Virus Research Articles

Reverse Genetics System for Crimean-Congo Hemorrhagic Fever Virus.

Reverse genetic systems are powerful tools in molecular virology that allow the generation of infectious recombinant virus and the manipulation of viral genomes. Reverse genetic systems enable the incorporation of reporter genes, facilitating many virological assays, including high-throughput screening. Additionally, reverse genetic systems can be used to introduce targeted mutations into the viral genome, allowing investigations of viral genetic elements and protein functions in virus pathogenesis and biology. Here we describe in detail the materials and methods required for the Crimean-Congo hemorrhagic fever virus (CCHFV) reverse genetic system. This system can be used to generate complete infectious recombinant virus, and virus-like replicon particles (VRPs) lacking the M segment but complemented with anexogenous source of glycoprotein precursor (GPC); resulting in single-round replicon particles that can be used to study components of the viral replicative cycle at a lower biosafety level.

The Presence of Two Distinct Lineages of the Foot-And-Mouth Disease Virus Type A in Russia in 2013–2014 Has Significant Implications for the Epidemiology of the Virus in the Region

Molecular surveillance of FMD epidemiology is a fundamental tool for advancing our understanding of virus biology, monitoring virus evolution, and guiding vaccine design. The accessibility of genetic data will facilitate a more comprehensive delineation of FMDV phylogeny on a global scale. In this study, we investigated the FMDV strains circulating in Russia during the 2013–2014 period in geographically distant regions utilizing whole genome sequencing followed by maximum-likelihood phylogenetic reconstruction of whole genome and VP1 gene sequences. Phylogenetic analysis showed congruence in the topology of the phylogenetic trees constructed using the complete genome and VP1 gene sequence, clearly demonstrating that the isolates analyzed belong to two distinct genetic lineages: A/SEA97 in the Far East and Iran-05 in the North Caucasus. The A/SEA97 isolates exhibited a close genetic identity to those from China and Mongolia, whereas the Iran-05 isolates demonstrated clusterization with those from Turkey. The vaccine-matching studies with isolates from the Far East and North Caucasus revealed no antigenic homology with A/SEA-97 (r1 = 0.015–0.29) and A/Iran 05 (r1 = 0.009–0.17). The close genetic relationship of FMDV in the reported outbreak waves to those from neighboring countries indicates that animal movement could contribute to spillover and virus dispersal. The phylogenetic data reported here provide insight into the molecular epidemiology of FMD in the Eurasia region, elucidating the circulation pattern, molecular evolution, and genetic diversity, which is highly valuable for guiding vaccine designs and improving regional eradication policies.

Chemical and Biological Investigations of Antiviral Agents Against Plant Viruses Conducted in China in the 21st Century.

Research into the biology of plant viruses, their mechanisms of pathogenicity, and the induction of host resistance has laid a solid foundation for the discovery of antiviral agents and their targets and the development of effective control technologies. Additionally, recent advancements in fields such as chemical biology, cheminformatics, bioinformatics, and synthetic biology have provided valuable methods and tools for the design of antiviral drugs, the synthesis of drug molecules, assessment of their activity, and investigation of their modes of action. Compared with drug development for human viral diseases, the control of plant viral diseases presents greater challenges, including the cost-benefit of agents, simplification of control technologies, and the effectiveness of treatments. Therefore, in the current context of complex outbreaks and severe damage caused by plant viral diseases, it is crucial to delve deeper into the research and development of antiviral agents. This review provides a detailed overview of the biological characteristics of current targets for antiviral agents, the mode of interaction between plant virus targets and antivirals, and insights for future drug development. We believe this review will not only facilitate the in-depth analysis of the development of antivirals for crops but also offer valuable perspectives for the development of antiviral agents for use in human and veterinary medicine.

From viral assembly to host interaction: AFM's contributions to virology.

