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The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.

SummaryBACKGROUNDVLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the SARS-CoV-2 spike protein.METHODSA phase 1 study of VLPCOV-01 was conducted at Medical Corporation Heishinkai OPHAC Hospital, Japan. Participants aged 18 to 55 or ≥65 years who had completed two doses of the BNT162b2 mRNA vaccine 6 to 12 months previously were randomised to receive one intramuscular vaccination of 0·3, 1·0, or 3·0 μg VLPCOV-01, 30 μg BNT162b2, or placebo between February 16, 2022, and March 17, 2022. Solicited adverse events were collected up to 6 days post-administration. Interim immunogenicity analyses included SARS-CoV-2 IgG and neutralising antibody titres. Follow-up for safety and immunogenicity evaluation is ongoing. (The trial is registered: jRCT2051210164).FINDINGS92 healthy adults were enrolled, with 60 participants receiving VLPCOV-01. No serious adverse events were reported up to 26 weeks, and no prespecified trial-halting events were met. VLPCOV-01 induced robust IgG titres against SARS-CoV-2 RBD protein that were maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5037 (95% CI 1272–19,940) at 0·3 μg to 12,873 (95% CI 937–17,686) at 3 μg, in comparison to 3166 (95% CI 1619–6191) with 30 μg BNT162b2. Among elderly participants, IgG titres at 26 weeks post-vaccination with 3 μg VLPCOV-01 were 9865 (95% CI 4396–22138) compared to 4183 (95% CI 1436–12180) following vaccination with 30 μg BNT162b2. Pseudovirus neutralising antibody responses were observed against multiple SARS-CoV-2 variants and strongly correlated with anti-SARS-CoV-2 IgG (r=0·950, pINTERPRETATIONVLPCOV-01 is immunogenic following low dose administration, with anti-SARS-CoV-2 immune responses comparable to BNT162b2. These findings support further development of VLPCOV-01 as a COVID-19 booster vaccine and the potential for saRNA vectors as a vaccine platform.FUNDINGSupported by AMED, Grant No. JP21nf0101627.

Background In vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the 3′-polypurine tract (3′-PPT). A clinical study also reported that mutations in the 3′-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3′-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs. Objectives To identify the specific mutation(s) in the G-tract of the 3′-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs. Methods The infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells. Results A single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3′-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or ‘T’ at the second position and ‘T’ at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose–response curves, consistent with a non-competitive mechanism of inhibition. Conclusions Mutations at the second and fifth positions in the G-tract of the 3′-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.

Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity.

In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape.

Macaque-tropic HIV-1 (HIV-1mt) variants have been developed to establish preferable primate models that are advantageous in understanding HIV-1 infection pathogenesis and in assessing the preclinical efficacy of novel prevention/treatment strategies. We previously reported that a CXCR4-tropic HIV-1mt, MN4Rh-3, efficiently replicates in peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques homozygous for TRIMCyp (CMsTC). However, the CMsTC challenged with MN4Rh-3 displayed low viral loads during the acute infection phase and subsequently exhibited short-term viremia. These virological phenotypes in vivo differed from those observed in most HIV-1-infected people. Therefore, further development of the HIV-1mt variant was needed. In this study, we first reconstructed the MN4Rh-3 clone to produce a CCR5-tropic HIV-1mt, AS38. In addition, serial in vivo passages allowed us to produce a highly adapted AS38-derived virus that exhibits high viral loads (up to approximately 106 copies ml−1) during the acute infection phase and prolonged periods of persistent viremia (lasting approximately 16 weeks postinfection) upon infection of CMsTC. Whole-genome sequencing of the viral genomes demonstrated that the emergence of a unique 15-nt deletion within the vif gene was associated with in vivo adaptation. The deletion resulted in a significant increase in Vpr protein expression but did not affect Vif-mediated antagonism of antiretroviral APOBEC3s, suggesting that Vpr is important for HIV-1mt adaptation to CMsTC. In summary, we developed a novel CCR5-tropic HIV-1mt that can induce high peak viral loads and long-term viremia and exhibits increased Vpr expression in CMsTC.

High genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1.

There were five epidemic waves of coronavirus disease 2019 in Japan between 2020 and 2021. It remains unclear how the domestic waves arose and abated. To better understand this, we analyzed the pangenomic sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and characterized the molecular epidemiological features of the five epidemic waves in Japan. In this study, we performed deep sequencing to determine the pangenomic SARS-CoV-2 sequences of 1,286 samples collected in two cities far from each other, Tokyo Metropolis and Nagoya. Then, the spatiotemporal genetic changes of the obtained sequences were compared with the sequences available in the Global Initiative on Sharing All Influenza Data (GISAID) database. A total of 873 genotypes carrying different sets of mutations were identified in the five epidemic waves. Phylogenetic analysis demonstrated that sharp displacements of lineages and genotypes occurred between consecutive waves over the 2 years. In addition, a wide variety of genotypes were observed in the early half of each wave, whereas a few genotypes were detected across Japan during an entire wave. Phylogenetically, putative descendant genotypes observed late in each wave displayed regional clustering and evolution in Japan. The genetic diversity of SARS-CoV-2 displayed uneven dynamics during each epidemic wave in Japan. Our findings provide an important molecular epidemiological basis to aid in controlling future SARS-CoV-2 epidemics.

Although the number of HIV-2-infected individuals is quite low in Japan, at least three groups of HIV-2 (A, B and CRF01_AB) have been detected thus far. In particular, CRF01_AB HIV-2 cases have been found only in limited areas, Cote d'Ivoire and Japan. Here, we demonstrate that Geenius HIV 1/2 Confirmatory Assay (Geenius, Bio-Rad Laboratories) is able to detect HIV-2 samples, including groups A, B and CRF01_AB, isolated in Japan.A total of 57 plasma samples, including three panels (Ⅰ: HIV-2-positive samples [n=9], Ⅱ: HIV-1 infection with HIV-2 antibody cross-reactivity samples [n=37], and Ⅲ: HIV negative with biological false-positive HIV-2 samples [n=11]) were tested by Geenius.Geenius determined Panel I to be "HIV-2 positive with/without HIV-1 cross-reactivity (n=4, respectively)", including HIV-2 group A and CRF01_AB. In the case with HIV-2 group B, all bands were detected, resulting in a Geenius interpretation of "HIV positive untypable". Geenius classified Panels II and III as "HIV-1 positive (n=37)" or "HIV negative (n=9)", "HIV indeterminate (n=1)" and "HIV-2 indeterminate (n=1)", suggesting 95.8% HIV-2 differentiation by Geenius.With Geenius, there were fewer false-positives for HIV-1/-2 negativity and fewer cross-reactions with HIV-2 among HIV-1-positive samples. Additionally, the assay could detect HIV-2 genetic group CRF01_AB. Geenius can be expected to be a useful diagnostic tool that is an alternative to conventional Western blotting.

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.