Isolation of a genotypically unique H5N1 influenza virus from duck meat imported into Japan from China.

Mase M, Eto M, Tanimura N, Imai K, Tsukamoto K, Horimoto T, Kawaoka Y, Yamaguchi S.

Department of Infectious Diseases, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.

An H5N1 influenza A virus was isolated from duck meat processed for human consumption, imported to Japan from Shandong Province, China in 2003. This virus was antigenically different from other H5 viruses, including the Hong Kong H5N1 viruses isolated from humans in 1997 and 2003. Sequence analysis revealed that six genes (PB1, PA, HA, NA, M, and NS) of this virus showed > 97% nucleotide identity with their counterparts from recent H5N1 viruses, but that the remaining two genes (PB2 and NP) were derived from other unknown viruses. This duck meat isolate was highly pathogenic to chickens upon intravenous or intranasal inoculation, replicated well in the lungs of mice and spread to the brain, but was not as pathogenic in mice as H5N1 human isolates (with a dose lethal to 50% of mice (MLD(50)) = 5 x 10(6) 50% egg infectious doses [EID(50)]). However, viruses isolated from the brain of mice previously infected with the virus were substantially more pathogenic (MLD(50) = approximately 10(2) EID(50)) and possessed some amino acid substitutions relative to the original virus. These results show that poultry products contaminated with influenza viruses of high pathogenic potential to mammals are a threat to public health even in countries where the virus is not enzootic and represent a possible source of influenza outbreaks in poultry.

To assess the genetic relationship of Dk/Yokohama/aq10/03 to other H5N1 viruses circulating in Asia since 1996, the sequences of all eight of its gene segments were determined, compared with those in GenBank (Table 2), and phylogenetically analyzed. Phylogenetic analysis of representative genes (i.e., the genes for HA, PB2, PA, and nucleoprotein [NP]) are shown in Fig. 1. We found that the genotype of Dk/Yokohama/aq10/03 was unique and distinct from any of the H5N1 virus genotypes previously reported, including a recent isolate in Japan (Table 2 and Fig. 1). Six genes (PB1, PA, HA, NA, M, and NS) of Dk/Yokohama/aq10/03 showed >97% nucleotide identity with those of the Asian H5N1 viruses isolated from 2000 to 2004. Phylogenetic analysis of the HA gene confirmed this similarity (Fig. 1a). However, the other genes of Dk/Yokohama/aq10/03 were distinct from those of known H5N1 viruses. For example, the PB2 gene was closely related to a Dutch H7N7 human isolate (A/Netherlands/1/2003), which was transmitted directly from a chicken. Similarly, the PA gene was most closely related to a A/chicken/Shantou/4231/2004 strain and to a recent H5N1 isolate from Japan, and the NP gene most closely related to an H3N8 isolate from an aquatic bird (A/aquatic bird/Hong Kong/399/99) (Table 2 and Figs. 1b杁). These results indicate that an H5N1 virus with a novel, previously unreported genotype was circulating in China.

Table 2.

Genetic homology of A/duck/Yokohama/aq10/2003 to other influenza A viruses Segment Number of nucleotides examined Viruses with the highest homologya % Homology
PB2 2280 A/chicken/Netherlands/1/2003 (H7N7) 96.9
PB1 2274 A/duck/Zhejiang/52/2000 (H5N1) 98.8
PA 2151 A/chicken/Shantou/4231/2004 (H5N1) 97.5
HA 1707 A/chicken/Jilin/9/2004 (H5N1) 97.0
NP 1497 A/aquatic bird/Hong Kong/399/99 (H3N8) 97.2
NA 1410 A/egret/Hong Kong/757.2/2003 (H5N1) 98.9
M 982 A/goose/Hong Kong/3014.8/2000 (H5N1) 99.1
NS 823 A/duck/Hong Kong/380.5/2001 (H5N1) 98.9