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Background: A limitation of approved oncolytic viruses is their requirement for intratumoral (i.t.) injection. TILT-123 (igrelimogene litadenorepvec, Ad5/3-E2F-D24-hTNFα-IRES-hIL-2) is a chimeric oncolytic adenovirus suitable for intravenous (i.v.) delivery due to its capsid modification and dual selectivity devices. It is armed with tumor necrosis alpha and interleukin-2 for promoting T-cell activation and lymphocyte trafficking to tumors, thereby enhancing the antitumor immune response. Here, we present the findings after a single i.v. administration of TILT-123 in three phase I dose escalation clinical trials. Methods: Patients with advanced solid tumors initially received a single i.v. dose of TILT-123 ranging from 3 × 109 to 4 × 1012 viral particles (VP). Blood was collected at baseline, 1, 16, and 192 h (7 days) post-treatment for bioavailability and serum analysis. Tumor biopsies were collected prior to treatment and 7 days post-treatment for analysis of viral presence and immunological effects. Patients did not receive any other cancer therapies during this period. Results: Across all three trials (TUNIMO, TUNINTIL, and PROTA), 52 total patients were treated with i.v. TILT-123. Overall, TILT-123 was found to be well-tolerated, with no dose-limiting toxicities observed. Post-treatment tumor biopsies showed expression of viral genes, presence of TILT-123 adenovirus proteins or DNA, and changes in immune cell infiltration from baseline. Increased virus dose did not lead to increased virus detection in tumors. Median overall survival was longer in patients with confirmed presence of TILT-123 in post-treatment biopsies (280 versus 190 days, p = 0.0405). Conclusion: TILT-123 demonstrated safety and significant intratumoral immunomodulation following a single i.v. administration, warranting further investigation. Trial registrations: TUNIMO—NCT04695327. Registered 4 January 2021, https://clinicaltrials.gov/study/NCT04695327. TUNINTIL—NCT04217473. Registered 19 December 2019, https://clinicaltrials.gov/study/NCT04217473. PROTA—NCT05271318. Registered 4 February 2022, https://clinicaltrials.gov/study/NCT05271318. [ABSTRACT FROM AUTHOR]

Lytic induction therapy was devised to selectively combat malignancies associated with Epstein–Barr virus (EBV) by triggering viral reactivation from latency. At present, the major challenges of lytic induction therapy are to maximize reactivating efficiencies and meanwhile minimize infectious virion production. C210, a novel curcumin derivative with potent Hsp90 inhibitory activity, was explored for EBV-reactivating and virion-producing effects in EBV-positive nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC) cell lines. And the molecular mechanisms underlying these effects were determined. Follow C210 treatment, EBV lytic RNAs and proteins were upregulated, but infectious virions were not produced. Knockdown of heat shock protein 90 (Hsp90) induced expression of lytic RNAs and proteins, and diminished C210-driven EBV lytic induction. Pretreatment with an X box binding protein 1 (XBP1) inhibitor reduced C210-induced EBV lytic RNA. Furthermore, we demonstrated that C210 inhibited the binding of Hsp90 with its clients, signal transducer and activator of transcription 3 (STAT3) and xeroderma pigmentosum group B-complementing protein (XPB), which subsequently promoted their proteasomal degradation. Degradation of STAT3 by C210 enhanced the EBV-reactivating and anticancer capacity of suberoylanilide hydroxamic acid (SAHA). Depletion of XPB blocked SAHA-induced expression of late viral genes and production of infectious virions. These results elucidate a novel Hsp90 inhibitor targeting EBV lytic phase and extend the research on lytic induction strategy, which may offer reference value in the treatment of EBV-positive malignancies. [ABSTRACT FROM AUTHOR]

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a crucial role in the viral immune processes of organisms, with STAT being a key transcription factor downstream in this pathway. The STAT gene of Bombyx mori has two splicing forms, named BmSTAT-S and BmSTAT-L. This study compared the effects of the two splicing forms on Bombyx mori nucleopolyhedrovirus (BmNPV) infection through cell-level interference and further explored whether BmSTAT participates in the immune response to BmNPV infection via transgenic intervention at the individual level. Our research results indicated that BmNPV upregulates the expression of BmSTAT-S and BmSTAT-L in Bombyx mori BmE cells and larvae. Furthermore, BmE cells with interference of BmSTAT-S or BmSTAT-L displayed significantly higher expression levels of the viral gene GP41 and increased viral fluorescence compared to the control group after 48 h of infection with BmNPV. Then, we constructed transgenic silkworms with genetic interference, and the results showed that both the transgenic silkworms with systemic interference and midgut-specific interference of the two splice forms of BmSTAT exhibited significantly reduced survival rates and increased viral replication numbers after infection with BmNPV. The above results indicated that the BmSTAT gene is involved in the immune response of Bombyx mori to BmNPV and these findings lay the foundation for further research on the mechanism of JAK/STAT signaling pathway involvement in BmNPV infection. [ABSTRACT FROM AUTHOR]

