Development of an RNA strand-specific hybridization assay to differentiate replicating versus non-replicating influenza A virus

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J Clin Microbiol. 2020 Apr 3. pii: JCM.00252-20. doi: 10.1128/JCM.00252-20. [Epub ahead of print]
Development of an RNA strand-specific hybridization assay to differentiate replicating versus non-replicating influenza A virus.


Yang G¹, Hodges EN², Winter J², Zanders N², Shcherbik S², Bousse T², Murray JR², Muraduzzaman AKM³, Rahman M³, Alamgir ASM³, Sabrina Flora M³, Blanton L², Barnes JR², Wentworth DE², Davis CT².

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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 RT-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 6.0x10² copies of molecules per reaction. No signal was detected in samples with a high load of non-target template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected at 2-4 hours post-inoculation 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.
Copyright ? 2020 American Society for Microbiology.



PMID:32245834DOI:10.1128/JCM.00252-20