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Rabies is a fatal neurological disorder caused by rabies virus (RABV) infection. Approximately 60,000 patients die from rabies annually, and there are no effective treatments for this disease. Nucleoside analogs are employed as antiviral drugs based on their broad antiviral spectrum, and certain nucleoside analogs have been reported to exhibit anti-RABV activity. The nucleoside analog β-d-N 4 -hydroxycytidine (NHC) has antiviral effects against a range of RNA viruses. Molnupiravir (MPV), a prodrug of NHC, is clinically used as an oral antiviral drug for coronavirus infections. Despite its broad-spectrum activity, the antiviral activity of NHC against RABV remains unclear. In this study, we reveal that NHC exhibits comparable in vitro anti-RABV activity as ribavirin and favipiravir (also known as T-705) with a 90% effective concentration of 6 μM in mouse neuroblastoma cells. NHC reduced viral loads in neuronal and nonneuronal cells in a dose-dependent manner. Both laboratory and field RABVs (fixed and street strains, respectively) were susceptible to NHC. However, no increase in survival or reduction in viral titers in the brain was observed in RABV-infected mice treated prophylactically with MPV. These findings highlight the potential and challenges of NHC in the treatment of RABV infection., Competing Interests: Declaration of competing interest All experiments reported in this paper was financially supported by Shionogi & Co., Ltd. The authors K.K., S.K., S.T. and A.S. are employees of Shionogi & Co., Ltd. The remaining authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health., Competing Interests: Declaration of competing interest Authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objectives: Human Leukocyte Antigen HLA-B*57:01 and B*58:01 are considered anti-HIV-1 protective alleles. HLA-B*57:01/58:01-restricted HIV-1 Gag TW10 (TSTLQEQIGW, Gag residues 240-249) epitope-specific CD8 + T cell responses that frequently select for a Gag escape mutation, T242N, with viral fitness cost are crucial for HIV-1 control. Although this finding has been observed in cohorts where HIV-1 subtype B or C predominates, the protective impact of HLA-B*57:01/58:01 has not been reported in Southeast Asian countries where HIV-1 CRF01_AE is the major circulating strain. Here, the effect of HLA-B*57:01/58:01 on CRF01_AE infection was investigated., Methods: The correlation of HLA-B*57:01/58:01 with viral load and CD4 counts were analyzed in the CRF01_AE-infected Vietnamese cohort (N = 280). The impact of the T242N mutation on CRF01_AE replication capacity was assessed., Results: HLA-B*57:01/58:01-positive individuals mostly had HIV-1 with T242N (62/63) but showed neither a significant reduction in viral load nor increased CD4 counts relative to B*57:01/58:01-negative participants. In vitro and in vivo analyses revealed a significant reduction in viral fitness of CRF01_AE with T242N. In silico analysis indicated reduced presentation of epitopes in the context of CRF01_AE compared to subtype B or C in 10/16 HLA-B*57:01/58:01-restricted HIV-1 epitopes., Conclusion: The protective impact of HLA-B*57:01/58:01 on CRF01_AE infection is impaired despite strong suppressive pressure by TW10-specific CD8 + T cells., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The genus Flavivirus includes pathogenic tick- and mosquito-borne flaviviruses as well as non-pathogenic insect-specific flaviviruses (ISFVs). Phylogenetic analysis based on whole amino acid sequences has indicated that lineage II ISFVs have similarities to pathogenic flaviviruses. In this study, we used reactive analysis with immune serum against Psorophora flavivirus (PSFV) as a lineage IIa ISFV, and Barkeji virus (BJV) as a lineage IIb ISFV, to evaluate the antigenic similarity among lineage IIa and IIb ISFVs, and pathogenic mosquito-borne flaviviruses (MBFVs). Binding and antibody-dependent enhancement assays showed that anti-PSFV sera had broad cross-reactivity with MBFV antigens, while anti-BJV sera had low cross-reactivity. Both of the lineage II ISFV antisera were rarely observed to neutralize MBFVs. These results suggest that lineage IIa ISFV PSFV has more antigenic similarity to MBFVs than lineage IIb ISFV BJV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2., Competing Interests: Declaration of competing interest Kentaro Uemura, Shinsuke Toba, Takao Sanaki and Akihiko Sato are employees of Shionogi & Co., Ltd. Other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The extensive sequence data generated from SARS-CoV-2 during the 2020 pandemic has facilitated the study of viral genome evolution over a brief period of time. This has highlighted instances of directional mutation pressures exerted on the SARS-CoV-2 genome from host antiviral defense systems. In this brief review we describe three such human defense mechanisms, the apolipoprotein B mRNA editing catalytic polypeptide-like proteins (APOBEC), adenosine deaminase acting on RNA proteins (ADAR), and reactive oxygen species (ROS), and discuss their potential implications on SARS-CoV-2 evolution., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC 50 s of Compound-X and Compound-Y against DENV2 were 3.9 μM and 9.2 μM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The SARS-CoV-2 pathogen has established endemicity in humans. This necessitates the development of rapid genetic surveillance methodologies to serve as an adjunct with existing comprehensive, albeit though slower, genome sequencing-driven approaches., Methods: A total of 21,789 complete genomes were downloaded from GISAID on May 28, 2020 for analyses. We have defined the major clades and subclades of circulating SARS-CoV-2 genomes. A rapid sequencing-based genotyping protocol was developed and tested on SARS-CoV-2-positive RNA samples by next-generation sequencing., Results: We describe 11 major mutations which defined five major clades (G 614 , S 84 , V 251 , I 378 and D 392 ) of globally circulating viral populations. The clades can specifically identify using an 11-nucleotide genetic barcode. An analysis of amino acid variation in SARS-CoV-2 proteins provided evidence of substitution events in the viral proteins involved in both host entry and genome replication., Conclusion: Globally circulating SARS-CoV-2 genomes could be classified into 5 major clades based on mutational profiles defined by an 11-nucleotide barcode. We have successfully developed a multiplexed sequencing-based, rapid genotyping protocol for high-throughput classification of major clade types of SARS-CoV-2 in clinical samples. This barcoding strategy will be required to monitor decreases in genetic diversity as treatment and vaccine approaches become widely available., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host major histocompatibility complex class I (MHC-I) genotypes have a great impact on viral replication and MHC-I-associated viral genome mutations are selected under CD8 + T-cell pressure. Association of MHC-I genotypes with HIV/SIV control has been investigated at MHC-I allele levels but not fully at haplotype levels. We previously established groups of rhesus macaques sharing individual MHC-I haplotypes. In the present study, we compared viral genome diversification after SIV infection in macaques possessing a protective MHC-I haplotype, 90-010-Id, with those possessing a non-protective MHC-I haplotype, 90-010-Ie. These two MHC-I haplotypes are associated with immunodominant CD8 + T-cell responses targeting similar regions of viral Nef antigen. Analyses of viral genome sequences and antigen-specific T-cell responses showed four and two candidates of viral CD8 + T-cell targets associated with 90-010-Id and 90-010-Ie, respectively, in addition to the Nef targets. In these CD8 + T-cell target regions, higher numbers of mutations were detected at the setpoint after SIV infection in macaques possessing 90-010-Id than those possessing 90-010-Ie. These results indicate higher selective pressure on overall CD8 + T-cell targets associated with the protective MHC-I haplotype, suggesting a pattern of HIV/SIV control by multiple target-specific CD8 + T-cell responses., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever/dengue shock syndrome. At present, no antiviral drugs are available for treatment DENV infections. In this study, a screening system based on a DENV-infected cell-based assay identified a novel anti-DENV agent with a benzimidazole skeleton, named Compound-B, which demonstrated antiviral activity specific to four DENV serotypes (EC 50 : 1.32-4.12 μM). Analysis of a single amino acid substitution of Compound-B-resistant DENV2 revealed that mutation C87S in the non-structural protein 4A (NS4A) contributes to resistance to Compound-B., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The HTLV-1 oncoprotein Tax plays an important role in ATL pathogenesis. ATL carries a poor prognosis due to chemotherapy resistance, stressing the need for alternative therapies. Here, we investigate the preclinical efficacy of the synthetic retinoid ST1926 in ATL and peripheral T-cell lymphomas. Clinically achievable concentrations of ST1926 induced a dramatic inhibition of cell proliferation in malignant T-cell lines and primary ATL cells with minimal effect on resting or activated normal lymphocytes. ST1926 induced apoptosis, DNA damage, and upregulation of p53 proteins in malignant T cells, whereas it caused an early downregulation of Tax proteins in HTLV-1-positive cells. In murine ATL, oral treatment with ST1926 prolonged survival and reduced leukemia cell infiltration, white blood cell counts, and spleen mass. In spleens of ST1926-treated animals, p53 and p21 proteins were upregulated, poly (ADP-ribose) polymerase was cleaved, and Tax transcripts were reduced. These results highlight the promising use of ST1926 as a targeted therapy for ATL., (© 2014 by The American Society of Hematology.)

