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.

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate.

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Generation of recombinant viruses directly from clinical specimens of COVID-19 patients.

Rapid characterization of the causative agent(s) during a disease outbreak can aid in the implementation of effective control measures. However, isolation of the agent(s) from crude clinical samples can be challenging and time-consuming, hindering the establishment of countermeasures. In the present study, we used saliva specimens collected for the diagnosis of SARS-CoV-2-a good example of a practical target-and attempted to characterize the virus within the specimens without virus isolation. Thirty-four saliva samples from coronavirus disease 2019 patients were used to extract RNA and synthesize DNA amplicons by PCR. New primer sets were designed to generate DNA amplicons of the full-length spike (S) gene for subsequent use in a circular polymerase extension reaction (CPER), a simple method for deriving recombinant viral genomes. According to the S sequence, four clinical specimens were classified as BA. 1, BA.2, BA.5, and XBB.1 and were used for the de novo generation of recombinant viruses carrying the entire S gene. Additionally, chimeric viruses carrying the gene encoding GFP were generated to evaluate viral propagation using a plate reader. We successfully used the RNA purified directly from clinical saliva samples to generate chimeric viruses carrying the entire S gene by our updated CPER method. The chimeric viruses exhibited robust replication in cell cultures with similar properties. Using the recombinant GFP viruses, we also successfully characterized the efficacy of the licensed antiviral AZD7442. Our proof-of-concept demonstrates the novel utility of CPER to allow rapid characterization of viruses from clinical specimens. IMPORTANCE: Characterization of the causative agent(s) for infectious diseases helps in implementing effective control measurements, especially in outbreaks. However, the isolation of the agent(s) from clinical specimens is often challenging and time-consuming. In this study, saliva samples from coronavirus disease 2019 patients were directly subjected to purifying viral RNA, synthesizing DNA amplicons for sequencing, and generating recombinant viruses. Utilizing an updated circular polymerase extension reaction method, we successfully generated chimeric SARS-CoV-2 viruses with sufficient in vitro replication capacity and antigenicity. Thus, the recombinant viruses generated in this study were applicable for evaluating the antivirals. Collectively, our developed method facilitates rapid characterization of specimens circulating in hosts, aiding in the establishment of control measurements. Additionally, this approach offers an advanced strategy for controlling other (re-)emerging viral infectious diseases.

Involvement of SARS-CoV-2 accessory proteins in immunopathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the largest single-stranded RNA virus known to date. Its genome contains multiple accessory protein genes that act against host immune responses but are not required for progeny virus production. The functions of the accessory proteins in the viral life cycle have been examined, but their involvement in viral pathogenicity remains unclear. Here, we investigated the roles of the accessory proteins in viral immunopathogenicity. To this end, recombinant SARS-CoV-2 possessing nonsense mutations in the seven accessory protein open reading frames (ORFs) (ORF3a, ORF3b, ORF6, ORF7a, ORF8, ORF9b, and ORF10) was de novo generated using an early pandemic SARS-CoV-2 strain as a backbone. We confirmed that the resultant virus (termed ORF3-10 KO) did not express accessory proteins in infected cells and retained the desired mutations in the viral genome. In cell culture, the ORF3-10 KO virus exhibited similar virus growth kinetics as the parental virus. In hamsters, ORF3-10 KO virus infection resulted in mild weight loss and reduced viral replication in the oral cavity and lung tissue. ORF3-10 KO virus infection led to mild inflammation, indicating that an inability to evade innate immune sensing because of a lack of accessory proteins impairs virus growth in vivo and results in quick elimination from the body. Overall, we showed that SARS-CoV-2 accessory proteins are involved in immunopathogenicity.

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.

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.

Conformational change in ricin toxin A-Chain: A critical factor for inhibitor binding to the secondary pocket.

Ricin toxin A-chain (RTA), a toxic protein from Ricinus communis, inactivates ribosomes to induce toxicity. The active site of RTA consists of two binding pockets. Many studies have focused on developing RTA inhibitors that can simultaneously bind to these critical pockets; however, almost all the inhibitors developed so far interact with only one pocket. In the present study, we discovered that pterin-7-carboxamides with aromatic l-amino acid pendants interacted with the active site of the enzyme in a 2-to-1 mode, where one inhibitor molecule bound to the primary pocket and the second one entered the secondary pocket in the active site of RTA. X-ray crystallographic analysis of inhibitor/RTA complexes revealed that the conformational changes of Tyr80 and Asn122 in RTA were critical for triggering the entry of inhibitor molecules into the secondary pocket of the RTA active site.

Efficacy of a Cap-Dependent Endonuclease Inhibitor and Neuraminidase Inhibitors against H7N9 Highly Pathogenic Avian Influenza Virus Causing Severe Viral Pneumonia in Cynomolgus Macaques.

