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Herpes Simplex Virus Type 1 (HSV-1) is a widespread human pathogen known for causing a spectrum of clinical manifestations, ranging from mild cold sores to severe complications like encephalitis. Understanding the strain-specific variations of HSV-1 is crucial for elucidating its pathogenesis and developing targeted therapeutic interventions. In this multifaceted study, we investigated the strain-specific characteristics of HSV-1 using an in vivo rat model. Firstly, a pilot study was conducted to assess the capacity of three HSV-1 strains (Fisher (F), KOS (K), and MacIntyre (M)) to induce cold sores in rats. Remarkably, the F strain exhibited pronounced pathogenicity, inducing erythema, swelling, and disrupted epidermis with ulceration, distinguishing it from the K and M strains. Subsequently, the treatment capability of intravenous acyclovir injection in HSV-1 F strain-infected rats was evaluated. Acyclovir treatment resulted in a significant reduction in HSV-1 viral copy numbers in serum and dissected neuronal tissues, particularly in the spinal cord, brain, and lower lip. Lastly, whole genome sequencing data revealed that high-impact mutations occurred in the K and M strains within the UL49, US2, and US3 genes. These mutations may play a pivotal role in influencing viral replication, dissemination, pathogenesis, and infectivity. In contrast, the moderate missense variant mutations detected in the US12, US8, UL3, UL30, UL31, and UL36 genes appeared to have no effect on viral pathogenesis and infectivity, based on RT-PCR data for spinal cord, trigeminal nerve, brain, and the lower lip. These strain-specific mutations underscore the dynamic nature of HSV-1 evolution. Collectively, our findings contribute to a deeper understanding of HSV-1 strain diversity and pave the way for the development of targeted therapeutic strategies against this medically significant virus.

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Currently, the analyses of and prediction using COVID-19-related data extracted from patient information repositories compiled by hospitals and health organizations are of paramount importance. These efforts significantly contribute to vaccine development and the formulation of contingency techniques, providing essential tools to prevent resurgence and to effectively manage the spread of the disease. In this context, the present research focuses on analyzing the biological information of the SARS-CoV-2 viral gene sequences and the clinical data of COVID-19-affected patients using publicly accessible data from Ecuador. This involves considering variables such as age, gender, and geographical location to understand the evolution of mutations and their distributions across Ecuadorian provinces. The Cross-Industry Standard Process for Data Mining (CRISP-DM) methodology is applied for data analysis. Various data preprocessing and statistical analysis techniques are employed, including Pearson correlation, the chi-square test, and analysis of variance (ANOVA). Statistical diagrams and charts are used to facilitate a better visualization of the results. The results illuminate the genetic diversity of the virus and its correlation with clinical variables, offering a comprehensive understanding of the dynamics of COVID-19 spread in Ecuador. Critical variables influencing population vulnerability are highlighted, and the findings underscore the significance of mutation monitoring and indicate a need for global expansion of the research area.

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Ultraviolet-C (UVC) irradiation is being used as an effective approach for the disinfection of pathogenic viruses present in air, surfaces, and water. Recently, far-UVC radiation (222 nm) emitted by KrCl* (krypton-chloride) excimer lamps have been recommended for disinfecting high-risk public spaces to reduce the presence and transmission of infectious viruses owing to limited human health exposure risks as compared to germicidal UVC (254 nm). In this study, the UVC inactivation performances of individual filtered KrCl* excimer lamp (222 nm) and germicidal UVC lamp (254 nm) were determined against four viruses, bacteriophages MS2, Phi6, M13, and T4, having different genome compositions (ssRNA, dsRNA, ssDNA and dsDNA, respectively) and shapes (i.e., spherical (Phi6), linear (M13), and icosahedral (MS2 and T4)). Here, the disinfection efficacies of filtered KrCl* excimer lamp (222 nm) and germicidal UVC lamp (254 nm) were evaluated for highly concentrated virus droplets that mimic the virus-laden droplets released from the infected person and deposited on surfaces as fomites. Filtered KrCl* excimer (222 nm) showed significantly better inactivation against all viruses having different genome compositions and structures compared to germicidal UVC (254 nm). The obtained sensitivity against the filtered KrCl* excimer (222 nm) was found to be in the order, T4 > M13 > Phi6 > MS2 whereas for the germicidal UVC (254 nm) it was T4 > M13 > MS2 > Phi6. These results provide a strong basis to promote the use of filtered KrCl* excimer lamps (222 nm) in disinfecting contagious viruses and to limit the associated disease spread in public places and other high-risk areas.

