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

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.

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

“…Detection of subgenomic RNAs in late infection, as described here up to 11 and 17 days after first detection by PCR and NGS, respectively, although in contrast to the studies by Wölfel et al 16 , is consistent with the recent findings described by van Kampen et al 17 , which detected the E gene subgenomic RNA by PCR in respiratory swabs up to 22 days after first day of onset of clinical symptoms. The participants in their study likely had more severe disease than the ones included in our study, as their study focused on hospitalized patients, many of which were in intensive care units, while our study subjects only had minor clinical symptoms and all self-isolated at home 26 . Nevertheless, although their study detected the E gene subgenomic RNA by PCR while we focused on the 7a subgenomic RNA by PCR and all the subgenomic RNAs by NGS, these studies support each other and were also supported by results presented in additional studies using the same subgenomic E gene PCR [18][19][20][21] .…”

“…The study included NGS of 12 SARS-CoV-2 PCR-positive samples and 1 negative sample as control. The 12 PCR-positive samples were obtained from eight individuals as NGS was done on samples taken at three time points from two individuals to monitor their infection 26 . Summary details of the samples included are shown in Table 1.…”

“…Nucleic acid extraction, cDNA synthesis, and SARS-CoV-2 Ampliseq NGS. Nucleic acid extraction and cDNA synthesis was performed by heating extracted nucleic acids at 70°C for 5 min and rapid cooling on ice before cDNA synthesis using SuperScript™ VILO™ Master Mix (Thermofisher Scientific, Victoria, Australia) as per manufacturers' instructions 26,32 . Prepared cDNA samples were then amplified using the Ion Ampliseq™ Library Kit 2.0 (Thermofisher Scientific, Victoria, Australia) 33,34 and a commercially available SARS-CoV-2 Ampliseq panel [https:// www.thermofisher.com/au/en/home/life-science/sequencing/dna-sequencing/ microbial-sequencing/microbial-identification-ion-torrent-next-generationsequencing/viral-typing/coronavirus-research.html] kindly provided by Thermofisher Scientific, Victoria, Australia.…”

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

“…That as NGS was done on samples taken at three time points from two individuals to monitor their infection 28 . For the studies described here we added one additional positive sample collected on the 24 th of April 2020.…”

“…In addition to the in-house PCR assays described above, we also used the commercial RT-PCR kit assay or the Ct reported from the original diagnostic laboratory as described previously 28 .…”