Virus Genus Research Articles

Experimental evolution at ecological scales allows linking of viral genotypes to specific host strains.

Viruses shape microbial community structure and activity through the control of population diversity and cell abundances. Identifying and monitoring the dynamics of specific virus-host pairs in nature is hampered by the limitations of culture-independent approaches such as metagenomics, which do not always provide strain-level resolution, and culture-based analyses, which eliminate the ecological background and in-situ interactions. Here, we have explored the interaction of a specific "autochthonous" host strain and its viruses within a natural community. Bacterium Salinibacter ruber strain M8 was spiked into its environment of isolation, a crystallizer pond from a coastal saltern, and the viral and cellular communities were monitored for one month using culture, metagenomics, and microscopy. Metagenome sequencing indicated that the M8 abundance decreased sharply after being added to the pond, likely due to forces other than viral predation. However, the presence of M8 selected for two species of a new viral genus, Phoenicisalinivirus, for which 120 strains were isolated. During this experiment, an assemblage of closely related viral genomic variants was replaced by a single population with the ability to infect M8, a scenario which was compatible with the selection of a genomic variant from the rare biosphere. Further analysis implicated a viral genomic region putatively coding for a tail fiber protein to be responsible for M8 specificity. Our results indicate that low abundance viral genotypes provide a viral seed bank that allows for a highly specialized virus-host response within a complex ecological background.

A novel Alteromonas phage with tail fiber containing six potential iron-binding domains.

Viruses play a vital role in regulating microbial communities, contributing to biogeochemical cycles of carbon, nitrogen, and essential metals. Alteromonas is widespread and plays an essential role in marine microbial ecology. However, there is limited knowledge about the interactions of Alteromonas and its viruses (alterophages). This study isolated a novel podovirus, vB_AmeP-R22Y (R22Y), which infects Alteromonas marina SW-47 (T). Phylogenetic analysis suggested that R22Y represented a novel viral genus within the Schitoviridae family. R22Y exhibited a broad host range and a relatively large burst size, exerting an important impact on the adaptability and dynamics of host populations. Two auxiliary metabolic genes, encoding Acyl carrier protein and AAA domain-containing protein, were predicted in R22Y, which may potentially assist in host fatty acid metabolism and VB12 biosynthesis, respectively. Remarkably, the prediction of the R22Y tail fiber structure revealed six conserved histidine residues (HxH motifs) that could potentially bind iron ions, suggesting that alterophages may function as organic iron-binding ligands in the marine environment. Our isolation and characterization of R22Y complements the Trojan Horse hypothesis, proposes the possible role of alterophages for marine iron biogeochemical cycling, and provides new insights into phage-host interactions in the iron-limited ocean.IMPORTANCEIron (Fe), as an essential micronutrient, is often a limiting factor for microbial growth in marine ecosystems. The Trojan Horse hypothesis suggests that iron in the phage tail fibers is recognized by the host's siderophore-bound iron receptor, enabling the phage to attach and initiate infection. The potential role of phages as iron-binding ligands has significant implications for oceanic trace metal biogeochemistry. In this study, we isolated a new phage R22Y with the potential to bind iron ions, using Alteromonas, a major siderophore producer, as the host. The tail fiber structure of R22Y exhibits six conserved HxH motifs, suggesting that each phage could potentially bind up to 36 iron ions. R22Y may contribute to colloidal organically complexed dissolved iron in the marine environment. This finding provides further insights into the Trojan Horse hypothesis, suggesting that alterophages may act as natural iron-binding ligands in the marine environment.

Recent Advances in Therapeutic Approaches Against Ebola Virus Infection.