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BACKGROUND: External quality assessment (EQA) schemes provide objective feedback to participating laboratories about the performance of their analytical systems and information about overall regional analytical performance. The EQAs are particularly important during pandemics as they also assess the reliability of individual test results and show opportunities to improve test strategies. With the end of the COVID-19 pandemic, the testing frequency significantly decreased in Austria. Here, we analyzed whether this decrease had an effect on participation and/or performance in SARS-CoV­2 virus detection EQAs, as compared to the pandemic era. MATERIAL AND METHODS: Identical samples were sent to all participating laboratories, and the EQA provider evaluated the agreement of the reported results with defined targets. The EQA was operated under two schemes with identical samples and therefore we analyzed it as a single EQA round. The performance of testing was reported as true positive ratios, comparing the post-pandemic data to previous rounds. Furthermore, subgroups of participants were analyzed stratified by laboratory type (medical or nonmedical) and the test system format (fully automated or requiring manual steps). RESULTS: While the frequency of false negative results per sample did not change during the 3 years of the pandemic (5.7%, 95% confidence interval [CI] 3.1-8.4%), an average per sample false negative ratio of 4.3% was observed in the first post-pandemic EQA (0%, 1.8%, and 11% for the 3 positive samples included in the test panel, n = 109 test results per sample). In this first post-pandemic EQA medical laboratories (average 0.4% false negative across 3 samples, n = 90) and automated test systems (average 1.2% false negative, n = 261) had lower false negative ratios than nonmedical laboratories (22.8%, n = 19) and manual test systems (16.7%, n = 22). These lower average ratios were due to a low concentration sample, where nonmedical laboratories reported 36.8% and manual test systems 54.5% true positive results. CONCLUSION: Overall ratios of true positive results were below the mean of all results during the pandemic but were similar to the first round of the pandemic. A lower post-pandemic true positive ratio was associated with specific laboratory types and assay formats, particularly for samples with low concentration. The EQAs will continue to monitor the laboratory performance to ensure the same quality of epidemiological data after the pandemic, even if vigilance has decreased.

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Pigs are natural host to various zoonotic pathogens including viruses. In this study, we analyzed the viral communities in the feces of 89 piglets with diarrhea under one month old which were collected from six farms in Jiangsu Province of the Eastern China, using the unbiased virus metagenomic method. A total of 89 libraries were constructed, and 46937894 unique sequence reads were generated by Illumina sequencing. Overall, the family Picornaviridae accounted for the majority of the total reads of putative mammalian viruses. Ten novel virus genomes from different family members were discovered, including Parvoviridae (n = 2), Picobirnaviridae (n = 4) and CRESS DNA viruses (n = 4). A large number of phages were identified, which mainly belonged to the order Caudovirales and the family Microviridae. Moreover, some identified viruses were closely related to viruses found in non-porcine hosts, highlighting the potential for cross-species virus dissemination. This study increased our understanding of the fecal virus communities of diarrhea piglets and provided valuable information for virus monitoring and preventing.

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Influenza A virus (IAV) represents a constant public health threat. The single-stranded, segmented RNA genome of IAV is replicated in host cell nuclei as a series of 8 ribonucleoprotein complexes (vRNPs) with RNA structures known to exert essential function to support viral replication. Here, we investigate RNA secondary structures and RNA interactions networks of the IAV genome and construct an in vivo structure model for each of the 8 IAV genome segments. Our analyses reveal an overall in vivo and in virio resemblance of the IAV genome conformation but also wide disparities among long-range and intersegment interactions. Moreover, we identify a long-range RNA interaction that exerts an essential role in genome packaging. Disrupting this structure displays reduced infectivity, attenuating virus pathogenicity in mice. Our findings characterize the in vivo RNA structural landscape of the IAV genome and reveal viral RNA structures that can be targeted to develop antiviral interventions.

