Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 17 Oct 2018 14:59:39 Enhanced pathogenicity and neurotropism of mouse-adapted H10N7 influenza virus are mediated by novel PB2 and NA mutations Xuxiao Zhang, 1 Guanlong Xu, 2 Chenxi Wang, 1 Ming Jiang, 1 Weihua Gao, 1 Mingyang Wang, 1 Honglei Sun, 1 Yipeng Sun, 1 Kin-Chow Chang, 3 Jinhua Liu 1 and Juan Pu 1, * Abstract The H10 subtype of avian influenza viruses (AIVs) circulates globally in wild birds and poultry, and this subtype has been shown to be increasingly prevalent in China. Among the various H10 viruses, H10N7 AIVs have caused repeated mammal and human infections. To investigate their genetic adaptation in mammals, we generated a mouse-adapted avian H10N7 variant (A/mallard/Beijing/27/2011-MA; BJ27-MA) which exhibited increased virulence in mice compared to wild-type virus and acquired neurotropism. Sequencing showed the absence of the widely recognized mammalian adaptation markers of E627K and D701N in PB2 in the mouse-adapted strain; instead, five amino acid mutations were identified: E158G and M631L in PB2; G218E in haemagglutinin (H3 numbering); and K110E and S453I in neuraminidase (NA). The neurovirulence of the BJ27-MA virus necessitated the combined presence of the PB2 and NA mutations. Mutations M631L and E158G of PB2 and K110E of NA were required to mediate increased virus replication and severity of infection in mice and mammalian cells. PB2-M631L was functionally the most dominant mutation in that it strongly upregulated viral polymerase activity and played a critical role in the enhancement of virus replication and disease severity in mice. K110E mutation in NA, on the other hand, significantly promoted NA enzymatic activity. These results indicate that the novel mutations in PB2 and NA genes are critical for the adaptation of H10N7 AIV in mice, and they could serve as molecular signatures of virus transmission to mammalian hosts, including humans. INTRODUCTION At present, avian influenza viruses (AIVs) – which contrib- uted to the 1918 H1N1, 1957 H2N2 and 1968 H3N2 virus pandemics [1] through viral reassortment – cause great eco- nomic loss to the global poultry industry. H5N1 and H9N2 influenza viruses, as the two major subtypes circulating in poultry, are prime candidates for potentially causing another human influenza outbreak [2, 3]. In addition, recently reported avian H7N9 virus infections in humans raised fur- ther concerns about the potential subtypes that could cause possible pandemics [4]. Thus, contingency planning for the prevention and management of AIV infections in humans should be based on a broad range of possible subtypes. The H10 subtype of AIV was first isolated from chickens in Germany in 1949 [5, 6]. Subsequently, viruses bearing the H10 haemagglutinin (HA) and a different neuraminidase (NA) started to circulate widely in wild birds and domestic poultry around the world [7–9]. In recent years, H10 sub- type AIVs have become increasingly prevalent in China [10]. Since 1984, repeated infections or deaths of mammals with this subtype have been reported [11–13]. Human cases of H10 virus infections have also been documented Received 6 February 2017; Accepted 9 March 2017 Author affiliations: 1 Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, PR China; 2 China Institute of Veterinary Drug Control, Beijing, PR China; 3 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, UK. *Correspondence: Juan Pu, pujuan@cau.edu.cn Keywords: H10N7 influenza virus; mouse-adaptation; pathogenicity; neurovirulence. Abbreviations: AIV, avian influenza virus; CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; E, eosin; EID 50 , 50 % egg infectious dose; H, haematoxylin; HA, haemagglutinin; HRP, horseradish peroxidase; MDCK, Madin–Darby canine kidney; MLD50, 50% mouse lethal dose; NA, neuraminidase; NP, nucleoprotein; p.i., post-inoculation; RNP, ribonucleoprotein; RT-PCR, reverse-transcription PCR; SPF, specific- pathogen-free. The nucleotide sequences of the eight gene segments of H10N7 and H10N7-MA are available from the ‘additional material for reviewers’. The nucleotide sequences of these segments will be released from the GenBank when the paper is published, and the accession numbers are listed as follows: KX898962 for PB2, KX898963 for PB1, KX898964 for PA, KX898965 for HA, KX898966 for NP, KX898967 for NA, KX898968 for M, KX898969 for NS, KY688108 for PB2 (MA), KY688107 for PB1 (MA), KY688106 for PA (MA), KY688101 for HA (MA), KY688104 for NP (MA), KY688103 for NA (MA), KY688102 for M (MA) and KY688105 for NS (MA). RESEARCH ARTICLE Zhang et al., Journal of General Virology 2017;98:1185–1195 DOI 10.1099/jgv.0.000770 000770 ã 2017 The Authors 1185