Ebola virus (EBOV) is a genus of negative-strand RNA viruses belonging to the family Filoviradae that was first described in 1976 in the present-day Democratic Republic of the Congo. It has intermittently affected substantial human populations in West Africa and presents itself as a global health menace due to the high mortality rate of patients, high transmission rate, difficult patient management, and the emergence of complicated autoimmune disease-like conditions post-infection. EBOV or other EBOV-like species as a biochemical weapon pose a significant risk; hence, the need to develop both prophylactic and therapeutic medications to combat the virus is unquestionable. In this review work, we have compiled the literature pertaining to transmission, pathogenesis, immune response, and diagnosis of EBOV infection. We included detailed structural details of EBOV along with all the available therapeutics against EBOV disease. We have also highlighted current developments and recent advances in therapeutic approaches against Ebola virus disease (EVD). The development of preventive vaccines against the virus is proving to be a successful effort as of now; however, problems concerning logistics, product stability, multi- dosing, and patient tracking are prominent in West Africa. Monoclonal antibodies that target EBOV proteins have also been developed and approved in the clinic; however, no small drug molecules that target these viral proteins have cleared clinical trials. An understanding of clinically approved vaccines and their shortcomings also serves an important purpose for researchers in vaccine design in choosing the right vector, antigen, and particular physicochemical properties that are critical for the vaccine's success against the virus across the world. Our work brings together a comprehensive review of all available prophylactic and therapeutic medications developed and under development against the EBOV, which will serve as a guide for researchers in pursuing the most promising drug discovery strategies against the EBOV and also explore novel mechanisms of fighting against EBOV infection.

Phages ZC01 and ZC03 require type-IV pilus for Pseudomonas aeruginosa infection and have a potential for therapeutic applications.

There has been a growing interest in bacteriophages as therapeutic agents to treat multidrug-resistant bacterial infections. The present work aimed at expanding the microbiological and molecular characterization of lytic phages ZC01 and ZC03 and investigating their efficacy in the control of Pseudomonas aeruginosa infection in an invertebrate animal model. These two phages were previously isolated from composting using P. aeruginosa strain PA14 as the enrichment host and had their genomes sequenced. ZC01 and ZC03 present, respectively, siphovirus and podovirus morphotypes. ZC01 was recently classified into the genus Abidjanvirus, while ZC03 belongs to Zicotriavirus genus of the Schitoviridae N4-like viruses. Through proteomics analysis, we identified virion structural proteins of ZC01 and ZC03, including a large virion-associated RNA polymerase that is characteristic of N4-like viruses, some hypothetical proteins whose annotation should be changed to virion structural proteins and a putative peptidoglycan hydrolase. Phages ZC01 and ZC03 exhibit a limited yet distinct host range, with moderate to high efficiency of plating (EOP) values observed for a few P. aeruginosa clinical isolates. Phage susceptibility assays in PA14 mutant strains point to the type-IV pilus (T4P) as the primary receptor for phages ZC01 and ZC03, and the major pilin (PilAPA14) is the T4P component recognized by these phages. Moreover, both phages significantly increase survival of Galleria mellonella larvae infected with PA14 strain. Taken together, these results underpin the therapeutic potential of these phages to treat infections by P. aeruginosa and lay the groundwork for a more detailed investigation of phage-bacteria-specific recognition mechanisms.IMPORTANCEPhage therapy is gaining increasing interest in cases of difficult-to-treat bacterial human infections, such as carbapenem-resistant Pseudomonas aeruginosa. In this work, we investigated the molecular mechanism underlying the interaction of the lytic phages ZC01 and ZC03 with the highly virulent P. aeruginosa PA14 strain and their efficacy to treat PA14 infection in Galleria mellonella larvae, a commonly used invertebrate model for phage therapy. We depicted the protein composition of ZC01 and ZC03 viral particles and identified pilin A, the major component of type-4 pilus, as the receptor recognized by these phages. Our findings indicate that phages ZC01 and ZC03 may be further used for developing therapies to treat multidrug-resistant P. aeruginosa infections.

