sites of HA,NA,PB1,PB2,PA,and the virus still belongs to the genetic characteristics of avian influenza virus.3.Experimental results of avian influenza virus infection in BALB/c mice:In the mouse infection experiment,the mice showed no obvious clinical symptoms after the 3 strains of virus infection,and no mice died during the infection process.After inoculation of mice with KM1135 and GY1774 virus strains,the virus isolation and culture were positive in the mouse throat swab specimens,grinding fluid specimens of turbinate,trachea,and lung tissue.Histopathological examination showed that there were inflammatory reactions in the turbinate,trachea,and lung tissues of mice.The influenza virus NP antigen in the tissues was detected.KM1135 was the most typical.After the GY6952 virus strain was inoculated in mice,the lung tissue grinding fluid was positive for virus isolation and culture,while the throat swab specimen,turbinate,and tracheal tissue grinding fluid specimens were negative for virus isolation and culture.Histopathological examination showed that there was inflammation reaction of the mouse lung tissue but pathological changes of the turbinate and tracheal tissues was not observed.Influenza virus NP antigen in the turbinate,trachea,and lung tissues was not detected.After the infection of the three viruses,the influenza virus-specific antibodies in the mouse serum samples were positive,and the antibody HI titer increased with the number of days after infection.4.Replication of avian influenza virus in human lung tissues in vitro:In the in vitro infection experiment of human lung tissue,human bronchus,lung tissue culture fluid,and grinding fluid specimens infected with the KM1135 virus strain were positive for virus isolation and culture.Histopathological examination showed that there was inflammation in the human bronchus and lung tissues.The influenza virus NP antigen in the alveolar tissue was detected,while the influenza virus NP antigen in the bronchus tissue was not detected.After infection with the GY1774 virus strain,human bronchus,lung tissue culture fluid,and grinding fluid specimens were positive for virus isolation and culture.Histopathological examination showed that there was inflammation in human bronchus and lung tissue,and the influenza virus NP antigen in bronchus and alveolar tissue was detected.Human bronchus,lung tissue culture fluid,and grinding fluid specimens infected with the GY6952 virus strain were positive for virus isolation and culture;Histopathological examination showed that there was inflammation in the human bronchus and lung tissues.The influenza virus NP antigen in the alveolar tissue was detected and the influenza virus NP antigen in the bronchial tissue was not detected.Conclusions:1.The HA cleavage site of the duck-originated H6N6 subtype avian influenza virus still belongs to the characteristics of low pathogenic avian influenza virus.H156K mutation of HA were found in three viruses,but no mutations in key amino acid sites of HA,NA,PB1,PB2,PA,and the virus still belongs to the genetic characteristics of avian influenza virus.2.Duck-originated H6N6 subtype avian influenza virus could directly infect BALB/c mice and the main sites of infection were the turbinate,trachea,and lung tissues of mice,indicating that these H6N6 subtype avian influenza virus could cross the species barrier to infect mammalian without adaptation.3.Duck-originated H6N6 subtype avian influenza virus can effectively replicate in human lung tissues and the main site of the virus replication was lung tissue,suggesting that these H6N6 subtype avian influenza virus could cross the species barrier to infect human potential.