Isolation,Identification And Serological Investigation And Transmission Characterization Of Mink H9N2 Influenza Virus

H9N2 influenza A virus(IAV)can cause low pathogenic respiratory disease in a variety of animals and humans.Mink are susceptible to IAV infection,and because the characteristics of mink farming is that foxes and raccoon dogs are raised together with mink in our country,which increases the chance of foxes and raccoon dogs infection with H9N2 IAV.To study the prevalence and evolution of H9N2 IAV in the mink and whether it caused infection in fox and raccoon dog population.In 2015,The lung tissues of 105 mink,32 foxes and 20 raccoon dogs were collected from Shandong province,China.Six strains of H9N2 influenza virus were isolated from the lung tissue of mink,named A/Mink/Shandong/Z1/2015,A/Mink/Shandong/Z2/2015,A/Mink/Shandong/Z3/2015,A/Mink/Shandong/Z4/2015,A/Mink/Shandong/Z5/2015 and A/Mink/Shandong/Z6/2015.But attempts at IAV isolation from foxes and raccoon dogs were unsuccessful.In this study,the complete gene segments of six isolated were analyzed in comparison with the sequences of H9N2 subtype influenza A virus in GenBank.Based on sequence analysis,the eight nucleotide sequences showed 99.2-100% identity among the six isolated.The PB1 ? PA ? HA ? NP ? NA ? M and NS genes of six isolates were close to A/mink/Shandong/F10/2013(H9N2)(Mk/SD/F10/13),more than 97.0%,However,the PB2 genes of six isolates exhibited high homology with the PB2 gene of A/environment/Suzhou/14/2013(H7N9),up to 99.4%.To identify the molecular characteristics of the six isolates in detail,the deduced amino acid sequences of each protein were analyzed and aligned using DNASTAR software.The eight deduced amino acid sequences homology were 98.2-100% among the six isolates,respectively.Analysis of key sites of HA in six isolates indicated that the 226 th amino acid for leucine(Leu)had the receptor SA ?-2,6-Gal specificity.The amino acids at the HA cleavage site of the six isolates possessed an RSSR motif,indicating that six isolates were low pathogenic influenza viruses.Analysis of the potential HA N-glycosylation sites of six isolates revealed that eight sites were found in the HA domain.Compared to the potential HAN-glycosylation sites of reference strains,six isolates lost a potential glycosylation site at position 218 and presented an additional potential site at position 313.The six isolates contained avian-like signature 627 E remains instead of the mammalian-like signature PB2-627 K,the PB2-701 N of the six isolates maybe compensate for the lack of PB2-627 K,enhanced the replication of some strains of IAV in mammalian hosts.To investigate the genetic origin of the gene segments of the virus more precisely,the phylogenetic trees were conducted using the nucleotide sequences of the six isolates and the corresponding genes of the reference viruses.The phylogenetic analysis results showed that the HA genes of the six isolates all fell into the Y280-like lineage,the NA genes and the“internal'' genes(PA,PB1,NP and NS)of the six isolates all fell into the Shanghai/F/98-like lineage,However,the M genes of the six isolates fell into G1-like lineage.and the seven genes of the six isolates had a close relationship with the genes of Mk/SD/F10/13.Besides,the PB2 genes of the six isolates fell into Korean-like lineage and had a close relationship with the genes of A/environment/Suzhou/14/2013(H7N9).To study whether H9N2 IAV can transmit horizontally in the mink population,cross the species barrier from mink to foxes and raccoon dogs.In this study,animal experiments were carried out using Mk / SD / F10 / 13 strain stored in the laboratory.The sentinel mink showed the clinical signs,including loss of appetite,dullness,lethargy,mild coughing and sneezing on days 4-8 post-inoculation(p.i.).The inoculated mink and sentinel mink were recovered finally.And no mink was dead.The virus was detected in the nose swab sample of the exposure mink on days 4-6 p.i..Autopsy could find lung hemorrhage,trachea rin hemorrhage,spleen hemorrhage.Histologic lesions show large area of bleeding in lung,thickening of the alveolar septa.The serum samples that collected from the sentinel mink were antibody-positive for H9N2 IAV on day 14 p.i..The sentinel foxes and raccoon dogs shown no clinic signs and virus shedding.But the serum samples that collected from the sentinel foxes and raccoon dog were antibody-positive for H9N2 influenza A virus on day 14 p.i..The results indicate that the mink H9N2 IAV can transmit horizontally in the mink population,cross the species barrier from mink to foxes and raccoon dogs under the experimental conditions.Besides,serological survey was performed to further investigate the prevalence of H9N2 IAV in mink,fox and raccoon dog population.The seroprevalence of antibodies toH9N2 in mink was 31%(97/313),more than 20% of 2013.The seroprevalence of antibodies to H9N2 in foxes was 59.4%(76/128).The seroprevalence of antibodies to H9N2 in raccoon dogs was 41.4%(106/256).The results show that the H9N2 IAV in the mink population is becoming more and more prevalent,and also widely prevalent in the fox and raccoon dog population.This study indicated the mink H9N2 IAV can transmit horizontally in the mink population,cross the species barrier from mink to foxes and raccoon dogs,and become prevalent in mink,fox and raccoon dog population.In addition,the H9N2 IAV was continued to evolve in the mink group.It maybe pose a threat to the fur animal breeding industry,and the fur animal may also infect other animals as intermediate hosts of H9N2 IAV,even human beings.This study provides important molecular epidemiological data for the prevention and monitoring of H9N2 IAV,which has important public health significance.