Viruses represent a diverse pool of obligate parasites that infect virtually every known organism, as such, their study is incredibly valuable for a range of fields including public health, medicine, agriculture, and ecology, and the development of biomedical technologies. Having evolved over millions of years, each virus has a unique and often complicated biology, that must be characterized on a case-by-case basis, even between strains of the same taxon. Owing to its nanoscale spatial resolution, atomic force microscopy (AFM) represents a powerful tool for exploring virus biology, including structural features, kinetics of binding to host cell ligands, virion self-assembly, and budding behaviors. Through the availability of numerous chemistries and advances in imaging modes, AFM is able to explore the complex web of host-virus interactions and life-cycle at a single virus level, exploring features at the level of individual bonds and molecules. Due to the wide array of techniques developed and data analysis approaches available, AFM can provide information that cannot be furnished by other modalities, especially at a single virus level. Here, we highlight the unique methods and information that can be obtained through the use of AFM, demonstrating both its utility and versatility in the study of viruses. As the technology continues to rapidly evolve, AFM is likely to remain an integral part of research, providing unique and important insight into many aspects of virology.

Evaluating the Impact of N-Glycan Sequon Removal in the p27 Peptide on RSV F Protein Immunogenicity and Functionality.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children, elderly and immunocompromised patients worldwide. The RSV fusion (F) protein, which has 5-6 N-glycosylation sites depending on the strain, is a major target for vaccine development. Two to three of these sites are located in the p27 peptide, which is considered absent in virions. Prior research from our group showed that removing the N-glycan at position 116 (N116) in p27 led to higher neutralizing antibody responses and better protection against RSV. In this study, the effect of single, double and triple N-glycan deletion mutations in F p27 was evaluated. Surprisingly, all mutants exhibited similar expressions and functionality to the wild-type F protein. All F p27 glycomutants induced neutralizing antibodies and lowered lung viral loads after an RSV challenge in a mouse model. Although N-glycans in p27 influence immune responses, their exact role in RSV biology remains unclear. Possibly, these glycans, which are mostly conserved, play a role in other aspects of virus replication and biology.

Rapid generation and characterization of recombinant HCoV-OC43-VR1558 infectious clones expressing reporter Renilla luciferase

Viral infectious clones (ICs) serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics. However, the molecular profiles and complex limitations of human coronaviruses (HCoVs) pose a challenge to ICs development. In this study, we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination (TAR) technology. Recombinant HCoV-OC43-VR1558, named as rOC43(1558)-WT, was rapidly generated by TAR. In addition, recombinant HCoV-OC43-VR1558-expressing reporter genes, named as rOC43(1558)-ns2FusionRluc, was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase (Rluc). We further characterized their replication through virus titration using 50% tissue culture infective dose (TCID50) and indirect immunofluorescence assay (IFA), luciferase reporter assay, and western blotting (WB) assay. The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells. These reporter viruses were validated as screening tools for inhibitors in vitro by evaluating the antiviral activities of remdesivir and chloroquine. The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were compared in vitro and in vivo. The TAR-based one-step assembly of IC was successfully applied, facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms, development of vaccines and diagnostic tests, and screening inhibitors of HCoVs.

Promotion of order Bunyavirales to class Bunyaviricetes to accommodate a rapidly increasing number of related polyploviricotine viruses.

Prior to 2017, the family Bunyaviridae included five genera of arthropod and rodent viruses with tri-segmented negative-sense RNA genomes related to the Bunyamwera virus. In 2017, the International Committee on Taxonomy of Viruses (ICTV) promoted the family to order Bunyavirales and subsequently greatly expanded its composition by adding multiple families for non-segmented to polysegmented viruses of animals, fungi, plants, and protists. The continued and accelerated discovery of bunyavirals highlighted that an order would not suffice to depict the evolutionary relationships of these viruses. Thus, in April 2024, the order was promoted to class Bunyaviricetes. This class currently includes two major orders, Elliovirales (Cruliviridae, Fimoviridae, Hantaviridae, Peribunyaviridae, Phasmaviridae, Tospoviridae, and Tulasviridae) and Hareavirales (Arenaviridae, Discoviridae, Konkoviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Phenuiviridae, and Wupedeviridae), for hundreds of viruses, many of which are pathogenic for humans and other animals, plants, and fungi.

