Optimising the detectability of H5N1 and H5N6 highly pathogenic avian influenza viruses in Vietnamese live-bird markets

Live bird markets (LBMs) are major targets for avian influenza virus (AIV) surveillance programmes. While sampling the LBM environment has become a widely used alternative to the labour-intensive sampling of live poultry, the design of surveillance programmes and the interpretation of their results are compromised by the lack of knowledge about the effectiveness of these sampling strategies. We used latent class models and a unique empirical dataset collated in Vietnamese LBMs to estimate the sensitivity and specificity of five different sample types for detecting AIVs subtypes H5N1 and H5N6: oropharyngeal duck samples, solid and liquid wastes, poultry drinking water and faeces. Results suggest that the sensitivity of environmental samples for detecting H5N1 viruses is equivalent to that of oropharyngeal duck samples; however, taking oropharyngeal duck samples was estimated to be more effective in detecting H5N6 viruses than taking any of the four environmental samples. This study also stressed that the specificity of the current surveillance strategy in LBMs was not optimal leading to some false positive LBMs. Using simulations, we identified 42 sampling strategies more parsimonious than the current strategy and expected to be highly sensitive for both viruses at the LBM level. All of these strategies involved the collection of both environmental and oropharyngeal duck samples

Genetic characterization of 2008 reassortant influenza A virus (H5N1), Thailand

In January and November 2008, outbreaks of avian influenza have been reported in 4 provinces of Thailand. Eight Influenza A H5N1 viruses were recovered from these 2008 AI outbreaks and comprehensively characterized and analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment. The results show that the 2008 H5N1 viruses displayed genetic drift characteristics (less than 3% genetic differences), as commonly found in influenza A viruses. Based on phylogenetic analysis, clade 1 viruses in Thailand were divided into 3 distinct branches (subclades 1, 1.1 and 1.2). Six out of 8 H5N1 isolates have been identified as reassorted H5N1 viruses, while other isolates belong to an original H5N1 clade. These viruses have undergone inter-lineage reassortment between subclades 1.1 and 1.2 and thus represent new reassorted 2008 H5N1 viruses. The reassorted viruses have acquired gene segments from H5N1, subclade 1.1 (PA, HA, NP and M) and subclade 1.2 (PB2, PB1, NA and NS) in Thailand. Bootscan analysis of concatenated whole genome sequences of the 2008 H5N1 viruses supported the reassortment sites between subclade 1.1 and 1.2 viruses. Based on estimating of the time of the most recent common ancestors of the 2008 H5N1 viruses, the potential point of genetic reassortment of the viruses could be traced back to 2006. Genetic analysis of the 2008 H5N1 viruses has shown that most virulence determinants in all 8 genes of the viruses have remained unchanged. In summary, two predominant H5N1 lineages were circulating in 2008. The original CUK2-like lineage mainly circulated in central Thailand and the reassorted lineage (subclades 1.1 and 1.2) predominantly circulated in lower-north Thailand. To prevent new reassortment, emphasis should be put on prevention of H5N1 viruses circulating in high risk areas. In addition, surveillance and whole genome sequencing of H5N1 viruses should be routinely performed for monitoring the genetic drift of the virus and new reassorted strains, especially in light of potential reassortment between avian and mammalian H5N1 viruses