Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA ₀ Cleavage Site

Abstract

Although a polybasic HA 0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA 0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA 0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCETo date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA 0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.A vian influenza viruses (AIV) belong to the genus Influenzavirus A in the family Orthomyxoviridae. Although wild waterfowl are considered the natural reservoir for all influenza A viruses, sporadic transmission to poultry and mammals can result in viruses that are adapted to a new host. The roles that the different viral genes play in this process, especially as it relates to tropism and virulence for domestic poultry species, are still not well understood.Influenza A viruses contain a negative-sense, single-stranded, eight-segmented RNA genome that codes for at least 11 proteins. Viruses are categorized based on the hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins. Sixt...

Influenza as a molecular walker

“…[3][4][5] This functional balance between HA and NA is reected in the evolution of HA and NA. [6][7][8][9][10] It has therefore been suggested that all changes in activity of HA or NA must be followed by an adjustment of the activity of the other to maintain a functional balance. 6,7,11,12 In vivo studies have shown that lower NA activity leads to less efficient virus replication, but stronger binding by HA can have the same effect.…”

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

“…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.…”

From low to high pathogenicity-Characterization of H7N7 avian influenza viruses in two epidemiologically linked outbreaks

“…Several attempts failed to convert an LP virus into the HP phenotype by merely grafting, by reverse genetics, a polybasic HACS into the HA protein of an LP H5 or H7 virus (Schrauwen et al., ; Stech et al., ). These investigations gave evidence that further, as yet ill‐defined, arrangements in either the HA or other viral proteins are required, at least in some viruses, in addition to a polybasic HACS (Abdelwhab et al., ; Diederich et al., ; Soda et al., ). Scattered throughout the genome of the currently studied LP/HP pair, ten amino acid substitutions outside the HACS were detected in the consensus sequence of the HPAI virus isolates, which allowed distinguishing the HP inheritor from its LP progenitor virus (Table ).…”