Leveraging Synthetic Virology for the Rapid Engineering of Vesicular Stomatitis Virus (VSV)

Vesicular stomatitis virus (VSV) is a prototype RNA virus that has been instrumental in advancing our understanding of viral molecular biology and has applications in vaccine development, cancer therapy, antiviral screening, and more. Current VSV genome plasmids for purchase or contract virus services provide limited options for modification, restricted to predefined cloning sites and insert locations. Improved methods and tools to engineer VSV will unlock further insights into long-standing virology questions and new opportunities for innovative therapies. Here, we report the design and construction of a full-length VSV genome. The 11,161 base pair synthetic VSV (synVSV) was assembled from four modularized DNA fragments. Following rescue and titration, phenotypic analysis showed no significant differences between natural and synthetic viruses. To demonstrate the utility of a synthetic virology platform, we then engineered VSV with a foreign glycoprotein, a common use case for studying viral entry and developing anti-virals. To show the freedom of design afforded by this platform, we then modified the genome of VSV by rearranging the gene order, switching the positions of VSV-P and VSV-M genes. This work represents a significant technical advance, providing a flexible, cost-efficient platform for the rapid construction of VSV genomes, facilitating the development of innovative therapies.

Synthesis of Novel Indolyl Aryl Sulfone-clubbed Hydrazone Derivatives as Potential HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: Molecular Modeling and QSAR Studies.

Non-Nucleoside Reverse Transcriptases Inhibitors (NNRTIs) are among the most extensively studied enzymes for understanding the biology of Human Immunodeficiency Viruses (HIV) and designing inhibitors for managing HIV infections. Indolyl aryl sulfones (IASs), an underexplored class of potent NNRTIs, require further exploration for the development of newer drugs for HIV. In this context, we synthesized a series of novels by Indolyl Aryl Sulfones with a hydrazone moiety at the carboxylate site of the indole nucleus. A 2D-QSAR model was developed to predict Reverse Transcriptase inhibitory activity against wild-type RT (WT-RT) enzyme. The model was successfully applied to predict the HIV-1 inhibitory activity of known Indolyl Aryl Sulfones. Considering the reliability, robustness, and reproducibility of the 2D-QSAR model, we made an in-silico prediction of the RT inhibition for our synthesized compounds (1-14). Molecular docking and dynamics simulations established our synthesized Indolyl Aryl Sulfones, particularly compounds 23, 24, and 28, as effective NNRTIs by stabilizing HIV reverse transcriptase's structure. Binding energy calculations revealed compound 28 as the strongest inhibitor (-43.21 ± 0.09 kcal/mol), followed by 23 (-40.94 ± 0.10 kcal/mol) and 24 (-39.18±0.08 kcal/mol), emphasizing their binding affinity towards HIV reverse transcriptase. In summary, the synthesized Indolyl Aryl Sulfones, particularly compounds 23, 24, and 28, demonstrate significant potential as Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) against HIV. These results highlight the promising role of these compounds in developing novel NNRTIs for managing HIV infections.

AAV2 can replicate its DNA by a rolling hairpin or rolling circle mechanism, depending on the helper virus.

AAV is a small helper virus-dependent, non-pathogenic parvovirus. The AAV genome replication mechanism was extensively studied in the presence of AdV as the helper virus and described to proceed using RHR. Surprisingly, HSV-1 co-infection facilitates RCR of the AAV2 DNA. We directly compared AdV5 and HSV-1 supported AAV2 DNA replication and showed that AAV2 can adapt its replication mechanism to the helper virus. A detailed understanding of the AAV replication mechanism expands our knowledge of virus biology and can contribute to increase gene therapy vector production.

Viral coexistence and insertional mutations in the ORF8 region of SARS-CoV-2: A possible mechanism of nucleotide insertion

The virus obtained from a swab sample ID: S66 in Hiroshima was reported to have a single T-base insertion in the ORF8 coding region. However, no T insertion was observed when we determined the genomic sequence using another method. We then extracted RNA from the S66 swab sample and sequenced the insertion site using the Sanger method. The resulting waveform was disrupted beyond the insertion site, suggesting the presence of a mixed population of viruses with different sequences. Through plasmid cloning of RT-PCR amplification fragments and virus cloning by limiting dilution, along with TIDE analysis to determine the ratio of components from the Sanger sequencing waveform, it was confirmed that the sample contained a mixture of viruses with varying numbers of T-base insertions. The virus with one T insertion (T1+) was predominant in 70–75 % of the genomes, and genomes with T0, T2+, T3+, T4+, and T5+ were also detected. No T-base insertion mutations were observed in the ORF8 region in three other SARS-CoV-2 samples. In the S66 sample, a C27911T point mutation near the insertion site in the ORF8 region resulted in a sequence of seven or more consecutive T bases, which was the cause of the T-base insertion. When the cloned S66 virus (T1+) was passaged in cultured cells, there was a tendency for viruses with more insertion bases to become dominant with successive generations, suggesting that the T-base insertion was due to polymerase stuttering. The insertion of T bases resulted in synthesis of deletion mutants of the ORF8 protein, but no significant change was observed in the proliferation of the viruses in cultured cells. A search of the GenBank database using NCBI BLAST for viruses similar to S66 with T-base insertion mutations revealed hundreds of viruses widely distributed on the molecular phylogenetic tree. These base insertion viruses were thought to have occasionally arisen during the virus infection process. This study suggests one mechanism of insertion mutations in SARS-CoV-2, and it is important to consider the emergence of future mutant strains.

