Transmission dynamics of SARS-CoV-2 within-host diversity in two major hospital outbreaks in South Africa.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in humans and a wide range of animal species. Its rapid global spread has resulted in a major public health emergency, necessitating commensurately rapid research to improve control strategies. In particular, the ability to effectively retrace transmission chains in outbreaks remains a major challenge, partly due to our limited understanding of the virus' underlying evolutionary dynamics within and between hosts. We used high-throughput sequencing whole-genome data coupled with bottleneck analysis to retrace the pathways of viral transmission in two nosocomial outbreaks that were previously characterised by epidemiological and phylogenetic methods. Additionally, we assessed the mutational landscape, selection pressures, and diversity at the within-host level for both outbreaks. Our findings show evidence of within-host selection and transmission of variants between samples. Both bottleneck and diversity analyses highlight within-host and consensus-level variants shared by putative source-recipient pairs in both outbreaks, suggesting that certain within-host variants in these outbreaks may have been transmitted upon infection rather than arising de novo independently within multiple hosts. Overall, our findings demonstrate the utility of combining within-host diversity and bottleneck estimations for elucidating transmission events in SARS-CoV-2 outbreaks, provide insight into the maintenance of viral genetic diversity, provide a list of candidate targets of positive selection for further investigation, and demonstrate that within-host variants can be transferred between patients. Together these results will help in developing strategies to understand the nature of transmission events and curtail the spread of SARS-CoV-2.

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.

Dynamically evolving novel overlapping gene as a factor in the SARS-CoV-2 pandemic.

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.