Biology subjects

Basile, K.

Publications and source records attributed to Basile, K..

7 recordsLinked to original sources

bioRxiv2024

STECode: an automated virulence barcode generator to aid clinical and public health risk assessment of Shiga toxin-producing Escherichia coli

Shiga Toxin (Stx) producing Escherichia coli (STEC) is a subset of pathogenic E. coli that can produce two types of Stx, Stx1 and Stx2, which can be further subtyped into four and 15 subtypes respectively. Not all subtypes, however, are equal in virulence potential, and the risk of severe disease including haemolytic uraemic syndrome has been linked to certain Stx2 subtypes e.g. Stx2a, Stx2d, highlighting the importance to survey stx subtypes. Previously, we developed a STEC virulence barcode to capture pertinent information on virulence genes to infer pathogenic potential. However, the process required multiple manual curation steps to determine the barcode. Here we introduce STECode, a bioinformatic tool to automate the STEC virulence barcode generation from sequencing reads or genomic assemblies. The development, and validation of STECode is described using a set of publicly available completed STEC genomes, along with their corresponding short reads. STECode was applied to interrogate the virulence landscape and molecular epidemiology of human STEC isolated during the period of the international border closures related to COVID-19 in the state of New South Wales, Australia. Impact statementWhole genome sequencing has been used to great effect in the genomic surveillance of STEC for public health purposes via the tracking of outbreaks. With STECode, we present a method to generate a STEC virulence barcode which captures pertinent subtyping information, useful for genomic inference of pathogenic potential. A key blind spot generated in short-read sequencing is the inability to detect the presence of multiple, isogenic stx copies in STEC. STECode mitigates this by inferring and reporting on the possibility of this occurrence. We envisage that this tool will value-add current genomic surveillance workflows through the ability to infer pathogenic potential.

microbiology↗

bioRxiv2023

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

AimsTo 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 SARS-CoV-2 variant of concern (VOC) Delta (B 1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. MethodsUsing a live-virus SARS-CoV-2 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 29-65], median time after second dose = 21 days [IQR 19-28] against three SARS-CoV-2 strains: wild-type, Beta and Delta. The determination of nAbT was performed by visual inspection of cytopathic effect (CPE) and in-house quantitative reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR) 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). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/558921v1_fig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f04650org.highwire.dtl.DTLVardef@1986fceorg.highwire.dtl.DTLVardef@d47ab9org.highwire.dtl.DTLVardef@1c524d5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Outline of virus neutralisation assays of emerging SARS-CoV-2 variants of concern C_FIG ResultsThere was a significant reduction in nAbT observed against the Delta and Beta VOC compared with wild-type, 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 SARS-CoV-2 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/558921v1_fig2.gif" ALT="Figure 2"> View larger version (18K): org.highwire.dtl.DTLVardef@1f51ee8org.highwire.dtl.DTLVardef@1c03b61org.highwire.dtl.DTLVardef@17a77d2org.highwire.dtl.DTLVardef@15084f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2:C_FLOATNO Neutralising Antibody Titres Figure description: Differences in neutralising antibody titres (nAbT) against the Wildtype, Beta and Delta SARS-CoV-2 lineages median of 21 days (IQR 19-28) after receiving the second dose of Pfizer-BioNTech (BNT162b2) vaccine measured by visual inspection of cytopathic effect (CPE) with confirmation of viral replication by SARS-CoV-2 by inhouse reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR). Results are reported in the box-whiskers plots as medians and upper and lower quartiles. There was a significant fold reduction in nAbT observed between both the Delta (M = 4.4, SD = 2), t(11) =-4.9, p = .00059, and Beta (M=2.3, SD=2) t(11) =-3 p = .01397 compared with wild-type. There was also a significant fold reduction in nAbT between Beta (M = 2.6, SD = 1.4), t(11) =-2.5, p = .02897 and Delta. Key: Delta - Delta (B.1.617.2) lineage; Beta - Beta (B.1.351) lineage; Wildtype - Wildtype (A.2.2) lineage; C_FIG DiscussionSignificant 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 SARS-CoV-2, as seen in our cohort. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 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.

microbiology↗

bioRxiv2023

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

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.

genomics↗

bioRxiv2021

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

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.

microbiology↗

bioRxiv2021

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

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.

microbiology↗

bioRxiv2020

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

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.

microbiology↗

bioRxiv2020

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

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.

microbiology↗