Molecular characterization and molecular diagnosis of recently emerged influenza a viruses (H5N1&H3N8)

Abstract

In this report, the genome of A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04 (H5N1), the virus responsible for the avian influenza outbreak during January 2004 in Thailand, was sequenced followed by the phylogenetic analysis. Molecular characterization of this virus showed evidence of highly pathogenic based on polybasic amino acids within the cleavage site of the hemagglutinin (H5), a 20-codon deletion in the neuraminidase (N1) and a 5-codon deletion in the non-structural protein, respectively. Molecular diagnosis for H5N1 influenza detection based on multiplex RT-PCR, multiplex real-time RT-PCR and real-time PCR with melting curve analysis were developed and validated. We also investigated two outbreaks of respiratory disease in an animal shelter and veterinary clinic in the State of Florida that involved dog breeds other than greyhounds. Influenza A virus subtype H3N8 was isolated and characterized genetically. Comparison of the nucleotide sequence of the hemagglutinin (H3) revealed that the new canine influenza isolates were closely related but distinguishable from earlier virus isolates from greyhound dogs. Moreover, a single step real-time RT-PCR for rapid detection of H3N8 canine influenza A virus was also evaluated. In conclusion, this study reported the molecular characterization providing crucial information of the nucleotide and deduced amino acid sequences while the molecular diagnosis based of RT-PCR and real-time RT-PCR were rapid, specific and sensitive assays suitable for immediate detection and large scale screening in order to control and prevent severe outbreaks of H5N1 avian influenza and H3N8 canine influenza viruses.