Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants.

In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.

Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant.

In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.

The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant

The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5. Graphical Saito and G2P-Japan Consortium et al. elucidate the virological properties of SARS-CoV-2 Omicron BA.2.75 variant. BA.2.75 is more transmissible than BA.5, and exhibits different antigenicity than BA.2 and BA.5. The BA.2.75 spike exhibits higher affinity to ACE2 and higher fusogenicity, and BA.2.75 is more pathogenic than BA.2 in hamsters.

Virological characteristics of the SARS-CoV-2 Omicron BA.2 subvariants, including BA.4 and BA.5.

After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.

Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.

The emergence of the Omicron variant of SARS-CoV-2 is an urgent global health concern1. In this study, our statistical modelling suggests that Omicron has spread more rapidly than the Delta variant in several countries including South Africa. Cell culture experiments showed Omicron to be less fusogenic than Delta and than an ancestral strain of SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into two subunits, which facilitates cell-cell fusion2,3, the Omicron S protein was less efficiently cleaved compared to the S proteins of Delta and ancestral SARS-CoV-2. Furthermore, in a hamster model, Omicron showed decreased lung infectivity and was less pathogenic compared to Delta and ancestral SARS-CoV-2. Our multiscale investigations reveal the virological characteristics of Omicron, including rapid growth in the human population, lower fusogenicity and attenuated pathogenicity.

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.

An Ultra-Rapid Real-Time RT-PCR Method Using PCR1100 for Detecting Human Orthopneumovirus.

Human orthopneumovirus, also known as the respiratory syncytial virus (RSV), is a leading cause of respiratory tract infections in children worldwide. The World Health Organization has taken steps toward establishing a global surveillance system for RSV, based on the global influenza surveillance and response system initiated in 2015. The US Centers for Disease Control and Prevention (CDC) has developed a genetic detection method based on real-time reverse transcription polymerase chain reaction (RT-PCR), which is used in global RSV surveillance. In Japan, immunoassay-based rapid antigen detection kits are widely used for the detection of RSV. In this study, an ultra-rapid real-time RT-PCR method for the rapid detection of RSV was developed using the PCR1100 device based on the US CDC assay in order to detect RSV in comparable time to rapid test kits. The ultra-rapid real-time RT-PCR could detect RSV viral RNA in less than 20 min while maintaining sensitivity and specificity comparable to conventional real-time RT-PCR using large installed instruments. Furthermore, combining ultra-rapid real-time RT-PCR with the M1 Sample Prep kit reduced the total working time for the detection of RSV from clinical specimen to less than 25 min, suggesting this method could be used for point-of-care RSV testing.

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.

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.

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.

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.

Severe Acute Respiratory Syndrome Coronavirus 2 Infection among Returnees to Japan from Wuhan, China, 2020.

In early 2020, Japan repatriated 566 nationals from China. Universal laboratory testing and 14-day monitoring of returnees detected 12 cases of severe acute respiratory syndrome coronavirus 2 infection; initial screening results were negative for 5. Common outcomes were remaining asymptomatic (n = 4) and pneumonia (n = 6). Overall, screening performed poorly.

Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak (preprint)

The Diamond Princess (DP) cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a COVID-19 case. We performed whole genome sequencing of SARS-CoV-2 directly from PCR-positive clinical specimens and conducted a haplotype network analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the DP originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters were 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 analysis in identifying potential infection routes.

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.