Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2

Publisher Copyright: © 2021 O'Toole Á et al.Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.Peer reviewe

Patterns of dispersal in the Tasman Sea and the South Pacific Ocean : population genetics and the role of environmental constraints

The interconnected nature of the oceans, the small size and passive nature of larvae has resulted in the presumption that marine populations are large and open. However, this paradigm has been challenged in the last few of decades, where genetic studies have found significant structure at very fine-scales (of the order of 10 km). Given that many sedentary marine organisms can only disperse via a larval phase, it is intuitive that the duration of this phase (pelagic larval duration, PLD) should correspond with a species dispersal capacity and hence predict population genetic structure. Furthermore, if PLD predicts dispersal capacity then PLD should correlate with a species distribution. In this thesis, the population genetic structure of three sedentary marine organisms, endemic to the south-east Australia Pacific region, was examined to explore the relationships between dispersal capacity, life-history and geographical distribution. In chapter 2 the influence of an oceanic frontal system, the Subtropical Front (STF), on the genetic structure (mitochondrial region cytochrome c oxidase subunit I), trophic status (stable isotopes δ15N and δ13C) and condition (RNA:DNA, protein:DNA, DNA:dry weight) of the benthic gastropod Fusitriton magellanicus was examined across the Chatham Rise, New Zealand. The front acted as barrier to the transport of material between flanks, such that populations of F. magellanicus from the northern flank were in poorer condition than those from the southern flank. In addition, δ15N signatures were different between flanks, the northern flank being more enriched than the southern flank, indicating that F. magellanicus on the north flank where either starving or occupying a higher trophic level. Despite the STF shaping the trophic ecology of F. magellanicus, no differences in genetic structure were found. This indicates that there is larval exchange between or around the STF and hence larval biology may override environmental factors. To explore if a limited dispersal capacity, in contrast to its PLD (~30 days), was responsible for driving the unusual and abbreviated distribution of the sea urchin Heliocidaris tuberculata, its population genetic structure was characterised in chapter 3. Examination of the genetic structure using three different genetic markers (COI, 16S and 12 microsatellites) across the geographical distribution of the species (New South Wales coast and across the Tasman Sea to Lord Howe, Norfolk and the Kermadec Islands) was made, revealing a complete absence of structure (microsatellites FST = 0.003, COI PhiPT = 0.0021, and 16S PhiPT = 0.029). To elucidate the role of other important environmental factors in shaping genetic patterns, correlations between genetic measures and key environmental variables were explored, but no significant relationships were found. Therefore, H. tuberculata is panmictic across its range and its unusual restricted latitudinal distribution, despite it wide longitudinal distribution, is not explained by a limited dispersal capacity. The population genetics of H. erythrogramma was examined across south-eastern Australia, to explore the influence of historical versus contemporary forces on the structure of a species with a short PLD (3-5 days), in chapter 4. Using two different markers (cytochrome c oxidase subunit 1 (COI) and microsatellites), the population genetic structure of H. erythrogramma was examined from New South Wales to South Australia inclusive of Victoria and Tasmania. Analysis of the COI data revealed two strong eastern and southern genetic groups, diverging across a known phylogeographical barrier, the Bassian Isthmus. Within the two genetic groups, the signature of contemporary processes was evident with the direction of migration consistent with the influence of the ocean currents on larval dispersal. A combination of contemporary oceanography, and habitat restrictions appear to maintain these signatures in the present day. Whilst PLD places some restrictions on dispersal capacity, it does not prevent an extensive geographical range and/or high levels of gene flow at extensive spatial scales. In chapter 5, it was postulated that temperature may restrict the southern range limit of H. tuberculata. Examination of the thermal tolerance of the two species revealed that the upper temperature tolerance for both species was the same, however, H. erythrogramma had a lower cold thermal tolerance than H. tuberculata. Furthermore, the temperature tolerances of both species matched sea surface temperatures that their larvae would normally encounter. Hence, an inability to tolerate cold temperatures explains why H. tuberculata has a restricted southern range limit, whereas the wider thermal envelope of H. erythrogramma ensures its distribution around temperate Australia. In summary, the thesis reveals that whilst life history considerations can shape the genetic structure of a species, oceanography and environmental considerations (e.g. temperature) can play a more important role in determining how populations are connected and species range limits