The unintegrated HIV-1 DNAs formed by reverse transcription in the early hours after infection are subject to profound transcriptional silencing. The repression of expression of foreign DNA, as an aspect of the innate immune system, serves to restrict the activity of many invading pathogens. Newly formed retroviral DNAs are rapidly loaded with histones upon entry into the nucleus, and the repression of their expression is mediated by an array of host proteins that introduce histone modifications characteristic of heterochromatin, including histone methylation and histone deacetylation. Knockout or knockdown of expression or inhibition of these host factors can relieve the silencing, allowing for viral gene expression even in settings where HIV-1 DNA integration is blocked. When viral DNA integration is allowed, forming the integrated provirus, the silencing in most cases is dramatically relieved, leading to high levels of expression and formation of progeny virus. In some settings and cell types, silencing of the integrated DNA is maintained, or re-established, such that the infected cells retain a silent copy of the viral DNA without production of progeny virus. The basis for the typical switch from silent DNA to actively expressed DNA upon integration is not yet fully clear. This review will summarize the current understanding of the regulation of expression of unintegrated HIV-1 DNAs and the nature of the chromatin that is formed on the viral DNA, and will especially focus on the host machinery that establishes repressive heterochromatin-like structures on the unintegrated DNA. The activation of expression that normally occurs upon integration, and the special circumstances when viral DNA expression is not activated, will also be discussed. These cases can result in the formation of populations of infected cells carrying silent proviruses, which persist for decades in infected individuals in spite of antiviral therapy. This pool of latently infected cells can be stochastically reactivated to give rise to spreading virus whenever antiviral drugs are withdrawn, and constitute the barrier to a true "cure" of AIDS. The hope is that a deeper understanding of the regulation of expression of viral DNAs will lead to new means to prevent or control viremia and disease. [ABSTRACT FROM AUTHOR]

Marek's disease (MD), an immunosuppressive disease induced by the Marek's disease virus (MDV), is regarded as an ideal model for lymphoma research to elucidate oncogenic and anti-oncogene genes. Using this model, we found that circRUNX2.2, derived from exon 6 of RUNX2, was significantly upregulated in MDV-infected tumorous spleens. In this study, we deeply analyzed the potential role of circRUNX2.2 in lymphoma cells. An open reading frame (ORF) in circRUNX2.2 with no stop codon was predicted, and small peptides (named circRUNX2.2-rt) presenting multiple ladder-like bands with different molecular weights encoded by circRUNX2.2 were detected via Western blotting assay. The polysome fraction assay reconfirmed the translation ability of circRUNX2.2, which could be detected in polysome fractions. Subsequent analysis verified that it translated in a rolling circle manner, rather than being assisted by the internal ribosome entry site (IRES) or m6A-mediated mechanism. Furthermore, we found that circRUNX2.2-rt was potently induced in MSB1 cells treated with sodium butyrate (NaB), which reactivated MDV and forced the MDV transition from the latent to reactivation phase. During this phase, MDV particles were clearly observed by electron microscopy, and the viral gene pp38 was also significantly upregulated. A biological function study showed that circRUNX2.2-rt promoted cell proliferation and cell cycle transition from the S to G2 phase and inhibited the apoptosis of MSB1. Further immunoprecipitation and mass spectrometry assays showed that 168 proteins potentially interacting with circRUNX2.2-rt were involved in multiple pathways related to cell cycle regulation, which proved that circRUNX2.2-rt could bind or recruit proteins to mediate the cell cycle. [ABSTRACT FROM AUTHOR]

Cervical cancer screening programs, including triage tests, need redesigning as human papillomavirus (HPV)‐vaccinated women are entering the programs. Methylation markers offer a potential solution to reduce false‐positive rates by identifying clinically relevant cervical lesions with progressive potential. In a nested case–control study, 9242 women who received the three‐dose HPV16/18‐vaccine at ages 12–15 or 18 in a community‐randomized trial were included. Subsequently, they were re‐randomized for either frequent or infrequent cervical cancer screening trials. Over a 15‐year post‐vaccination follow‐up until 2022, 17 high‐grade squamous intraepithelial lesion (HSIL) and 15 low‐grade (LSIL) cases were identified at the 25‐year screening round, alongside 371 age and community‐matched HPV16/18‐vaccinated controls. Methylation analyses were performed on cervical samples collected at age 25, preceding histologically confirmed LSIL or HSIL diagnoses. DNA methylation of viral (HPV16/18/31/33) and host‐cell genes (EPB41L3, FAM19A4, and miR124‐2) was measured, along with HPV‐genotyping. No HPV16/18 HSIL cases were observed. The predominant HPV‐genotypes were HPV52 (29.4%), HPV59/HPV51/HPV58 (each 23.5%), and HPV33 (17.7%). Methylation levels were generally low, with no significant differences in mean methylation levels of viral or host‐cell genes between the LSIL/HSIL and controls. However, a significant difference in methylation levels was found between HSIL cases and controls when considering a combination of viral genes and EPB41L3 (p value =.0001). HPV‐vaccinated women with HSIL had HPV infections with uncommon HPV types that very rarely cause cancer and displayed low methylation levels. Further investigation is warranted to understand the likely regressive nature of HSIL among HPV‐vaccinated women and its implications for management. [ABSTRACT FROM AUTHOR]

