| Summary: | Outbreak of avian Influenza (AI) in Indonesia has been reported since the mid
of 2003, affected to layer commercial farm in Province of Central and West Java.
Clinical sign observation since in the early outbreak in the year 2003 to 2008
indicated some variation of symptoms of AI virus infection. Moreover, the AI
infections are still existing in the field sporadically, both in farms with and without
AI vaccination practices. This study was a retrospective study which was designed to
molecularly characterize of hemagglutinin (HA) gene of AI virus, particularly in the
fragment of cleavage site (CS), fusion site (FS), receptor binding site (RBS), and
antigenic sites (AS). Avian influenza viruses isolated from various poultry since 2003
until 2008 outbreak in Indonesia, which exhibited variation of symptoms and were
obtained from farm with and without AI vaccination practices. Isolation and
propagation were done on the chicken embryonated egg specific pathogen free. For
virus detection and subtyping, serological identification and reverse transcriptase
polymerase chain reaction (RT-PCR) method of H5 and N1 gene fragment was
performed. Further characterizations were amplification of CS, FS, RBS, and AS
domain of HA gene. All of RT-PCR HA gene positive products were sequenced to
determine the nucleotide composition at the targeted fragment. Obtained sequences
were analyzed by program Mega 4.0 versions, included multiple alignment, deductive
amino acid prediction, phylogenetic tree and pair wise distance calculation. The result
showed that all AIV isolates were identified as H5N1 subtype. Sequence analysis of
CS area determined five motives of multiple basic amino acids which were classified
as highly pathogenic avian influenza virus. The features of the motive were observed
have some mutations and deletions. Multiple alignment of FS domain of all viruses
isolated since 2003 to 2008 showed a conserved domain. Further characterizations on
amino acid responsible for RBS indicated a binding preference to avian like receptor,
sialic acid α 2, 3 Gal. Antigenic sites analysis determined some mutations of amino
acid in certain antigenic sites. Phylogenetic study showed that clustering of AIV did
not based on species of bird or geographic origin. Generally, AIV created two clusters
of viruses based on years of AIV isolation. Viruses isolated at the beginning of
outbreak in the year 2003, 2004, and 2005 tended to cluster which was different from
that of isolated from 2006 to 2008. Pair wise distance calculation demonstrated the
increase of amino acids substitution were observed at the AIV isolated in the year
2003 to 2008. Chi-square analyses indicated that the lesion type character and
vaccination status were not associated with the mutation of HA gene of AIV H5N1
subtype, at CS area, FS, RBS and AS which were studied. Our finding demonstrated
that the AIV H5N1 subtype in this study showed molecular dynamics of HA gene and
suggests that the AIV H5N1 subtype is evolving continuously.
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