Streamlining large-scale oceanic biomonitoring using passive eDNA samplers integrated into vessel's continuous pump underway seawater systems.

Passive samplers are enabling the scaling of environmental DNA (eDNA) biomonitoring in our oceans, by circumventing the time-consuming process of water filtration. Designing a novel passive sampler that does not require extensive sample handling time and can be connected to ocean-going vessels without impeding normal underway activities has potential to rapidly upscale global biomonitoring efforts onboard the world's oceanic fleet. Here, we demonstrate the utility of an artificial sponge sampler connected to the continuous pump underway seawater system as a means to enable oceanic biomonitoring. We compared the performance of this passive sampling protocol with standard water filtration at six locations during a research voyage from New Zealand to Antarctica in early 2023. Eukaryote metabarcoding of the mitochondrial COI gene revealed no significant difference in phylogenetic α-diversity between sampling methods and both methods delineated a progressive reduction in number of Zero-Radius Operational Taxonomic Units (ZOTUs) with increased latitudes. While both sampling methods revealed comparable trends in geographical community compositions, distinct clusters were identified for passive samplers and water filtration at each location. Additionally, greater variability between replicates was observed for passive samplers, resulting in an increased estimated level of replication needed to recover 90 % of the biodiversity. Furthermore, traditional water filtration failed to detect three phyla observed by passive samplers and extrapolation analysis estimated passive samplers recover a larger number of ZOTUs compared to water filtration for all six locations. Our results demonstrate the potential of this passive eDNA sampler protocol and highlight areas where this emerging technology could be improved, thereby enabling large-scale offshore marine eDNA biomonitoring by leveraging the world's oceanic fleet without interfering with onboard activities.

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and ß-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq.

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

Rapid Response to SARS-CoV-2 in Aotearoa New Zealand: Implementation of a Diagnostic Test and Characterization of the First COVID-19 Cases in the South Island.

It has been 20 months since we first heard of SARS-CoV-2, the novel coronavirus detected in the Hubei province, China, in December 2019, responsible for the ongoing COVID-19 pandemic. Since then, a myriad of studies aimed at understanding and controlling SARS-CoV-2 have been published at a pace that has outshined the original effort to combat HIV during the beginning of the AIDS epidemic. This massive response started by developing strategies to not only diagnose individual SARS-CoV-2 infections but to monitor the transmission, evolution, and global spread of this new virus. We currently have hundreds of commercial diagnostic tests; however, that was not the case in early 2020, when just a handful of protocols were available, and few whole-genome SARS-CoV-2 sequences had been described. It was mid-January 2020 when several District Health Boards across New Zealand started planning the implementation of diagnostic testing for this emerging virus. Here, we describe our experience implementing a molecular test to detect SARS-CoV-2 infection, adapting the RT-qPCR assay to be used in a random-access platform (Hologic Panther Fusion® System) in a clinical laboratory, and characterizing the first whole-genome SARS-CoV-2 sequences obtained in the South Island, right at the beginning of the SARS-CoV-2 outbreak in New Zealand. We expect that this work will help us and others prepare for the unequivocal risk of similar viral outbreaks in the future.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.