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

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011.
During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition.

Complete Genome Sequences of Newcastle Disease Virus Strains Circulating in Chicken Populations of Indonesia

Newcastle disease (ND) is a highly contagious viral disease of poultry. ND was first reported in Java, Indonesia, in 1926. Although all commercial chickens in Indonesia are routinely vaccinated with live Newcastle disease virus (NDV) vaccines, ND continues to be a major problem for the poultry industry (7). NDV is a member of the genus Avulavirus in the family Paramyxoviridae. The genome of NDV is single-stranded, negative-sense, nonsegmented RNA with six genes encoding seven major viral proteins (3). NDV strains are divided into two classes based on genetic analysis: class I strains have been isolated mainly from wild birds and are generally avirulent, whereas class II strains have been recovered from wild and domestic birds and include virulent and avirulent isolates (2). Class I and II viruses are divided into 9 and 11 genotypes, respectively (4). To date, no complete genome sequences have been reported for NDV strains isolated from chickens in Indonesia. Only two partial F gene sequences (315 and 395 nucleotides) were available (1).

In 2009 and 2010, outbreaks of ND occurred in commercial chickens in Indonesia, causing up to 70% to 80% mortality. The sequences of the F and HN genes of eight NDV isolates were determined by reverse transcription (RT)-PCR using overlapped consensus primers and direct sequencing. The 3= and 5= termini were determined by rapid amplification of cDNA ends (RACE) (8). The amino acid sequence identities of the F and HN proteins among these eight strains ranged from 96% to 100% and 93% to 100%, respectively, compared to 87% and 89% between the Indonesian strains and the vaccine strains B1 and LaSota. This indicates that the circulating strains were substantially distinct from the vaccine strains in use and suggests that antigenic differences contributed to poor vaccine protection.

The sequence of the F protein cleavage site is a major determinant of NDV pathogenicity. The cleavage sites of virulent NDV strains usually contain multiple basic residues, whereas avirulent strains have fewer basic residues (5, 6). The eight Indonesian strains had two cleavage-site sequences that were each consistent with a virulent pathotype: RRQKR2F and RRRKR2F (underlining represents the basic residue; arrow represents the site of cleavage). Phylogenetic analysis of the F and HN genes of the eight strains constructed by MEGA4.0 indicated that they represent two new subgroups within genotype VII in class II.

Complete genome sequences were determined for two of these Indonesian NDV strains, namely, Banjarmasin/010/10 and Sukorejo/019/10, each representing one of the two new subgroups. The genomes of both Indonesian strains are 15,192 nucleotides in length. Comparison of genome sequences demonstrated 92% nucleotide sequence identity between the two Indonesian strains compared with 82% identity between either Indonesian strain and the vaccine strain B1 or LaSota (note that both vaccine strains are genotype II of class II and share 99% identity). The data described here provide evidence of divergence in circulating virulent strains within an outbreak as well as between these circulating and vaccine strains.

Nucleotide sequence accession numbers. The following genome sequences have been deposited in GenBank: Banjarmasin/010/10 (HQ697254), Sukorejo/019/10 (HQ697255), Makassar/003/09 (HQ697256), Gianyar/013/10 (HQ697257), Sragen/014/10(HQ697258), Kudus/017/10 (HQ697259), Kudus/018/10 (HQ697260), and Bali/020/10 (HQ697261).

Minute excretion of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) from experimentally infected domestic pigeons (Columbia livia) and lack of transmission to sentinel Chickens

Five out of sixteen domestic pigeons, inoculated oculo-nasally with a high dose of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1), developed clinical signs and neurological lesions leading to death of three pigeons 5–7 days after inoculation [ Klopfleisch, R., Werner, O., Mundt, E., Harder, T. & Teifke, J. P. (2006). Vet Pathol 43, 463–470 ] . H5N1 virus was recovered from all organs sampled from two apparently healthy pigeons at 3 days post-infection and from the three pigeons which died spontaneously. All surviving birds shed  virus via the oropharynx and the cloaca at minimal titres and seroconverted. Sentinel chickens reared in direct contact to the pigeons neither developed clinical signs nor seroconverted to the H5N1 virus.