Emergence and evolution of avian H5N2 influenza viruses in chickens in Taiwan

Abstract

Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available information suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multiple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the prevalence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003.

Importance: H5N2 avian influenza viruses first appeared in chickens in Taiwan in 2003 and caused a series of outbreaks afterwards. Phylogenetic analyses show that the chicken H5N2 viruses have H5 and N2 genes that are closely related to those of a vaccine strain originating from Mexico in 1994, while the contemporary duck H5N2 viruses in Taiwan belong to the Eurasian gene pool. The unusually high similarity of the chicken H5N2 viruses to the Mexican vaccine strain suggests that these viruses might have been introduced to Taiwan by using inadequately inactivated or attenuated vaccines. These chicken H5N2 viruses are developing varying levels of pathogenicity that could lead to significant consequences for the local poultry industry. These findings emphasize the need for strict quality control and competent oversight in the manufacture and usage of avian influenza virus vaccines and indicate that alternatives to widespread vaccination may be desirable.

FIG 1

Timeline of the Mexican-like H5N2 lineage viruses and the H6N1 viruses from Taiwan. Events discussed in the text are marked on the timeline with the duration of H5N2 outbreaks in Mexico and Taiwan. Arrows below the timeline indicate the length of persistence of the main lineages in their regions.

FIG 2

Phylogenies of the H5 and N2 genes. (A and B) Maximum-likelihood phylogenies of H5 and N2 genes showing the chicken H5N2 viruses from Taiwan. Bootstrap support and Bayesian posterior clade probabilities of selected lineages are shown. Taxa from the H5N2 outbreak in Mexico and the related viruses found in Japan are indicated. The most recent common ancestor (MRCA) of the Mexican-origin H5N2 lineage is indicated by the open circles in the trees. Changes of the amino acid at position -3 of HA1 (Table 3) are indicated in orange and a connecting thin bar. In the HA tree, the long branch leading to the Taiwan H5N2 duck viruses reported in this study has a length of 0.249 substitutions/site. (C and D) Linear regression plots of genetic distances from the MRCA of the Mexican-origin H5N2 lineage against isolation time of the taxa. The estimate and 95% confidence intervals for the evolutionary rate are shown by solid and dashed lines, respectively. Green arrows indicate the isolation dates of Mexican viruses that have similar genetic distances to the MRCA as the 2003 isolate from Taiwan.

FIG 3

Maximum-likelihood phylogenies of the PB2 and PB1 genes. Taxon names of sequences reported in this study are shown in blue (chicken H5N2 lineage), green (enzootic chicken H6N1 lineage), and purple (duck H5N2). The chicken H5N2 viruses initially isolated from Taiwan are in red. Virus subtypes are indicated in parentheses. Bootstrap support values (percents) from 1,000 pseudoreplicates are shown for selected lineages. The scale bar to the left of each tree represents 0.01 substitutions per site. Abbreviations for host species: Ck, chicken; Dk, duck; WDk, wild duck; Qa, quail; Gs, Goose; BHG, bar-headed goose. Human isolates do not use any host specification. HK, Hong Kong.

FIG 4

Maximum-likelihood phylogenies of the PA and NP gene segments. Other details are as described in the legend of Fig. 3.

FIG 5

Maximum-likelihood phylogenies of the M and NS gene segments. Other details are as described in the legend of Fig. 3.