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

Abstract

Acquisition of a polybasic cleavage site (pCS) in the hemagglutinin (HA) is a prerequisite for the shift of low pathogenic (LP) avian influenza virus (AIV) to the highly pathogenic (HP) form in chickens. Whereas presence of a pCS is required for high pathogenicity, less is known about the effect of composition of pCS on virulence of AIV particularly H7N7. Here, we investigated the virulence of four avian H7N7 viruses after insertion of different naturally occurring pCS from two HPAIV H7N7 (designated pCSGE and pCSUK) or from H7N1 (pCSIT). In vitro, the different pCS motifs modulated viral replication and the HA cleavability independent on the HA background. However, in vivo, the level of virulence conferred by the different pCS varied significantly. Within the respective viral backgrounds viruses with pCSIT and pCSGE were more virulent than those coding for pCSUK. The latter showed also the most restricted spread in inoculated birds. Besides the pCS, other gene segments modulated virulence of these H7N7 viruses. Together, the specific composition of the pCS significantly influences virulence of H7N7 viruses. Eurasian LPAIV H7N7 may shift to high pathogenicity after acquisition of “specific” pCS motifs and/or other gene segments from HPAIV.

Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)—Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV

“…Matriptase activated H9N2 AIVs with R-X-X-R or R-X-R-R motifs [32], similar to the HACS of H4N2 viruses generated in this study. Moreover, some LPAIVs (e.g., H6N1 and H7N7) were able to replicate in MDCK, MDCKII and/or CEK cells without exogenous trypsin [28,35]. In addition to the unidentified endogenous proteases in these cells, an impact of proteases in the allantoic fluid in virus stocks on activation in different cells cannot be excluded [36].…”

Parallel Evolution in the Emergence of Highly Pathogenic Avian Influenza A Viruses

“…whereas a minimal consensus -PEXPKXnK/RnRGLF-was identified for the HP viruses (Bosch, et al 1981) (Abdelwhab, et al 2016). Nine different genetically distinct HP clusters (C1-C9) were selected from the inferred HA phylogeny ( Figure 1B).…”

“…Thus, understanding the mechanisms driving the evolution of virulence in avian influenza viruses is critical. Although virulent phenotypes are complex traits (Hatta, et al 2001) (Jackson, et al 2008) (Conenello, et al 2007) (Schmolke, et al 2011) (Qi, et al 2014), one of the best characterized molecular markers of pathogenicity for avian influenza A viruses is a polybasic cleavage site (pCS) within the hemagglutinin (HA) protein, rendering the viral protein a target for broadly expressed host proteases that facilitate a systemic spread (Horimoto and Kawaoka 1994) (Abdelwhab, et al 2016) (Hulse, et al 2004).…”

Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV) Reduced Virus Fitness in Chickens which is Restored by Reassortment with Highly Pathogenic H5N1 AIV

“…Therefore, the negative impact of additional basic aa in the CS on virus replication and transmission in chickens probably precludes their emergence in nature. Moreover, the increased number of basic aa in the presence of other gene segments from H4N2 did not result in HP phenotype after ON or IV infections resembling H5/H7 viruses [23,45]. In other studies, the insertion of a polybasic CS conferred high virulence to a low-pathogenic H6N1 virus (IVPI= 1.4) [27] but not an H3N8 virus [48].…”

“…Moreover, some LPAIVs (e.g. H6N1 and H7N7) were able to replicate in MDCK, MDCKII and/or CEK cells without exogenous trypsin [27,45]. In addition to the unidentified endogenous proteases in these cells, an impact of proteases in the allantoic fluid in virus stocks on activation in different cells cannot be excluded [46].…”