Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay

Abstract

Background: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. Methods: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. Results: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3′ termini of each genomic segment and subsequent qPCR of the 5′ regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the longrange RT-qPCR is highly correlated with plaque assay (Adjusted R 2 = 0.931, P = 0.000066). Conclusions: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.

Differentiating between infectious and non-infectious influenza A virus and coronavirus RNA levels using long-range RT-qPCR

“…2C). These results suggest that a long-range RT-qPCR protocol provides the best estimate of the infectious influenza A virus titer than a protocol based on random hexamers, in line with previous influenza A virus studies (7, 8).…”

“…We here used primers that bind to the 3′ terminus of full-length influenza A virus, HCoV 229E, or SARS-CoV-2 genome RNA molecules to reduce the detection of non-infectious viral RNA by RT-qPCR. Our results confirm previous observations with influenza A viruses (7, 8) showing that long-range RT-qPCR data more closely match measurements of infectious virus levels than RT-qPCR data obtained with random hexamers. Moreover, we demonstrate that the long-range RT-qPCR has a similar sensitivity as a commercially available and clinically approved, high-throughput detection assays.…”

“…1). We find that the long-range RT-qPCR method facilitates the differential detection of infectious and non-infectious influenza A and human coronavirus (HCoV) 229E, in agreement with previous influenza A virus studies (7, 8). Moreover, the RT-qPCR provides the same sensitivity as commercially available Taq-Path RT-qPCR kits at detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples, but the assay yields significantly fewer positive results in clinical samples obtained from clinically recovered COVID-19 patients.…”

“…Ultraviolet C (UVC) can create cross-links or breaks in nucleic acid strands and inactivate influenza A virus (7,9,10), HCoV 229E, and SARS-CoV-2 (11)(12)(13)(14)(15)(16)(17). Following calibration of our UVC instrument (Fig.…”

UVC disinfects SARS-CoV-2 by induction of viral genome damage without apparent effects on viral morphology and proteins

“…Third, UVC-induced virus protein-genome crosslinking is observed in poliovirus 21 . Fourth, UVC irradiation damages the genome of influenza virus 22 . However, UVC-induced damage varies in different virus types, as UVC damages the viral genome, but not viral proteins in adenovirus 23 .…”