SARS-CoV-2 intra-host diversity, antibody response, and disease severity after reinfection by the variant of concern Gamma in Brazil.

The rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma in Amazonas during early 2021 fueled a second large COVID-19 epidemic wave and raised concern about the potential role of reinfections. Very few cases of reinfection associated with the VOC Gamma have been reported to date, and their potential impact on clinical, immunological, and virological parameters remains largely unexplored. Here we describe 25 cases of SARS-CoV-2 reinfection in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected with distinct viral lineages between March and December 2020 (B.1.1, B.1.1.28, B.1.1.33, B.1.195, and P.2) and reinfected with the VOC Gamma between 3 to 12 months after primo-infection. We found a similar mean cycle threshold (Ct) value and limited intra-host viral diversity in both primo-infection and reinfection samples. Sera of 14 patients tested 10-75 days after reinfection displayed detectable neutralizing antibodies (NAb) titers against SARS-CoV-2 variants that circulated before (B.1.*), during (Gamma), and after (Delta and Omicron) the second epidemic wave in Brazil. All individuals had milder or no symptoms after reinfection, and none required hospitalization. These findings demonstrate that individuals reinfected with the VOC Gamma may display relatively high RNA viral loads at the upper respiratory tract after reinfection, thus contributing to onward viral transmissions. Despite this, our study points to a low overall risk of severe Gamma reinfections, supporting that the abrupt increase in hospital admissions and deaths observed in Amazonas and other Brazilian states during the Gamma wave was mostly driven by primary infections. Our findings also indicate that most individuals analyzed developed a high anti-SARS-CoV-2 NAb response after reinfection that may provide some protection against reinfection or disease by different SARS-CoV-2 variants.

Detection of SARS-CoV-2 Genome for over 100 Days after COVID-19 Onset.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is spreading globally. Generally, the viral genome becomes undetectable within a couple of weeks after infection. Herein, we report a case of long-term detection of the SARS-CoV-2 genome in the same individual for 106 days. Whole genome sequencing was performed on specimens taken at the onset of the disease and 2 months after onset, and the B.1.1.7 lineage was detected in both samples. A comparison of the full-length sequences revealed a single-base difference and no amino acid mutations. This is the first case in Japan where the virus was detected over a long period, and the full-length sequences were compared.

SARS-CoV-2 is localized in cardiomyocytes: a postmortem biopsy case.

A 72-year-old patient was admitted to the intensive care unit due to acute respiratory distress syndrome caused by COVID-19. On day 20, the patient experienced shock. The electrocardiogram showed ST segment elevation in leads V3-V6 and severe left ventricular dysfunction with an ejection fraction of 35%-40%. The left ventricle showed basal hypokinesis and apical akinesis, while the creatine kinase level was normal, indicating Takotsubo cardiomyopathy. On day 24, the patient died of multiple organ failure. In post-mortem biopsy, SARS-CoV-2 antigen was detected in cardiomyocytes by immunostaining. Moreover, SARS-CoV-2 RNA was detected in heart tissue. We need to further analyse the direct link between SARS-CoV-2 and cardiomyocytes.

Genome Recombination between the Delta and Alpha Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Prominent genomic recombination has been observed between the Delta and Alpha variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), isolated from clinical specimens in Japan. Interestingly, the recombination variant detected in this study carries a spike protein identical to that in the domestic Delta variant, thereby suggesting that further risks would not be associated with infectivity and immune escape. The recombinant was classified as an XC lineage in the PANGOLIN database. It is necessary to intensively study such marked genetic variations and characterize emerging variants after careful verification of their lineage and clade assignment.

Singing Is a Risk Factor for Severe Acute Respiratory Syndrome Coronavirus 2 Infection: A Case-Control Study of Karaoke-Related Coronavirus Disease 2019 Outbreaks in 2 Cities in Hokkaido, Japan, Linked by Whole Genome Analysis.

