Identification of SARS-CoV-2 variants in wastewater using targeted amplicon sequencing during a low COVID-19 prevalence period in Japan.

Wastewater-based epidemiology is expected to be able to identify SARS-CoV-2 variants at an early stage via next-generation sequencing. In the present study, we developed a highly sensitive amplicon sequencing method targeting the spike gene of SARS-CoV-2, which allows for sequencing viral genomes from wastewater containing a low amount of virus. Primers were designed to amplify a relatively long region (599 bp) around the receptor-binding domain in the SARS-CoV-2 spike gene, which could distinguish initial major variants of concern. To validate the methodology, we retrospectively analyzed wastewater samples collected from a septic tank installed in a COVID-19 quarantine facility between October and December 2020. The relative abundance of D614G mutant in SARS-CoV-2 genomes in the facility wastewater increased from 47.5 % to 83.1 % during the study period. The N501Y mutant, which is the characteristic mutation of the Alpha-like strain, was detected from wastewater collected on December 24, 2020, which agreed with the fact that a patient infected with the Alpha-like strain was quarantined in the facility on this date. We then analyzed archived municipal wastewater samples collected between November 2020 and January 2021 that contained low SARS-CoV-2 concentrations ranging from 0.23 to 0.43 copies/qPCR reaction (corresponding to 3.30 to 4.15 log10 copies/L). The targeted amplicon sequencing revealed that the Alpha-like variant with D614G and N501Y mutations was present in municipal wastewater collected on December 4, 2020 and later, suggesting that the variant had already spread in the community before its first clinical confirmation in Japan on December 25, 2020. These results demonstrate that targeted amplicon sequencing of wastewater samples is a powerful surveillance tool applicable to low COVID-19 prevalence periods and may contribute to the early detection of emerging variants.

Cross-Border Transmissions of the Delta Substrain AY.29 During Tokyo Olympic and Paralympic Games.

Tokyo Olympic and Paralympic Games, postponed for the COVID-19 pandemic, were finally held in the summer of 2021. Just before the games, the Alpha variant was being replaced with the more contagious Delta variant. AY.4 substrain AY.29, which harbors two additional characteristic mutations of 5239C > T (NSP3 Y840Y) and 5514T > C (NSP3 V932A), emerged in Japan and became dominant in Tokyo by the time of the Olympic Games. Variants of SARS-CoV-2 genomes were performed to extract AY.29 Delta substrain samples with 5239C > T and 5514T > C. Phylogenetic analysis was performed to illustrate how AY.29 strains evolved and were introduced into countries abroad. Simultaneously, ancestral searches were performed for the overseas AY.29 samples to identify their origins in Japan using the maximum variant approach. As of January 10, 2022, 118 samples were identified in 20 countries. Phylogenetic analysis and ancestral searches identified 55 distinct introductions into those countries. The United States had 50 samples with 10 distinct introductions, and the United Kingdom had 13 distinct strains introduced in 18 samples. Other countries or regions with multiple introductions were Canada, Germany, South Korea, Hong Kong, Thailand, and the Philippines. Among the 20 countries, most European and North American countries have vaccination rates over 50% and sufficient genomic surveillances are conducted; transmissions seem contained. However, propagation to unvaccinated regions might have caused unfathomable damages. Since samples in those unvaccinated countries are also undersampled with a longer lead time for data sharing, it will take longer to grasp the whole picture. More rigorous departure screenings for the participants from the unvaccinated countries might have been necessary.

The detectability and removal efficiency of SARS-CoV-2 in a large-scale septic tank of a COVID-19 quarantine facility in Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to be present in sewage, and wastewater-based epidemiology has attracted much attention. However, the physical partitioning of SARS-CoV-2 in wastewater and the removal efficiency of treatment systems require further investigation. This study aimed to investigate the detectability and physical partitioning of SARS-CoV-2 in wastewater and assess its removal in a large-scale septic tank employing anaerobic, anoxic, and oxic processes in a sequential batch reactor, which was installed in a coronavirus disease 2019 (COVID-19) quarantine facility. The amount of SARS-CoV-2 RNA in wastewater was determined with polyethylene glycol (PEG) precipitation followed by quantitative polymerase chain reaction (qPCR), and the association of SARS-CoV-2 with wastewater solids was evaluated by the effect of filtration prior to PEG precipitation (pre-filtration). The amount of SARS-CoV-2 RNA detected from pre-filtered samples was substantially lower than that of samples without pre-filtration. These results suggest that most SARS-CoV-2 particles in wastewater are associated with the suspended solids excluded by pre-filtration. The removal efficiency of SARS-CoV-2 in the septic tank was evaluated based on the SARS-CoV-2 RNA concentrations in untreated and treated wastewater, which was determined by the detection method optimized in this study. Escherichia coli and pepper mild mottle virus (PMMoV) were also quantified to validate the wastewater treatment system's performance. The mean log10 reduction values of SARS-CoV-2, E. coli, and PMMoV were 2.47 (range, 2.25-2.68), 2.81 (range, 2.45-3.18), and 0.66 (range, 0.61-0.70), respectively, demonstrating that SARS-CoV-2 removal by the wastewater treatment system was comparable to or better than the removal of fecal indicators. These results suggest that SARS-CoV-2 can be readily removed by the septic tank. This is the first study to determine the removal efficiency of SARS-CoV-2 in a facility-level sequencing batch activated sludge system.

