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 Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations.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. IMPORTANCEIn 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. PB1-F2) (1, 2). Currently, AIV are classified into 16 HA (H1 to H16) and 9 NA (N1 to N9) subtypes, which are known to infect birds, whereas influenza viruses of subtypes H17N10 and H18N11 have been identified in bats (3). While AIV of all subtypes induce subclinical or only mild disease in poultry, the virulence of H5 and H7 subtypes varies from asymptomatic to highly lethal infections (4). Major outbreaks of fowl plague are caused by highly pathogenic (HP) AIV subtypes that evolve ...

Composition of the Hemagglutinin Polybasic Proteolytic Cleavage Motif Mediates Variable Virulence of H7N7 Avian Influenza Viruses

“…In this study, pCSGE and pCSIT conferred high virulence in chickens to LPAI H7N7 viruses, wide dissemination in different organs and increased virus excretion from both inoculated and contact birds. In contrast, pCSGE did not confer high virulence to LPAIV H7N112 or LPAIV H3N835 and reduced transmissibility of HPAIV H7N1 in chickens (Abdelwhab et al ., unpublished data). Likewise, pCSIT was insufficient to support full virulence of LPAIV H7N112.…”

“…In contrast, pCSGE did not confer high virulence to LPAIV H7N112 or LPAIV H3N835 and reduced transmissibility of HPAIV H7N1 in chickens (Abdelwhab et al ., unpublished data). Likewise, pCSIT was insufficient to support full virulence of LPAIV H7N112. A modification of the monobasic CS of LPAIV H7N2 to different pCS motifs modulated the virulence in chickens which varied according to the number of basic amino acids in the CS36.…”

“…This alteration results in HA cleavage-activation not only by tissue-restricted trypsin-like proteases that cleave LPAIV in the respiratory and digestive tracts, but also by ubiquitous furin-like proteases found throughout the host resulting in systemic infection6. Nevertheless, several H5 and H7 viruses with pCS exhibited relatively low virulence in chickens and further mutations within or beyond the HA were required for high virulence and/or efficient transmissibility in birds891011121314. To date little is known about the impact of different pCS sequences on virulence of AIV in chickens and modulation of virulence by the reassortment of different gene segments.…”

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens

“…This is supported by our recent findings that virulence of this HPAIV H7N1 is modulated by amino acids in the HA2 domain in addition to the multibasic cleavage site. 25 Likewise, truncation of NS1 from an Italian LPAIV H7N1 did not increase virulence of the virus in chickens 20 and extension of the C-terminal end of NS1 of an HPAIV H5N1 did not decrease virulence in chickens. 13 In our study, compared to the Hp virus, the NS1 mutants exhibited impaired virus excretion from infected birds at 4dpi particularly via the cloacal route.…”

Stop-codon variations in non-structural protein NS1 of avian influenza viruses

“…The minor role, if any, of NS1 modifications on the viral phenotype is in line with the findings of a previous article by the same group of authors, who showed that the HA protein is the major virulence determinant of the H7N1 virus in chickens: the multibasic cleavage site of the HA, along with 3 mutations in the HA2 domain, suffice to confer the HP phenotype in chickens. 18 What is the fate of viruses harboring Ns1-Truncations?…”