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Development of an RNA Strand-Specific Hybridization Assay To Differentiate Replicating versus Nonreplicating Influenza A Viruses

Development of an RNA Strand-Specific Hybridization Assay To Differentiate Replicating versus Nonreplicating Influenza A Viruses

Authors :
Meerjady Sabrina Flora
Genyan Yang
Lenee Blanton
Erin Hodges
Janna R. Murray
A. S. M. Alamgir
David E. Wentworth
Jörn Winter
Natosha Zanders
Tatiana Bousse
John Barnes
A. K. M. Muraduzzaman
Svetlana Shcherbik
Mahbubur Rahman
C. Todd Davis
Source :
J Clin Microbiol
Publication Year :
2020
Publisher :
American Society for Microbiology, 2020.

Abstract

Replication of influenza A virus (IAV) from negative-sense viral RNA (vRNA) requires the generation of positive-sense RNA (+RNA). Most molecular assays, such as conventional real-time reverse transcriptase PCR (rRT-PCR), detect total RNA in a sample without differentiating vRNA from +RNA. These assays are not designed to distinguish IAV infection versus exposure of an individual to an environment enriched with IAVs but wherein no viral replication occurs. We therefore developed a strand-specific hybridization (SSH) assay that differentiates between vRNA and +RNA and quantifies relative levels of each RNA species. The SSH assay exhibited a linearity of 7 logs with a lower limit of detection of 6.0 × 102 copies of molecules per reaction. No signal was detected in samples with a high load of nontarget template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected 2 to 4 h postinoculation of MDCK cells, whereas synthesis of cold-adapted IAV +RNA was significantly impaired at 37°C. The SSH assay was then used to test IAV rRT-PCR positive nasopharyngeal specimens collected from individuals exposed to IAV at swine exhibitions (n = 7) or while working at live bird markets (n = 2). The SSH assay was able to differentiate vRNA and +RNA in samples collected from infected, symptomatic individuals versus individuals who were exposed to IAV in the environment but had no active viral replication. Data generated with this technique, especially when coupled with clinical data and assessment of seroconversion, will facilitate differentiation of actual IAV infection with replicating virus versus individuals exposed to high levels of environmental contamination but without virus infection.

Details

ISSN :
1098660X and 00951137
Volume :
58
Database :
OpenAIRE
Journal :
Journal of Clinical Microbiology
Accession number :
edsair.doi.dedup.....f8e61ee9143c04c80e617c0cc4f7f441
Full Text :
https://doi.org/10.1128/jcm.00252-20