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force.

Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection.

Cross-Border Transmissions of the Delta Substrain AY.29 During Tokyo Olympic and Paralympic Games

Tokyo Olympic and Paralympic Games, postponed for the COVID-19 pandemic, were finally held in the summer of 2021. Just before the games, the Alpha variant was being replaced with the more contagious Delta variant. AY.4 substrain AY.29, which harbors two additional characteristic mutations of 5239C > T (NSP3 Y840Y) and 5514T > C (NSP3 V932A), emerged in Japan and became dominant in Tokyo by the time of the Olympic Games. Variants of SARS-CoV-2 genomes were performed to extract AY.29 Delta substrain samples with 5239C > T and 5514T > C. Phylogenetic analysis was performed to illustrate how AY.29 strains evolved and were introduced into countries abroad. Simultaneously, ancestral searches were performed for the overseas AY.29 samples to identify their origins in Japan using the maximum variant approach. As of January 10, 2022, 118 samples were identified in 20 countries. Phylogenetic analysis and ancestral searches identified 55 distinct introductions into those countries. The United States had 50 samples with 10 distinct introductions, and the United Kingdom had 13 distinct strains introduced in 18 samples. Other countries or regions with multiple introductions were Canada, Germany, South Korea, Hong Kong, Thailand, and the Philippines. Among the 20 countries, most European and North American countries have vaccination rates over 50% and sufficient genomic surveillances are conducted;transmissions seem contained. However, propagation to unvaccinated regions might have caused unfathomable damages. Since samples in those unvaccinated countries are also undersampled with a longer lead time for data sharing, it will take longer to grasp the whole picture. More rigorous departure screenings for the participants from the unvaccinated countries might have been necessary.

DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

COVID-19 risk assessment at the opening ceremony of the Tokyo 2020 Olympic Games.

The 2020 Olympic/Paralympic Games have been postponed to 2021, due to the COVID-19 pandemic. We developed a model that integrated source-environment-receptor pathways to evaluate how preventive efforts can reduce the infection risk among spectators at the opening ceremony of Tokyo Olympic Games. We simulated viral loads of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emitted from infectors through talking/coughing/sneezing and modeled temporal environmental behaviors, including virus inactivation and transfer. We performed Monte Carlo simulations to estimate the expected number of newly infected individuals with and without preventive measures, yielding the crude probability of a spectator being an infector among the 60,000 people expected to attend the opening ceremony. Two indicators, i.e., the expected number of newly infected individuals and the newly infected individuals per infector entry, were proposed to demonstrate the extent of achievable infection risk reduction levels by implementing possible preventive measures. A no-prevention scenario produced 1.5-1.7 newly infected individuals per infector entry, whereas a combination of cooperative preventive measures by organizers and the spectators achieved a 99% risk reduction, corresponding to 0.009-0.012 newly infected individuals per infector entry. The expected number of newly infected individuals was calculated as 0.005 for the combination of cooperative preventive scenarios with the crude probability of a spectator being an infector of 1 × 10-5. Based on our estimates, a combination of cooperative preventions between organizers and spectators is required to prevent a viral spread at the Tokyo Olympic/Paralympic Games. Further, under the assumption that society accepts < 10 newly infected persons traced to events held during the entire Olympic/Paralympic Games, we propose a crude probability of infectors of < 5 × 10-5 as a benchmark for the suppression of the infection. This is the first study to develop a model that can assess the infection risk among spectators due to exposure pathways at a mass gathering event.