The anti-influenza virus drug favipiravir has little effect on replication of SARS-CoV-2 in cultured cells.

Favipiravir (T-705, commercial name Avigan), a drug developed to treat influenza virus infection, has been used in some countries as an oral treatment for COVID-19; however, its clinical efficacy in this context is controversial.….

Use of the particle agglutination/particle agglutination inhibition test for antigenic analysis of SARS-CoV-2.

Background: The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell-based assays using special equipment such as plate reader or ELISPOT analyzer. Methods: Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus-infected hamster antisera in addition to the beads and virus (PAI test). Results: The PA titer was positively correlated with the plaque-forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods. Conclusions: The PAI test is an easy and rapid method to analyze the antigenicity of SARS-CoV-2.

Frequent occurrence of mutations in nsp3 and nsp4 of SARS-CoV-2, presumably caused by the inhaled asthma drug ciclesonide.

Mutations in nonstructural protein 3 (nsp3) and nsp4 of SARS-CoV-2, presumably induced by the asthma drug ciclesonide (which also has anti-SARS-CoV-2 activity), were counted 5,851 cases in the GISAID EpiCoV genome database. Sporadic occurrence of mutants not linked to each other in the phylogenetic tree were identified at least 88 times; of which, 58 had one or more descendants in the same branch. Five of these had spread to more than 100 cases, and one had expanded to 4,748 cases, suggesting the mutations are frequent, selected in individual patients, and transmitted to form clusters of cases. Clinical trials of ciclesonide as a treatment for COVID-19 are the presumed cause of the frequent occurrence of mutations between 2020 June and 2021 November. In addition, because ciclesonide is a common treatment for asthma, it can drive mutations in asthmatics suffering from COVID-19. Ciclesonide-resistant mutations, which have unpredictable effects in humans, are likely to continue to emerge because SARS-CoV-2 remains prevalent globally.

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

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.

Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

Less Frequent Sequence Mismatches in Variants of Concern (VOCs) of SARS-CoV-2 in the Real-Time RT-PCR Assays Developed by the National Institute of Infectious Diseases, Japan.

Various variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began emerging worldwide from the end of 2020 to the beginning of 2021. The variants GRY/VOC202012/01 (B1.1.7), GH/N501Y.V2 (B1.351), and GR/N501Y.V3 (P1) are characterized by N to Y amino acid substitution at position 501 in the S protein. The variant containing L to R substitution at position 452 in the S protein G/L452R.V3 (B1.617) was endemic to India. The heightened concern regarding these variants is related to their increased viral infectivity. Information about nucleotide mismatch(es) on the primer/probe sequence is important for maintaining good performance of real-time PCR assays. In this study, real-time RT-PCR assays developed by the National Institute of Infectious Diseases, Japan (NIID-N2 and NIID-S2 assays), were reviewed to analyze nucleotide mismatches of variants in primer/probe sequences. The frequency of mismatched sequences in three variants (GRY/VOC202012/01, GH/N501Y.V2, and GR/N501Y.V3) was lower than that in all SARS-CoV-2 sequences. The mismatch, that G to C substitution at nucleotide 8 in reverse primer of S2 set, elevated to about 16.3% in G/L452R.V3, however the substitution did not affect the analytical sensitivity of assay. Therefore, the study indicates that the NIID-N2 and NIID-S2 sets detect VOCs of SARS-CoV-2 with reliable efficiency.

Performance Evaluation of Real-Time RT-PCR Assays for the Detection of Severe Acute Respiratory Syndrome Coronavirus-2 Developed by the National Institute of Infectious Diseases, Japan.

Soon after the 2019 outbreak of coronavirus disease 2019 in Wuhan, China, a protocol for real-time RT-PCR assay detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was established by the National Institute of Infectious Diseases (NIID) in Japan. The protocol used Charité's nucleocapsid (Sarbeco-N) and NIID nucleocapsid (NIID-N2) assays. During the following months, SARS-CoV-2 spread and caused a global pandemic, and various SARS-CoV-2 sequences were registered in public databases, such as the Global Initiative on Sharing All Influenza Data (GISAID). In this study, we evaluated the S2 assay (NIID-S2) that was newly developed to replace the Sarbeco-N assay and the performance of the NIID-N2 and NIID-S2 assays, referring to mismatches in the primer/probe targeted region. We found that the analytical sensitivity and specificity of the NIID-S2 set were comparable to those of the NIID-N2 assay, and the detection rate for clinical specimens was identical to that of the NIID-N2 assay. Furthermore, among the available sequences (approximately 192,000), the NIID-N2 and NIID-S2 sets had 2.6% and 1.2% mismatched sequences, respectively, although most of these mismatches did not affect the amplification efficiency, except the 3' end of the NIID-N2 forward primer. These findings indicate that the previously developed NIID-N2 assay is suitable for the detection of SARS-CoV-2 with support from the newly developed NIID-S2 set.

An Ultra-Rapid Real-Time RT-PCR Method Using the PCR1100 to Detect Severe Acute Respiratory Syndrome Coronavirus-2.

The disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Wuhan, China, in December 2019, has rapidly spread worldwide. SARS-CoV-2 is usually detected via real-time reverse-transcription polymerase chain reaction (RT-PCR). However, the increase in specimen load in institutions/hospitals necessitates a simpler detection system. Here, we present an ultra-rapid, real-time RT-PCR assay for SARS-CoV-2 detection using PCR1100 device. Although PCR1100 tests only one specimen at a time, the amplification period is less than 20 min and the sensitivity and specificity match those of conventional real-time RT-PCR performed on large instruments. The method is potentially helpful when daily multiple SARS-CoV-2 testing is needed, for example to confirm virus-free status prior to patient discharge.

The Inhaled Steroid Ciclesonide Blocks SARS-CoV-2 RNA Replication by Targeting the Viral Replication-Transcription Complex in Cultured Cells.

Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), in cultured cells. The 90% effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 µM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double-stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients.IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses the replication of coronaviruses, including betacoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphacoronavirus (human coronavirus 229E [HCoV-229E]), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS and COVID-19.

Development of Genetic Diagnostic Methods for Detection for Novel Coronavirus 2019(nCoV-2019) in Japan.

During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.

Environmental Sampling for Severe Acute Respiratory Syndrome Coronavirus 2 During a COVID-19 Outbreak on the Diamond Princess Cruise Ship.

During a COVID-19 outbreak on the Diamond Princess cruise ship we sampled environmental surfaces after passengers and crew vacated cabins. SARS-CoV-2 RNA was detected in 58 of 601 samples (10%) from case cabins 1-17 days after cabins were vacated but not from noncase cabins. There was no difference in detection proportion between cabins of symptomatic (15%, 28/189; cycle quantification [Cq], 29.79-38.86) and asymptomatic cases (21%, 28/131; Cq, 26.21-38.99). No SARS-CoV-2 virus was isolated from any of the samples. Transmission risk of SARS-CoV-2 from symptomatic and asymptomatic patients may be similar and surfaces could be involved in transmission.

Ultra-Rapid Real-Time RT-PCR Method for Detecting Middle East Respiratory Syndrome Coronavirus Using a Mobile PCR Device, PCR1100.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used, and these are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These disadvantages limit rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established comprising a multiplex assay for upE and ORF1a running on a mobile PCR1100 device. As few as five copies of the MERS-CoV RNA can be detected within 20 min using the standard WHO assays in the mobile PCR device, with the sensitivity and specificity being similar to those of a conventional real-time PCR instrument such as the LightCyler, thereby enabling timely intervention to control MERS-CoV infection.

Antiviral activities of mycophenolic acid and IMD-0354 against SARS-CoV-2.

In this study, the anti-severe acute respiratory syndrome coronavirus-2 (anti-SARS-CoV-2) activity of mycophenolic acid (MPA) and IMD-0354 was analyzed. These compounds were chosen based on their antiviral activities against other coronaviruses. Because they also inhibit dengue virus (DENV) infection, other anti-DENV compounds/drugs were also assessed. On SARS-CoV-2-infected VeroE6/TMPRSS2 monolayers, both MPA and IMD-0354, but not other anti-DENV compounds/drugs, showed significant anti-SARS-CoV-2 activity. Although MPA reduced the viral RNA level by only approximately 100-fold, its half maximal effective concentration was as low as 0.87 µ m, which is easily achievable at therapeutic doses of mycophenolate mofetil. MPA targets the coronaviral papain-like protease and an in-depth study on its mechanism of action would be useful in the development of novel anti-SARS-CoV-2 drugs.

Assessment of Real-Time RT-PCR Kits for SARS-CoV-2 Detection.

The coronavirus induced disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan (China) in December 2019 is currently spreading rapidly worldwide. We recently reported a laboratory protocol for the diagnosis of SARS-CoV-2 based on real-time reverse transcriptase PCR (RT-PCR) assays using two primer sets, N and N2. On January 30-31, 2020, the protocol and the reagents for these assays were distributed to the local public health institutes and quarantine depots in Japan nationwide, and thereafter SARS-CoV-2 diagnostic testing was initiated. For further validation, the assays were compared with the commercially available kits using the SARS-CoV-2 viral RNA and clinical specimens obtained from COVID19-suspected individuals. The LightMix Modular SARS and Wuhan CoV E-gene (LM S&W-E) assay was highly sensitive for the SARS-CoV-2, as was the N2 set, as both the assays showed consistent results for the clinical specimens. While the LM S&W-E set targets the highly conserved region of E gene in the SARS-CoV and SARS-CoV-2, the N2 set was designed to target specifically the unique region in the SARS-CoV-2 N gene. Therefore, the N2 set exhibits high specificity and sensitivity for SARS-CoV-2 detection. These results indicate that the protocol using the N and N2 sets is comparable to the commercially available kits, and thus is reliable for laboratory diagnosis of COVID-19.

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.