Surface microlayer-mediated virome dissemination in the Central Arctic

BackgroundAquatic viruses act as key players in shaping microbial communities. In polar environments, they face significant challenges such as limited host availability and harsh conditions. However, due to the restricted accessibility of these ecosystems, our understanding of viral diversity, abundance, adaptations, and host interactions remains limited.ResultsTo fill this knowledge gap, we studied viruses from atmosphere-close aquatic ecosystems in the Central Arctic and Northern Greenland. Aquatic samples for virus-host analysis were collected from ~60 cm depth and the submillimeter surface microlayer (SML) during the Synoptic Arctic Survey 2021 on icebreaker Oden in the Arctic summer. Water was sampled from a melt pond and open water before undergoing size-fractioned filtration, followed by genome-resolved metagenomic and cultivation investigations. The prokaryotic diversity in the melt pond was considerably lower compared to that of open water. The melt pond was dominated by a Flavobacterium sp. and Aquiluna sp., the latter having a relatively small genome size of 1.2 Mb and the metabolic potential to generate ATP using the phosphate acetyltransferase-acetate kinase pathway. Viral diversity on the host fraction (0.2–5 µm) of the melt pond was strikingly limited compared to that of open water. From the 1154 viral operational taxonomic units (vOTUs), of which two-thirds were predicted bacteriophages, 17.2% encoded for auxiliary metabolic genes (AMGs) with metabolic functions. Some AMGs like glycerol-3-phosphate cytidylyltransferase and ice-binding like proteins might serve to provide cryoprotection for the host. Prophages were often associated with SML genomes, and two active prophages of new viral genera from the Arctic SML strain Leeuwenhoekiella aequorea Arc30 were induced. We found evidence that vOTU abundance in the SML compared to that of ~60 cm depth was more positively correlated with the distribution of a vOTU across five different Arctic stations.ConclusionsThe results indicate that viruses employ elaborate strategies to endure in extreme, host-limited environments. Moreover, our observations suggest that the immediate air-sea interface serves as a platform for viral distribution in the Central Arctic.Dvb_jZZ1H7pg55rxrDSVb2Video

Viral Diversity in Mixed Tree Fruit Production Systems Determined through Bee-Mediated Pollen Collection

Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple (Malus domestica), another member of the Rosacea and distantly related to Prunus, can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee (Apis mellifera) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus, family Bromoviridae), Secoviridae family members tomato necrotic ringspot virus (genus Nepovirus), tobacco necrotic ringspot virus (genus Nepovirus), prunus virus F (genus Fabavirus), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

HuR (ELAVL1) regulates the CCHFV minigenome and HAZV replication by associating with viral genomic RNA.

Crimean-Congo Hemorrhagic Fever virus (CCHFV) is a tick-borne pathogen that causes severe acute fever disease in humans and requires a biosafety level 4 laboratory for handling. Hazara virus (HAZV), belonging to the same virus genus as CCHFV, does not exhibit pathogenesis in humans. To investigate host RNA-binding proteins (RBPs) that regulate CCHFV replication, we generated a series of mutant RAW264.7 cells by CRISPR/Cas9 system and these cells were infected with HAZV. The viral titers in the supernatant of these cells was investigated, and HuR (ELAVL1) was identified. HuR KO RAW264.7 cells reduced HAZV replication. HuR is an RBP that enhances mRNA stability by binding to adenyl-uridine (AU)-rich regions in their 3' non-coding region (NCR). HuR regulates innate immune response by binding to host mRNAs of signaling molecules. The expression of cytokine genes such as Ifnb, Il6, and Tnf was reduced in HuR KO cells after HAZV infection. Although HuR supports the innate immune response during HAZV infection, we found that innate immune activation by HAZV infection did not affect its replication. We then investigated whether HuR regulates HAZV genome RNA stability. HAZV RNA genome was precipitated with an anti-HuR antibody, and HAZV genome RNA stability was lowered in HuR KO cells. We found that HuR associated with HAZV RNA and stabilized it to enhance HAZV replication. Furthermore, HuR-deficiency reduced CCHFV minigenome replication. CCHFV is a negative-strand RNA virus and positive-strand RNA is produced during replication. HuR was associated with positive-strand RNA rather than negative-strand RNA, and AU-rich region in 3'-NCR of S segment was responsible for immunoprecipitation with anti-HuR antibody and minigenome replication. Additionally, HuR inhibitor treatment reduced CCHFV minigenome replication. Our results indicate that HuR aids replication of the CCHFV minigenome by associating with the AU-rich region in the 3'-NCR.

