Substitution Arg140Gly in HA/H7 attenuated highly pathogenic avian influenza virus FPV/Rostock/34 (H7N1)

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. H7 viruses circulate in ducks, poultry, equine and have repeatedly caused outbreaks of disease in humans. In order to study the pathogenicity factors of H7N1 viruses, several laboratory variants of the A/FPV/Rostock/34 (H7N1) strain were obtained by passages in the chicken lungs. After 10 such passages, a variant was obtained that differed from the parent virus by amino acid substitutions Val109Phe in PB2, Gln621Lys in PB1, Thr32Ala and Leu586Phe in PA Gly140Arg in HA1 and Ala101Thr in HA2 (numbering by H3), Ser82Arg in M2, Arg118Lys and Met124Arg in NS1. No differences were found in proteins NA, NP, M1 and NS2. The resulting variant was hundreds of times more pathogenic for chickens than the original laboratory variant of the virus. The study of intermediate passages showed that the jump in pathogenicity occurs sharply between the fifth and sixth passage through the chicken lungs. By cloning these variants, a pair of strains (R5p and R6p) were obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely Gly140Arg in HA1. It is important to emphasize that this substitution is a reversion, since Arg is located in position 140 HA1 of original the A/FPV/Rostock/34 (H7N1) virus (GenBank). This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity, R6p differs from R5p by an increased affinity for a negatively charged receptor analogue, an increased affinity for MDCK cells, while maintaining a receptor specificity profile.

A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo . Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo . The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

An Avian H7N1 Gain-of-Function Experiment of Great Concern

ABSTRACT Inappropriately named gain-of-function influenza research seeks to confer airborne transmission on avian influenza A viruses that otherwise cause only dead-end infections in humans. A recent study has succeeded in doing this with a highly pathogenic ostrich H7N1 virus in a ferret model without loss of virulence. If transposable to humans, this would constitute a novel virus with a case fatality rate ~30 greater than that of Spanish flu. A commentary from three distinguished virologists considered the benefits of this work to outweigh potential risks. I beg to disagree with conclusions in both papers, for the underlying science is not as strong as it appears.

Species-Specific Contribution of the Four C-Terminal Amino Acids of Influenza A Virus NS1 Protein to Virulence

ABSTRACT Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian C-terminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.