Recently, clade 2.3.2 viruses have been repeatedly detected in wild birds in Hong Kong, Japan, Russia and Mongolia and it was suggested that this clade may be established in migrating birds . More recently, clade 2.3.2 has been repeatedly detected in wild birds in Europe and there has been an increased prevalence of this virus clade in poultry outbreaks in South East Asia , .
The first introduction of clade 2.3.2 H5N1 virus to South Asia was reported in Nepal in February, 2010 , . Outbreaks in Eastern India and Bangladesh during the same period were due to clade 2.2 H5N1 viruses . Here we report the first detection and the genetic and antigenic characterization of clade 2.3.2 H5N1 viruses in Indian poultry.
Results and Discussion
Phylogenetic analysis of the HA genes (Figure 1) showed that the chicken and duck isolates of 2011 clustered with clade 2.3.2 viruses rather than with the clade 2.2 viruses reported earlier in India. Phylogenetically the 2011 Tripura isolates from the two farms clustered tightly together with 100% bootstrap value indicating a single introduction event. While they clustered within clade 220.127.116.11 with contemporary isolates from China, Vietnam, Hong Kong SAR, Japan, Mongolia, Nepal and Russia, the 2011 Tripura isolates were clearly distinct from these other viruses including those detected in Nepal (Figure 1). The Nepal isolates of 2010 shared only 97.6% similarity with 2011 Tripura isolates, and were phylogenetically much closer to those in Qinghai Lake and Mongolia in 2009. The phylogenetic analysis of the other seven genes shows similar evolutionary relationships to clade 2.3.2 viruses. The NA (Figure 2) and internal genes of the Indian isolates grouped with the recently isolated clade 2.3.2 viruses in genotype V based on classification described in  and  and were separate from the Indian and Bangladesh clade 2.2 viruses. The PA gene of the Indian isolates of 2011 formed a well supported group distinguishing genotype V and other clade 2.3.2 viruses from other subclades (Figure 3).
N- linked glycosylation masks the oligosaccharides on the hemagglutinin (HA) and neuraminidase of influenza A virus from recognition by lectins of the innate immune system and thereby inhibit antibody mediated neutralization. Site specific glycosylation in the HA gene affects cleavage of the HA and thus the virulence of the influenza viruses . Seven potential glycosylation sites (N-X-S/T where X is any amino acid except Proline) were present in the HA protein of the 2011 Tripura viruses at positions 11NST13, 23NVT25, 140NSS142, 165NNT167, 286NSS288, 484NGT486 and 543NGS545. There was one additional glycosylation site compared with Indian clade 2.2 viruses due to R140N mutation. This additional glycosylation site might indicate increased adaptation of the virus in land based poultry compared to clade 2.2 viruses  with no significant effect on the virulence of the virus as it is not located on the HA globular head .
The S129L substitution which is characteristic of clade 2.3.2 viruses  is present in all 2011 Tripura isolates. The amino acid mutations V223I and M230I in receptor binding domain of clade 2.2 viruses isolated from the largest human cluster in Egypt (Gharbya cluster) and the two human cases of Bangladesh are present in 2011 Tripura clade 2.3.2 isolates (http://www.recombinomics.com/News/03161103/H5N1_Dhaka_Cluster.html). Acquiring of these amino acid markers are significant since they are absent in Indian clade 2.2 viruses and are dominant in seasonal H1N1, H3N2 and influenza B viruses. The acquisition of additional glycosylation site and accumulation of these mutations might indicate that the H5N1 viruses are evolving in land based poultry and might acquire the ability to support human transmission . Hence, there is a need to monitor the evolution of these clade 18.104.22.168 viruses.
The intravenous pathogenicity index calculated for all the seven isolates ranged from 2.80–2.95 indicating high pathogenicity to chickens. However, the 61% mortality observed in the outbreak at State Duck Breeding Farm was unusually high for duck species. Further investigations are needed to ascertain whether the presence of concurrent infection with other infectious agents such as duck viral enteritis or duck viral hepatitis contributed to the increased mortality.
The identification of new clade 22.214.171.124 H5N1 viruses in South Asia (India, Bangladesh and Nepal) that are phylogenetically closely related to those isolated in Qinghai Lake, China and Mongolia in 2009 and 2010 is reminiscent of the introduction of clade 2.2 viruses in this region in 2006/7 . It is now important to monitor whether the clade 126.96.36.199 is replacing the previous clade 2.2 in this region or co-circulating with it. The WHO report on antigenic and genetic characteristics of zoonotic influenza viruses and development of candidate vaccine viruses for pandemic preparedness (http://www.who.int/influenza/resources/documents/2011_09_h5_h9_vaccinevirusupdate.pdf) also reveals isolation of clade 188.8.131.52 viruses in Myanmar and Bangladesh which showed reduced reactivity with post-infection ferret antisera against the clade 2.3.4 viruses. Continued circulation of the H5N1 viruses of various subclades which are more adapted to land based poultry in a highly populated region such as South Asia might lead to evolution of pandemic strains with devastating consequences. Hence, there is an urgent need for faster sharing of data in public domain or through bilateral/international agencies for better management of control of the virus spread and its evolution in South Asia.
PCR amplification and sequencing
Sequencing of complete genome (all 8 segments) of 7 H5N1 viruses isolated from various sample types in both the outbreaks (one viral allantoic fluid of duck origin, 1 virus from duck carcass, 3 from chicken carcass and 1 each from cloacal and tracheal swab pools) was carried out. RT-PCR for amplification of various gene fragments with overlapping segment-specific primers was carried out using Platinum Taq High Fidelity (Invitrogen, USA) as described previously . The PCR products were gel purified using QIAquick gel extraction kit (Qiagen, Germany) and sequenced using specific PCR primers with BigDye® Terminator v3.1 Cycle Sequencing Kit (Cat. 4337455, Applied Biosystems, USA) in 3130- Genetic Analyzer (Applied Biosystems, USA). Nucleotide sequences reported in this study have been submitted to the GenBank (accession numbers CY089410-CY089425, CY089468- CY089477, CY092115- CY092120, CY092121- CY092132, CY092133- CY092144, CY089468- CY089477).