Coxsackievirus B3 Activates Macrophages Independently of CAR-Mediated Viral Entry.

Enteroviruses are a genus of small RNA viruses that are responsible for approximately one billion global infections annually. These infections range in severity from the common cold and flu-like symptoms to more severe diseases, such as viral myocarditis, pancreatitis, and neurological disorders, that continue to pose a global health challenge with limited therapeutic strategies currently available. In the current study, we sought to understand the interaction between coxsackievirus B3 (CVB3), which is a model enterovirus, and macrophage cells, as there is limited understanding of how this virus interacts with macrophage innate immune cells. Our study demonstrated that CVB3 can robustly activate macrophages without apparent viral replication in these cells. We also showed that myeloid cells lacked the viral entry receptor coxsackievirus and adenovirus receptor (CAR). However, the expression of exogenous CAR in RAW264.7 macrophages was unable to overcome the viral replication deficit. Interestingly, the CAR expression was associated with altered inflammatory responses during prolonged infection. Additionally, we identified the autophagy protein LC3 as a novel stimulus for macrophage activation. These findings provide new insights into the mechanisms of CVB3-induced macrophage activation and its implications for viral pathogenesis.

274 Evaluation of recombinant Capripoxvirus antigens in the development of indirect enzyme linked immunosorbent assays

Abstract Capripoxvirus is a genus of DNA viruses that includes sheep pox virus, goat pox virus and lumpy skin disease virus (LSDV). These viruses are considered high consequence viruses due to the severe clinical disease leading to economic losses to production and impact on trade. The recent spread of LSDV in Europe and Asia has demonstrated the transboundary nature of LSDV. The vaccines used to control Capripoxvirus infections currently do not have the ability to differentiate infected from vaccinated animals (DIVA) and the enzyme linked immunosorbent assays (ELISA) available for detection of antibodies to Capripoxvirus infections are not ideal. Previous research has been done to evaluate different Capripoxvirus antigens using E. coli expressed antigens; however, there has been no prior evaluation of baculovirus expressed Capripoxvirus antigens for use in ELISA. The objective of the current research was to produce 6 recombinant Capripoxvirus antigens using a baculovirus expression vector system (BEVS) and to evaluate these antigens in ELISAs to test bovine, sheep, and goat serum. The sequences used to produce the recombinant antigens were based on a consensus obtained using reference genomes consisting of all three viruses within the genus. All six antigens were produced and purified in-house using BEVS followed by benchtop or FPLC purification (Figure 1). To date, the antigens have been fully validated for performance in indirect ELISA using sheep sera (Figure 2) and partially using cattle sera, with goat sera validation to follow. The ELISAs were developed and optimized using known reference sera as controls. The performance of the ELISAs developed was assessed in comparison with the only commercially available Capripoxvirus serological test. The results demonstrate that some of the antigens used for the ELISAs completely outperformed the commercial test. Several antigens were found to be useful for ELISAs to detect responses in sheep and cattle sera. The sensitivities and specificities of the different ELISAs for sheep sera ranged from 99.54% to 53.57% sensitivity and 99.54 to 94.95% specificity with likelihood ratios above 10 using receiver operating characteristic curve analysis, while the commercial kit had a 42.85% sensitivity using the same positive sheep serum panel evaluated. The development of these serological assays allows for the simultaneous evaluation of immunogenicity of Capripoxvirus antigens through the testing of serum from experimentally infected animals. This study demonstrates the proof of principle, that sensitive and specific ELISAs can be developed for Capripoxviruses.