Adult T-cell leukemia/lymphoma (ATL) is a highly invasive and intractable T-cell malignancy caused by human T-cell leukemia virus-1 infection. We demonstrate herein that normal tissue-derived epithelial cells (NECs) exert protective effects on the survival of leukemic cells, which may partially account for high resistance to antileukemic therapies in patients with ATL. Viral gene-silenced, ATL-derived cell lines (ATL cells) dramatically escaped from histone deacetylase inhibitor-induced apoptosis by direct co-culture with NECs. Adhesions to NECs suppressed p21(Cip1) expression and increased a proportion of resting G0/G1 phase cells in trichostatin A (TSA)-treated ATL cells. ATL cells adhering to NECs down-regulated CD25 expression and enhanced vimentin expression, suggesting that most ATL cells acquired a quiescent state by cell-cell interactions with NECs. ATL cells adhering to NECs displayed highly elevated expression of the cancer stem cell marker CD44. Blockade of CD44 signaling diminished the NEC-conferred resistance of ATL cells to TSA-induced apoptosis. Co-culture with NECs also suppressed the expression of NKG2D ligands on TSA-treated ATL cells, resulting in decreased natural killer cell-mediated cytotoxicity. Combined evidence suggests that interactions with normal epithelial cells augment the resistance of ATL cells to TSA-induced apoptosis and facilitate immune evasion by ATL cells., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine.