Background: Singing in an indoor space may increase the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We conducted a case-control study of karaoke-related coronavirus disease 2019 (COVID-19) outbreaks to reveal the risk factors for SARS-CoV-2 infection among individuals who participate in karaoke. Methods: Cases were defined as people who enjoyed karaoke at a bar and who tested positive for SARS-CoV-2 by reverse-transcription polymerase chain reaction between 16 May and 3 July 2020. Controls were defined as people who enjoyed karaoke at the same bar during the same period as the cases and tested negative. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. ORs of key variables adjusted for each other were also estimated (aOR). Results: We identified 81 cases, the majority of whom were active elderly individuals (median age, 75 years). Six cases died (case fatality ratio, 7%). Among the cases, 68 (84%) were guests, 18 of whom had visited ≧2 karaoke bars. A genome analysis conducted in 30 cases showed 6 types of isolates within 4 single-nucleotide variation difference. The case-control study revealed that singing (aOR, 11.0 [95% CI, 1.2-101.0]), not wearing a mask (aOR, 3.7 [95% CI, 1.2-11.2]), and additional hour spent per visit (aOR, 1.7 [95% CI, 1.1-2.7]) were associated with COVID-19 infection. Conclusions: A karaoke-related COVID-19 outbreak that occurred in 2 different cities was confirmed by the results of genome analysis. Singing in less-ventilated, indoor and crowded environments increases the risk of acquiring SARS-CoV-2 infection. Wearing a mask and staying for only a short time can reduce the risk of infection during karaoke.

Singing Is a Risk Factor for SARS-CoV-2 Infection: A Case-control Study of Karaoke-related COVID-19 Outbreaks in Two Cities in Hokkaido, Japan, Linked by Whole Genome Analysis

Background Singing in an indoor space may increase the risk of SARS-CoV-2 infection. We conducted a case-control study of karaoke-related COVID-19 outbreaks to reveal the risk factors for SARS-CoV-2 infection among individuals who participate in karaoke. Methods Cases were defined as people who enjoyed karaoke at a bar and who tested positive for SARS-CoV-2 by RT-PCR between May 16 and July 3, 2020. Controls were defined as people who enjoyed karaoke at the same bar during the same period as the cases and tested negative. Odds ratio (OR) and confidence interval (CI) were calculated. ORs were adjusted by variables with significantly high odds ratio (aOR). Results We identified 81 cases, the majority of whom were active elderly individuals (median age: 75 years). Six cases died (case fatality ratio: 7%). Among the cases, 68 (84%) were guests, 18 of whom had visited more than two karaoke bars. A genome analysis conducted in 30 cases showed six types of isolates within four single-nucleotide variations difference. The case-control study revealed that singing (aOR 11.0, 95% CI, 1.2-101.0), not wearing a mask (aOR 3.7, 95% CI 1.2-11.2) and time spent per visit (aOR 1.7, 95% CI 1.1-2.7) were associated with COVID-19 infection. Conclusions A karaoke-related COVID-19 outbreak that occurred in two different cities was confirmed by the results of genome analysis. Singing in less-ventilated, indoor and crowded environments increases the risk of acquiring SARS-CoV-2 infection. Wearing a mask and staying for only a short time can reduce the risk of infection during karaoke.

A lethal mouse model for evaluating vaccine-associated enhanced respiratory disease during SARS-CoV-2 infection.

One safety concern during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine development has been the vaccine-associated enhanced disease, which is characterized by eosinophilic immunopathology and T helper cell type 2 (TH2)­biased immune responses with insufficient neutralizing antibodies. In this study, we established a lethal animal model using BALB/c mice and a mouse-passaged isolate (QHmusX) from a European lineage of SARS-CoV-2. The QHmusX strain induced acute respiratory illness, associated with diffuse alveolar damage and pulmonary edema, in TH2-prone adult BALB/c mice, but not in young mice or TH1-prone C57BL/6 mice. We also showed that immunization of adult BALB/c mice with recombinant spike protein without appropriate adjuvant caused eosinophilic immunopathology with TH2-shifted immune response and insufficient neutralizing antibodies after QHmusX infection. This lethal mouse model is useful for evaluating vaccine-associated enhanced respiratory disease during SARS-CoV-2 infection and may provide new insights into the disease pathogenesis of SARS-CoV-2.