Hantavirus Pulmonary Syndrome Outbreak Anticipation by a Rapid Synchronous Increase in Rodent Abundance in the Northwestern Argentina Endemic Region: Towards an Early Warning System for Disease Based on Climate and Rodent Surveillance Data.

Hantavirus pulmonary syndrome (HPS) is an American emerging disease caused by the rodent-borne virus genus Orthohantavirus (Family: Hantaviridae: Order: Elliovirales Class: Bunyaviricetes). In Argentina, almost half of the HPS infections occur in the northwestern endemic region. In this study, we monitored rodent abundance during 2022 and 2023 in three sites with different sampling methods (removal trapping, live trapping and hunted rodents by domestic cats) to evaluate their relationship with human infections. We found a similar pattern of variation in rodent abundance across time, and particularly a synchronous rise of rodent abundance that anticipated an HPS outbreak in 2023. Our dynamic regression models revealed a positive relationship between HPS cases and rodent abundance with a three-month lag, as well as rainfall with an eight-month lag. Our results provide a framework for the planning and implementation of public health prevention campaigns based on climatology and rodent monitoring. Domestic cats bringing rodents into houses can be an overlooked risk factor, particularly if viral shedding of infected rodents is magnified by stress. HPS is a disease of public health concern due to its high mortality rate, the lack of a specific therapeutic treatment and no vaccine. Thus, prevention of infections is of the utmost importance.

Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers

Single-stranded, positive-sense RNA ((+)RNA) viruses replicate their genomes in virus-induced intracellular membrane compartments. (+)RNA viruses dedicate a significant part of their small genomes (a few thousands to a few tens of thousands of bases) to the generation of these compartments by encoding membrane-interacting proteins and/or protein domains. Noroviruses are a very diverse genus of (+)RNA viruses including human and animal pathogens. Human noroviruses are the major cause of acute gastroenteritis worldwide, with genogroup II genotype 4 (GII.4) noroviruses accounting for the vast majority of infections. Three viral proteins encoded in the N-terminus of the viral replication polyprotein direct intracellular membrane rearrangements associated with norovirus replication. Of these three, nonstructural protein 4 (NS4) seems to be the most important, although its exact functions in replication organelle formation are unknown. Here we produce, purify and characterize GII.4 NS4. AlphaFold modeling combined with experimental data refine and correct our previous crude structural model of NS4. Using simple artificial liposomes, we report an extensive characterization of the membrane properties of NS4. We find that NS4 self-assembles and thereby bridges liposomes together. Cryo-EM, NMR and membrane flotation show formation of several distinct NS4 assemblies, at least two of them bridging pairs of membranes together in different fashions. Noroviruses belong to (+)RNA viruses whose replication compartment is extruded from the target endomembrane and generates double-membrane vesicles. Our data establish that the 21-kDa GII.4 human norovirus NS4 can, in the absence of any other factor, recapitulate in tubo several features, including membrane apposition, that occur in such processes.

Linc20486 promotes BmCPV replication through inhibiting the transcription of AGO2 and Dicers

The silkworm holds pivotal economic importance, serving not only as a primary source of silk but also as a prominent model organism in scientific research. Nonetheless, silkworm farming remains vulnerable to diverse factors, with viral infections posing the gravest threat to the sericulture industry. Among these, the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a member of the Reoviridae family and the cytoplasmic polyhedrosis virus genus, emerges as a significant pathogen in silkworm production. BmCPV infection primarily induces midgut sepsis in silkworms, spreads rapidly, and can inflict substantial economic losses on sericulture production. Presently, effective strategies for preventing and treating BmCPV infections are lacking.Long non-coding RNA (lncRNA) constitutes a class of RNA molecules with transcripts exceeding 200 nt, playing a crucial role in mediating the interplay between pathogens and host cells. Investigation through high-throughput technology has unveiled that BmCPV infection markedly upregulates the expression of Linc20486. This observation suggests potential involvement of Linc20486 in regulating virus replication. Indeed, as anticipated, knockdown of Linc20486 in cells profoundly impedes BmCPV replication, whereas overexpression significantly enhances virus propagation.To probe into the mechanism underlying Linc20486′s impact on virus replication, its effects on autophagy, innate immunity, and RNAi-related pathways were scrutinized. The findings revealed that Linc20486 exerts significant influence on the expression of RNAi pathway-related genes, such as Dicer1, Dicer2 and AGO2. This discovery holds promise for unveiling novel avenues to comprehend and combat BmCPV infections in silkworms.