Summit disease, in which infected hosts seek heights (gravitropism), first noted in modern times by nineteenth-century naturalists, has been shown to be induced by disparate pathogens ranging from viruses to fungi. Infection results in dramatic changes in normal activity patterns, and such parasite manipulation of host behaviors suggests a strong selection for convergent outcomes albeit evolved via widely divergent mechanisms. The two best-studied examples involve a subset of viral and fungal pathogens of insects that induce "summiting" in infected hosts. Summiting presumably functions as a means for increasing the dispersal of the pathogen, thus significantly increasing fitness. Here, we review current advances in our understanding of viral- and fungal-induced summit disease and the host behavioral manipulation involved. Viral genes implicated in this process include a host hormone-targeting ecdysteroid UDP-glucosyltransferase (apparently essential for mediating summit disease induced by some viruses but not all) and a protein tyrosine phosphatase, with light dependance implicated. For summit disease-causing fungi, though much remains obscure, targeting of molting, circadian rhythms, sleep, and responses to light patterns appear involved. Targeting of host neuronal pathways by summit-inducing fungi also appears to involve the production of effector molecules and secondary metabolites that affect host muscular, immune, and/or neurological processes. It is hypothesized that host brain structures, particularly Mushroom Bodies (no relation to the fungus itself), important for olfactory association learning and control of locomotor activity, are critical targets for mediating summiting during infection. This phenomenon expands the diversity of microbial pathogen-interactions and host dynamics. Key points: • Summit disease or height seeking (gravitropism) results from viral and fungal pathogens manipulating insect host behaviors presumably to increase pathogen dispersal. • Insect baculoviruses and select fungal pathogens exhibit convergent evolution in host behavioral manipulation but use disparate molecular mechanisms. • Targets for affecting host behavior include manipulation of host hormones, feeding, locomotion, and immune, circadian, and neurological pathways. [ABSTRACT FROM AUTHOR]

The escalating severity of Bombyx mori nuclear polyhedrosis virus (BmNPV) infections poses significant challenges to the silkworm industry, especially when massive production shifts occur from the eastern regions to western regions with lower labor costs. Education and experience levels are different and disease control is badly needed. To solve the problems, we have developed an innovative CRISPR/Cas9 system specifically targeting BmNPV to enhance viral resistance. For the system, we selected BmNPV genes linked to virus replication and proliferation as targets, designing 2 sites for each gene. Mutating the target sequence renders the system incapable of efficiently cleaving the virus genome, hence decreasing cleavage efficiency. We conducted a search for “NGG” or “CCN” target sequences in the BmNPV genome, excluding non‐recurring and potential targets in the B. mori genome. We successfully identified 2 distinct target sequences in the BmNPV genome—one being repeated 12 times and the other three times. These sequences lead to fragmentation of virus genome into multiple large segments that are difficult to repair. Transgenic silkworms demonstrate robust resistance to viruses, significantly boosting their survival rates compared with wild‐type silkworms under various virus infection concentrations. Our system efficiently targets dozens of viral genomes with just 2 sequences, minimizing transposable elements while ensuring cutting effectiveness. This marks a pioneering advancement by using repetitive elements within the virus genome for targeted CRISPR cleavage, aiming for antiviral effects through genome fragmentation rather than disrupting essential viral genes. Our research introduces innovative concepts to CRISPR antiviral investigations and shows promise for the practical application of gene editing in industrial silkworm strains. [ABSTRACT FROM AUTHOR]

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 μm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per μL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification. [ABSTRACT FROM AUTHOR]

