Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection.

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

The development of a rapid, high-throughput neutralization assay using a SARS-CoV-2 reporter.

Many methods have been developed to measure the neutralizing capacity of antibodies to SARS-CoV-2. However, these methods are low throughput and can be difficult to quickly modify in response to emerging variants. Therefore, an experimental system for rapid and easy measurement of the neutralizing capacity of antibodies against various variants is needed. In this study, we developed an experimental system that can efficiently measure the neutralizing capacity of sera by using a GFP-carrying recombinant SARS-CoV-2 with spike proteins of multiple variants (B.1.1, BA.5, or XBB.1.5). For all 3 recombinant chimeric genomes generated, neutralizing antibody titers determined by measuring GFP fluorescence intensity correlated significantly with those calculated from viral RNA levels measured by RT-qPCR in the supernatant of infected cells. Furthermore, neutralizing antibody titers determined by visually assessing GFP fluorescence using microscopy were also significantly correlated with those determined by RT-qPCR. By using this high-throughput method, it is now possible to quickly and easily determine the neutralizing capacity of antibodies against SARS-CoV-2 variants.

Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant.

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5.

A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

Virological characteristics of the SARS-CoV-2 BA.2.86 variant.

In late 2023, several SARS-CoV-2 XBB descendants, notably EG.5.1, were predominant worldwide. However, a distinct SARS-CoV-2 lineage, the BA.2.86 variant, also emerged. BA.2.86 is phylogenetically distinct from other Omicron sublineages, accumulating over 30 amino acid mutations in its spike protein. Here, we examined the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Additionally, four clinically available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 are significantly lower than those of BA.2 both in vitro and in vivo, the attenuated pathogenicity of BA.2.86 is likely due to its decreased replication capacity. These findings uncover the features of BA.2.86, providing insights for control and treatment.

Prolonged shedding of viable SARS-CoV-2 in immunocompromised patients with haematological malignancies: A prospective study.

Prolonged SARS-CoV-2 infection in immunocompromised individuals has been scattered, but the details remain unclear. We conducted a prospective study with 26 COVID-19 patients with haematological malignancies to determine viral shedding kinetics and characteristics. We obtained nasopharyngeal swabs from the patients 21-28 days post-onset for a PCR test and performed virus isolation from the PCR-positive samples. A viable virus was detected in five patients (19.2%), all of whom had malignant lymphoma. Those patients had significantly lower CD4+ T-cell counts than the PCR-negative patients. A comparison of previous chemotherapy showed that anti-CD20 antibodies and bendamustine may be risk factors for prolonged viral shedding.

Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics.

IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. A phase 1 study of VLPCOV-01 is conducted (jRCT2051210164). Participants who completed two doses of the BNT162b2 mRNA vaccine previously are randomized to receive one intramuscular vaccination of 0.3, 1.0, or 3.0 µg VLPCOV-01, 30 µg BNT162b2, or placebo. No serious adverse events have been reported. VLPCOV-01 induces robust immunoglobulin G (IgG) titers against the RBD protein that are maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5,037 (95% confidence interval [CI] 1,272-19,940) at 0.3 µg to 12,873 (95% CI 937-17,686) at 3 µg compared with 3,166 (95% CI 1,619-6,191) with 30 µg BNT162b2. Neutralizing antibody titers against all variants of SARS-CoV-2 tested are induced. VLPCOV-01 is immunogenic following low-dose administration. These findings support the potential for saRNA as a vaccine platform.

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5.

The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.

A third dose of the BNT162b2 mRNA vaccine sufficiently improves the neutralizing activity against SARS-CoV-2 variants in liver transplant recipients.

