Outbreaks of H5N1 avian influenza show strong seasonality. It is not clear where the source of virus originates from in each new outbreak season. This study sought to understand the nature of viral resurgence in recent outbreak seasons in Thailand, where the epidemic is relatively well controlled. In such a situation, indigenous viruses surviving the inter-outbreak season would have to pass through a bottleneck. In order to look for evidence of the bottleneck effect, viral genome sequences from recent outbreaks in the country were analysed. H5N1 avian influenza viruses were isolated from six outbreaks in the rainy season and winter of 2007 through to early 2008. Most of the outbreaks were in the Yom-Nan River basin in the southern part of the northern region of the country. Sequences of these viral isolates were identified as clade 1, genotype Z, similar to viruses from previous years in the central region of the country. The sequences clustered into two groups, one of which was closely related to viruses isolated from the same area in July 2006. These analyses indicated that there was a strong bottleneck effect on the virus population and that only a few lineages remained in the area. In addition, evidence of reassortment among these viruses was found. These indicated re-emergence of viruses from a small pool of indigenous sources that had been silently perpetuated over the dry summer months. Therefore, an approach to eradicate H5N1 avian influenza from the area by eliminating these local reservoirs may be feasible and should be seriously considered.

Highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype has been reported to infect pigeons asymptomatically or induce mild symptoms. However, host immune responses of pigeons inoculated with HPAIVs have not been well documented. To assess host responses of pigeons against HPAIV infection, we compared lethality, viral distribution and mRNA expression of immune related genes of pigeons infected with two HPAIVs (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) isolated from wild birds in Thailand. The survival experiment showed that 25% of pigeons died within 2 weeks after the inoculation of two HPAIVs or medium only, suggesting that these viruses did not cause lethal infection in pigeons. Pigeon04 replicated in the lungs more efficiently than T.sparrow05 and spread to multiple extrapulmonary organs such as the brain, spleen, liver, kidney and rectum on days 2, 5 and 9 post infection. No severe lesion was observed in the lungs infected with Pigeon04 as well as T.sparrow05 throughout the collection periods. Encephalitis was occasionally observed in Pigeon04- or T.sparrow05-infected brain, the severity, however was mostly mild. To analyze the expression of immune-related genes in the infected pigeons, we established a quantitative real-time PCR analysis for 14 genes of pigeons. On day 2 post infection, Pigeon04 induced mRNA expression of Mx1, PKR and OAS to a greater extent than T.sparrow05 in the lungs, however their expressions were not up-regulated concomitantly on day 5 post infection when the peak viral replication was observed. Expressions of TLR3, IFNα, IL6, IL8 and CCL5 in the lungs following infection with the two HPAIVs were low. In sum, Pigeon04 exhibited efficient replication in the lungs compared to T.sparrow05, but did not induce excessive host cytokine expressions. Our study has provided the first insight into host immune responses of pigeons against HPAIV infection.

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Wild bird surveillance for highly pathogenic avian influenza (HPAI) H5N1 virus from 2004 to 2007 in Thailand indicated that the prevalence of infection with avian influenza H5N1 virus in wild birds was low (1.0%, 95% confidence interval [CI]: 0.7-1.2, 60/6,263 pooled samples). However, the annual prevalence varied considerably over this period, with a peak of 2.7% (95% CI: 1.4, 4.1) in 2004. Prevalence dropped to 0.5% (95% CI: 0.3, 0.8]) and 0.6% (95% CI: 0.3, 1.0) in 2005 and 2006, respectively, and then increased to 1.8% (95% CI: 1.0, 2.6) in 2007. During this period, 16 species from 12 families of wild birds tested positive for H5N1 virus infection. All samples from juvenile birds were negative for H5N1 virus, whereas 0.6% (95% CI: 0.4, 0.9) of pooled samples from adult birds were positive. Most positive samples originated from peridomestic resident species. Infected wild bird samples were only found in provinces where poultry outbreaks had occurred. Detection of H5N1 virus infection in wild birds was reported up to 3 yr after eradication of the poultry outbreaks in those provinces. As observed with outbreaks in poultry, the frequencies of H5N1 outbreaks in wild birds were significantly higher in winter. Further understanding of the mechanisms of persistence and ongoing HPAI H5N1 transmission between wild birds and domestic poultry is needed.

Five erythrocyte species (horse, goose, chicken, guinea pig, and human) were used to agglutinate avian influenza H5N1 viruses by hemagglutination assay and to detect specific antibody by hemagglutination inhibition test. We found that goose erythrocytes confer a greater advantage over other erythrocyte species in both assays.