Characterization and comparative genomic analysis of a marine Bacillus phage reveal a novel viral genus.

Although recent metagenomics research has obtained a wealth of phage genetic information, much of it is considered "dark matter" because of the lack of similarity with known sequences in the database. Therefore, the isolation and characterization of novel phages will help to interpret the vast unknown viral metagenome data and improve our understanding of phage diversity and phage-host interactions. Bacillus pumilus shows high economic relevance due to its wide applications in biotechnology, industry, biopharma, and environmental sectors. Since phages influence the abundance, metabolism, evolution, fitness, and ecological functions of bacteria through complex interactions, the significance of isolation and characterization of novel phages infecting B. pumilus is apparent. In this study, we isolated and characterized a B. pumilus phage belonging to a novel viral genus, which provides essential knowledge for phage biology as well as the industrial application of B. pumilus.

In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

Unveiling the genomic diversity and ecological impact of phage communities in hospital wastewater

Phages are pivotal in shaping microbial communities and biogeochemical cycles, while our understanding of the diversity, functions potential, and resistance gene carriage of phages in hospital wastewater (HWW) remains limited. We collected influent and effluent samples from the 3 hospital wastewater treatment plants (HWTPs) to assess the diversity and fate of phages, the interactions between phages and hosts, and the presence of resistance genes and auxiliary metabolic genes (AMGs) encoded by phages. Compared to influent, effluent showed reduced phage abundance and altered composition, with decreases in Microviridae and Inoviridae. The gene-sharing network highlights that many phages in HWW are not classified in known viral genera, suggesting HWW as a rich source of new viruses. There was a significant association between phages and microorganisms, with approximately 32.57 % of phages expected to be capable of infecting microbial hosts, characterized primarily by lytic activity. A total of 8 unique antibiotic resistance genes, 13 unique metal resistance genes, and 5 mobile genetic elements were detected in 3 HWTPs phageomes. Phage AMGs have the potential to influence carbon, nitrogen, phosphorus, and sulfur metabolism, impacting biogeochemical cycles. This study reveals the genomic diversity and ecological role of phages in HWTPs, highlighting their environmental and ecosystem impact.

Development of a Crystallographic Screening to Identify Sudan Virus VP40 Ligands.

The matrix protein VP40 of the highly pathogenic Sudan virus (genus Orthoebolavirus) is a multifunctional protein responsible for the recruitment of viral nucleocapsids to the plasma membrane and the budding of infectious virions. In addition to its role in assembly, VP40 also downregulates viral genome replication and transcription. VP40's existence in various homo-oligomeric states is presumed to underpin its diverse functional capabilities during the viral life cycle. Given the absence of licensed therapeutics targeting the Sudan virus, our study focused on inhibiting VP40 dimers, the structural precursors to critical higher-order oligomers, as a novel antiviral strategy. We have established a crystallographic screening pipeline for the identification of small-molecule fragments capable of binding to VP40. Dimeric VP40 of the Sudan virus was recombinantly expressed in bacteria, purified, crystallized, and soaked in a solution of 96 different preselected fragments. Salicylic acid was identified as a crystallographic hit with clear electron density in the pocket between the N- and the C-termini of the VP40 dimer. The binding interaction is predominantly coordinated by amino acid residues leucine 158 (L158) and arginine 214 (R214), which are key in stabilizing salicylic acid within the binding pocket. While salicylic acid displayed minimal impact on the functional aspects of VP40, we delved deeper into characterizing the druggability of the identified binding pocket. We analyzed the influence of residues L158 and R214 on the formation of virus-like particles and viral RNA synthesis. Site-directed mutagenesis of these residues to alanine markedly affected both VP40's budding activity and its effect on viral RNA synthesis, underscoring the potential of the salicylic acid binding pocket as a drug target. In summary, our findings lay the foundation for structure-guided drug design to provide lead compounds against Sudan virus VP40.