Rampant C-to-U RNA editing drives the mutation and evolution of SARS-CoV-2. While much attention has been paid to missense mutations, the C-to-U events leading to AUG and thus creating novel ORFs were uninvestigated. By utilizing the public time-course mutation data from the worldwide SARS-CoV-2 population, we systematically identified the "AUG-gain mutations" caused by C-to-U RNA editing. Synonymous mutations were of special focus. A total of 58 synonymous C-to-U sites are able to create out-of-frame AUG in coding sequence (CDS). These 58 synonymous sites showed significantly higher allele frequency (AF) and increasing rate (dAF/dt) than other C-to-U synonymous sites in the SARS-CoV-2 population, suggesting that these 58 AUG-gain events conferred additional benefits to the virus and are subjected to positive selection. The 58 predicted new ORFs created by AUG-gain events showed the following advantages compared to random expectation: they have longer lengths, higher codon adaptation index (CAI), higher Kozak scores, and higher tRNA adaptation index (tAI). The 58 putatively novel ORFs have high expressibility and are very likely to be functional, providing an explanation for the positive selection on the 58 AUG-gain mutations. Our study proposed a possible mechanism of the emergence of de novo genes in SARS-CoV-2. This idea should be helpful in studying the mutation and evolution of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Introduction: PRV infection in swine can cause devastating disease and pose a potential threat to humans. Advancing the interplay between PRV and host is essential to elucidate the pathogenic mechanism of PRV and identify novel anti-PRV targets. Methods: PARP11-KO PK-15 cells were firstly constructed by CRISPR/Cas9 technology. Next, the effect of PARP11-KO on PRV infection was determined by RT-qPCR, TCID50 assay, RNA-seq, and western blot. Results and discussion: In this study, we identified PARP11 as a host factor that can significantly affect PRV infection. Inhibition of PARP11 and knockout of PARP11 can significantly promoted PRV infection. Subsequently, we further found that PARP11 knockout upregulated the transcription of NXF1 and CRM1, resulting in enhanced transcription of viral genes. Furthermore, we also found that PARP11 knockout could activate the autophagy pathway and suppress the mTOR pathway during PRV infection. These findings could provide insight into the mechanism in which PARP11 participated during PRV infection and offer a potential target to develop anti-PRV therapies. [ABSTRACT FROM AUTHOR]

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. There is currently no clinically approved therapeutic strategy able to efficiently target cccDNA (Lucifora J 2016). The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins, could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program. [ABSTRACT FROM AUTHOR]

Infectious laryngotracheitis (ILT) remains a significant concern for the poultry industry worldwide due to its impact on animal welfare and its substantial economic consequences. The disease is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This study investigated in vitro host-virus interactions of a glycoprotein G (gG) deletion mutant vaccine strain of ILTV (ΔgG ILTV), and its parent wild-type strain (CSW-1 ILTV). Inoculations were performed separately for the two strains of ILTV using both a primary (chicken embryonic kidney, CEK) and a continuous culture (leghorn male hepatoma, LMH) of chicken cells. Transcriptome analysis was performed at 12 hours post infection. Each cell-type displayed distinct effects on host and viral gene transcription, with a greater number of viral and host genes differentially transcribed in CEK cells and LMH cells, respectively. Both cell-types infected with either strain demonstrated enrichment of pathways related to signalling, and gene ontologies (GO) associated with chemotaxis. Infection with either strain upregulated both SOCS proteins and certain proto-oncogenes, which may contribute to prolonged viral persistence by promoting immunosuppression and preventing apoptosis, respectively. Patterns of gene transcription related to cytokines, chemokines, endosomal TLRs, and interferon responses, as well as pathways associated with histone acetylation, transport, and extracellular matrix organization were similar within each cell type, regardless of the viral strain. In CEK cells, GO terms and pathways were downregulated uniquely after CSW-1 ILTV infection, indicating a viral-strain specific effect in this cell-type. Overall, this study highlights that the observed differences in host and ILTV gene transcription in vitro were more strongly influenced by the cell-types used rather than the presence or absence of gG. This underscores the importance of cell-line selection in studying host-virus interactions and interpreting experimental results. [ABSTRACT FROM AUTHOR]

Disinfection with LED lamps is a promising ecological and economical substitute for mercury lamps. However, the optimal time/dose relationship needs to be established. Pathogen inactivation by UV-A primarily relies on induced reactive oxygen species (ROS) formation and subsequent oxidative damage. While effective against bacteria and enveloped viruses, non-enveloped viruses are less sensitive. In this study, we explored the disinfection properties of 10 W UV-A LED, emitting in the 365–375 nm range. UV-A at high values of irradiance (~ 0.46 W/cm2) can potentially induce ROS formation and direct photochemical damage of the pathogen nucleic acids, thus improving the disinfection. The UV-A inactivation was evaluated for the bacterium Escherichia coli (E. coli), non-enveloped RNA bacteriophage MS2, and enveloped mammalian RNA virus—Semliki Forest virus (SFV). The 4 log10 reduction doses for E. coli and SFV were 268 and 241 J/cm2, respectively. Furthermore, in irradiated E. coli, ROS production positively correlated with the inactivation rate. In the case of MS2 bacteriophage, the 2.5 log10 inactivation was achieved by 679 J/cm2 within 30 min of irradiation. The results demonstrate significant disinfection efficiency of non-enveloped virus MS2 using high-irradiance UV-A. This suggests a potential strategy for improving the inactivation of UV-A-unsusceptible pathogens, particularly non-enveloped viruses. Additionally, the direct UV-A irradiation of self-replicating viral RNA from SFV led to a significant loss of viral gene expression in cells transfected with the irradiated RNA. Therefore, the virus inactivation mechanism of high-irradiance UV-A LED can be partially determined by the direct damage of viral RNA. [ABSTRACT FROM AUTHOR]