Genomic Epidemiology Reveals Multiple Introductions of Severe Acute Respiratory Syndrome Coronavirus 2 in Niigata City, Japan, Between February and May 2020.

The coronavirus disease 2019 (COVID-19) has caused a serious disease burden and poses a tremendous public health challenge worldwide. Here, we report a comprehensive epidemiological and genomic analysis of SARS-CoV-2 from 63 patients in Niigata City, a medium-sized Japanese city, during the early phase of the pandemic, between February and May 2020. Among the 63 patients, 32 (51%) were female, with a mean (±standard deviation) age of 47.9 ± 22.3 years. Fever (65%, 41/63), malaise (51%, 32/63), and cough (35%, 22/63) were the most common clinical symptoms. The median C t value after the onset of symptoms lowered within 9 days at 20.9 cycles (interquartile range, 17-26 cycles), but after 10 days, the median C t value exceeded 30 cycles (p < 0.001). Of the 63 cases, 27 were distributed in the first epidemic wave and 33 in the second, and between the two waves, three cases from abroad were identified. The first wave was epidemiologically characterized by a single cluster related to indoor sports activity spread in closed settings, which included mixing indoors with families, relatives, and colleagues. The second wave showed more epidemiologically diversified events, with most index cases not related to each other. Almost all secondary cases were infected by droplets or aerosols from closed indoor settings, but at least two cases in the first wave were suspected to be contact infections. Results of the genomic analysis identified two possible clusters in Niigata City, the first of which was attributed to clade S (19B by Nexstrain clade) with a monophyletic group derived from the Wuhan prototype strain but that of the second wave was polyphyletic suggesting multiple introductions, and the clade was changed to GR (20B), which mainly spread in Europe in early 2020. These findings depict characteristics of SARS-CoV-2 transmission in the early stages in local community settings during February to May 2020 in Japan, and this integrated approach of epidemiological and genomic analysis may provide valuable information for public health policy decision-making for successful containment of chains of infection.

Transmission of SARS-CoV-2 during a 2-h domestic flight to Okinawa, Japan, March 2020.

BACKGROUND: Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has rapidly spread globally. Potentially infected individuals travel on commercial aircraft. Thus, this study aimed to investigate and test the association between the use of face masks, physical distance, and COVID-19 among passengers and flight attendants exposed to a COVID-19 passenger in a domestic flight. METHODS: This observational study investigated passengers and flight attendants exposed to COVID-19 on March 23, 2020, on board a flight to Naha City, Japan. Secondary attack rates were calculated. Whole-genome sequencing of SARS-CoV-2 was used to identify the infectious linkage between confirmed cases in this clustering. The association between confirmed COVID-19 and proximity of passengers' seats to the index case and/or the use of face masks was estimated using logistic regression. RESULTS: Fourteen confirmed and six probable cases were identified among passengers and flight attendants. The secondary attack rate was 9.7%. Twelve of 14 SARS-CoV-2 genome sequences in confirmed cases were identical to that of the index case or showed only one nucleotide mutation. Risk factors for infection included not using a face mask (adjusted odds ratio [aOR]: 7.29, 95% confidence interval [95% CI]: 1.86-28.6), partial face mask use (aOR: 3.0, 95% CI: 0.83-10.8), and being seated within two rows from the index patient (aOR: 7.47, 95% CI: 2.06-27.2). CONCLUSION: SARS-CoV-2 was transmitted on the airplane. Nonuse of face masks was identified as an independent risk factor for contracting COVID-19 on the airplane.

A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site.

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.

A discernable increase in the severe acute respiratory syndrome coronavirus 2 R.1 lineage carrying an E484K spike protein mutation in Japan.

