An increased ratio of SARS-CoV-2 positive to negative sense genomic and subgenomic RNAs within routine diagnostic upper respiratory tract swabs may be a marker of virion shedding (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly in the global population since its emergence in humans in late 2019. Replication of SARS-CoV-2 is characterised by transcription and replication of genomic length RNA and shorter subgenomic RNAs to produce virus proteins and ultimately progeny virions. Here we explore the pattern of both genome-length and subgenomic RNAs and positive and negative strand SARS-CoV-2 RNAs in diagnostic nasopharyngeal swabs using sensitive probe based PCR assays as well as Ampliseq panels designed to target subgenomic RNAs. Using these assays, we measured the ratios of genomic to subgenomic RNAs as well as the ratios of positive to negative strand RNAs in SARS-CoV-2 positive nasopharyngeal swab samples. We found that while subgenomic RNAs and negative strand RNA can be readily detected in swab samples taken up to 19 and 17 days post symptom onset respectively, and therefore their detection alone is not likely an indicator of active SARS-CoV-2 replication. However, the ratios of genomic-length to subgenomic RNA and also of positive to negative strand RNA were elevated in some swabs, particularly those collected around the onset of clinical symptoms or in an individual with decreasing PCR Cts in successive swab samples. We tentatively conclude that it may be possible to refine such molecular assays to help determine if active replication of virus is occurring and progeny virions likely present in a SARS-CoV-2 positive individual. Assays targeting subgenomic N or ORF7a RNAs as well as strand specific ORF7a total genome-length and subgenomic RNAs may be the most sensitive for this purpose as these targets were consistently the most abundant in the swab samples.

Sequence analysis of travel-related SARS-CoV-2 cases in the Greater Geelong region, Australia (preprint)

This study reports the sequence analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from infected individuals within the Greater Geelong region, Victoria, Australia. All but one individual had recently returned from travelling abroad, and all had clinical signs consistent with SARS-CoV-2 infection. SARS-CoV-2 belonging to three lineages were detected and represent separate introductions of the virus into the region. Sequence data were consistent with the recent travel history of each case. Full virus genome sequencing can play an important role in supporting local epidemiological tracing and monitoring for community transmission. Quality of the SARS-CoV-2 sequences obtained was highly dependent on appropriate sample collection and handling.

SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication (preprint)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in China in late December 2019 and has spread worldwide. Coronaviruses are enveloped, positive sense, single-stranded RNA viruses and employ a complicated pattern of virus genome length RNA replication as well as transcription of genome length and leader containing subgenomic RNAs. Although not fully understood, both replication and transcription are thought to take place in so-called double-membrane vesicles in the cytoplasm of infected cells. We here describe detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection, and provide a likely explanation not only for extended PCR positivity of such samples, but also for discrepancies in results of different PCR methods described by others. Overall, we present evidence that subgenomic RNAs may not be an indicator of active coronavirus replication/infection, but that these RNAs, similar to the virus genome RNA, may be rather stable, and thus detectable for an extended period, most likely due to their close association with cellular membranes.