SARS-CoV-2 RNA-dependent RNA Polymerase is Linked to Mitochondrial Dysfunction in Mouse Heart (preprint)

Little is known about specific viral factors responsible for the pathogenesis and pathophysiology of long COVID. Here we describe a conditional knock-in (cKI) mouse strain inducibly expressing Nsp12, an essential component of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Induction of Nsp12 translation was dependent on co-treatment with inhibitors of the integrated stress response in vitro and in vivo. We show that Nsp12 has a biologically significant link to mitochondrial dysfunction in vivo. In vitro, ectopic Nsp12 expression suppressed mitochondrial function in primary lung epithelial cells isolated from Nsp12 cKI mice. This functionality was physiologically relevant because, although ectopic Nsp12 expression in mouse lungs did not induce pneumonia, it did decrease mitochondrial activity in the hearts of Nsp12 cKI mice over the short and long terms. Administration in vivo of an RdRp inhibitor, EIDD-2801, restored mitochondrial function in cardiomyocytes of Nsp12 cKI mice. Our data demonstrate that SARS-CoV-2 RdRp activity in the lungs leads to cardiac mitochondrial dysfunction in vivo, generating a phenotype resembling aspects of long COVID in humans. Therapeutic targeting of SARS-CoV-2 RdRp may thus represent a novel means of preventing or mitigating intense fatigue and/or myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)-like disease caused by mitochondrial dysfunction in long COVID patients.

Germinal center-derived broadly neutralizing antibodies adapt to SARS-CoV-2 antigenic drift (preprint)

The outbreak of SARS-CoV-2 variant Omicron which harbors a striking number of mutations in the spike protein has been raising concerns about the effectiveness of vaccines and antibody treatment1. Here, we confirmed a substantial reduction in neutralizing potency against Omicron in all convalescent and vaccinated sera. However, we found that some people infected by the early strain show relatively higher neutralization to Omicron. From those B cells, we developed neutralizing antibodies inhibiting broad variants including Delta and Omicron. Unlike reported antibodies, one had an extremely large interface and widely covered receptor binding motif of spike, thereby interfering with diversified variants. Somatic mutations introduced by long-term germinal center reaction contributed to the key structure of antibodies and the universal interaction with spike variants. Recalling such rare B cells may confer sustainable protection against SARS-CoV-2 variants emerging one after another.

SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion (preprint)

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and at least five variants of concerns (VOCs) have been considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2 lineage (delta variant), closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617 variants are highly fusogenic and form prominent syncytia. Bioinformatic analyses reveal that the P681R mutation in the spike protein is highly conserved in this lineage. Although the P681R mutation decreases viral infectivity, this mutation confers the neutralizing antibody resistance. Notably, we demonstrate that the P681R mutation facilitates the furin-mediated spike cleavage and enhances and accelerates cell-cell fusion. Our data suggest that the P681R mutation is a hallmark characterizing the virological phenotype of this newest VOC, which may associate with viral pathogenicity. HighlightsO_LIP681R mutation is highly conserved in the B.1.617 lineages C_LIO_LIP681R mutation accelerates and enhances SARS-CoV-2 S-mediated fusion C_LIO_LIPromotion of viral fusion by P681R mutation is augmented by TMPRSS2 C_LI

Duration of Infectious Virus Shedding in Patients With Severe Coronavirus Disease 2019 Who Required Mechanical Ventilation (preprint)

Approximately 5% of patients with coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 develop severe COVID-19. Severe COVID-19 requires respiratory management with mechanical ventilation and an extended period of treatment. Prolonged infectious virus shedding is a concern in severe COVID-19 cases, but few reports have examined the duration of infectious virus shedding. Therefore, we investigated the duration of infectious virus shedding in patients transferred to Hiroshima University Hospital with severe COVID-19 requiring mechanical ventilation. Nasopharyngeal swab specimens were collected and analyzed using both viral culture and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) tests between December 2020 and February 2021. Of the 23 patients tested, the proportions of those with positive test results at first specimen collection on RT-qPCR and viral culture tests were 95·7% and 30·4%, respectively. All six patients with positive viral culture test results who were followed-up tested negative 24 days after symptom onset but remained positive on RT-qPCR. The longest negative conversion time was observed in a dialysis patient on immunosuppressive drugs. This study indicated that patients with severe COVID-19 remain culture positive for ≥ 10 days after symptom onset. Additionally, immunosuppressed patients with severe COVID-19 could consider isolation for ≥ 20 days.

Ethanol susceptibility of SARS-CoV-2 and other enveloped viruses (preprint)

Ethanol is an effective disinfectant against the novel coronavirus SARS-CoV-2. However, its effective concentration has not been shown, and we therefore analyzed the effects of different concentrations of ethanol on SARS-CoV-2. When SARS-CoV-2 was treated with varying ethanol concentrations and examined for changes in infectivity, the ethanol concentration at which 99% of the infectious titers were reduced was 24.1%(w/w) [29.3%(v/v)]. For reference, ethanol susceptibility was also examined with other envelope viruses, including influenza virus, vesicular stomatitis virus in the family Rhabdoviridae, and Newcastle disease virus in the family Paramyxoviridae, and the 99% inhibitory concentrations were found to be 28.8%(w/w) [34.8%(v/v)], 24.0%(w/w) [29.2%(v/v)], and 13.3%(w/w) [16.4%(v/v)], respectively. Some differences from SARS-CoV-2 were observed, but the differences were not significant. It was concluded that ethanol at a concentration of 30%(w/w) [36.2%(v/v)] almost completely inactivates SARS-CoV-2.

Duration of Infectious Virus Shedding in Patients with Severe Coronavirus Disease 2019 Who Required Mechanical Ventilation (preprint)

Background: Around 5% of patients with coronavirus disease ( COVID-19) caused by severe acute respiratory syndrome coronavirus 2 develop severe COVID-19. Severe COVID-19 requires respiratory management with mechanical ventilation and an extended period of treatment. Prolonged infectious virus shedding is a concern in severe COVID-19 cases, but few reports have examined the duration of infectious virus shedding. We investigated the duration of infectious virus shedding in patients transferred to Hiroshima University Hospital with severe COVID-19 requiring mechanical ventilation. Methods: Nasopharyngeal swab specimens were collected and analyzed using both viral culture and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) tests between December 2020 and February 2021.Findings: Of the 23 patients tested, the percentage of those with positive test results at first specimen collection (the median number of days to first specimen collection after symptom onset was ten) on RT-qPCR and viral culture was 95 ·7% (n = 22) and 30·4% (n = 7), respectively. All six patients with positive viral culture test results who were followed-up tested negative by day 24 after symptom onset but remained positive on RT-qPCR. Specimen viral loads based on PCR testing did not decrease over time, but viral loads determined via culture tests loads decreased over time. The longest negative conversion time was observed in a dialysis patient on immunosuppressive drugs.Interpretation: This study indicated that patients with severe COVID-19 remain culture positive ≥ ten days after symptom onset. The work also suggests that immunosuppressed patients with severe COVID-19 could consider isolation for ≥ 20 days.Funding: NoneDeclaration of Interests: We declare no competing interests.Ethics Approval Statement: This research was approved by the Ethical Committee for Epidemiology of Hiroshima University (approval number: E-2157).

Prolyl isomerase Pin1 plays an essential role in SARS-CoV-2 proliferation, indicating its possibility as a novel therapeutic target (preprint)

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC50 below 5 µM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)—a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1—similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19.