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Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

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Both influenza A virus genome transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the influenza RNA polymerase (FluPol), are dynamically regulated across the virus life cycle. It has been reported that the last amino acid I121 of the viral NS2 protein plays a critical role in promoting viral genome replication in influenza mini-replicon systems. Here, we performed a 20 natural amino acid substitution screening at residue NS2-I121 in the context of virus infection. We found that the hydrophobicity of the residue 121 is essential for virus survival. Interestingly, through serial passage of the rescued mutant viruses, we further identified adaptive mutations PA-K19E and PB1-S713N on FluPol which could effectively compensate for the replication-promoting defect caused by NS2-I121 mutation in the both mini-replicon and virus infection systems. Structural analysis of different functional states of FluPol indicates that PA-K19E and PB1-S713N could stabilize the replicase conformation of FluPol. By using a cell-based NanoBiT complementary reporter assay, we further demonstrate that both wild-type NS2 and PA-K19E/PB1-S713N could enhance FluPol dimerization, which is necessary for genome replication. These results reveal the critical role NS2 plays in promoting viral genome replication by coordinating with FluPol.IMPORTANCEThe intrinsic mechanisms of influenza RNA polymerase (FluPol) in catalyzing viral genome transcription and replication have been largely resolved. However, the mechanisms of how transcription and replication are dynamically regulated remain elusive. We recently reported that the last amino acid of the viral NS2 protein plays a critical role in promoting viral genome replication in an influenza mini-replicon system. Here, we conducted a 20 amino acid substitution screening at the last residue 121 in virus rescue and serial passage. Our results demonstrate that the replication-promoting function of NS2 is important for virus survival and efficient multiplication. We further show evidence that NS2 and NS2-I121 adaptive mutations PA-K19E/PB1-S713N regulate virus genome replication by promoting FluPol dimerization. This work highlights the coordination between NS2 and FluPol in fulfilling efficient genome replication. It further advances our understanding of the regulation of viral RNA synthesis for influenza A virus.

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Huge phages have genomes larger than 200 kilobases, which are particularly interesting for their genetic inventory and evolution. We screened 165 wastewater metagenomes for the presence of viral sequences. After identifying over 600 potential huge phage genomes, we reduced the dataset using manual curation by excluding viral contigs that did not contain viral protein-coding genes or consisted of concatemers of several small phage genomes. This dataset showed seven fully annotated huge phage genomes. The phages grouped into distinct phylogenetic clades, likely forming new genera and families. A phylogenomic analysis between our huge phages and phages with smaller genomes, i.e., less than 200 kb, supported the hypothesis that huge phages have undergone convergent evolution. The genomes contained typical phage protein-coding genes, sequential gene cassettes for metabolic pathways, and complete inventories of tRNA genes covering all standard and rare amino acids. Our study showed a pipeline for huge phage analyses that may lead to new enzymes for therapeutic or biotechnological applications.

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Baculovirus expression system1s are a widely used tool in recombinant protein and biologics production. To enable the possibility of genome modifications unconstrained through low-throughput and bespoke classical genome manipulation techniques, we set out to construct a baculovirus vector (>130 kb dsDNA) built from modular, chemically synthesized DNA parts. We constructed a synthetic version of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) through two steps of hierarchical Golden Gate assembly. Over 140 restriction endonuclease sites were removed to enable the discrimination of the synthetic genome from native baculovirus genomes. A head-to-head comparison of our modular, synthetic AcMNPV genome with native baculovirus vectors showed no significant difference in baculovirus growth kinetics or recombinant adeno-associated virus production-suggesting that neither baculovirus replication nor very-late gene expression were compromised by our design or assembly method. With unprecedented control over the AcMNPV genome at the single-nucleotide level, we hope to ambitiously explore novel AcMNPV vectors streamlined for biologics production and development.

