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Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/24/2004 Publication Date: 6/15/2004 Citation: Lee, C.W., Suarez, D.L. 2004. Application Of Real-Time RT-PCR For The Quantification And Competitive Replication Study Of H5 and H7 Subtype Avian Influenza Virus. Journal of Virological Methods, 2004 v.119, p.151-158. Interpretive Summary: Avian influenza can cause a severe disease in poultry, including chickens and turkeys. Because of its economic importance, scientific studies to produce new and better vaccines and to understand how the virus causes disease need to be conducted. For these studies it is important to measure how much virus is being shed by the bird, which affects the abilities of other birds to be infected. Currently, the amount of virus being shed was measured by inoculating the sample into embryonating chickens eggs. This technique although accurate is slow and requires a large number of eggs. This paper describes a new technique that can measure the amount of virus in a sample using quantitative realtime PCR. The technique was shown to be accurate, reproducible, fast, and it had an equal sensitivity to the standard egg inoculation method. This technique should allow researchers to have better data from their experiments and greatly reduce the number of chicken eggs required for these studies. Technical Abstract: Avian influenza (AI) viruses are endemic in wild birds and if transmitted to poultry can cause serious economic losses. In the study of AI, the quantification of virus shed from infected birds is valuable in pathogenesis studies and to determine the effectiveness of vaccines, and is performed routinely by cultivation of virus containing samples using embryonating chicken eggs (ECE) and expressed by 50% egg infectious dose (EID50). Although, this assay is accurate and is the standard test for infectious virus titration, the method is laborious, requires a large number of ECE, and takes at least 7 days to determine results. In this study, a one-tube hydrolysis fluorescent probe based real-time RT-PCR (RRT-PCR) was applied for the quantification of AI virus and compared with conventional virus titration method. A strong positive correlation was observed between the amount of RNA determined by quantitative RRT-PCR and the EID50s determined by conventional methods. This RRT-PCR test was further applied in the study of competitive replication of co-infected H5 and H7 subtype viruses in chickens. Using hemagglutinin subtype specific probes, we were able to determine the amount of individual subtype virus, which could not have been done with conventional methods. This RRT-PCR based quantification of AI virus, which is specific, sensitive, easy to perform, and rapid, will be useful for virological, pathogenesis, and protection studies. |
