Characterization of the influenza A H5N1 viruses of the 2008-09 outbreaks in India reveals a third introduction and possible endemicity

Abstract

Widespread infection of highly pathogenic avian influenza A H5N1 was reported from backyard and commercial poultry in West Bengal (WB), an eastern state of India in early 2008. Infection gradually spread to Tripura, Assam and Sikkim, the northeastern states, with 70 outbreaks reported between January 2008 and May 2009. Whole genome sequence analysis of three isolates from WB, one isolate from Tripura along with the analysis of hemagglutinin (HA) and neuraminidase (NA) genes of 17 other isolates was performed during this study. In the HA gene phylogenetic tree, all the 2008-09 Indian isolates belonged to EMA3 sublineage of clade 2.2. The closest phylogenetic relationship was found to be with the 2007-09 isolates from Bangladesh and not with the earlier 2006 and 2007 Indian isolates implying a third introduction into the country. The receptor-binding pocket of HA1 of two isolates from WB showed S221P mutation, one of the markers predicted to be associated with human receptor specificity. Two substitutions E119A (2 isolates of WB) and N294S (2 other isolates of WB) known to confer resistance to NA inhibitors were observed in the active site of neuraminidase. Several additional mutations were observed within the 2008-09 Indian isolates indicating genetic diversification. Overall, the study is indicative of a possible endemicity in the eastern and northeastern parts of the country, demanding active surveillance specifically in view of the critical mutations that have been observed in the influenza A H5N1 viruses.

Figure 1. Location of highly pathogenic avian influenza (HPAI) Influenza A H5N1 virus outbreaks during 2008-09 in India.

Indian states shown as underlined uppercase, districts in lowercase and neighbouring countries in uppercase font. The phase wise spread is as indicated in the colour scheme. Inset shows the earliest Indian H5N1 outbreak of 2006 (grey oval). Black box in the inset shows the area of enlargement. Rectangular boxes attached to different locations in the map denote the place of isolation and include the isolation dates along with the number in the parenthesis denoting the number of isolates. * indicates the isolates whose full genome has been sequenced.

Figure 2. The HA (A) and NA (B) phylogenetic trees constructed by using the neighbour-joining method as implemented in MEGA.

Scale bar indicates number of nucleotide substitutions per site. Gs/Guangdong/1/96 was used as out group sequence. Gs/Guangdong/1/96 was used as out group sequence. Abbreviations: Ck-Chicken, Dk-Duck, Gs-Goose, Ws-Whooper swan, Tk-Turkey, Pg-Peregrine, HB-Houbara Bustard, CO-Cygnus Olor, DGs-Domestic Goose.

Figure 3. HA gene phylogeny of the 2008-09 Indian isolates and 2007-09 Bangladesh isolates using the Bayesian method.