Three COVID-19 waves in Japan have been characterized by the presence of distinct PANGO lineages (B.1.1. 162, B.1.1.284, and B.1.1.214). Recently, in addition to the B.1.1.7 lineage, which shows 25% abundance, an R.1 lineage carrying the E484K mutation in the spike protein was found to show up to 40% predominance. E484K could be a pivotal amino acid substitution with the potential to mediate immune escape; thus, more attention should be paid to such potential variants of concern to avoid the emergence of mutants of concern. Such comprehensive real-time genome surveillance has become essential for the containment of COVID-19 clusters.

Haplotype Explorer: an infection cluster visualization tool for spatiotemporal dissection of the COVID-19 pandemic.

The worldwide eruption of coronavirus disease 2019 (COVID-19) that began in Wuhan, China in late 2019 reached 10 million cases by late June 2020. In order to understand the epidemiological landscape of the COVID-19 pandemic, many studies have attempted to elucidate phylogenetic relationships between collected viral genome sequences using haplotype networks. However, currently available applications for network visualization are not suited to understand the COVID-19 epidemic spatiotemporally due to functional limitations that motivated us to develop Haplotype Explorer, an intuitive tool for visualizing and exploring haplotype networks. Haplotype Explorer enables to dissect epidemiological consequences via interactive node filters and provides the perspective on infectious disease dynamics depend on regions and time, such as introduction, outbreak, expansion, and containment. Here, we demonstrate the effectiveness of Haplotype Explorer by showing features and an example of visualization. The demo using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes are available at https://github.com/TKSjp/HaplotypeExplorer/blob/master/Example/. There are several examples using SARS-CoV-2 genomes and Dengue virus serotype 1 E-genes sequence.

A Genome Epidemiological Study of SARS-CoV-2 Introduction into Japan.

After the first case of coronavirus disease 2019 (COVID-19) in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating the emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases continued to appear until early April 2020, many with unclear infection routes and no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from the COVID-19 cases that appeared until early April 2020 and to characterize their genealogical networks in order to demonstrate possible routes of spread in Japan. Nasopharyngeal specimens were collected from patients, and reverse transcription-quantitative PCR tests for SARS-CoV-2 were performed. Positive RNA samples were subjected to whole-genome sequencing, and a haplotype network analysis was performed. Some of the primary clusters identified during January and February 2020 in Japan descended directly from the Wuhan-Hu-1-related isolates from China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that the COVID-19 cases from late March through early April may have created an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. In conclusion, genome surveillance has suggested that there were at least two distinct SARS-CoV-2 introductions into Japan from China and other countries.IMPORTANCE This study aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases and to characterize their genealogical networks to demonstrate possible routes of spread in Japan. We found that there were at least two distinct SARS-CoV-2 introductions into Japan, initially from China and subsequently from other countries, including Europe. Our findings can help understand how SARS-CoV-2 entered Japan and contribute to increased knowledge of SARS-CoV-2 in Asia and its association with implemented stay-at-home/shelter-in-place/self-restraint/lockdown measures. This study suggested that it is necessary to formulate a more efficient containment strategy using real-time genome surveillance to support epidemiological field investigations in order to highlight potential infection linkages and mitigate the next wave of COVID-19 in Japan.

Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR.

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.

Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak.

The Diamond Princess cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a coronavirus disease 2019 case. We performed whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from PCR+ clinical specimens and conducted a phylogenetic analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the Diamond Princess originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters could be linked to transmission through mass-gathering events in the recreational areas and direct transmission among passengers who shared cabins during the quarantine. This study demonstrates the usefulness of haplotype network/phylogeny analysis in identifying potential infection routes.

SARS-CoV-2 Genome Analysis of Japanese Travelers in Nile River Cruise.

Japan has reported 26 cases of coronavirus disease 2019 (COVID-19) linked to cruise tours on the River Nile in Egypt between March 5 and 15, 2020. Here, we characterized the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome of isolates from 10 travelers who returned from Egypt and from patients possibly associated with these travelers. We performed haplotype network analysis of SARS-CoV-2 isolates using genome-wide single-nucleotide variations. Our analysis identified two potential Egypt-related clusters from these imported cases, and these clusters were related to globally detected viruses in different countries.