Effect of the PB2 and M Genes on the Replication of H6 Influenza Virus in Chickens

Abstract

H6 subtype influenza viruses are commonly isolated from wild aquatic birds. However, limited information is available regarding H6 influenza virus isolated from chickens. We compared the viral genome segment between A/chicken/Hong Kong/W312/97 (H6N1), which was able to grow in chicken trachea, and A/duck/Shantou/5540/01 (H6N2), which was isolated from wild aquatic duck, to explore the factors for effective replication in chicken. When chickens were inoculated with 7 + 1 reassortants (W312 background), the replication of viruses with PB2 and M genes derived from the duck strain was significantly reduced. Chimeras of PB2 and M proteins, encoding the C-terminal region of the PB2 protein and the M2 protein from W312, were required for efficient replication in canine-derived (MDCK) cells and in chicken trachea. These results indicate that host range may be determined by some types of internal proteins such as PB2 and M2, as well as by surface glycoprotein like hemagglutinin.

Figure 1

Representative characteristics of plaque formed on MDCK cell cultures. Typical large plaques were induced by the parental W312 strain (a). The plaque size of each reassortant virus with ST-PB1 (d), ST-PA (e), ST-NP (g), ST-NA (h), and ST-NS (j) was similar to that of the W312 strain. The reassortant virus with ST-PB2 (c) and ST-M (i) formed obviously smaller plaque compared with the other reassortants. Countable clear plaques were not detected when parental ST strain (b) and the virus harbored the ST-HA gene (f) resulting in forming the same characteristics in the cells as that of ST strain.

Figure 2

Diagram of the PB2 and M proteins expressed by recombinant viral genomes. Each number indicates the position of amino acid residues that differ between A/teal/Hong Kong/W312/97 (H6N1) and A/duck/Shantou/5540/01 (H6N2) strains. Chimeric PB2 genes were generated by digesting DNAs with AflII, exchanging the fragments, and then ligating them to the pHW2000 vector. To create a chimeric M gene, specifically designed primer sets were used to amplify M1 and M2 sequences, and then the exchanged fragments were ligated to the pHW2000 vector.

Figure 3

Replication of the parental and reassortant viruses in MDCK cells. MDCK cells were infected with each virus at an MOI = 0.001. Each plot displays average results of three independent experiments. Panel (a) shows the growth kinetics of parental W312 strain versus the virus with ST-PB2 gene or the viruses with chimeric PB2 genes. Panel (b) shows the growth kinetics of parental W312 strain versus the virus with ST-M gene or the viruses with chimeric M genes.