Long-term serial passaging of SARS-CoV-2 reveals signatures of convergent evolution (preprint)

Measures to control the COVID-19 pandemic such as antiviral therapy and vaccination have been challenged by ongoing virus evolution under antiviral and immune pressures. Understanding viral evolutionary dynamics is crucial for responding to SARS-CoV-2, and preparing for the next pandemic, by informing prediction of virus adaptation, public health strategies, and design of broadly effective therapies. Whole-genome sequencing (WGS) of SARS-CoV-2 during the pandemic enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. Nine virus lineages, including four "variants of concern" and three former "variants under investigation" as designated by the World Health Organisation, were chosen to investigate intra- and inter-lineage evolution through long-term serial passaging in Vero E6 cells. Viruses were sampled over at least 33 passages (range 33-100) and analysed using WGS to examine evolutionary dynamics and identify key mutations with implications for virus fitness, transmissibility, and immune evasion. All passages continued to replicate in culture, despite regular accumulation of mutations. There was evidence of convergent acquisition of mutations both across passage lines and compared with contemporaneous SARS CoV-2 clinical sequences from population studies. Some of these convergent mutations are hypothesised to be important in proliferation of SARS-CoV-2 lineages, such as by evading host immune responses (e.g. S:A67V, S:H655Y). Given these mutations arose in vitro, in the absence of a multicellular host immune response, this suggests virus genome mutation resulted from stochastic events, rather than immune-driven mutation. There was a regular gain and loss of low-frequency variants during serial passaging, but some became fixed in subsequent multiple passages, suggesting either a benefit of the mutation in vitro, or at least a lack of deleterious effect. Our findings reveal valuable insights into the evolution of SARS-CoV-2 by quantitatively investigating evolutionary dynamics of the virus over the greatest number of serial passages to date. Knowledge of these evolutionary trends will be useful for public health and the development of antiviral and vaccine measures to reduce the effects of SARS CoV-2 infection on the human population.

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance (preprint)

The capacity to undertake whole genome sequencing (WGS) in public health laboratories (PHLs) has grown rapidly in response to COVID-19, and SARS-CoV-2 genomic data has been invaluable for managing the pandemic. The public health response has been further supported by the rapid upgrade and implementation of laboratory and bioinformatic resources. However, there remains a high degree of variability in methods and capabilities between laboratories. In addition to evolving methodology and improved understanding of SARS-CoV-2, public health laboratories have become strained during surges in case numbers, adding to the difficulty of ensuring the highest data accuracy. Here, we formed a national working group comprised of laboratory scientists and bioinformaticians from Australia and New Zealand to improve data concordance across PHLs. Through investigating discordant sequence data from Australia's first external SARS-CoV-2 WGS proficiency testing program (PTP), we show that most discrepancies in genome assessment arose from intrahost variation. While others could be remedied using reasonable, parsimonious bioinformatic quality control. Furthermore, we demonstrate how multidisciplinary national working groups can inform guidelines in real time for bioinformatic quality acceptance criteria. Provision of technical feedback allows laboratory improvement during a pandemic in real time, enhancing public health responses.

Emergence and antibody evasion of BQ and BA.2.75 SARS-CoV-2 sublineages in the face of maturing antibody breadth at the population level (preprint)

The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and related sub-lineages. Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants at two levels: (i) we tracked over 400,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using antibody pools. (ii) we mapped the antibody response at the individual level using blood from strigently curated vaccine and convalescent cohorts. In pooled antibody samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases we observed increased antibody breadth to variants that were yet to be in circulation. Resolution of viral neutralisation at the cohort level supported equivalent coverage across prior and emerging variants with emerging isolates BQ.1.1, XBB.1 and BR.2.1 the most evasive. Further, these emerging variants were resistant to Evusheld, whilst neutralization resistance to Sotrovimab was restricted to BQ.1.1 and further supported by lack of Spike glycoprotein binding to this variant. An outgrowth advantage through better utilization of TMPRSS2 was observed across BQ lineages and not those derived from BA.2.75. We conclude at this current point in time that variants derived from BQ lineages can evade antibodies at levels equivalent to their most evasive BA.2.75 counterparts but sustain an entry phenotype that would promote an additional outgrowth advantage.

Persistence of a SARS-CoV-2 variant with a frameshifting deletion for the duration of a major outbreak (preprint)

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. Whole-genome sequencing of virus from an early case revealed a sub-consensus level of sequencing reads supporting a 17-nucleotide frameshift-inducing deletion in ORF7a that truncated the peptide sequence. The variant rapidly became represented at the consensus level (Delta-ORF7a{Delta}17del) in most of the outbreak cases in Australia. Retrospective analysis of ORF7a deletions in all GISAID clade GK Delta genomes showed that of 4,018,216 genomes, 134,751 (~3.35%) possessed a deletion in ORF7a, with the ORF7a{Delta}17del mutation by far the most common. Approximately 99.05% of Delta-ORF7a{Delta}17del genomes on GISAID originated from the Australian Delta outbreak, and comprised 87% of genomes in the outbreak. In vitro comparison of lineages in cell culture showed a significantly greater proportion of cells were infected with Delta-ORF7a{Delta}17del than with a contemporaneous Delta variant without ORF7a{Delta}17del (Delta-ORF7aintact), and the proportion was also measurably higher than an early SARS-CoV-2 strain (A.2.2). These results showed that Delta-ORF7a{Delta}17del potentially has a slight growth advantage compared to Delta-ORF7aintact. Delta-ORF7a{Delta}17del viruses still produced ORF7a protein, but significantly less than A.2.2, in a different cellular distribution with a more diffuse expression throughout the cytoplasm of infected cells. These data suggest that the proliferation of Delta-ORF7a{Delta}17del genomes during the Australian Delta outbreak was likely not a result of an intrinsic benefit of the ORF7a{Delta}17del mutation, but rather a chance founder effect. Nonetheless, the abundance of different ORF7a deletions in genomes worldwide suggests these have some benefit to virus transmission. IMPORTANCE Deletions in the ORF7a region of SARS-CoV-2 have been noted since early in the COVID-19 pandemic, but are generally reported as transient mutations that are quickly lost in the population. Consequently, ORF7a deletions are considered disadvantageous to the virus through possible loss-of-function effects. In constrast to these earlier reports, we present the first report of a SARS-CoV-2 variant with an ORF7a deletion that dominated for the entirety of a protracted outbreak, and found no associated fitness disadvantage or advantage in cell culture. The relatively common rise and fall of ORF7a deletion variants over time likely represent chance founder events followed by proliferation until a more fit variant(s) is introduced to the population. Our global clade-level survey of ORF7a deletions will be a useful resource for future studies into this gene region.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern. (preprint)

