ß-d-N4-hydroxycytidine, a metabolite of molnupiravir, exhibits in vitro antiviral activity against rabies virus.

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 ß-d-N4-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.

Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19.

One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs for given therapeutic targets. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified therapeutics for cancer, lifestyle related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. We envision that LIGHTHOUSE will help accelerate drug discovery and fill the gap between bench side and bedside.

Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for Severe Acute Respiratory Syndrome Coronavirus 2 3CL Protease.

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLpro that possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a (YH-6) with the R configuration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6 formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6 suggest its potential as a lead compound for the treatment of COVID-19.

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

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

Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

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.

RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung cells.

Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor-mediated innate immunity1,2. Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid-inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3' untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all-trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells.

Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin.

The genus Flavivirus includes a range of mosquito-specific viruses in addition to well-known medically important arboviruses. Isolation and comprehensive genomic analyses of viruses in mosquitoes collected in Bolivia resulted in the identification of three novel flavivirus species. Psorophora flavivirus (PSFV) was isolated from Psorophora albigenu. The coding sequence of the PSFV polyprotein shares 60 % identity with that of the Aedes-associated lineage II insect-specific flavivirus (ISF), Marisma virus. Isolated PSFV replicates in both Aedes albopictus- and Aedes aegypti-derived cells, but not in mammalian Vero or BHK-21 cell lines. Two other flaviviruses, Ochlerotatus scapularis flavivirus (OSFV) and Mansonia flavivirus (MAFV), which were identified from Ochlerotatus scapularis and Mansonia titillans, respectively, group with the classical lineage I ISFs. The protein coding sequences of these viruses share only 60 and 40 % identity with the most closely related of known lineage I ISFs, including Xishuangbanna aedes flavivirus and Sabethes flavivirus, respectively. Phylogenetic analysis suggests that MAFV is clearly distinct from the groups of the current known Culicinae-associated lineage I ISFs. Interestingly, the predicted amino acid sequence of the MAFV capsid protein is approximately two times longer than that of any of the other known flaviviruses. Our results indicate that flaviviruses with distinct features can be found at the edge of the Bolivian Amazon basin at sites that are also home to dense populations of human-biting mosquitoes.

Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells.

Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E-18; false discovery rate [FDR] 3.11E-15) and the ER unfolded protein response (P value: 7.58E-18; FDR 3.11E-15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection.

Genetic diversity of rabies virus in different host species and geographic regions of Zambia and Zimbabwe.

Rabies is endemic in Zambia and Zimbabwe. The previously investigated strains of rabies virus in central Zambia belong to the Africa 1b lineage, with similar circulating virus strains found in the various tested hosts and regions. However, prior work assessed only limited regions and host species. Thus, this study aimed to more comprehensively determine the genetic diversity of rabies virus across regions of Zambia and Zimbabwe. RNA (n = 76) was extracted from positive direct fluorescent antibody test brain tissues from dog, cow, goat, cat, pig, human, and jackal collected from Zambia and Zimbabwe. The amplicons of the nucleoprotein and glycoprotein genes were obtained from all examined samples by nested RT-PCR and subsequently sequenced. A phylogenetic analysis of the N gene confirmed that all the endemic strains of rabies virus in Zambia and Zimbabwe belong to the Africa 1b lineage. The obtained viral gene sequences were phylogenetically divided into two clusters. Cluster II comprised only Zambian strains. In contrast, cluster I comprised both Zambia and Zimbabwe strains, with strains from Zimbabwe forming a distinct lineage from Zambian strains, implying viral genetic divergence due to geographical barriers. However, no evidence of clustering based on host or region was observed, implying the circulation of similar virus strains occurs in different hosts and regions of Zambia and Zimbabwe. The clustering of rabies virus strains from jackals with those from domestic animals provides evidence of similar virus strains circulating in both wildlife and domestic animals, and that the jackal might be one of the potential reservoirs of rabies virus infection. In this study, no strains circulating in Zimbabwe were detected in Zambia.

The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection.

Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.

Ribavirin-related compounds exert in vitro inhibitory effects toward rabies virus.

Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.

Detection of novel gammaherpesviruses from fruit bats in Indonesia.

Bats are an important natural reservoir of zoonotic viral pathogens. We previously isolated an alphaherpesvirus in fruit bats in Indonesia, and here establish the presence of viruses belonging to other taxa of the family Herpesviridae. We screened the same fruit bat population with pan-herpesvirus PCR and discovered 68 sequences of novel gammaherpesvirus, designated 'megabat gammaherpesvirus' (MgGHV). A phylogenetic analysis of approximately 3.4 kbp of continuous MgGHV sequences encompassing the glycoprotein B gene and DNA polymerase gene revealed that the MgGHV sequences are distinct from those of other reported gammaherpesviruses. Further analysis suggested the existence of co-infections of herpesviruses in Indonesian fruit bats. Our findings extend our understanding of the infectious cycles of herpesviruses in bats in Indonesia and the phylogenetic diversity of the gammaherpesviruses.

Identification of the same polyomavirus species in different African horseshoe bat species is indicative of short-range host-switching events.

Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.

Discovery of African bat polyomaviruses and infrequent recombination in the large T antigen in the Polyomaviridae.

Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.

Detection of novel polyomaviruses in fruit bats in Indonesia.

Bats are an important natural reservoir for a variety of viral pathogens, including polyomaviruses (PyVs). The aims of this study were: (i) to determine which PyVs are present in bats in Indonesia and (ii) to analyze the evolutionary relationships between bat PyVs and other known PyVs. Using broad-spectrum polymerase chain reaction (PCR)-based assays, we screened PyV DNA isolated from spleen samples from 82 wild fruit bats captured in Indonesia. Fragments of the PyV genome were detected in 10 of the 82 spleen samples screened, and eight full-length viral genome sequences were obtained using an inverse PCR method. A phylogenetic analysis of eight whole viral genome sequences showed that BatPyVs form two distinct genetic clusters within the proposed genus Orthopolyomavirus that are genetically different from previously described BatPyVs. Interestingly, one group of BatPyVs is genetically related to the primate PyVs, including human PyV9 and trichodysplasia spinulosa-associated PyV. This study has identified the presence of novel PyVs in fruit bats in Indonesia and provides genetic information about these BatPyVs.

Role of the C-terminal region of vervet monkey polyomavirus 1 VP1 in virion formation.

Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (ΔC VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and ΔC VP1 VLPs were similar in size, but the number of ΔC VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation.

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection.

Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcAß1-3GalNAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV.

Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D.

The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.