|CHRZASTEK, KLAUDIA - Orise Fellow|
|LEE, DONG-HUN - Orise Fellow|
|SHARMA, POONAM - Orise Fellow|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2017
Publication Date: 6/19/2017
Publication URL: http://handle.nal.usda.gov/10113/5729157
Citation: Chrzastek, K., Lee, D., Smith, D.M., Sharma, P., Suarez, D.L., Pantin Jackwood, M.J., Kapczynski, D.R. 2017. Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses. Virology. 509:159-166. doi:10.1016/j.virol.2017.06.019.
Interpretive Summary: Avian influenza, Newcastle disease and infectious bronchitis are severe systemic, highly transmissible disease of poultry that can cause high mortality rates and severe economic losses. Genetic analysis of these viruses has been non-systematic, leaving critical gaps on genetics in our knowledge base. Traditionally, diagnosis had been slow requiring virus isolation and identification. This delayed diagnosis has hampered efforts for early detection and elimination, resulting in devastating natural outbreaks. Rapid real-time diagnostics have increased our ability to identify these viruses in birds and differentiate these viral infections from “look-a-like” respiratory diseases. However, these techniques require sequence specific reagents for detection. We have developed a diagnostic technique using next generation sequencing that is not dependent of specific primers and generates a complete profile of microbes within a sample. In addition, full length genome sequencing is obtained during analysis, reducing the need for further preparation after virus identification. Taken together, this new technique can be applied for poultry diagnostics to rapidly and thoroughly detect and qualify the type and lineage of viral isolates in clinical samples. This technique can also be applied across many etiologic agents as well as different agriculture animals.
Technical Abstract: Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combination with MiSeq platform was applied to target negative- and positive-sense single-stranded RNA viral sequences. This method allowed successful sequence assembly of full or near full length avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) viral genome. Moreover, SISPA analysis applied to unknown clinical cases of mixed viral infections produced genome assemblies comprising 98% NDV and 99% of IBV genomes. Complete or near complete virus genome sequence was obtained with titers at or above 104.5 EID50/ml (50% embryo infectious dose), and virus identification could be detected with titers at or above 103 EID50/ml. Taken together, these studies demonstrate a simple template enrichment protocol for rapid detection and accurate characterization of avian RNA viruses.