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Genomic studies of viral diseases in aquaculture have received more and more attention with the growth of the aquaculture industry, especially the emerging and re-emerging viruses whose genome could contain recombination, mutation, insertion, and so on, and may lead to more severe diseases and more widespread infections in aquaculture animals. The present review is focused on aquaculture viruses, which is belonged to two clades, Varidnaviria and Duplodnaviria, and one class Naldaviricetes, and respectively three families: Iridoviridae (ranaviruses), Alloherpesviridae (fish herpesviruses), and Nimaviridae (whispoviruses). The viruses possessed DNA genomes nearly or larger than 100 kbp with gene numbers more than 100 and were considered large DNA viruses. Genome analysis and experimental investigation have identified several genes involved in genome replication, transcription, and virus-host interactions. In addition, some genes involved in virus genetic variation or specificity were also discussed. A summary of these advances would provide reference to future discovery and research on emerging or re-emerging aquaculture viruses.

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Mountain and polar glaciers cover 10% of the Earth's surface and are typically extreme environments that challenge life of all forms. Viruses are abundant and active in supraglacial ecosystems and play a crucial role in controlling the supraglacial microbial communities. However, our understanding of virus ecology on glacier surfaces and their potential impacts on downstream ecosystems remains limited. Here, we present the supraglacial virus genome (SgVG) catalog, a 15-fold expanded genomic inventory of 10,840 DNA-virus species from 38 mountain and polar glaciers, spanning habitats such as snow, ice, meltwater, and cryoconite. Supraglacial DNA-viruses were highly specific compared to viruses in other ecosystems yet exhibited low public health risks. Supraglacial viral communities were primarily constrained by habitat, with cryoconite displaying the highest viral activity levels. We observed a prevalence of lytic viruses in all habitats, especially in cryoconite, but a high level of lysogenic viruses in snow and ice. Additionally, we found that supraglacial viruses could be linked to ∼83% of obtained prokaryotic phyla/classes and possessed the genetic potential to promote metabolism and increase cold adaptation, cell mobility, and phenolic carbon use of hosts in hostile environmental conditions using diverse auxiliary metabolic genes. Our results provide the first systematic characterization of the diversity, function, and public health risks evaluation of mountain and polar supraglacial DNA viruses. This understanding of glacial viruses is crucial for function assessments and ecological modeling of glacier ecosystems, especially for the Tibetan Plateau's Mountain glaciers, which support ∼20% of the human populations on Earth.

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BACKGROUND: The outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in the global COVID-19 pandemic. The urgency for an effective SARS-CoV-2 vaccine has led to the development of the first series of vaccines at unprecedented speed. The discovery of SARS-CoV-2 spike-glycoprotein mutants, however, and consequentially the potential to escape vaccine-induced protection and increased infectivity, demonstrates the persisting importance of monitoring SARS-CoV-2 mutations to enable early detection and tracking of genomic variants of concern. RESULTS: We developed the CoVigator tool with three components: (1) a knowledge base that collects new SARS-CoV-2 genomic data, processes it and stores its results; (2) a comprehensive variant calling pipeline; (3) an interactive dashboard highlighting the most relevant findings. The knowledge base routinely downloads and processes virus genome assemblies or raw sequencing data from the COVID-19 Data Portal (C19DP) and the European Nucleotide Archive (ENA), respectively. The results of variant calling are visualized through the dashboard in the form of tables and customizable graphs, making it a versatile tool for tracking SARS-CoV-2 variants. We put a special emphasis on the identification of intrahost mutations and make available to the community what is, to the best of our knowledge, the largest dataset on SARS-CoV-2 intrahost mutations. In the spirit of open data, all CoVigator results are available for download. The CoVigator dashboard is accessible via covigator.tron-mainz.de. CONCLUSIONS: With increasing demand worldwide in genome surveillance for tracking the spread of SARS-CoV-2, CoVigator will be a valuable resource of an up-to-date list of mutations, which can be incorporated into global efforts.