Tolerance and the assessment of heavy metal pollution in sessile invertebrates

Heavy metals are powerful agents of disturbance, and it is therefore important that stringent and effective monitoring practices be in place. This thesis examines the ecological-relevance of metal accumulation in a biomonitor to sessile invertebrate fauna. However there is the potential for effects of metals to be different depending on the level of tolerance of the population being sampled. Therefore, differential tolerance across four estuaries was examined in a common sessile species.The levels of metals were quantified around an industrialised harbour, Port Kembla, using the common native oyster, Saccostrea glomerata. Settlement plates were concurrently deployed at each site to capture the level of sessile invertebrate recruitment at each site. Increasing levels of lead and copper were associated with severe decreases in the abundance of the native barnacle, Amphibalanus variegatus. In striking comparison, positive correlations were found between the abundance of a cosmopolitan worm and lead and copper. The biomonitor was found to provide a sensitive measure of metals which were found to be of ecological-relevance.Organisms have been shown to develop tolerance to heavy metals, therefore, the effects measured may have been different depending on the level of tolerance in the community. Populations of the native barnacle, Amphibalanus variegatus, collected from within heavily-modified estuaries (including Port Kembla) were found to be more tolerant to a copper challenge than populations from within un-modified estuaries. High levels of copper were recorded within the heavily-modified estuaries compared to the un-modified estuaries suggesting tolerance had developed in response to this contaminant.In summary, metals were found to be correlated with severe effects in the abundance of anative barnacle, however, populations of this species were found to be more tolerant to copper than populations from heavily-modified estuaries. This suggests that the same level of contaminants in pristine estuaries could potentially have much greater impacts than measured in this study. It may also suggest that a greater tolerance may not equate to a greater ecological success

Effects of marine heatwave conditions across the metamorphic transition to the juvenile sea urchin (Heliocidaris erythrogramma)

For short development species, like the sea urchin Heliocidaris erythrogramma, the entire planktonic duration can be impacted by marine heatwaves (MHW). Developmental thermal tolerance of this species through metamorphosis was investigated over a broad range (7.6–28.0 ◦C), including temperatures across its distribution and MHW conditions. In controls (19.5–21.0 ◦C), 80% of individuals developed to metamorphosis at day 5, doubling to 10 days at 14.0 ◦C. The thermal range (14.4–21.2 ◦C) of metamorphosis on day 7 reflected the realised thermal niche with 25.9 ◦C the upper temperature for success (T40). By day 10, juvenile tolerance narrowed to the local range (16.2–19.0 ◦C), similar to levels tolerated by adults, indicating negative carryover effects across the metamorphic transition. Without phenotypic adjustment or adaptation, regional warming will be detrimental, although populations may be sustained by thermotolerant offspring. Our results show the importance of the metamorphic transition in understanding the cumulative sensitivity of species to MHW

[In Press] Staying in place and moving in space : contrasting larval thermal sensitivity explains distributional changes of sympatric sea urchin species to habitat warming

For marine ectotherms, larval success, planktonic larval duration and dispersal trajectories are strongly influenced by temperature, and therefore, ocean warming and heatwaves have profound impacts on these sensitive stages. Warming, through increased poleward flow in regions with western boundary currents, such as the East Australia Current (EAC), provides opportunities for range extension as propagules track preferred conditions. Two sea urchin species, Centrostephanus rodgersii and Heliocidaris tuberculata, sympatric in the EAC warming hotspot, exhibit contrasting responses to warming. Over half a century, C. rodgersii has undergone marked poleward range extension, but the range of H. tuberculata has not changed. We constructed thermal performance curves (TPC) to determine if contrasting developmental thermal tolerance can explain this difference. The temperatures tested encompassed present-day distribution and forecast ocean warming/heatwave conditions. The broad and narrow thermal optimum (Topt) ranges for C. rodgersii and H. tuberculata larvae (7.2 and 4.7°C range, respectively) matched their realized (adult distribution) thermal niches. The cool and warm temperatures for 50% development to the feeding larva approximated temperatures at adult poleward range limits. Larval cool tolerances with respect to mean local temperature differed, 6.0 and 3.8°C respectively. Larval warm tolerances were similar for both species as are the adult warm range edges. The larvae of both species would be sensitive to heatwaves. Centrostephanus rodgersii has stayed in place and shifted in space, likely due to its broad cold–warm larval thermal tolerance and large thermal safety margins. Phenotypic plasticity of the planktonic stage of C. rodgersii facilitated its range extension. In contrast, larval cold intolerance of H. tuberculata explains its restricted range and will delay poleward extension as the region warms. In a warming ocean, we show that intrinsic thermal biology traits of the pelagic stage provide an integrative tool to explain species-specific variation in range shift patterns