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (FT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology. IMPORTANCE Functional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes. [ABSTRACT FROM AUTHOR]

Bovine herpesvirus type 1 (BoHV-1) causes severe diseases in bovine species and great economic burden to the cattle industry worldwide. Due to its complex life cycle, many host factors that affect BoHV-1 replication remain to be explored. To understand the possible roles that the Oct1 cellular protein could play in this process, we first created Oct1-deficient MDBK cells using CRISPR/Cas9-mediated genome editing. Upon infection, the absence of Oct1 in MDBK cells significantly impacted BoHV-1 replication, a phenotype rescued by over-expressing the wild-type Oct1 protein in the deficient cells. We further found that the expression of all three classes of temporal genes, including essential and non-essential viral genes, were significantly reduced in Oct1 knockout MDBK cells, following both high and low multiplicity of infection. In summary, our findings confirm that the bovine Oct1 protein acts as a pro-viral factor for BoHV-1 replication by promoting its viral gene transcription in MDBK cells. [ABSTRACT FROM AUTHOR]

Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations. [ABSTRACT FROM AUTHOR]

Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro -infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription. [Display omitted] • We uncovered a heterogeneous transcriptional termination of HBV transcripts in cellular models and in patients with CHB. • HBV transcription termination is functionally linked to HBV replication. • RNA helicases DDX5 and DDX17 promote HBV transcriptional read-through and viral RNA destabilisation. • DDX5 and DDX17 are host HBV restriction factors and are downregulated in patients with CHB. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a common herpesvirus that persistently infects a large portion of the world's population. Despite the robust host immune response, HCMV is able to replicate, evade host defenses, and establish latency throughout the lifespan by developing multiple immunomodulatory strategies, making the studies on the interaction between HCMV infection and host response particularly important. HCMV has a strict host specificity that specifically infects humans. Therefore, most of the in vivo researches of HCMV rely on clinical samples. Fortunately, the establishment of humanized mouse models allows for convenient in-lab animal experiments involving HCMV infection. Single-cell RNA sequencing enables the study of the relationship between viral and host gene expressions at the single-cell level within host cells. In this study, we assessed the gene expression alterations of PBMCs at the single-cell level within HCMV-infected humanized mice, which sheds light onto the virus-host interactions in the context of HCMV infection of humanized mice and provides a valuable dataset for the related researches. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we reported a peste des petits ruminants virus infection case in wild animals, and a virus strain was subsequently sequenced. Our results showed the existence of peste des petits ruminants virus in wild small ruminants in China, which was found to be closely related to the peste des petits ruminants virus isolated in China between 2013 and 2014. Our findings indicated that more attention should be paid to the prevention and control of wildlife diseases because there is the possibility of wild animals infecting domestic animals. In 2013, the second outbreak of peste des petits ruminants occurred in China, leading to a spillover in more than 20 provinces and municipalities over the next few months. Thereafter, the epidemic situation was stable owing to strict prevention and control measures. In February 2024, several bharals and argali with suspected symptoms of PPR were discovered in Rutog country, Tibet Autonomous Region. Samples collected from these animals were delivered to our laboratory for diagnosis; the results of fluorescence quantitative reverse-transcription (RT) PCR indicated that all samples were positive for PPR viral RNA. The N and F gene fragments were amplified successfully via RT-PCR, and these results confirmed that these animals were infected with PPRV. A PPRV strain (subsequently named ChinaTibet2024) was sequenced, and its genome length was 15,954 nucleotides. A phylogenetic tree analysis using N and F genes and viral genomes showed that the ChinaTibet2024 genome was classified into lineage IV of the PRRV genotypes. The genome of the ChinaTibet2024 strain was found to be closely related to PPRVs isolated in China between 2013 and 2014. A base insertion and a base deletion were detected in the M gene 5′ untranslated region. Results indicated that the prevalent PPRV strains in China did not show significant changes and that special attention should be paid to the surveillance of wild animals as an important part of PPR prevention and control. [ABSTRACT FROM AUTHOR]

