Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/30/2007
Publication Date: 8/1/2007
Citation: Afonso, C.L. 2007. Sequencing of avian influenza virus genomes following random amplification. Biotechniques. 43(2):188-192. Interpretive Summary: Avian Influenza (AI) is a significant disease of birds and a threat to humans. Low pathogenic (LP) avian influenza viruses (AIV) are naturally found in wild birds and recently Asian H5N1 highly pathogenic (HP) AIVs have also been found in wild birds. This wild bird reservoir presents an ongoing threat of introduction into poultry flocks. Recently, primarily as a result of the emergence of the Asian H5N1 viruses capable of zoonotic spread, wild and domestic bird surveillance for AIV has increased worldwide which requires the development of fast and precise methods to characterize these isolates. The high capacity for avian influenza to change in wild birds make it impossible to predict the genetic composition of the new isolates, and in some cases it is not easy to spot immediately the differences between pathogenic and non-pathogenic viruses using current available methods. Here we describe a new approach to characterize AIV in 48 hours without the need of a previous hemaglutination test. This procedure also has the potential to detect mixed infections or other contaminants viruses.
Technical Abstract: Avian Influenza (AI) is a significant disease of birds and a threat to humans. Recently, as a result of the emergence of Asian H5N1 viruses capable of zoonotic spread, wild and domestic bird surveillance for Avian Influenza viruses (AIV) has increased worldwide, requiring the development of fast and precise methods to characterize these isolates. The high capacity for mutation (genetic drift) and re-assortments (genetic shift) in wild birds make it impossible to predict the genetic composition of new isolates. Methods such as DNA microarrays, real time PCR, and rapid nucleotide sequencing of PCR products occasionally fail or produce biased results primarily because the detection relies upon existing primers. These approaches presume that not yet characterized viruses will resemble previously sequenced viruses. In addition, with the exception of complete genomic sequencing, most AIV characterization methods are based on very small genomic regions. We have developed a random sequencing approach that utilizes non-purified viruses obtained from allantoic fluids as the source of RNA and allows the fast and precise characterization of AIV. This approach can be completed from RNA isolation to characterization in less than 2 days.