Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine (preprint)

Aims: To explore viral evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer BioNTech vaccine (BNT162b2) are as effective at neutralising the SARSCoV2 variant of concern (VOC) Delta (B 1.617.2) compared to the earlier lineages Beta (B.1.351) and wildtype (lineage A.2.2) virus. Methods: Using a live virus SARSCoV2 neutralisation assay in Vero E6 cells we determined neutralising antibody titres (nAbT) in 14 participants (vaccine naive (n=2) and post second dose of BNT162b2 vaccination (n=12), median age 45 years [IQR 29 to 65], median time after second dose = 21 days [IQR 19 to 28] against three SARSCoV2 strains: wild-type, Beta and Delta. The determination of nAbT was performed by visual inspection of cytopathic effect (CPE) and inhouse quantitative reverse transcriptase real time quantitative polymerase chain reaction (RTqPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 hrs, negative and positive controls underwent genome sequencing using the Respiratory Viral Oligo Panel version 2 (RVOP) (Illumina Inc. (San Diego, United States of America)) viral enrichment and short read sequencing using (Illumina Inc. (San Diego, United States of America)),(Figure 1). Results: There was a significant reduction in nAbT observed against the Delta and Beta VOC compared with wildtype, 4.4 fold (p = >0.0006) and 2.3 fold (p = 0.0140), respectively (Figure 2). Neutralizing antibodies were not detected in one vaccinated immunosuppressed participant nor the vaccine naive participants (n=2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARSCoV2 infection 12 months prior (Table S1). Limited consensus level mutations occurred in the SARS-CoV-2 genome of any lineage during in vitro neutralisation, however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S) and membrane protein. Discussion: Significant reductions in nAbT post vaccination were identified, with Delta demonstrating a 4.4 fold reduction. The reduction in nAbT for the VOC Beta has been previously documented, however, limited data is available on vaccine evasion for the Delta VOC, the predominant strain currently circulating worldwide at the time. Studies in high incidence countries may not be applicable to low incidence settings such as Australia as nAbT may be significantly higher in vaccine recipients previously infected with SARSCoV2, as seen in our cohort. Monitoring viral evolution is critical to evaluate the impact of novel SARSCoV2 variants on vaccine effectiveness as mutational profiles in the sub-consensus genome could indicate increases in transmissibility, virulence or allow the development of antiviral resistance.

A laboratory framework for ongoing optimisation of amplification based genomic surveillance programs (preprint)

Constantly evolving viral populations affect the specificity of primers and quality of genomic surveillance. This study presents a framework for continuous optimisation of sequencing efficiency for public health surveillance based on the ongoing evolution of the COVID-19 pandemic. SARS-CoV-2 genomic clustering capacity based on three amplification based whole genome sequencing schemes was assessed using decreasing thresholds of genome coverage and measured against epidemiologically linked cases. Overall genome coverage depth and individual amplicon depth were used to calculate an amplification efficiency metric. Significant loss of genome coverage over time was documented which was recovered by optimisation of primer pooling or implementation of new primer sets. A minimum of 95% genome coverage was required to cluster 94% of epidemiologically defined SARS-CoV-2 transmission events. Clustering resolution fell to 70% when only 85% of genome coverage was achieved. The framework presented in this study can provide public health genomic surveillance programs a systematic process to ensure an agile and effective laboratory response during rapidly evolving viral outbreaks.

SABRes: A tool for in silico detection of drug resistance conferring mutations in SARS-CoV-2 genomes (preprint)

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in over 25,000 genomes generated over the course of the SARS-CoV-2 pandemic in Australia.

Co-infection with SARS-COV-2 Omicron and Delta Variants Revealed by Genomic Surveillance (preprint)

We identified the co-infection of the SARS-CoV-2 Omicron and Delta variants in two epidemiologically unrelated patients with chronic kidney disease requiring haemodialysis. Both SARS-CoV-2 variants were co-circulating locally at the time of detection. Amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified Omicron and Delta subpopulations in respiratory samples from the two patients. These findings highlight the importance of genomic surveillance in vulnerable populations.

RESISTANCE CONFERRING MUTATIONS IN SARS-CoV-2 DELTA FOLLOWING SOTROVIMAB INFUSION (preprint)

Several Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralising monoclonal antibodies (mAbs) have received emergency use authorisation by regulatory agencies for treatment and prevention of Coronavirus Disease 2019 (COVID-19), including in patients at risk for progression to severe disease. Here we report the persistence of viable SARS-CoV-2 in patients treated with sotrovimab and the rapid development of spike gene mutations that have been shown to confer high level resistance to sotrovimab in vitro. We highlight the need for SARS-CoV-2 genomic surveillance in at risk individuals to inform stewardship of mAbs use and prevent potential treatment failures.

Improved neutralization of the SARS-CoV-2 Omicron variant after Pfizer-BioNTech BNT162b2 COVID-19 vaccine boosting (preprint)

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired 15 mutations in the receptor binding domain of the spike protein, raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three and six months post-two doses of Pfizer-BioNTech BNT162b2 has a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres are boosted. Despite this increase, neutralising antibody titres are reduced four-fold for Omicron compared to lineage A.2.2 SARS-CoV-2.

