Changes in the Length of the Neuraminidase Stalk Region Impact H7N9 Virulence in Mice

Abstract

The neuraminidase stalk of the newly emerged H7N9 influenza virus possesses a 5-amino-acid deletion. This study focuses on characterizing the biological functions of H7N9 with varied neuraminidase stalk lengths. Results indicate that the 5-amino-acid deletion had no impact on virus infectivity or replication in vitro or in vivo compared to that of a virus with a full-length stalk, but enhanced virulence in mice was observed for H7N9 encoding a 19-to 20-amino-acid deletion, suggesting that N9 stalk length impacts virulence in mammals, as N1 stalk length does. In February and March 2013, a novel influenza A (H7N9) virus emerged and spread quickly across China, resulting in a total of 672 laboratory-confirmed cases in humans, with at least 271 deaths as of 23 June 2015 (http://www.who.int/influenza/human _animal_interface/HAI_Risk_Assessment/en/) occurring during the subsequent years. Influenza A virus is an enveloped, negative-sense, single-stranded RNA virus, with a segmented genome comprising eight gene segments: hemagglutinin (HA), neuraminidase (NA), basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA), nucleoprotein (NP), matrix protein (M), and nonstructural protein (NS). Influenza A viruses may encode up to 16 proteins: HA, NA, PB2, PB1, PB1-F2, PB1-N40, PA, PA-X, PA-N155, PA-N182, NP, M1, M2, M42, NS1, and NS2 (1, 2). Cleavage of the HA precursor polypeptide (HA0) into subunits HA1 and HA2 is required for virus infection, and the amino acid residues at the HA0 cleavage site are a molecular hallmark for virulence in chickens. Almost all of the highly pathogenic avian influenza viruses (HPAIVs) possess a multibasic amino acid motif in the HA cleavage site, unlike with low-pathogenicity avian influenza viruses (LPAIVs) (3-5). Based on the molecular characteristics of the HA0 cleavage site and asymptomatic infections in chickens, the new H7N9 virus was confirmed as an LPAIV (6, 7). Nevertheless, this H7N9 virus caused severe disease in humans, with a high mortality rate (ϳ40%). The underlying mechanisms behind the virulence of H7N9 influenza virus in humans are yet not fully understood.Apart from the human-strain-like mutations in HA and PB2, a 5-amino-acid deletion in the NA stalk region (69th to 73rd amino acids) was detected in this novel H7N9 virus (6). Notably, this deletion appeared for the first time in N9, based on NA sequences deposited in GenBank. Apart from that of H7N9, NAs with shortened stalks have previously been reported with different influenza subtypes, such as H5N1, H6N1, H7N1, H7N3, H9N2, and H2N2 (8-14). The shortened NA stalk was considered a relic of molecular evolution which resulted from the early adaptation of influenza virus from wild aquatic birds to terrestrial poultry (8, 10, 11). Viruses containing short-stalk NA were shown to have altered rates of virus

Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China

“…However, a T160A mutation in the HA protein, which prevents glycosylation at residue 158N, was observed in eight of nine cases (except for A/Guangzhou/39715/2014) (Tables S9 and S10) as well as the majority of avian H5N6 strains, suggesting a possible increased viral affinity for human-like (a2-6-SA) receptor binding (Shi et al, 2014;Zhang et al, 2013;Russell et al, 2012;Herfst et al, 2012). Deletions in the NA stalk region (11 amino acids in N6, positions 58-68) were observed in eight of the nine clinical cases (except for A/Sichuan/26221/2014), which were often observed in AIV lineages adapted to terrestrial poultry and may increase the virulence to mammals (Bi et al, 2015b). The E627K substitution in PB2, normally seen in mammal-adapted AIVs, was found in five human cases (A/Guangzhou/39715/2014, A/Yunnan/ DQ001/2015 and A/Yunnan/DQ002/2015, A/Shenzhen/TH002/ 2016, A/Shenzhen/TH003/2016), and D701N in PB2 was found in one human isolate (A/Sichuan/26221/2014).…”

Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses

“…More recently, the NA stalk truncation was also recognized in H7N9 human strains, which have been posing severe public health threats in China since 2013 27 . Intriguingly, the NA stalk truncation appears related with the pathogenicity increases of avian IAVs 19 , 20 , 24 , 25 , 28 – 31 , and some of these studies noted the pathogenic contribution of N -linked glycosylation (NLG) pattern changes in the globular head region of hemagglutinin (HA) protein 20 , 24 . In fact, the NLG pattern changes in the HA of IAVs have been suggested for their differential effects on viral antigenicity, pathogenicity, transmissibility, and/or immune evasion from host immunity 14 , 32 , 33 .…”

Genetic Characteristics and Pathogenicity Analysis in Chickens and Mice of Three H9N2 Avian Influenza Viruses

“…In our study, all of the three H9N2 viruses had a Q216L mutation, which indicated that these viruses had the characteristics of human influenza virus-like receptor specificity. All of the three H9N2 viruses encoded a 3 aa deletion at the NA stalk region, which has been shown to alter the rates of virus growth, virulence, and transmission in chickens and mammals [35][36][37][38][39][40]. They all had an S31N mutation in the M2 protein, which is associated with amantadine resistance [24].…”