Respiratory Viral Sequencing Panel identifies SARS-CoV-2 variants, transmission and other co-circulating viruses in Georgia, USA: A Diagnostic and Epidemiologic Tool for Mass Surveillance in COVID-19 Pandemic (preprint)

ABSTRACT

Background In the current phase of COVID-19 pandemic, we are facing two serious public health challenges that include deficits in SARS-CoV-2 variant monitoring, and neglect of other co-circulating respiratory viruses. Additionally, accurate assessment of the evolution, extent and dynamics of the outbreak are required to understand the transmission of the virus amongst seemingly unrelated cases and provide critical epidemiological information. To address these challenges, we evaluated a new high-throughput next-generation sequencing (NGS), respiratory viral panel (RVP) that includes 40 viral pathogens with the aim of analyzing viral subtypes, mutational variants of SARS-CoV-2, model to understand the spread of the virus in the state of Georgia, USA, and to assess other circulating viruses in the same population. Methods This study evaluated a total of 522 samples that included 483 patient samples and 42 synthetic positive control material. The performance metrics were calculated for both clinical and reference control samples by comparing detection results with the RT-PCR assay. The limit of detection (LoD) studies were conducted as per the FDA guidelines. Inference and visualization of the phylogeny of the SARS-CoV-2 sequences were performed through the Nextstrain Command-Line Interface (CLI) tool, utilizing the associated augur and auspice toolkits. Results The performance metrics calculated using both the clinical samples and the reference controls revealed a PPA, NPA and accuracy of 95.98%, 85.96% and 94.4%, respectively. The LoD was determined to be 10 copies/ml with all 25 replicates detected across two different runs. The clade for pangolin lineage B that contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF 3a and, S84L in ORF8 covarying with the D614G spike protein mutation were the most prevalent, early in the pandemic, in Georgia, USA. In our analysis, isolates from the same county formed paraphyletic groups, which indicated virus transmission between counties. Conclusion The study demonstrates the clinical and public health utility of the NGS-RVP to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats, models that provide insights into viral transmission patterns and predict transmission/ resurgence of regional outbreaks and provide critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.