Prevalencia e infección mixta de virus en cultivos de maracuyá amarillo (Passiflora edulis f. flavicarpa) en el Valle del Cauca, Colombia

Yellow passion fruit crop is affected by multiple phytosanitary problems. Among the main ones are viruses of the genera Potyvirus (soybean mosaic virus, SMV), Cucumovirus (cucumber mosaic virus, CMV), Tymovirus (tymovirus passiflorae, PYMV) and Begomovirus (passionfruit leaf distortion virus, PLDV). Information about the prevalence and interaction between these viruses is scarce. The objective of this study was to verify the prevalence and identify the types of mixed viral infections of potyvirus, cucumovirus, tymovirus, and begomovirus in passion fruit crops in Valle del Cauca, Colombia. Passion fruit leaf samples with symptoms were collected and their nucleic acids were purified. Virus identification was performed by PCR using universal primers by viral genus, and specific primers for the SMV, CMV, PFYMV and PLDV viruses. The incidence of each virus by municipality was analyzed and the severity of the symptoms was compared with the diagnosis made. In total, 66 samples were collected in the municipalities of Toro, Dagua, Roldanillo, Bolívar, La Unión and El Cerrito. It was established that the most prevalent viruses are PLDV followed by SMV and CMV. The presence of PYMV was not detected in the samples analyzed. The presence of double and triple infections among potyvirus, cucumovirus and begomovirus were identified for the first time, finding greater severity of symptoms with a greater number of viruses identified per sample. This research provides key results for the design of control strategies for viral diseases in passion fruit.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

A parasite odyssey: An RNA virus concealed in Toxoplasma gondii.

We are entering a 'Platinum Age of Virus Discovery', an era marked by exponential growth in the discovery of virus biodiversity, and driven by advances in metagenomics and computational analysis. In the ecosystem of a human (or any animal) there are more species of viruses than simply those directly infecting the animal cells. Viruses can infect all organisms constituting the microbiome, including bacteria, fungi, and unicellular parasites. Thus the complexity of possible interactions between host, microbe, and viruses is unfathomable. To understand this interaction network we must employ computationally assisted virology as a means of analyzing and interpreting the millions of available samples to make inferences about the ways in which viruses may intersect human health. From a computational viral screen of human neuronal datasets, we identified a novel narnavirus Apocryptovirus odysseus (Ao) which likely infects the neurotropic parasite Toxoplasma gondii. Previously, several parasitic protozoan viruses (PPVs) have been mechanistically established as triggers of host innate responses, and here we present in silico evidence that Ao is a plausible pro-inflammatory factor in human and mouse cells infected by T. gondii. T. gondii infects billions of people worldwide, yet the prognosis of toxoplasmosis disease is highly variable, and PPVs like Ao could function as a hitherto undescribed hypervirulence factor. In a broader screen of over 7.6 million samples, we explored phylogenetically proximal viruses to Ao and discovered nineteen Apocryptovirus species, all found in libraries annotated as vertebrate transcriptome or metatranscriptomes. While samples containing this genus of narnaviruses are derived from sheep, goat, bat, rabbit, chicken, and pigeon samples, the presence of virus is strongly predictive of parasitic Apicomplexa nucleic acid co-occurrence, supporting the fact that Apocryptovirus is a genus of parasite-infecting viruses. This is a computational proof-of-concept study in which we rapidly analyze millions of datasets from which we distilled a mechanistically, ecologically, and phylogenetically refined hypothesis. We predict that this highly diverged Ao RNA virus is biologically a T. gondii infection, and that Ao, and other viruses like it, will modulate this disease which afflicts billions worldwide.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.