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53–Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it. [ABSTRACT FROM AUTHOR]

Adenoviruses are commonly utilized as viral vectors for gene therapy, genetic vaccines, and recombinant protein expression. To generate replication-defective adenoviruses, E1-complementing cell lines such as HEK293A are utilized; however, limitations remain. Repeated passage of E1-deleted virus in HEK293A cells increases the occurrence of replication-competent adenoviruses (RCAs). In the present study, we developed a novel cell line originating from human primary cells. L132 cells were transduced two times with E1-encoded retrovirus and three times with E1A-encoded retrovirus. Finally, we selected the most productive L132 cell line for generation of RCA-free adenovirus, GT541. GT541 can serve as an alternative cell line to HEK293A and other adenovirus-producing cells. [ABSTRACT FROM AUTHOR]

The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis. [ABSTRACT FROM AUTHOR]

The persistent infection caused by foot‐and‐mouth disease virus (FMDV) still lacks a reliable explanation, as its etiology and maintenance are intricate and potentially involve concurrent infections with multiple pathogens. In this study, we utilized the nanopore platform for direct sequencing of clinical samples obtained from cattle persistently infected with FMDV serotype O and investigated the distribution characteristics of coinfecting pathogens in their pharyngeal region. Briefly, we exploited Oxford Nanopore sequencing technology to generate high‐quality and sufficient sequence data for the comprehensive characterization of microbial genomes. Furthermore, we performed sequence comparison, alignment, and phylogenetic tree construction. Our findings revealed a total of 23 viruses in FMDV carrier bovine, with FMDV, bovine orthopneumovirus, and Choristoneura fumiferana granulovirus emerging as the top three identified pathogens. The analysis unexpectedly revealed the presence of porcine circovirus type 2 and pepper mild mottle virus among the viral genes detected in the bovine FMDV carrier. Compared to noncarrier, carrier bovine of FMDV exhibited a greater diversity and abundance of mycoplasma types as well as reads counts. Therefore, we propose that the establishment and perpetuation of persistent FMDV infection may be attributed to the simultaneous presence of other viral agents and mycoplasmas. These findings highlight the significance of investigating multipathogen coinfection in elucidating the etiology of persistent FMD virus infection. [ABSTRACT FROM AUTHOR]

Gene drives are genetic modifications designed to propagate efficiently through a population. Most applications rely on homologous recombination during sexual reproduction in diploid organisms such as insects, but we recently developed a gene drive in herpesviruses that relies on co-infection of cells by wild-type and engineered viruses. Here, we report on a viral gene drive against human herpes simplex virus 1 (HSV-1) and show that it propagates efficiently in cell culture and during HSV-1 infection in mice. We describe high levels of co-infection and gene drive-mediated recombination in neuronal tissues during herpes encephalitis as the infection progresses from the site of inoculation to the peripheral and central nervous systems. In addition, we show evidence that a superinfecting gene drive virus could recombine with wild-type viruses during latent infection. These findings indicate that HSV-1 achieves high rates of co-infection and recombination during viral infection, a phenomenon that is currently underappreciated. Overall, this study shows that a viral gene drive could spread in vivo during HSV-1 infection, paving the way toward therapeutic applications. Gene drives are genetic modifications designed to propagate efficiently through a population. Here, the authors develop a viral gene drive against herpes simplex virus 1 (HSV-1) and show that it propagates efficiently during HSV-1 infection in mice. [ABSTRACT FROM AUTHOR]

Gene therapy is the technique of inserting foreign genetic elements into host cells to achieve a therapeutic effect. Although gene therapy was initially formulated as a potential remedy for specific genetic problems, it currently offers solutions for many diseases with varying inheritance patterns and acquired diseases. There are two major groups of vectors for gene therapy: viral vector gene therapy and non-viral vector gene therapy. This review examines the role of a macromolecule's chemical and physical architecture in non-viral gene delivery, including their design and synthesis. Polymers can boost circulation, improve delivery, and control cargo release through various methods. The prominent examples discussed include poly-L-lysine, polyethyleneimine, comb polymers, brush polymers, and star polymers, as well as hydrogels and natural polymers and their modifications. While significant progress has been made, challenges still exist in gene stabilization, targeting specificity, and cellular uptake. Overcoming cytotoxicity, improving delivery efficiency, and utilizing natural polymers and hybrid systems are vital factors for prospects. This comprehensive review provides an illuminating overview of the field, guiding the way toward innovative non-viral-based gene delivery solutions. [ABSTRACT FROM AUTHOR]