Abortive Infection of Animal Cells: What Goes Wrong.

Even if a virus successfully binds to a cell, defects in any of the downstream steps of the viral life cycle can preclude the production of infectious virus particles. Such abortive infections are likely common in nature and can provide fundamental insights into the cell and host tropism of viral pathogens. Research over the past 60 years has revealed an incredible diversity of abortive infections by DNA and RNA viruses in various animal cell types. Here we discuss the general causes of abortive infections and provide specific examples from the literature to illustrate the range of abortive infections that have been reported. We also discuss how abortive infections can have critical roles in shaping host immune responses and in the development of virus-induced cancers. Finally, we describe how abortive infections can be applied to basic and clinical research, underscoring the importance of understanding these fascinating aspects of virus biology.

Review of Association Between Urinary Tract Infections and Immunosuppressive Drugs after Heart Transplantation.

Management of infections in heart transplant recipients is complex and crucial. In this population, there is a need for a better understanding of immunosuppressive trough levels (C0), infectious complications, and urinary tract infections (UTIs). The purpose of this review was to understand the association between immunosuppressive trough levels and UTIs after heart transplantation. A review of scientific literature (n= 100) was conducted based on the topic of interest by searching PUBMED.Gov (https://pubmed.ncbi.nlm.nih.gov/), Web of Science, and Scopus. The analysis of bacterial pulmonary infection required the occurrence of new or deteriorating pulmonary infiltrates and the development of organisms in cultures of sputum specimens. The diagnosis of UTIs was based on the result of related signs, pyuria, and a positive urine culture. The incidence of UTIs was reported as 0.07 episodes/1000 regarding heart transplantation days. An eightfold increase in the rate of rejection was noted in heart transplant recipients with higher variability in tacrolimus C0. There are associations between C0 of immunosuppressive drugs and clinical presentation of infection complications. Recipients with a low metabolism of immunosuppressive drugs are more susceptible to infectious complications. Attention to the biology of herpes viruses, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Staphylococcus saprophyticus after heart transplantation are important, in which some of them are the most common pathogens responsible for UTIs. Pneumocystis and cytomegalovirus affect all transplant recipients. Pneumonia due to bacterial, viral, protozoa, and fungal infections, in addition to UTIs, are more specific reported types of infections in heart transplant recipients. Bacterial infections produced by extensively drug-resistant Enterobacteriaceae, vancomycin-resistant enterococci, and non-fermenting gramnegative bacteria were reported to increase after transplantation.

Suitable Mouse Model to Study Dynamics of West Nile Virus Infection in Culex quinquefasciatus Mosquitoes.

West Nile Virus (WNV) poses a significant global public health threat as a mosquito-borne pathogen. While laboratory mouse models have historically played a crucial role in understanding virus biology, recent research has focused on utilizing immunocompromised models to study arboviruses like dengue and Zika viruses, particularly their interactions with Aedes aegypti mosquitoes. However, there has been a shortage of suitable mouse models for investigating WNV and St. Louis encephalitis virus interactions with their primary vectors, Culex spp. mosquitoes. Here, we establish the AG129 mouse (IFN α/β/γ R-/-) as an effective vertebrate model for examining mosquito-WNV interactions. Following intraperitoneal injection, AG129 mice exhibited transient viremia lasting several days, peaking on the second or third day post-infection, which is sufficient to infect Culex quinquefasciatus mosquitoes during a blood meal. We also observed WNV replication in the midgut and dissemination to other tissues, including the fat body, in infected mosquitoes. Notably, infectious virions were present in the saliva of a viremic AG129 mouse 16 days post-exposure, indicating successful transmission capacity. These findings highlight the utility of AG129 mice for studying vector competence and WNV-mosquito interactions.

