Molecular Characterization Of H9N2 Influenza Virus Isolated From Mink And Its Pathogenesis In Mink

In mid-August 2013, two H9N2 influenza viruses, named A/mink/Shandong/F6/2013(Mk/SD/F6/13) and A/mink/Shandong/F10/2013(Mk/SD/F10/13), were isolated from lung samples of 2 of 45 farmed mink exhibiting respiratory signs in mideastern Shandong province,China. The seroprevalence of antibodies to H9N2 in mink was 20%(53/265).In this study, the complete gene segments(PB2、PB1、PA、HA、NP、NA、NS、M)of Mk/SD/F6/13 and Mk/SD/F10/13 were obtained by RT-PCR using eight specific primers which were designed and synthesized according to the sequences of H9N2 subtype influenza A virus in GenBank. Based on sequence analysis, the eight nucleotide sequences showed99.7-100% identity between Mk/SD/F6/13 and Mk/SD/F10/13. The HA, NP and NS genes of two isolates were close to A/chicken/Zhejiang/329/2011(H9N2), the NA and PB1 genes, to A/duck/Hunan/S4111/2011(H9N2), the PA and M genes to A/chicken/Shanghai/C1/2012(H9N2). However, the PB2 genes of two isolates exhibited high homology with the PB2 gene of A/Turkey/California/189/66(H9N2).To investigate more precisely the genetic origin of the gene segments of the H9N2 influenza A viruses, the phylogenetic trees were conducted using the nucleotide sequences of reference viruses available in GenBank. Phylogenetic analysis of HA genes revealed that Mk/SD/F6/13 and Mk/SD/F10/13 belonged to Y280-like sublineage and had a close relationship with A/chicken/Shanghai/C1/2012, indicating the two isolates belonged to the Eurasian lineage. The NA gene phylogenetic analysis shown that the two strains clustered with Y280-like viruses and the NA genes of two isolates had a close relationship with the NA gene of A/duck/Hunan/S4111/2011(H9N2). Phylogenetic analysis of the “internal”genes(PB1, PA, NP, M and NS) demonstrated that two strains were similar to the Shanghai/F/98-like viruses and had a close relationship with A/chicken/Shanghai/C1/2012(H9N2). However, phylogenetic analysis of PB2 genes revealed that two isolates belong to A/Turkey/Wisconsin/1/66-like viruses, which belong to the North American lineage.To try to identify the molecular characteristics of Mk/SD/F6/13 and Mk/SD/F10/13 indetail, the deduced amino acid sequences of each protein were analyzed and aligned using DNASTAR software. The eight deduced amino acid sequences homology were 100%between Mk/SD/F6/13 and Mk/SD/F10/13, respectively. Analysis of key sites of HA in two isolates indicated that the 226 th amino acid for leucine(Leu) had the receptor SA α-2,6-Gal specificity. The amino acids at the HA cleavage site of Mk/SD/F6/13 and Mk/SD/F10/13 possessed an RSSR motif identical to that of G1 and Y280-like viruses, indicating that two isolates were low pathogenic influenza viruses. Analysis of the potential HA N-glycosylation sites of Mk/SD/F6/13 and Mk/SD/F10/13 revealed that eight sites were found in the HA domain.Compared to the potential HA N-glycosylation sites of A/Chicken/Shanghai/F/98 and A/Chicken/Beijing/1/94, two isolates lost a potential glycosylation site at position 218 and presented an additional potential site at position 313. The PB2 protein of Mk/SD/F6/13 and Mk/SD/F10/13 encoded for Lys at position 627, which is a characteristic of all of the mammalian influenza A viruses.To determine the pathogenicity of the isolates, the healthy mink were inoculated intranasally with Mk/SD/F6/13. The infected mink presented mild respiratory signs. Virus shedding from the inoculated mink was confirmed by virus isolation. Mild multifocal or coalescing pulmonary lesions were observed in the infected mink. The tissues of heart and spleen presented hemorrhage and infiltration of macrophages and neutrophils. Trachea, lung,colon and heart tissues from the infected mink were positive for influenza A virus using RT-PCR, the other tissue samples negative. The serum samples that collected from the inoculated mink were antibody-positive for H9N2 influenza A virus, HI titre 1:64-1:512. The inoculated mink gradually recovered from the disease and no mink died of these disease.Experimental infection of mink demonstrated that mink could be infected by Mk/SD/F6/13.This is the first evidence of a natural H9N2 infection in mink. Although we did not draw a conclusion whether mink could also serve as an intermediate host for influenza A viruses with pandemic potential for the other animals in this study, it is important to monitor H9N2 inflienza A viruses in all species and acquire great precautions from the pandemic preparedness point of view.