Background/Objectives: Adeno-associated viruses (AAVs) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of the transduction of AAV2/5, 2/6, 2/8 and 2/9 expressing GFP in preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytometry. Methods: Three transduction modes in 3T3-L1 preadipocyte cells assessed: AAV transduction in 3T3-L1 preadipocyte cells, transduction with further differentiation into mature adipocyte-like cells and the transduction of differentiated 3T3-L1 adipocytes. For the in vivo study, we injected AAV2/6, AAV2/8 and AAV2/9 in adipose tissue of C57BL6 mice, and the transduction capacity of AAV2/6, along with AAV2/8 and AAV2/9 was evaluated. Results: AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5–2-fold more effective than AAV2/5, and AAV2/8 in the range of viral concentrations from 2 × 104 to 1.6 × 105 VG/cell. AAV2/5 and AAV2/8 showed transduction efficiencies similar to each other. The expression of GFP under the CMV promoter remained stable for up to 20 days. The induction of 3T3-L1 differentiation in three days after AAV transduction did not alter the GFP expression level, and AAV2/6 showed the best transduction efficiency. AAV2/6 demonstrated the ability to transduce mature adipocytes. These results were confirmed by in vivo studies on C57BL6 mice. AAV2/6 had the highest transducing activity on both inguinal and interscapular adipose tissue. Conclusions: Thus, AAV2/6 has demonstrated higher transduction efficacy compared to AAV2/5, AAV2/8 and AAV2/9 both in 3T3-L1 adipocytes and adipose tissue in vivo, which proves its usability along with AAV2/8 and AAV2/9 for gene delivery to adipocytes. [ABSTRACT FROM AUTHOR]

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism. [ABSTRACT FROM AUTHOR]

The unprecedented research effort associated with the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) included several extensive proteomic studies that identified host proteins that interact with individual viral gene products. However, in most cases, the consequences of those virus–host interactions for virus replication were not experimentally pursued, which is a necessary step in determining whether the interactions represent pro- or anti-viral events. One putative interaction commonly identified in multiple studies was between the host adaptor protein complex 3 (AP-3) subunit B1 (AP3B1) and the SARS-CoV-2 envelope protein (E). AP3B1 is one subunit of AP-3 required for the biogenesis of lysosomal-related organelles (LROs), and its function impacts important disease processes including inflammation and vascular health. Thus, interactions between AP3B1 and SARS-CoV-2 might influence the clinical outcomes of infection. To determine if AP3B1 has a role in the SARS-CoV-2 replication cycle, we first confirmed the interaction in virus-infected cells using immunoprecipitation (IP) and immunofluorescence assays (IFA). AP3B1 is required by multiple viruses to aid in the replication cycle and therefore may be a therapeutic target. However, we found that the overexpression of AP3B1 suppressed SARS-CoV-2 replication, whereas the siRNA-mediated depletion of AP3B1 increased the release of infectious virus, suggesting an antiviral role for AP3B1. Together, our findings suggest that AP3B1 is an intrinsic barrier to SARS-CoV-2 replication through interactions with the viral E protein. Our work justifies further investigations of LRO trafficking in SARS-CoV-2 target cells and their role in viral pathogenesis. [ABSTRACT FROM AUTHOR]

The productive replication of human immunodeficiency virus type 1 (HIV-1) involves intricate interactions between viral proteins and host cell machinery. However, the contributions of the lysosomal pathways for HIV-1 replication are not fully understood. The goal of this study was to determine the impact of lysosome-targeting compounds on HIV-1 replication and identify the cellular changes that are linked to HIV-1 inhibition using cell culture models of HIV-1 infection. Here, we demonstrate that the treatment of cells with various pharmacological agents known to inhibit lysosomal functions interfere with HIV-1 replication. The vacuolar ATPase (V-ATPase) inhibitor bafilomycin A1 exerted a potent inhibition of HIV-1 replication. Bafilomycin A1 inhibition of HIV-1 was independent of coreceptor tropism of HIV-1. Our data suggest that bafilomycin A1 inhibits HIV-1 at the post-integration steps of the virus life cycle, which include viral gene expression, virus assembly, and/or egress. Analysis of the cellular alterations following bafilomycin A1 treatment indicates that bafilomycin A1 causes a disruption in lysosome structure and functions. Treatment of cells with bafilomycin A1 caused an accumulation of unesterified cholesterol in lysosomes along with the expansion of the lysosomal compartments. Interestingly, the overexpression of the lysosomal cholesterol transporter Niemann–Pick type C 1 (NPC1) partially relieved bafilomycin A1 inhibition of HIV-1. Collectively, our data suggest that bafilomycin A1 inhibits HIV-1 replication in part by disrupting the lysosomal cholesterol trafficking pathway. [ABSTRACT FROM AUTHOR]