H7N9 highly pathogenic avian influenza virus (HPAIV) infection in a human was first reported in 2017. A/duck/Japan/AQ-HE29-22/2017 (H7N9) (Dk/HE29-22), found in imported duck meat at an airport in Japan, possesses a hemagglutinin with a multibasic cleavage site, indicating high pathogenicity in chickens, as in the case of other H7 HPAIVs. In the present study, we examined the pathogenicity of Dk/HE29-22 and the effectiveness of a cap-dependent endonuclease inhibitor (baloxavir) and neuraminidase inhibitors (oseltamivir and zanamivir) against infection with this strain in a macaque model (n = 3 for each group). All of the macaques infected with Dk/HE29-22 showed severe signs of disease and pneumonia even after the virus had disappeared from lung samples. Virus titers in macaques treated with baloxavir were significantly lower than those in the other treated groups. After infection, levels of interferon alpha and beta (IFN-α and IFN-ß) in the blood of macaques in the baloxavir group were the highest among the groups, whereas levels of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13) were slightly increased in the untreated group. In addition, immune checkpoint proteins, including programmed death 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), were expressed at high levels in the untreated group, especially in one macaque that showed severe signs of disease, indicating that negative feedback responses against vigorous inflammation may contribute to disease progression. In the group treated with baloxavir, the percentages of PD-1-, CTLA-4-, and TIGIT-positive T lymphocytes were lower than those in the untreated group, indicating that reduction in virus titers may prevent expression of immune checkpoint molecules from downregulation of T cell responses.

Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.

Efficacy of Neuraminidase Inhibitors against H5N6 Highly Pathogenic Avian Influenza Virus in a Nonhuman Primate Model.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used nonhuman primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe, with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in interferon alpha (IFN-α) showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and the severity of symptoms in the early stage. This study also showed pathologically severe lung injury states in cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and further studies on virus pathogenicity and new antiviral therapies.

Animal Models Used in Hepatitis C Virus Research.

The narrow range of species permissive to infection by hepatitis C virus (HCV) presents a unique challenge to the development of useful animal models for studying HCV, as well as host immune responses and development of chronic infection and disease. Following earlier studies in chimpanzees, several unique approaches have been pursued to develop useful animal models for research while avoiding the important ethical concerns and costs inherent in research with chimpanzees. Genetically related hepatotropic viruses that infect animals are being used as surrogates for HCV in research studies; chimeras of these surrogate viruses harboring specific regions of the HCV genome are being developed to improve their utility for vaccine testing. Concurrently, genetically humanized mice are being developed and continually advanced using human factors known to be involved in virus entry and replication. Further, xenotransplantation of human hepatocytes into mice allows for the direct study of HCV infection in human liver tissue in a small animal model. The current advances in each of these approaches are discussed in the present review.

Induction of humoural and cellular immunity by immunisation with HCV particle vaccine in a non-human primate model.

OBJECTIVE: Although HCV is a major cause of chronic liver disease worldwide, there is currently no prophylactic vaccine for this virus. Thus, the development of an HCV vaccine that can induce both humoural and cellular immunity is urgently needed. To create an effective HCV vaccine, we evaluated neutralising antibody induction and cellular immune responses following the immunisation of a non-human primate model with cell culture-generated HCV (HCVcc). DESIGN: To accomplish this, 10 common marmosets were immunised with purified, inactivated HCVcc in combination with two different adjuvants: the classically used aluminum hydroxide (Alum) and the recently established adjuvant: CpG oligodeoxynucleotide (ODN) wrapped by schizophyllan (K3-SPG). RESULTS: The coadministration of HCVcc with K3-SPG efficiently induced immune responses against HCV, as demonstrated by the production of antibodies with specific neutralising activity against chimaeric HCVcc with structural proteins from multiple HCV genotypes (1a, 1b, 2a and 3a). The induction of cellular immunity was also demonstrated by the production of interferon-γ mRNA in spleen cells following stimulation with the HCV core protein. These changes were not observed following immunisation with HCVcc/Alum preparation. No vaccination-related abnormalities were detected in any of the immunised animals. CONCLUSIONS: The current preclinical study demonstrated that a vaccine included both HCVcc and K3-SPG induced humoural and cellular immunity in marmosets. Vaccination with this combination resulted in the production of antibodies exhibiting cross-neutralising activity against multiple HCV genotypes. Based on these findings, the vaccine created in this study represents a promising, potent and safe prophylactic option against HCV.

Pterin-7-carboxamides as a new class of aldose reductase inhibitors.

Aldose reductase is related to the onset and progression of diabetic complications, such as neuropathy, retinopathy, angiopathy, and so on: therefore molecules that are capable of inhibiting the enzyme are potential drugs for treatment of diabetic complications. Epalrestat is the sole aldose reductase inhibitor that is clinically used, but still has some drawbacks. Thus, the development of new aldose reductase inhibitors is still desired. We have synthesized a series of new pterin-7-carboxamides, and evaluated their in vitro inhibitory activities against human aldose reductase. All newly synthesized compounds exhibited the inhibitory activity. Among them, 1a having a glycine side chain exhibits the highest activity comparable to that of sorbinil, a highly active aldose reductase inhibitor. Molecular docking of 1a on the active site of the enzyme indicated this compound interacts with amino acid residues that are specific to the enzyme and related to suppressing side effects. Based on these results, we proved perin-7-carboxamides to be a new class of aldose reductase inhibitors, and particularly compound 1a was found to be a good candidate for further biological investigations as a drug for treatment of diabetic complications with fewer side effects.

Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome.

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.

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.

Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery.

Modification of RNA with N 6 -methyladenosine (m 6 A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m 6 A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m 6 A writers Mettl3 and Mettl14 , nor the m 6 A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro . To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236.

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

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.

Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle.

In the present study, we monitored Foxp3(+) T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3(+) CD4(+) cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3(+) CD4(+) cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3(+) CD4(+) T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.