Mapping disparities in viral infection rates using highly multiplexed serology.

Our understanding of population-level virus infection rates and associated health disparities is incomplete. In part, this is because of the high diversity of human-infecting viruses and the limited breadth and sensitivity of traditional approaches for detecting infection events. Here, we demonstrate the potential for modern, highly multiplexed antibody detection methods to greatly increase our understanding of disparities in rates of infection across subpopulations (e.g., different sexes or ethnic groups). The use of antibodies as biomarkers allows us to detect evidence of past infections over an extended period, and our approach for highly multiplexed serology (PepSeq) allows us to measure antibody responses against hundreds of viruses in an efficient and cost-effective manner.

The Study of Metalloproteome of DNA Viruses: Identification, Functional Annotation, and Diversity Analysis of Viral Metal-Binding Proteins.

Metalloproteins are fundamental to diverse biological processes but still lack extensive investigation in viral contexts. This study reveals the prevalence and functional diversity of metal-binding proteins in DNA viruses. Among a subset of 1432 metalloproteins, zinc and magnesium-binding proteins are notably abundant, indicating their importance in viral biology. Furthermore, significant numbers of proteins binding to iron, manganese, copper, nickel, mercury, and cadmium were also detected. Human-infecting viral proteins displayed a rich landscape of metalloproteins, with MeBiPred (964 proteins) and Pfam (666) yielding the highest numbers. Interestingly, many essential viral proteins exhibited metal-binding capabilities, including polymerases, DNA binding proteins, helicases, dUPTase, thymidine kinase, and various structural and accessory proteins. This study sheds light on the ubiquitous presence of metalloproteins, their functional signatures, subcellular placements, and metal-utilization patterns, providing valuable insights into viral biology. A similar metal utilization pattern was observed in similar functional proteins across the various DNA viruses. Furthermore, these findings provide a foundation for identifying potential drug targets for combating viral infections.

Epitranscriptomic m5C methylation of SARS-CoV-2 RNA regulates viral replication and the virulence of progeny viruses in the new infection.

While the significance of N6-methyladenosine (m6A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m5C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m5C is more abundant than m6A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m5C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m5C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m5C-MeRIP-seq identified the NSUN2-dependent m5C-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from Nsun2 deficiency mice with low levels of m5C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m5C modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of m5C methylation to SARS-CoV-2 RNA.

From viruses to humans – Exploring the structure–function relationship of the Kesv protein for the future of biomedicine

Viruses often use ion channel proteins to initialise host infections. Defects in ion channel proteins are also linked to several metabolic disorders in humans. In that instance, modulation of ion channel activities becomes central to development of antiviral therapies and drug design. Kesv, a potassium-selective ion channel protein expressed by Ectocarpus siliculosus virus (EsV), possesses remarkable properties which can help to characterise the molecular basis of the functional processes relevant to virus biology and human physiology. The small structural features of this ion channel could serve as a fundamental primer to study more complex ion channels from humans. Therefore, in spite of their evolutionary distance, the potential link between viral and human ion channel proteins could provide opportunities for therapeutic and biotechnological applications.

Dengue virus preferentially uses human and mosquito non-optimal codons

Codon optimality refers to the effect that codon composition has on messenger RNA (mRNA) stability and translation level and implies that synonymous codons are not silent from a regulatory point of view. Here, we investigated the adaptation of virus genomes to the host optimality code using mosquito-borne dengue virus (DENV) as a model. We demonstrated that codon optimality exists in mosquito cells and showed that DENV preferentially uses nonoptimal (destabilizing) codons and avoids codons that are defined as optimal (stabilizing) in either human or mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are upregulated during infection, and so is the tRNA decoding the nonoptimal and DENV preferentially used codon for arginine. We found that adaptation during single-host passaging in human or mosquito cells results in the selection of synonymous mutations towards DENV’s preferred nonoptimal codons that increase virus fitness. Finally, our analyses revealed that hundreds of viruses preferentially use nonoptimal codons, with those infecting a single host displaying an even stronger bias, suggesting that host–pathogen interaction shapes virus-synonymous codon choice.

A C-Degron Structure-Based Approach for the Development of Ligands Targeting the E3 Ligase TRIM7.

TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.