Insertion of a multibasic cleavage motif into the hemagglutinin of a low-pathogenic avian influenza H6N1 virus induces a highly pathogenic phenotype

Abstract

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 [downward arrow] 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 HA(0) (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.

FIG. 1.

In vitro phenotype of H6N1_MBCS in comparison with those of H6N1_WT, H5N1_WT, and H5N1_ΔMBSC influenza A viruses. (A) Western blots of transfected 293T cells with the H6N1_WT, H6N1_MBCS, H5N1_WT, or H5N1_ΔMBCS HA open reading frame, treated with (+) or without (−) trypsin. (B, C) Replication kinetics of the H6N1_WT (squares) and H6N1_MBCS (circles) viruses in MDCK cells in the presence (filled symbols) or absence (open symbols) of trypsin (B) or the H5N1_WT (squares) and H5N1_ΔMBCS (circles) viruses in MDCK cells in the presence (filled symbols) or absence (open symbols) of trypsin (C). Geometric mean titers were calculated from two independent experiments; error bars indicate standard deviations.

FIG. 2.

Clinical symptoms of chickens infected with H6N1_MBCS virus, consistent with those of chickens infected with HPAI virus, as follows: cyanosis of the comb and wattles (A); subcutaneous hemorrhage of the shanks (B); and severe depression, ruffled feathers with edema of head and neck, and cyanosis of the comb, wattles, and feet (C).

FIG. 3.

Virus shedding from the respiratory tract (A) and the intestinal tract (B) after intranasal inoculation of chickens the H6N1_WT (open circles) and H6N1_MBCS (closed circles) viruses. Oropharyngeal and cloacal swabs were taken daily, and virus titers in MDCK cells were determined by endpoint titration. The geometric mean titers, calculated per day per group, are displayed; error bars indicate 95% confidence interval values. The dotted line indicates the cutoff value of the assay.

FIG. 4.

Tissue distribution of the H6N1_WT (left) and H6N1_MBCS (right) viruses after intranasal inoculation of chickens. Expression of viral antigen could not be detected in serial sections of these tissues of chickens infected with the H6N1_WT virus in the lung, tracheal epithelium, nasal turbinate (NT), brain, liver, spleen, heart, intestine, and comb. Expression of viral antigen could be detected in all tissues of chickens infected with the H6N1_MBCS virus. The chickens were inoculated intranasally with 5 × 10⁶ TCID₅₀ of H6N1_WT and H6N1_MBCS viruses and euthanized 3 days postinfection. Tissue sections were stained with a monoclonal antibody against influenza A virus nucleoprotein, visible as red-brown staining.