Insertion of a multibasic cleavage site in the haemagglutinin of human influenza H3N2 virus does not increase pathogenicity in ferrets

Abstract

A multibasic cleavage site (MBCS) in the haemagglutinin (HA) protein of influenza A virus is a key determinant of pathogenicity in chickens, and distinguishes highly pathogenic avian influenza (HPAI) viruses from low pathogenic avian influenza viruses (LPAI). An MBCS has only been detected in viruses of the H5 and H7 subtypes. Here we investigated the phenotype of a human H3N2 virus with an MBCS in HA. Insertion of an MBCS in the H3N2 virus resulted in cleavage of HA and efficient replication in Madin-Darby canine kidney cells in the absence of exogenous trypsin in vitro, similar to HPAI H5N1 virus. However, studies in ferrets demonstrated that insertion of the MBCS into HA did not result in increased virus shedding, cellular host range, systemic replication or pathogenicity, as compared with wild-type virus. This study indicates that acquisition of an MBCS alone is insufficient to increase pathogenicity of a prototypical seasonal human H3N2 virus.In wild birds and poultry throughout the world, avian influenza viruses expressing 16 antigenically distinct subtypes of haemagglutinin (HA) and nine subtypes of neuraminidase have been described (Fouchier et al., 2005; Röhm et al., 1996). Only subtypes H1, H2 and H3 have circulated extensively in humans (Nicholson et al., 2003). Avian influenza viruses can be classified, based on pathogenicity in chickens, into highly pathogenic avian influenza (HPAI) and lowpathogenic avian influenza (LPAI) viruses. Virtually all known HPAI viruses are of the H5 or H7 subtypes. Direct transmission of HPAI H5N1 viruses to humans was first detected in 1997 (de Jong et al., 1997) and has continued to be reported since (WHO, 2010). In addition, various human cases of HPAI H7 virus infection have been reported Tweed et al., 2004;Webster et al., 1981).Pathogenicity of avian influenza viruses in chickens is directly associated with the cleavability of the HA glycoprotein (Chen et al., 1998;Horimoto & Kawaoka, 1994;Klenk & Garten, 1994;Webster & Rott, 1987). HA is initially synthesized as an HA0 precursor protein that is subsequently cleaved into the two functional subunits HA1 and HA2. This cleavage step is essential for virus infectivity since uncleaved HA is able to mediate virus attachment but is unable to perform the fusion step necessary for the initiation of infection (Steinhauer, 1999). HA proteins of LPAI viruses have a single arginine at the cleavage site that is recognized by trypsin-like proteases expressed predominantly in the respiratory and intestinal tract. HAs of HPAI viruses contain a multibasic cleavage site (MBCS) that can be cleaved by ubiquitously expressed furin and related furin-like proprotein convertases (PC5/6) (Bertram et al., 2010; Stieneke-Gröber et al., 1992). The cleavage properties of HA and the expression patterns of proteases in the host are considered to be the major determinants of systemic influenza virus replication and pathogenesis in chickens.The association between HA cleavability and replication outside the respiratory tract is less straightforward ...

The Multibasic Cleavage Site in H5N1 Virus Is Critical for Systemic Spread along the Olfactory and Hematogenous Routes in Ferrets

“…In contrast, in mammals-and some bird species-the presence of an MBCS in H5N1 virus results in systemic replication of H5N1 virus, without extensive replication in endothelial cells (18,21). Interestingly, the presence of an MBCS in a seasonal H3N2 virus did not result in systemic dissemination (34). This lack of systemic dissemination might be due in part to a difference in cell tropism in the respiratory tract.…”

“…6). Furthermore, low replication in the respiratory epithelium explains the relatively low virus titers found in the nasal swabs compared to the titers found after inoculation with a seasonal H3N2 virus (34). It is therefore tempting to speculate that a switch from replication in the olfactory epithelium to replication in the respiratory epithelium would result in higher virus titers in nasal swabs and possibly efficient respiratory droplet transmission (36a).…”

“…None of the human pandemic or seasonal influenza viruses harbor an MBCS, and introduction of an MBCS in a human seasonal H3N2 influenza virus neither increased pathogenicity nor induced systemic spread in ferrets (34). In contrast, removal of the MBCS from HA of an HPAI H5N1 virus resulted in a virus that caused respiratory tract infection without systemic spread in mice, indicating that the MBCS is a major virulence factor for mice (12).…”

Influenza A Virus Infections in Swine

“…Artificially inserting a multiple basic amino acid sequence by reverse genetics into the HA of mildly virulent viruses does not result in increased virulence or systemic infection in ferrets. 122 However, the HPAI H5N1 virus, which does have a multibasic cleavage site, can be lethal for mice and ferrets in which it induces increased cytokine levels and virus titers and results in systemic infection. 136 This does not occur in pigs, 82 which may reflect a difference in tissue proteases in this species.…”

Characterization of an H4N2 influenza virus from Quails with a multibasic motif in the hemagglutinin cleavage site

“…Non-H5/H7 subtypes (including H4) influenza viruses can support a MBCS but enhanced pathogenicity also depended on the internal gene constellation [7–10] However, with the exception of a canine H3N2 virus isolated from in 2009 in China [11], non-H5/H7 subtypes with MBCS-like motifs are not isolated in the field.…”