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.

Findings H5N1 influenza A virus has caused avian influenza (AI) outbreaks worldwide. In Thailand, 7 major AI outbreaks have been reported since early 2004 [1][2][3]. In January 2008, outbreaks of H5N1 virus occurred in two provinces, Nakhon Sawan and Phichit. The outbreak in Nakhon Sawan affected 60,000 birds in a broiler farm and chicken in nearby backyards, while the outbreak in Phichit occurred among backyard chicken. In November 2008, H5N1 outbreaks were also reported in two provinces, Sukhothai and Uthai Thani. Both outbreaks occurred among backyard poultry in villages (Fig 1). Currently, at least two clades of influenza A virus (H5N1) have been reported in Thailand including clade1 viruses which are predominant in lower-north and central Thailand and clade2.3.4 viruses which are predominant in northeast Thailand [1,3,4]. Clade1 H5N1 viruses in Thailand have been further divided into 3 distinct subclades including the original clade1 (CUK2-like), clade1.p1 (PC168-like) and clade1.p2 (PC170-like) [3,5]. One study has documented evidence of genetic reassortment of H5N1 viruses in Thailand in 2007 [6]. In this study, we have comprehensively characterized the 2008 H5N1 viruses recovered during the 6 th and 7 th waves of AI outbreaks in Thailand. The 2008 H5N1 viruses were compared with H5N1 isolates obtained from each wave of AI outbreaks in Thailand. The whole genome sequences of the viruses were analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment among H5N1 viruses.
Eight H5N1 viruses were isolated from Nakhon Sawan (n = 3), Phichit (n = 1), Sukhothai (n = 2) and Uthai Thani (n = 2) ( Table 1 and Fig 1). The viruses were isolated by embryonated egg inoculation [7]. All 8 viruses were confirmed as Influenza A virus subtype H5N1 by real-time-RT-PCR [8]. Whole genome sequences were obtained as previously described [9]. Phylogenetic and genetic relatedness analyses were conducted using the MEGA 4.0 program applying the neighbor-joining (NJ) approach. Bootstrapping support for tree topologies was also performed with 1000 replicates using MEGA 4.0 [10]. In addition, Bayesian trees were generated using MrBayes V.3.1.2 [11]. Nucleotide identities and genetic analyses were performed using the MegAlign program (DNASTAR). Genetic recombination analysis of H5N1 viruses was performed by Bootscan analysis [12]. Potential sites of genetic reassortment were examined by using tMRCA analysis in a Bayesian Markov chain Monte Carlo BMCMC [13] framework using BEAST version 1.5.4 [11]. Substitution model SRD06 was used. BMCMC runs contained 20 million generations and the convergence of the runs was inspected using Tracer version 1.5. LogCombiner V1.5.4 and Tree Annotator V.1.5.4 were used to combine the results of multiple runs and calculate the mean evolutionary rates and divergence times. Dated phylogeny trees were visualized with Figtree V.1.2.3 [11]. All 8 H5N1 viruses including   Table 2). The genetic distance among H5N1 viruses circulating in Thailand over the 5-year course has remained below 3% indicating genetic drift characteristics of the viruses.
In order to study the reassortment of the viruses, lineage identification and the time of the most recent common ancestor (tMRCA) of H5N1 isolates representing 7 waves of AI outbreaks were examined. Our result showed that the 2004 H5N1 isolates (NIAH7540 and NIAH 8246) had undergone genetic drift and resulted in a new lineage of PB2, HA, NP and NA genes (Table 3).     Fig 4).  (Fig 4). This result confirms that the new reassorted viruses had acquired four gene segments from group1.1 and other genes from group1.2 [3]. It is noted that Bootscan and tMRCA analysis have been used to confirm reassortment of H5N1viruses from Indonesia [14].       [25]. The 2008 H5N1 viruses contain ESEV residues at the C-terminus of NS1 indicating high virulence [26]. As for the PB2 gene, the viruses contain E627 indicating low virulence in mammalian species [27]. An E627K mutation in PB2 would indicate increased viral replication efficiency in mammals [28]. Analysis of amino acids associated with human and avian H5N1 viruses indicated that the reassorted 2008 H5N1 viruses possess both human and avian characteristics. It is noted that the virulence of these reassortant viruses will need to be further confirmed in an animal model to validate their pathogenicity.
In conclusion, the clade nomenclature of H5N1 viruses has been determined based on the HA gene [29]. The viruses examined in this study have undergone inter-lineage reassortment and thus represent the genetic parents of the new reassorted 2008 H5N1 viruses. The potential site of genetic reassortment of Thai 2008 H5N1 could be traced backed to 2006. Bootscan analysis of the 2008 H5N1 viruses supported the reassortment sites. The new reassorted viruses have acquired four gene segments (PA,HA,NP,M) from sub-clade1.1 and four genes (PB2,PB1,NA,NS) from sub-clade1.2. This finding raises concerns with regard to circulation of newly reassorted viruses in Thailand. Geographically, the 2008 reassorted lineages predominantly circulated in the lower-north provinces of Thailand, while the original CUK2 lineage is mainly found in the central provinces. This phenomenon may be due to the strong bottle-neck effect in the region especially in the lower north of Thailand. To prevent future reassortment, emphasis should be put on prevention and control of AI outbreaks in the lower north of Thailand, not only in avian species but also in mammals [30,31], since a human-animal interface could create any emerging virus similar to the recently emerged pandemic H1N1 [32]. In addition, surveillance and whole genome sequencing of H5N1 viruses should be routinely performed for monitoring the genetic drift of the virus and identifying new reassorted strains, especially in light of potential reassortment between avian and mammalian H5N1 viruses.

Additional material
Additional file 1: Estimated time of the most recent common ancestor (tMRCAs) for Thai H5N1 viruses. Table of the estimated time of the most recent common ancestor (tMRCAs) for Thai H5N1 viruses.