Uncovering strain- and age- dependent differences in innate immune response to SARS-CoV-2 infection in nasal epithelia using combined short and long-read scRNA-seq (preprint)

Assessing the impact of SARS-CoV-2 variants on host immune systems is crucial with the continuous arrival of novel variants. However, there is a lack of reported studies utilizing single-cell RNA-sequencing (scRNA-seq) to elucidate the age-dependent host-viral interactions with different SARS-CoV-2 strains. Therefore, we employed Full-Length Transcriptome Sequencing (FLT-Seq) combining Illumina and Oxford Nanopore Technologies (ONT) with 10X 3-prime single cell sequencing to investigate host transcriptomic changes during wild-type (WT) and Alpha-strain SARS-CoV-2 infections within air-liquid-interface (ALI)-cultured human nasal epithelial cells from adults and adolescents. The results revealed increased innate immune responses in cells with lower viral loads which were lacking in cells with higher viral load. Alpha-infections showed heightened expression of genes related to interferon (IFN)-responses and protein-folding compared with WT, implying the increased immune response and endoplasmic reticulum stress with the variant of a higher transmission rate. ACE2 expression was elevated in infected populations of secretory and goblet cells with high IFN-stimulation and a subpopulation of ciliated cells but was lacking in bystander cells and other infected-cell types, despite detectable IFN-stimulation and regardless of strain. We used long-read sequencing to identify differential transcript usage (DTU) of IFN-response and translational genes, including IFI27, RPL4 and RPL15 (padj < 0.05) in infected cells compared with control, and consistent DTU of RPL15 and MX1 between Alpha and WT strain-infected cells in various cell-types. Together, these results reveal the dynamic transcriptional/translational/post-translational activity upon SARS-CoV-2 infection, which appeared to be age and strain-dependent. Overall, this study highlights the complexity of cell-type, age and viral-strain-dependent host epithelial responses to SARS-CoV-2.

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.

Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection (preprint)

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA (preprint)

2.IntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 L of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD {+/-}7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.