A molecular and antigenic survey of H5N1 highly pathogenic avian influenza virus isolates from smallholder duck farms in Central Java, Indonesia during 2007-2008

Abstract

BackgroundIndonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI) virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed.ResultsAll 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1), clade 2.1.3 (80), and IDN/6/05-like viruses (3) that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA) and neuraminidase (NA) sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens.ConclusionsThe 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to chickens over the study period suggests that ducks are more likely to survive infection and they may better suit the role of long-term maintenance host for H5N1. As some viruses were isolated from dead birds, there was no clear correlation between genetic variations and pathogenicity of these viruses.

Antigenic Variation of Clade 2.1 H5N1 Virus Is Determined by a Few Amino Acid Substitutions Immediately Adjacent to the Receptor Binding Site

“…Of these 96 HA sequences, 79 were from viruses isolated during the currently underreported period since 2008. To evaluate recent genetic variation of clade 2.1 viruses, we constructed a maximum-likelihood phylogenetic tree using the sequences described in the present study and publically available clade 2.1 sequences (1–3, 19) (Fig. 1; see also Fig.…”

“…The clades as defined previously are specified (1–3). The tree contains sequences from the present study from 2008 to 2011 (in blue) and publically available sequences (1–3, 19). Red bars indicate viruses selected for antigenic characterization (Table 1), including the recommended poultry vaccine strain (A/Chicken/West Java/30/07) and the candidate vaccine strain for human use (A/Indonesia/5/05).…”

“…Following the initial detection of H5N1 clade 2.1 viruses in 2003 in Central Java, the viruses diversified into three distinct genetic lineages (clades 2.1.1, 2.1.2, and 2.1.3) from 2004 onwards and became enzootic in most parts of Indonesia (1–3, 5, 19). Since 2004, the ongoing genetic diversification of clade 2.1.3 viruses necessitated further classification into fourth-order clades (2.1.3.1, 2.1.3.2, and 2.1.3.3), while viruses of clades 2.1.1 and 2.1.2 have apparently disappeared.…”

“…The HA sequences of 96 H5N1 viruses isolated in Indonesia that were obtained as described above were combined with the publically available clade 2.1 HA sequences (1–3, 19). Phylogenetic tree construction of HA sequences was performed using the maximum-likelihood method available from the PAUP* software package (version 4.0b10) (32) under the GTR + I + Γ 4 model (the general time-reversible model with the proportion of invariant sites and the gamma distribution of among-site rate variation with four categories estimated from the empirical data) as determined by the ModelTest program (33).…”

A comparative evaluation of feathers, oropharyngeal swabs, and cloacal swabs for the detection of H5N1 highly pathogenic avian influenza virus infection in experimentally infected chickens and ducks

“…In addition, this virus is representative of the most prevalent HPAI present in Indonesia. 32 Twenty immature pectorosternal and 5 immature flight feathers were collected after euthanasia from 3 IDN 32226-infected ducks on 3, 4, and 5 dpi, to test for virus by culture in embryonated eggs and Vero cells. All ducks were confirmed as infected by the isolation of virus from tissues taken at autopsy (data not shown).…”