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Cutaneous human papillomavirus type 5 (HPV5) belongs to the supposedly oncogenic ß-HPVs associated with specific types of skin and oral cavity cancers. Three viral proteins, namely, helicase E1 and transcription factors E2 and E8^E2, are master regulators of the viral life cycle. HPV5 E2 is a transcriptional activator that also participates in the E1-dependent replication and nuclear retention of the viral genome, whereas E8^E2 counterbalances the activity of E2 and inhibits HPV transcription and replication. In the present study, we demonstrate that the HPV5 E2 protein is extensively phosphorylated by cellular protein kinases, and serine residue 402 (S402) is the highest scoring phosphoacceptor site. This residue is located within a motif conserved among many ß-HPVs and in the oncogenic HPV31 α-type. Using the nonphosphorylatable and phosphomimetic mutants, we demonstrate that phosphorylation of the E2 S402 residue is required for the transcription and replication of the HPV5 genome in U2OS cells and human primary keratinocytes. Mechanistically, the E2-S402-phopshodeficient protein is unable to trigger viral gene transcription and has an impaired ability to support E1-dependent replication, but the respective E8^E2-S213 mutant displays no phenotype. However, phosphorylation of the E2 S402 residue has no impact on the E2 stability, subcellular localization, self-assembly, DNA-binding capacity, and affinity to the E1 and BRD4 proteins. Further studies are needed to identify the protein kinase(s) responsible for this phosphorylation. IMPORTANCE Human papillomavirus type 5 (HPV5) may play a role in the development of specific types of cutaneous and head and neck cancers. The persistence of the HPV genome in host cells depends on the activity of its proteins, namely, a helicase E1 and transcription/replication factor E2. The latter also facilitates the attachment of episomal viral genomes to host cell chromosomes. In the present study, we show that the HPV5 E2 protein is extensively phosphorylated by host cell protein kinases, and we identify serine residue 402 as the highest scoring phosphoacceptor site of E2. We demonstrate that the replication of the HPV5 genome may be blocked by a single point mutation that prevents phosphorylation of this serine residue and switches off the transcriptional activity of the E2 protein. The present study contributes to a better understanding of ß-HPV5 replication and its regulation by host cell protein kinases.

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Poxviruses are known to evolve slower than RNA viruses with only 1-2 mutations/genome/year. Rather than single mutations, rearrangements such as gene gain and loss, which have been discussed as a possible driver for host adaption, were described in poxviruses. In 2022 and 2023 the world is being challenged by the largest global outbreak so far of Mpox virus, and the virus seems to have established itself in the human community for an extended period of time. Here, we report five Mpox virus genomes from Germany with extensive gene duplication and loss, leading to the expansion of the ITR regions from 6400 to up to 24,600 bp. We describe duplications of up to 18,200 bp to the opposed genome end, and deletions at the site of insertion of up to 16,900 bp. Deletions and duplications of genes with functions of supposed immune modulation, virulence and host adaption as B19R, B21R, B22R and D10L are described. In summary, we highlight the need for monitoring rearrangements of the Mpox virus genome rather than for monitoring single mutations only.

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Recent years have seen major changes in the classification criteria and taxonomy of viruses. The current classification scheme, also called "megataxonomy of viruses", recognizes six different viral realms, defined based on the presence of viral hallmark genes (VHGs). Within the realms, viruses are classified into hierarchical taxons, ideally defined by the phylogeny of their shared genes. To enable the detection of shared genes, viruses have first to be clustered, and there is currently a need for tools to assist with virus clustering and classification. Here, VirClust is presented. It is a novel, reference-free tool capable of performing: (i) protein clustering, based on BLASTp and Hidden Markov Models (HMMs) similarities; (ii) hierarchical clustering of viruses based on intergenomic distances calculated from their shared protein content; (iii) identification of core proteins and (iv) annotation of viral proteins. VirClust has flexible parameters both for protein clustering and for splitting the viral genome tree into smaller genome clusters, corresponding to different taxonomic levels. Benchmarking on a phage dataset showed that the genome trees produced by VirClust match the current ICTV classification at family, sub-family and genus levels. VirClust is freely available, as a web-service and stand-alone tool.

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All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~102-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.

