ABSTRACT
The newly found Omicron SARS-CoV-2 variant of concern has rapidly spread worldwide. Omicron carries numerous mutations in key regions and is associated with increased transmissibility and immune escape. The variant has recently been divided into four subvariants with substantial genomic differences, in particular between Omicron BA.1 and BA.2. With the surge of Omicron subvariants BA.1 and BA.2, a large number of reinfections from earlier cases has been observed, raising the question of whether BA.2 specifically can escape the natural immunity acquired shortly after a BA.1 infection. To investigate this, we selected a subset of samples from more than 1,8 million cases of infections in the period from November 22, 2021, until February 11, 2022. Here, individuals with two positive samples, more than 20 and less than 60 days apart, were selected. From a total of 187 reinfection cases, we identified 47 instances of BA.2 reinfections shortly after a BA.1 infection, mostly in young unvaccinated individuals with mild disease not resulting in hospitalization or death. In conclusion, we provide evidence that Omicron BA.2 reinfections do occur shortly after BA.1 infections but are rare.
ABSTRACT
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. We successfully developed a multiplex PCR assay to simultaneously measure the relative amount of SARS-CoV-2 full length genomic RNA as well as subgenomic N gene and subgenomic ORF7a RNA. We found that subgenomic RNAs and both positive and negative strand RNA can be readily detected in swab samples taken up to 19 and 17 days post symptom onset respectively, and are strongly correlated with the amount of genomic length RNA present within a sample. Their detection and measurement is therefore unlikely to provide anymore insight into the stage of infection and potential infectivity of an individual beyond what can already be inferred from the total viral RNA load measured by routine diagnostic SARS-CoV-2 PCRs. Using both an original commercial and two custom SARS-CoV-2 Ampliseq mini-panels, we identified that both ORF7a and N gene subgenomic RNAs were consistently the most abundant subgenomic RNAs. We were also able to identify several non-canonical subgenomic RNAs, including one which could potentially be used to translate the ORF7b protein and others which could be used to translate ORF9b and the ORF N* which has arisen from a new transcription regulatory sequence recently created by mutations after SARS-CoV-2 jumped into people. SARS-CoV-2 genomic length and subgenomic length RNAs were present in samples even if cellular RNA was degraded, further indicating that these molecules are likely protected from degradation by the membrane structures seen in SARS-CoV-2 infected cells.
ABSTRACT
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.
ABSTRACT
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, provide evidence for their nuclease resistance and likely protection by cellular membranes consistent with being part of virus-induced replication organelles. Furthermore, we show that the ratios of genomic to subgenomic RNA as well as the ratios of plus to negative strand RNA of genomic and subgenomic RNA are consistent with what have been detected for other coronaviruses in cell culture; albeit with the caveat that in vivo diagnostic samples, even in relatively early infection, the ratios of these RNAs are most reminiscent of late culture, semi-purified virus preparations shown to have a relatively constant ratio of genomic to subgenomic RNAs of around 5-10 or higher, while the ratios of positive to negative strands are more than 100 for the genomic RNA and around 20 for the subgenomic RNAs. Overall, our results may help explain the extended PCR positivity of some samples, and may also, at least in part, help explain discrepancies in results of different diagnostic PCR methods described by others; in particular for samples with a low virus load or of poor quality. 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.
