Insertion of a Multibasic Cleavage Motif into the Hemagglutinin of a Low-Pathogenic Avian Influenza H6N1 Virus Induces a Highly Pathogenic Phenotype

The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA₀ (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.Wild birds represent the natural reservoir of avian influenza A viruses in nature (43). Influenza A viruses are classified on the basis of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. In wild birds throughout the world, influenza A viruses representing 16 HA and 9 NA antigenic subtypes have been found in numerous combinations (also called subtypes, e.g., H1N1, H6N1) (12). Besides classification based on the antigenic properties of HA and NA, avian influenza A viruses can also be classified based on their pathogenic phenotype in chickens. Highly pathogenic avian influenza (HPAI) virus, an acute generalized disease of poultry in which mortality may be as high as 100%, is restricted to subtypes H5 and H7. Other avian influenza A virus subtypes are generally low-pathogenic avian influenza (LPAI) viruses that cause much milder, primarily respiratory disease in poultry, sometimes with loss of egg production (6).The HA protein of influenza A virus is initially synthesized as a single polypeptide precursor (HA₀), which is cleaved into HA₁ and HA₂ subunits by host cell proteases. The mature HA protein mediates binding of the virus to host cells, followed by endocytosis and HA-mediated fusion with endosomal membranes (43). Influenza viruses of subtypes H5 and H7 may become highly pathogenic after introduction into poultry and cause outbreaks of HPAI. The switch from an LPAI phenotype to the HPAI phenotype of these H5 and H7 influenza A viruses is achieved by the introduction of basic amino acid residues into the HA₀ cleavage site by substitution or insertion, resulting in the so-called multibasic cleavage site (MBCS), which facilitates systemic virus replication (4, 5, 14, 44). The cleavage of the HA₀ of LPAI viruses is restricted to trypsin-like proteases which recognize the XXX(R/K)↓G cleavage motif, where the downward arrow indicates the site of cleavage. Replication of these LPAI viruses is therefore restricted to sites in the host where these enzymes are expressed, i.e., the respiratory and intestinal tract (32, 38). The introduction of an RX(R/K)R↓G or R(R/K)XR↓G minimal MBCS motif into the H5 and H7 subtype viruses facilitates the recognition and cleavage of the HA₀ by ubiquitous proprotein convertases, such as furin (20, 32, 41, 45). H5 influenza A viruses with a minimal MBCS motif only have the highly pathogenic phenotype if the masking glycosylation site at position 11 in the HA is replaced by a nonglycosylation site. Otherwise, at least one additional basic amino acid has to be inserted to allow the shift from an LPAI virus phenotype to an HPAI virus phenotype to occur (15, 18, 21, 22, 28). No information is available on the minimal prerequisites of H7 influenza A viruses to become highly pathogenic, but all HPAI H7 viruses have at least 2 basic amino acid insertions in the HA₀ cleavage site (22). HA₀ with the MBCS is activated in a broad range of different host cells and therefore enables HPAI viruses to replicate systemically in poultry (46). To date, little is known about the apparent subtype-specific nature of the introduction of the MBCS into LPAI viruses and the evolutionary processes involved in the emergence of HPAI viruses. When an MBCS was introduced in a laboratory-adapted strain of influenza virus, A/Duck/Ukraine/1/1963 (H3N8), it did not result in a dramatic change in pathogenic phenotype (35). Here, the effect of the introduction of an MBCS into a primary LPAI H6N1 virus, A/Mallard/Sweden/81/2002, is described. The introduction of an MBCS resulted in trypsin-independent replication in vitro and enhanced pathogenesis in a chicken model. Understanding the basis of the HA subtype specificity of the introduction of an MBCS into avian influenza viruses will lead to a better understanding of potential molecular restrictions involved in emergence of HPAI outbreaks.