Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice

Abstract

The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD50) of >7.5 log10 50% egg infectious dose (EID50), whereas A/chicken/Hunan/S1220/2017 (CK/S1220) is virulent in mice, with an MLD50 of 3.2 log10 EID50. In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP, carrying the nucleoprotein (NP) of CK/SD098, was avirulent in mice, with an MLD50 of >107.5 EID50. The NPs of these two viruses differ by two amino acids, at positions 286 and 437. We further demonstrated that the amino acid mutations A286V and T437M of NP independently slowed the process of NP import to and export from the nucleus and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccines and antiviral drugs against influenza viruses. IMPORTANCE The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality rate of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggested that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice and the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza viruses is a polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China

“…H7N9 HPAIVs isolated from avian species have low-to-moderate pathogenicity in mice [7], but after replication in mammalian hosts, the H7N9 influenza viruses could easily acquire more mutations--primarily the PB2 627K or PB2 701N mutation--and then become more virulent in mice [6,[22][23][24]. Several other amino acids in PB2, PB1, PA, and NP were reported to affect H7N9 virus replication and virulence in mammalian cells or animals [5,[25][26][27][28][29][30]. The CK/SD010/IM/19 virus is more virulent in mice than other strains in the same genotype, but all of these viruses have conserved amino acids at positions that have been reported to affect the virulence of H7N9 viruses in mammals (S1 Table ), implying that some other unidentified molecular markers may have contributed to the increased virulence of CK/SD010/IM/19.…”

H7N9 Influenza Virus in China

“…In addition, other mutations in the viral polymerases-PB2 K526R, A588V, or a combination of PB2 482R, 588V, and PA 497R-also contribute to the pathogenicity of H7N9 virus in humans or other mammals (Song et al 2014;Xiao et al 2016;Yamayoshi et al 2018). A recent study by Ma et al (2020) identified two residues, 286A and 437T, in the NP protein as prerequisites for the virulence of H7N9 virus in mammals. The H7N9 viruses also harbored other known pathogenicity signatures, such as N30D and T215A in M1 and P42S in NS1, which have been shown to increase the pathogenicity of H5N1 viruses (Jiao et al 2008;Fan et al 2009).…”

Evolution and extensive reassortment of H5 influenza viruses isolated from wild birds in China over the past decade

“…The GBHG/ QH/9/2015(H5N1) virus had 627 K in its PB2, yet its virulence in mice was intermediate. We speculate that some other residues in its genome may have limited its virulence in mice, as observed in the H7N9 virus by Ma et al [23]. The NA stalk and NS deletions (Table 2) have been reported to increase the virulence of H5N1 viruses in mice [19].…”