Adult T-cell leukemia (ATL) is a T-cell malignancy caused by human T lymphotropic virus type I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have used mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice reproduces a leukemia and lymphoma that is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to stromal cell-derived factor-1alpha (SDF-1alpha). Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1alpha. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1alpha-induced migration and phosphorylation of extracellular signal-related kinase 1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Using the SCID mouse model, it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1alpha/CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL.

Adult T-cell leukemia/lymphoma (ATL) is a malignant lymphoproliferative disorder caused by HTLV-I infection. In ATL, chemotherapeutic responses are generally poor, which has suggested the existence of chemotherapy-resistant cancer stem cells (CSCs). To identify CSC candidates in ATL, we have focused on a Tax transgenic mouse (Tax-Tg) model, which reproduces ATL-like disease both in Tax-Tg animals and also after transfer of Tax-Tg splenic lymphomatous cells (SLCs) to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Using a limiting dilution transplantation, it was estimated that one CSC existed per 10(4) SLCs (0.01%). In agreement with this, we have successfully identified candidate CSCs in a side population (0.06%), which overlapped with a minor population of CD38(-)/CD71(-)/CD117(+) cells (0.03%). Whereas lymphoma did not develop after transplantation of 10(2) SLCs, 10(2) CSCs could consistently regenerate the original lymphoma. In addition, lymphoma and CSCs could also be demonstrated in the bone marrow and CD117(+) CSCs were observed in both osteoblastic and vascular niches. In the CSCs, Tax, Notch1, and Bmi1 expression was down-regulated, suggesting that the CSCs were derived from Pro-T cells or early hematopoietic progenitor cells. Taken together, our data demonstrate that CSCs certainly exist and have the potential to regenerate lymphoma in our mouse model.

HIV-1 genome has an AU-rich sequence and requires rapid nuclear export by Rev activity to prevent multiple splicing. HIV-1 infection occurs in activated CD4(+) T cells where the decay of mRNAs of cytokines and chemokines is regulated by the binding of AU-rich elements to the mRNA-destabilizing protein tristetraprolin. We here investigated the influence of tristetraprolin on the replication of HIV-1. Treatment of siRNA against tristetraprolin in a latently HIV-1 infected cell line increases HIV-1 production following stimulation. A chloramphenicol acetyltransferase and luciferase assay revealed that exogenous tristetraprolin reduced HIV-1 virion production and in contrast increased the multiply spliced products. Furthermore, quantitative RT-PCR analysis showed tristetraprolin increases the ratio of multiple-spliced RNAs to un-, single-spliced RNA. Moreover, an electrophoretic mobility shift assay showed that tristetraprolin binds to synthesized HIV-1 RNA with AU-rich sequence but not to RNA with less AU sequence. These results suggest that tristetraprolin is a regulator of HIV-1 replication and enhances splicing by direct binding to AU-rich sequence of HIV-1 RNAs.

AU-rich elements (AREs) in the 3'-untranslated region of mRNAs promote rapid decay of the mRNAs for certain cytokines, including that encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that an RNA molecule based on the ARE of GM-CSF mRNA is cleaved between U and A residues in the presence of bovine serum albumin of which cleavage effect is attenuated by acetylation. Furthermore, the expression of RNA molecule containing the ARE of GM-CSF mRNA in human cell lines was increased by inhibition of histone deacetylase activity and attenuation of Dicer expression. These findings suggest that degradation of mRNAs containing an ARE might be regulated by positive charge of polypeptides and Dicer.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1., ((c) 2002 Elsevier Science (USA).)

A human immunodeficiency virus type 1 (HIV-1) particle contains approximately 1200 molecules of gag proteins and two copies of a 9.2-kb genomic RNA which has been reported to be dimerized and rapidly cleaved and to form a complex with a nucleocapsid protein, p7 (NCp7), during viral budding. These suggest that the cleavage can be reconstituted with gag proteins in vitro. Here we show that the p15(gag) coding region of viral RNA is fragmented in viral particles and that in vitro-synthesized RNA transcripts of HIV-1 undergo cleavage which is activated by NCp7 and other factors. Single-stranded oligoribonucleotides were cleaved between C and A or U and A, leaving 2',3'-cyclic phosphate and 5'-hydroxyl termini. These findings might explain the rapid degradation of genomic RNAs in HIV-1 particles., ((c) 2002 Elsevier Science (USA).)