Introduction: We examined the neutralizing antibody production efficiency of the second and third severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine doses (2nd- and 3rd-dose) and neutralizing activity on mutant strains, including, the Ancestral, Beta and Omicron strains using green fluorescent protein-carrying recombinant SARS-CoV-2, in living-donor liver transplantation (LDLT) recipients. Methods: The patients who were administered vaccines other than Pfizer- BioNTechBNT162b2 and who had coronavirus disease 2019 in this study period were excluded. We enrolled 154 LDLT recipients and 50 healthy controls. Result: The median time were 21 days (between 1st and 2nd vaccination) and 244 days (between 2nd and 3rd vaccination). The median neutralizing antibody titer after 2nd-dose was lower in LDLT recipients than in controls (0.46 vs 1.00, P<0.0001). All controls had SARS-CoV-2 neutralizing antibodies, whereas 39 LDLT recipients (25.3%) had no neutralizing antibodies after 2nd-dose; age at vaccination, presence of ascites, multiple immunosuppressive treatments, and mycophenolate mofetil treatment were significant risk factors for nonresponder. The neutralizing activities of recipient sera were approximately 3-fold and 5-fold lower than those of control sera against the Ancestral and Beta strains, respectively. The median antibody titer after 3rd-dose was not significantly different between recipients and controls (1.02 vs 1.22, p=0.0758); only 5% recipients was non-responder. The neutralizing activity after third dose to Omicron strains were enhanced and had no significant difference between two groups. Conclusion: Only the 2nd-dose was not sufficiently effective in recipients; however, 3rd-dose had sufficient neutralizing activity against the mutant strain and was as effective as that in healthy controls.

Impact of Imprinted Immunity Induced by mRNA Vaccination in an Experimental Animal Model.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has led to concerns that ancestral SARS-CoV-2-based vaccines may not be effective against newly emerging Omicron subvariants. The concept of "imprinted immunity" suggests that individuals vaccinated with ancestral virus-based vaccines may not develop effective immunity against newly emerging Omicron subvariants, such as BQ.1.1 and XBB.1. In this study, we investigated this possibility using hamsters. Although natural infection induced effective antiviral immunity, breakthrough infections in hamsters with BQ.1.1 and XBB.1 Omicron subvariants after receiving the 3-dose mRNA-lipid nanoparticle vaccine resulted in only faintly induced humoral immunity, supporting the possibility of imprinted immunity.

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.

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.

saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern.

Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.

Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

Smoking enhances the expression of angiotensin-converting enzyme 2 involved in the efficiency of severe acute respiratory syndrome coronavirus 2 infection.

Smoking is one of the risk factors most closely related to the severity of coronavirus disease 2019 (COVID-19). However, the relationship between smoking history and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and nonsmokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of nonsmokers and ex-smokers with a long history of nonsmoking (cessation period longer than 6 months). We also showed that the efficiency of SARS-CoV-2 infection was enhanced in a manner dependent on the angiotensin-converting enzyme 2 (ACE2) expression level. Using RNA-seq analysis on the lungs of smokers, we identified that the expression of inflammatory signaling genes was correlated with ACE2 expression. Notably, with increasing duration of smoking cessation among ex-smokers, not only ACE2 expression level but also the expression levels of inflammatory signaling genes decreased. These results indicated that smoking enhances the expression levels of ACE2 and inflammatory signaling genes. Our data suggest that the efficiency of SARS-CoV-2 infection is enhanced by smoking-mediated upregulation of ACE2 expression level.

Rare case of intracerebral hemorrhage in anaphylactic shock following administration of intramuscular adrenaline: A case report.

Intracerebral hemorrhage should be considered as a possible adverse event in patients with anaphylactic shock who are treated with adrenaline administration, especially in those at high risk of serious bleeding events.

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.2.75 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.

Photochemical Identification of Auxiliary Severe Acute Respiratory Syndrome Coronavirus 2 Host Entry Factors Using µMap.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of the COVID-19 pandemic, remains a global medical problem. Angiotensin-converting enzyme 2 (ACE2) was identified as the primary viral entry receptor, and transmembrane serine protease 2 primes the spike protein for membrane fusion. However, ACE2 expression is generally low and variable across tissues, suggesting that auxiliary receptors facilitate viral entry. Identifying these factors is critical for understanding SARS-Cov-2 pathophysiology and developing new countermeasures. However, profiling host-virus interactomes involves extensive genetic screening or complex computational predictions. Here, we leverage the photocatalytic proximity labeling platform µMap to rapidly profile the spike interactome in human cells and identify eight novel candidate receptors. We systemically validate their functionality in SARS-CoV-2 pseudoviral uptake assays with both Wuhan and Delta spike variants and show that dual expression of ACE2 with either neuropilin-2, ephrin receptor A7, solute carrier family 6 member 15, or myelin and lymphocyte protein 2 significantly enhances viral uptake. Collectively, our data show that SARS-CoV-2 synergistically engages several host factors for cell entry and establishes µMap as a powerful tool for rapidly interrogating host-virus interactomes.