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Fish novirhabdoviruses, including viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), and infectious hematopoietic necrosis virus (IHNV), harbor a unique non-virion (NV) gene that is crucial for efficient replication and pathogenicity. The effective levels and the function of the N-terminal region of the NV protein, however, remain poorly understood. In the present study, several recombinant VHSVs, which completely lack (rVHSV-ΔNV) or harbor an additional (rVHSV-dNV) NV gene, were generated using reverse genetics. To confirm the function of the N-terminal region of the NV protein, recombinant VHSVs with the NV gene that gradually mutated from the start codon (ATG) to the stop codon (TGA), expressed as N-terminally truncated NV proteins (rVHSV-NV1, -NV2, and -NV3), were generated. CPE progression and viral growth analyses showed that epithelioma papulosum cyprini (EPC) cells infected with rVHSV-ΔNV or rVHSV-NV3-which did not express NV protein-rarely showed CPE and viral replication as opposed to EPC cells infected with rVHSV-wild. Interestingly, regardless of the presence of two NV genes in the rVHSV-dNV genome, EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP (control) failed to induce CPE and viral replication. In EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP, which harbored a longer VHSV genome than the wild-type, Mx gene expression levels, which were detected by luciferase activity assay, were particularly high; Mx gene expression levels were higher in EPC cells infected with rVHSV-ΔNV, -NV2, or -NV3 than in those infected with rVHSV-wild or rVHSV-NV1. The total amount of NV transcript produced in EPC cells infected with rVHSV-wild was much higher than that in EPC cells infected with rVHSV-dNV. However, the expression levels of the NV gene per viral particle were significantly higher in EPC cells infected with rVHSV-dNV than in cells infected with rVHSV-wild. These results suggest that the NV protein is an essential component in the inhibition of host type-I interferon (IFN) and the induction of viral replication. Most importantly, viral genome length might affect viral replication efficiency to a greater extent than does NV gene expression. In in vivo pathogenicity experiments, the cumulative mortality rates of olive flounder fingerlings infected with rVHSV-dNV or rVHSV-wild were similar (60-70%), while those of fingerlings infected with rVHSV-A-EGFP were lower. Moreover, the virulence of rVHSV-ΔNV and rVHSV, both harboring a truncated NV gene (rVHSV-NV1, -NV2, and -NV3), was completely attenuated in the olive flounder. These results suggest that viral pathogenicity is affected by the viral replication rate and NV gene expression. In conclusion, the genome length and NV gene (particularly the N-terminal region) expression of VHSVs are closely associated with viral replication in host type-I IFN response and the viral pathogenicity.

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OBJECTIVES: More than two years into the COVID-19 pandemic, SARS-CoV-2 still remains a global public health problem. Successive waves of infection have produced new SARS-CoV-2 variants with new mutations for which the impact on COVID-19 severity and patient survival is uncertain. METHODS: A total of 764 SARS-CoV-2 genomes, sequenced from COVID-19 patients, hospitalized from 19th February 2020 to 30 April 2021, along with their clinical data, were used for survival analysis. RESULTS: A significant association of B.1.1.7, the alpha lineage, with patient mortality (log hazard ratio (LHR) = 0.51, C.I. = [0.14,0.88]) was found upon adjustment by all the covariates known to affect COVID-19 prognosis. Moreover, survival analysis of mutations in the SARS-CoV-2 genome revealed 27 of them were significantly associated with higher mortality of patients. Most of these mutations were located in the genes coding for the S, ORF8, and N proteins. CONCLUSIONS: This study illustrates how a combination of genomic and clinical data can provide solid evidence for the impact of viral lineage on patient survival.

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Viruses have evolved numerous mechanisms to exploit the molecular machinery of their host cells, including the broad spectrum of host RNA-binding proteins (RBPs). However, the RBP interactomes of most viruses are largely unknown. To shed light on the interaction landscape of RNA viruses with human host cell RBPs, we have analysed 197 single-stranded RNA (ssRNA) viral genome sequences and found that the majority of ssRNA virus genomes are significantly enriched or depleted in motifs for specific human RBPs, suggesting selection pressure on these interactions. To facilitate tailored investigations and the analysis of genomes sequenced in future, we have released our methodology as a fast and user-friendly computational toolbox named SMEAGOL. Our resources will contribute to future studies of specific ssRNA virus-host cell interactions and support the identification of antiviral drug targets.

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