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, the Omicron BA.1 variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant demonstrated higher numbers of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5 has now started to dominate globally, with the potential to supplant BA.2. To address the relative threat of BA.5, we determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a well characterised, genetically engineered ACE2/TMPRSS2 cell line. We then assessed the impact of BA.5 infection on humoral neutralisation in vitro, in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. The infectivity of virus in primary swabs and expanded isolates revealed whilst BA.1 and BA.2 are attenuated through ACE2/TMPRSS2, BA.5 infectivity is equivalent to that of an early 2020 circulating clade and has greater sensitivity to the TMPRSS2 inhibitor Nafamostat. As with BA.1, we observed BA.5 to significantly reduce neutralisation titres across all donors. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 7-fold with BA.5. Of all therapeutic antibodies tested, we observed a 14.3-fold reduction using Evusheld and 16.8 reduction using Sotrovimab when neutralising a Clade A versus BA.5 isolate. These results have implications for ongoing tracking and management of Omicron waves globally.

SARS-CoV-2 Omicron: reduction of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern (preprint)

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in the first world. In late 2021, the Omicron (B.1.1.529) virus variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant demonstrated higher numbers of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. We assessed the impact of Omicron infection on the ability of: serum from vaccinated and / or previously infected individuals; concentrated human IgG from plasma donors, and licensed monoclonal antibody therapies to neutralise virus in vitro. There was a 17 to 22-fold reduction in neutralisation titres across all donors who had a detectable neutralising antibody titre to the Omicron variant. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 16-fold. Of all therapeutic antibodies tested, significant neutralisation of the Omicron variant was only observed for Sotrovimab, with other monoclonal antibodies unable to neutralise Omicron in vitro. These results have implications for ongoing therapy of individuals infected with the Omicron variant.

Assessment of inter-laboratory differences in SARS-CoV-2 consensus genome assemblies between public health laboratories in Australia (preprint)

Whole-genome sequencing of viral isolates is critical for informing transmission patterns and ongoing evolution of pathogens, especially during a pandemic. However, when genomes have low variability in the early stages of a pandemic, the impact of technical and/or sequencing errors increases. We quantitatively assessed inter-laboratory differences in consensus genome assemblies of 72 matched SARS-CoV-2-positive specimens sequenced at different laboratories in Sydney, Australia. Raw sequence data were assembled using two different bioinformatics pipelines in parallel, and resulting consensus genomes were compared to detect laboratory-specific differences. Matched genome sequences were predominantly concordant, with a median pairwise identity of 99.997%. Identified differences were predominantly driven by ambiguous site content. Ignoring these produced differences in only 2.3% (5/216) of pairwise comparisons, each differing by a single nucleotide. Matched samples were assigned the same Pango lineage in 98.2% (212/216) of pairwise comparisons, and were mostly assigned to the same phylogenetic clade. However, epidemiological inference based only on single nucleotide variant distances may lead to significant differences in the number of defined clusters if variant allele frequency thresholds for consensus genome generation differ between laboratories. These results underscore the need for a unified, best-practices approach to bioinformatics between laboratories working on a common outbreak problem.

Rapid spread of a SARS-CoV-2 Delta variant with a frameshift deletion in ORF7a (preprint)

Australia is currently experiencing COVID-19 outbreaks from infection with SARS-CoV-2 Delta variants (B.1.617.2, AY.3). Analysis of the index case reveals a sub-consensus level of sequencing reads ([~]25%) that support a 17-nucleotide deletion in ORF7a (ORF7a{Delta}17del). ORF7a{Delta}17del induces a frameshift mutation in ORF7a, which truncates the peptide and potentially leads to reduced suppression of host restriction factor BST-2/CD317/Tetherin. Despite this, the mutation has rapidly become represented at the consensus level in subsequent cases: approximately 72% of SARS-CoV-2 genomes in the Australian outbreak possess ORF7a{Delta}17del, and 99.7% (1534/1538) of Delta genomes on GISAID with ORF7a{Delta}17del originate from the current Australian outbreak (5 August 2021). The global abundance of this mutation might be underestimated given the difficulty of variant calling software correctly calling insertion/deletions (indels), the common inability of phylogenetics software to take indels into account, and the tendency of GISAID to not release submissions that contain a frameshift mutation (unless specifically requested). Overall, the rapid increase of persistent ORF7a{Delta}17del variants is concerning, and suggests either a chance founder effect with a neutral mutation yet to be purged, or that the ORF7a{Delta}17del mutation provides a direct selective advantage.