Differential tolerance to copper, but no evidence of population-level genetic differences in a widely-dispersing native barnacle

Despite many estuaries having high levels of metal pollution, species are found to persist in these stressful environments. Populations of estuarine invertebrates exposed to toxic concentrations of such metals may be under selection. However, in species with a wide-dispersal potential, any short-term results of localized selection may be counteracted by external recruitment from populations not under selection. The barnacle Amphibalanus variegatus is found in nearshore coastal environments as well as sheltered embayments and estuaries, including metal-impacted estuaries, from New South Wales, Australia to Western Australia. The fertilised eggs of A. variegatus are brooded internally and released as larvae (nauplii), which remain in the water-column for ~14 days before settling. Hence the species has a considerable dispersal capacity. The purpose of this study was to examine whether populations of A. variegatus from metal-impacted sites, displayed a greater tolerance to a toxicant (copper) than reference populations. Adult barnacles where collected from twenty sites within two metal-impacted and fourteen sites within two reference estuaries. Within 24 h, adults were induced to spawn and the offspring were exposed to copper in a laboratory assay. Larvae collected from the metal-impacted estuaries demonstrated a greater tolerance to copper compared to those from reference sites. To determine if selection/localised in the metal impacted sites was occurring, the genetic structure of populations at three sites was examined using an AFLP methodology. No evidence of unique population identity and or selection (outlier loci) was detected suggesting that: (1) the tolerance displayed in the assay was derived from acclimation during development; and/or (2) that the populations are open preventing the fixation of any unique alleles

Characterisation of thirteen polymorphic microsatellite markers for the red sea urchin Heliocidaris tuberculata (Lamarck, 1816) developed using a 454-sequencing approach

The sea urchin Heliocidaris tuberculata, is valued by commercial fisheries because of the high quality of its roe. Declines in abundance following a peak in the Australian fishery around the millennium, suggests that recruitment and hence the subsequent recovery of populations in this species, may be low. Despite this, because of its high commercial value the species continues to be exploited with little knowledge of sources and sinks for recruits between populations. Following nuclear microsatellite identification using a 454-sequencing approach, a set of 13 novel markers were optimised for H. tuberculata. The loci show clear banding patterns, with 2–14 alleles per locus and observed heterozygosity values ranging from 0.12 to 0.73. These markers will be used to assess genetic diversity and connectivity between populations across the geographical range of the species

Exploring programmatic indicators of tuberculosis control that incorporate routine Mycobacterium tuberculosis sequencing in low incidence settings: a comprehensive (2017–2021) patient cohort analysisResearch in context

Summary: Background: Routine whole genome sequencing of Mycobacterium tuberculosis has been implemented with increasing frequency. However, its value for tuberculosis (TB) control programs beyond individual case management and enhanced drug resistance detection has not yet been explored. Methods: We analysed routine sequencing data of culture-confirmed TB cases notified between 1st January 2017 and 31st December 2021 in New South Wales (NSW), Australia. Genomic surveillance included evidence of local TB transmission, defined by single nucleotide polymorphism (SNP) clustering over a variable (0–25) SNP threshold, and drug resistance conferring mutations. Findings: M. tuberculosis sequences from 1831 patients were examined, representing 64.8% of all notified TB cases and 96.2% of culture-confirmed cases. Applying a traditional 5-SNP cluster threshold identified 62 transmission clusters with 183 clustered cases; 101/183 (55.2%) had 0 SNP differences. Cluster assessment over a 5-year period, using a 5-SNP threshold, provided a comprehensive overview of likely recent transmission within NSW, Australia, as an indicator of local TB control. Genotypic drug susceptibility testing (DST) was highly concordant with phenotypic DST and provided a 6.8% increase in antimycobacterial resistance detection. Importantly, it detected mutations missed by routine molecular tests. Lineage 2 strains were more likely to be drug resistant (p < 0.0001) and locally transmitted if drug resistant (p < 0.0001). Interpretation: Performing routine prospective WGS in a low incidence country like Australia, provides genomically informed programmatic indicators of local TB control. A rolling 5-year cluster assessment reflects epidemic containment and progress towards ‘zero TB transmission’. Genomic DST also provides valuable information for clinical care and drug resistance surveillance. Funding: NHMRC Centre for Research Excellence in Tuberculosis (www.tbcre.org.au) and NSW Health Prevention Research Support Program

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture

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) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). 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: Intrahost single nucleotide variants 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 dilutions than the clinical cohorts. Discussion and Conclusion: 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