SARS-CoV-2 within-host and in-vitro genomic variability and sub-genomic RNA levels indicate differences in viral expression between clinical and in-vitro cohorts. (preprint)

Background: Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection benchmarked against an in-vitro infection model. Methods: Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33). Results: ISNVs were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. SgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture cohort than the clinical. Discussion and Conclusions: The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-Cov-2 variants may co-circulate. SgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

Off-season RSV epidemics in Australia after easing of COVID-19 restrictions (preprint)

Human respiratory syncytial virus (RSV) is an important cause of acute respiratory infection (ARI) with the most severe disease in the young and elderly. Non-pharmaceutical interventions (NPIs) and travel restrictions for controlling COVID-19 have impacted the circulation of most respiratory viruses including RSV globally, particularly in Australia, where during 2020 the normal winter epidemics were notably absent. However, in late 2020, unprecedented widespread RSV outbreaks occurred, beginning in spring, and extending into summer across two widely separated states of Australia, Western Australia (WA) and New South Wales (NSW) including the Australian Capital Territory (ACT). Genome sequencing revealed a significant reduction in RSV genetic diversity following COVID-19 emergence except for two genetically distinct RSV-A clades. These clades circulated cryptically, likely localized for several months prior to an epidemic surge in cases upon relaxation of COVID-19 control measures. The NSW/ACT clade subsequently spread to the neighbouring state of Victoria (VIC) and caused extensive outbreaks and hospitalisations in early 2021. These findings highlight the need for continued surveillance and sequencing of RSV and other respiratory viruses during and after the COVID-19 pandemic as mitigation measures introduced may result in unusual seasonality, along with larger or more severe outbreaks in the future.

Documenting Elimination of Co-Circulating Covid-19 Clusters Using Genomics in New South Wales, Australia (preprint)

Objective: To adapt ‘fishplots’ to describe SARS-CoV-2 genomic cluster evolution. Results: This novel analysis adapted the fishplot to depict the size and duration of circulating genomic clusters over time in New South Wales, Australia. It illuminated the effectiveness of interventions on the emergence, spread and eventual elimination of clusters and distilled genomic data into clear information to inform public health action.

SARS-CoV-2 Genome Sequencing Methods Differ In Their Ability To Detect Variants From Low Viral Load Samples (preprint)

SARS-CoV-2 genomic surveillance has been vital in understanding the spread of COVID-19, the emergence of viral escape mutants and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low frequency variants. Our analyses highlight that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina respiratory viral oligo panel, and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high quality sequence data from low viral load samples for public health and genomic surveillance purposes.

Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19 (preprint)

BackgroundThe detection of SARS-CoV-2 by real-time polymerase chain reaction (PCR) in respiratory samples collected from persons recovered from COVID-19 does not necessarily indicate shedding of infective virions. By contrast, the isolation of SARS-CoV-2 using cell-based culture likely indicates infectivity, but there are limited data on the correlation between SARS-CoV-2 culture and PCR. Here we review our experience using SARS-CoV-2 culture to determine infectivity and safe de-isolation of COVID-19 patients. Methods195 patients with diverse severity of COVID-19 were tested (outpatients [n=178]), inpatients [n=12] and ICU [n=5]). SARS-CoV-2 PCR positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect (CPE). SARS-CoV-2-induced CPE was confirmed by PCR of culture supernatant. Where no CPE was documented, PCR was performed on day four to confirm absence of virus replication. Cycle threshold (Ct) values of the day four PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined as a Ctsample - Ctculture value of [≥]3. FindingsOf 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was only successfully isolated from samples with Ctsample values <32, including in 28/181 (15%), 19/42 (45%) and 9/11 samples (82%) collected from outpatients, inpatients and ICU patients, respectively. The mean duration from symptom onset to culture positivity was 4.5 days (range 0-18 days). SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (p<0.001) and ICU patients (p<0.0001) compared with outpatients, and in samples with lower Ctsample values. ConclusionSARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.

REVEALING COVID-19 TRANSMISSION BY SARS-CoV-2 GENOME SEQUENCING AND AGENT BASED MODELLING (preprint)

Community transmission of the new coronavirus SARS-CoV-2 is a major public health concern that remains difficult to assess. We present a genomic survey of SARS-CoV-2 from a during the first 10 weeks of COVID-19 activity in New South Wales, Australia. Transmission events were monitored prospectively during the critical period of implementation of national control measures. SARS-CoV-2 genomes were sequenced from 209 patients diagnosed with COVID-19 infection between January and March 2020. Only a quarter of cases appeared to be locally acquired and genomic-based estimates of local transmission rates were concordant with predictions from a computational agent-based model. This convergent assessment indicates that genome sequencing provides key information to inform public health action and has improved our understanding of the COVID-19 evolution from outbreak to epidemic.

An emergent clade of SARS-CoV-2 linked to returned travellers from Iran (preprint)

The SARS-CoV-2 epidemic has rapidly spread outside China with major outbreaks occurring in Italy, South Korea and Iran. Phylogenetic analyses of whole genome sequencing data identified a distinct SARS-CoV-2 clade linked to travellers returning from Iran to Australia and New Zealand. This study highlights potential viral diversity driving the epidemic in Iran, and underscores the power of rapid genome sequencing and public data sharing to improve the detection and management of emerging infectious diseases.