Isolation and genetic characterization of H5N2 influenza viruses from pigs in Korea

Abstract

Due to dual susceptibility to both human and avian influenza A viruses, pigs are believed to be effective intermediate hosts for the spread and production of new viruses with pandemic potential. In early 2008, two swine H5N2 viruses were isolated from our routine swine surveillance in Korea. The sequencing and phylogenetic analysis of surface proteins revealed that the Sw/Korea/C12/08 and Sw/Korea/C13/08 viruses were derived from avian influenza viruses of the Eurasian lineage. However, although the Sw/Korea/C12/08 isolate is an entirely avian-like virus, the Sw/Korea/C13/08 isolate is an avian-swine-like reassortant with the PB2, PA, NP, and M genes coming from a 2006 Korean swine H3N1-like virus. The molecular characterization of the two viruses indicated an absence of significant mutations that could be associated with virulence or binding affinity. However, animal experiments showed that the reassortant Sw/Korea/C13/08 virus was more adapted and was more readily transmitted than the purely avian-like virus in a swine experimental model but not in ferrets. Furthermore, seroprevalence in swine sera from 2006 to 2008 suggested that avian H5 viruses have been infecting swine since 2006. Although there are no known potential clinical implications of the avian-swine reassortant virus for pathogenicity in pigs or other species, including humans, at present, the efficient transmissibility of the swine-adapted H5N2 virus could facilitate virus spread and could be a potential model for pandemic, highly pathogenic avian influenza (e.g., H5N1 and H7N7) virus outbreaks or a pandemic strain itself.

FIG. 1.

Immunoblotting using H5 antigen derived from the Sw/Korea/C13/07 (H5N2) virus as the primary antibody and purified by ultracentrifugation with a CaCl₂ cushion at 112,600 × g for 3 h at 4°C (Beckman). Thirty micrograms of H5 protein per lane was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and immunoblotted with pig sera at a dilution of 1:200. All of the sera giving a positive result in the HI and virus-neutralizing antibody tests were subjected to immunoblotting together with a random selection of sera giving negative HI results. A representative result indicating the different HI titers obtained for each respective serum is shown. M, 75-kDa protein marker (Bio-Rad); +C, positive control; −C, negative control.

FIG. 2.

(a and b) Phylogenetic tree based on the nucleotide sequences of the H5 HA gene (a) and the N2 NA gene (b). The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap replicates. Only bootstrap values ≥60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being characterized in this study. Ck, chicken; Dk, duck; Ga, garganey; Gs, goose; Ml, mallard; Qa, quail; RT, rudder turnstone; Tk, turkey; Tl, teal; and Wb, wild bird. Standard abbreviations are used for state names in the United States. (c to h) Phylogenetic tree based on the nucleotide sequences of the PB2, PA, NP, M, PB1, and NS genes. The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap replicates. Only bootstrap values ≥60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being characterized in this study. Ab, aquatic bird; Pr, parrot; Ps, pheasant; Sb, shorebird; Sl, shoveler; Sw, swine; and Te, tern. Standard abbreviations are used for state names in the United States.

FIG. 2.

(a and b) Phylogenetic tree based on the nucleotide sequences of the H5 HA gene (a) and the N2 NA gene (b). The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap replicates. Only bootstrap values ≥60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being characterized in this study. Ck, chicken; Dk, duck; Ga, garganey; Gs, goose; Ml, mallard; Qa, quail; RT, rudder turnstone; Tk, turkey; Tl, teal; and Wb, wild bird. Standard abbreviations are used for state names in the United States. (c to h) Phylogenetic tree based on the nucleotide sequences of the PB2, PA, NP, M, PB1, and NS genes. The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap replicates. Only bootstrap values ≥60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being characterized in this study. Ab, aquatic bird; Pr, parrot; Ps, pheasant; Sb, shorebird; Sl, shoveler; Sw, swine; and Te, tern. Standard abbreviations are used for state names in the United States.

FIG. 2.

(a and b) Phylogenetic tree based on the nucleotide sequences of the H5 HA gene (a) and the N2 NA gene (b). The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap replicates. Only bootstrap values ≥60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being characterized in this study. Ck, chicken; Dk, duck; Ga, garganey; Gs, goose; Ml, mallard; Qa, quail; RT, rudder turnstone; Tk, turkey; Tl, teal; and Wb, wild bird. Standard abbreviations are used for state names in the United States. (c to h) Phylogenetic tree based on the nucleotide sequences of the PB2, PA, NP, M, PB1, and NS genes. The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap replicates. Only bootstrap values ≥60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being characterized in this study. Ab, aquatic bird; Pr, parrot; Ps, pheasant; Sb, shorebird; Sl, shoveler; Sw, swine; and Te, tern. Standard abbreviations are used for state names in the United States.

FIG. 3.

Histologic lesions in pigs infected with porcine H5N2 viruses. Lung tissue sections were stained by immunohistochemistry using a polyclonal antibody against the nucleoprotein gene (anti-NP) of influenza virus to show influenza virus infection. (a) Sw/Kor/C12/08; (b) Sw/Kor/C13/08; (c) positive contact (Sw/Kor/CC13/08); (d) negative contact. Magnification, ×400. The arrows indicate influenza virus-infected cells.