Patient characteristics and public health office factors associated with long reporting delay of COVID-19 cases in Sapporo City, Japan.

BACKGROUND: For therapeutic efficacy, molnupiravir and nirmatrelvir-ritonavir must be started to treat patients within 5 days of disease onset to treat patients with COVID-19. However, some patients spend more than 5 days from disease onset before reporting to the Public Health Office. This study aimed to clarify the characteristics of patients with reporting delay. METHODS: This study included data from 12,399 patients with COVID-19 who reported to the Public Health Office from March 3rd, 2021 to June 30th, 2021. Patients were stratified into "linked" (n=7,814) and "unlinked" (n=4,585) cases depending on whether they were linked to other patients. A long reporting delay was defined as the difference between the onset and reporting dates of 5 days or more. Univariate and multivariate analyses were performed using log-binomial regression to identify factors related to long reporting delay, and prevalence ratios with corresponding 95% confidence intervals were calculated. RESULTS: The proportion of long reporting delay was 24.4% (1904/7814) and 29.3% (1344/4585) in linked and unlinked cases, respectively. Risks of long reporting delay among linked cases were living alone and onset on the day with a higher 7-day daily average confirmed cases or onset on weekends; whereas, risks for unlinked cases were age over 65 years, without occupation and living alone. CONCLUSION: Our results suggest the necessity to establish a Public Health Office system that is less susceptible to the rapid increase in the number of patients, promotes educational activities for people with fewer social connections, and improves access to health care.

Case-Control Study of Long COVID, Sapporo, Japan.

We conducted a cross-sectional survey among SARS-CoV-2-positive persons and negative controls in Sapporo, Japan, to clarify symptoms of long COVID. We collected responses from 8,018 participants, 3,694 case-patients and 3,672 controls. We calculated symptom prevalence for case-patients at 2-3, 4-6, 7-9, 10-12, and 13-18 months after illness onset. We used logistic regression, adjusted for age and sex, to estimate the odds ratio (OR) for each symptom and control reference. We calculated symptom prevalence by stratifying for disease severity, age, and sex. At 4-18 months from illness onset, ORs for anosmia, ageusia, dyspnea, alopecia, and brain fog were consistently >1, whereas ORs for common cold-like, gastrointestinal, and dermatologic symptoms were <1. Time trend ORs increased for diminished ability to concentrate, brain fog, sleep disturbance, eye symptoms, and tinnitus. Clinicians should focus on systemic, respiratory, and neuropsychiatric symptoms among long COVID patients.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

Facilitating the deployment of Japanese human resources for responding global outbreaks of emerging and Re-emerging infectious diseases: A cross-sectional study.

INTRODUCTION: In response to global outbreaks of infectious diseases, the need for support from organizations such as the World Health Organization Global Outbreak Alert and Response Network (GOARN) is increasing. Identifying the obstacles and support needs for applicants could increase GOARN deployments from Japan. METHODS: This cross-sectional study involved a web-based, self-administered questionnaire survey targeting Japanese participants in the GOARN Tier 1.5 training workshop, held in Tokyo in December 2019. RESULTS: All 47 Japanese participants in the workshop responded to the survey. Most responders were male and in their 30s and 40s. Participants specialized in case management (42.6%), infection prevention and control (25.6%), epidemiology and surveillance (19.1%). Only two participants (4.6%) had experienced a GOARN deployment. Their motivations for joining the GOARN training workshop were "Desire to be part of an international emerging infectious disease response team" (44.6%), "Interest in making an international contribution" (19.1%), and "Interest in working for the Japanese government in the field of international infectious diseases" (14.9%). Obstacles to GOARN deployments were "Making time for deployments" (45.7%) and "Lack of required professional skills and knowledge" (40.4%). The support needs for GOARN deployments constituted "Periodic simulation training" (51.1%), "Financial support during deployments" (44.7%), and "Technical support for deployments" (40.4%). CONCLUSIONS: Our study revealed the obstacles and support needs of Japanese candidates for GOARN deployment. Making time and upskilling for GOARN deployment were the main obstacles. More practical training (like GOARN Tier 2.0) with other supports are needed. The national framework is desirable to realize these supports.

Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections. METHODOLOGY/PRINCIPAL FINDINGS: The antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2'-fluoro-2'-deoxycytidine (2'-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2'-FdC were 41.0, 61.8, and 13.6 µM in Vero cells and 20.7, 25.8, and 8.8 µM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (-2 dpi-2 dpi) or on the day of infection (0 dpi-4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi-4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi). CONCLUSIONS/SIGNIFICANCE: Although the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment.

Antiviral treatments targeting the coronavirus disease 2019 are urgently required. We screened a panel of already approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine in VeroE6/TMPRSS2 cells: the anti-inflammatory drug cepharanthine and human immunodeficiency virus protease inhibitor nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry through the blocking of viral binding to target cells, while nelfinavir suppressed viral replication partly by protease inhibition. Consistent with their different modes of action, synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation was highlighted. Mathematical modeling in vitro antiviral activity coupled with the calculated total drug concentrations in the lung predicts that nelfinavir will shorten the period until viral clearance by 4.9 days and the combining cepharanthine/nelfinavir enhanced their predicted efficacy. These results warrant further evaluation of the potential anti-SARS-CoV-2 activity of cepharanthine and nelfinavir.

Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells.

BACKGROUND: An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19. METHODS: We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan. RESULTS: The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus. CONCLUSION: In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.

Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein.

BACKGROUND: SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. METHODS: To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). RESULTS: The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. CONCLUSIONS: In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

A Prospective, Randomized, Open-Label Trial of Early versus Late Favipiravir Therapy in Hospitalized Patients with COVID-19.

Favipiravir is an oral broad-spectrum inhibitor of viral RNA-dependent RNA polymerase that is approved for treatment of influenza in Japan. We conducted a prospective, randomized, open-label, multicenter trial of favipiravir for the treatment of COVID-19 at 25 hospitals across Japan. Eligible patients were adolescents and adults admitted with COVID-19 who were asymptomatic or mildly ill and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients were randomly assigned at a 1:1 ratio to early or late favipiravir therapy (in the latter case, the same regimen starting on day 6 instead of day 1). The primary endpoint was viral clearance by day 6. The secondary endpoint was change in viral load by day 6. Exploratory endpoints included time to defervescence and resolution of symptoms. Eighty-nine patients were enrolled, of whom 69 were virologically evaluable. Viral clearance occurred within 6 days in 66.7% and 56.1% of the early and late treatment groups (adjusted hazard ratio [aHR], 1.42; 95% confidence interval [95% CI], 0.76 to 2.62). Of 30 patients who had a fever (≥37.5°C) on day 1, times to defervescence were 2.1 days and 3.2 days in the early and late treatment groups (aHR, 1.88; 95% CI, 0.81 to 4.35). During therapy, 84.1% developed transient hyperuricemia. Favipiravir did not significantly improve viral clearance as measured by reverse transcription-PCR (RT-PCR) by day 6 but was associated with numerical reduction in time to defervescence. Neither disease progression nor death occurred in any of the patients in either treatment group during the 28-day participation. (This study has been registered with the Japan Registry of Clinical Trials under number jRCTs041190120.).