The highly abundant and stable antiviral small RNA derived from honeysuckle, known as miR2911, has been shown to play a key role in inhibiting influenza virus infection and SARS-CoV-2 infection. However, whether miR2911 inhibits the replication of porcine reproductive and respiratory syndrome virus (PRRSV) remains unknown. Hence, this study investigated the mechanisms underlying the action of miR2911 during PRRSV infection. Six targets of miR2911 within the PRRSV orf1 (Nsp2: 2459 to 2477, 1871 to 1892, 954 to 977, and 1271 to 1292; Nsp1: 274 to 296 and 822 to 841) were successfully identified by using the miRanda v1.0b software. The miR2911 target sequence was analyzed by target sequence comparison, and only individual base mutations existed in different prevalent strains, and the miR2911 target region was highly conserved among different strains. Subsequently, through the dual luciferase reporter gene assay and miR2911 overexpression assay, it was demonstrated that miR2911 significantly inhibits the replication of PRRSV by targeting regions of PRRSV Nsp1 and Nsp2. These findings offer new insights for the development of novel anti-PRRSV drugs. [ABSTRACT FROM AUTHOR]

Viruses sense cellular states and environmental signals by recruiting host transcription factors to cis -regulatory elements (CREs) in viral genomes. Viral CREs act as signal integration hubs that impart specificity to viral gene regulation. Viral CREs are highly evolvable due to their high mutation rate and ability to rapidly change sensing mechanisms, affecting infection spread and patient outcomes. Latent viral infections can be treated by targeting viral CREs for reactivation or permanent silencing. To withstand a hostile cellular environment and replicate, viruses must sense, interpret, and respond to many internal and external cues. Retroviruses and DNA viruses can intercept these cues impinging on host transcription factors via cis -regulatory elements (CREs) in viral genomes, allowing them to sense and coordinate context-specific responses to varied signals. Here, we explore the characteristics of viral CREs, the classes of signals and host transcription factors that regulate them, and how this informs outcomes of viral replication, immune evasion, and latency. We propose that viral CREs constitute central hubs for signal integration from multiple pathways and that sequence variation between viral isolates can rapidly rewire sensing mechanisms, contributing to the variability observed in patient outcomes. [ABSTRACT FROM AUTHOR]

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5′-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo. Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo. Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers. [ABSTRACT FROM AUTHOR]

Simple Summary: African swine fever (ASF) causes serious economic losses to the global pig industry. Although several commercial vaccines have been registered in some Asian countries, the development of ASF vaccines is still actively continuing. One of the current trends is the development of a safe DIVA vaccine against ASF, the use of which will allow differentiating between infected or vaccinated pigs. To date, commercial ASF vaccines are live vaccines based on the ASFV attenuated strains with the deletion of the viral genes responsible for virulence. However, some recombinant strains with the deletions of virulence genes may have residual virulence and be unsafe. In our work, we have shown that the infection of animals with the ASFV A137R-deletion mutant with a dose 10 times higher than the described safe dose leads to the death of 87.5% of the infected animals. We assume that the deletion of the A137R gene may be additionally introduced into the genomes of candidate vaccine strains in order to reduce their reactogenicity and as an antigenic and genetic marker for the differentiation of infected and vaccinated animals (DIVA strategy). African swine fever (ASF) is an emerging disease caused by the African swine fever virus (ASFV), which is a great threat to the swine industry worldwide. Currently registered vaccines that have demonstrated protection against the homologous ASFV strains are live attenuated vaccines based on recombinant ASFV strains with the deletions of virulence-associated genes. In this study, we evaluated the deletion of the A137R gene in the ASFV virulent Stavropol_01/08 strain isolated in Russia in 2008. Our animal experiment results demonstrated that the deletion of the A137R gene did not lead to the full attenuation of this strain, and increasing the dose of the A137R-deletion mutant during infection led to the death of 87.5% of the infected animals. In this report, we also demonstrated that immunofluorescence (IFA) and Western blotting assays based on the recombinant p11.5 protein can be used to detect antibodies in animals infected with the attenuated ASFV variants of several genotypes/serotypes. Both assays were specific to ASFV p11.5 protein and showed negative results when examining the sera of the non-infected animals or those infected with the A137R-deletion mutant. Therefore, we propose to use the p11.5 protein along with other previously proposed ASFV proteins, such as CD2v, as negative antigenic DIVA markers for an attenuated ASF vaccine. [ABSTRACT FROM AUTHOR]

Importance: Ovine pulmonary adenomatosis (OPA) and maedi-visna disease (MVD) are chronic and progressive infectious diseases in sheep caused by Jaagsiekte sheep retrovirus (JSRV) and maedi-visna virus (MVV), respectively. Objective: To investigate the pathological changes and conduct viral gene analysis of OPA and MVD co-occurrence in Inner Mongolia, China. Methods: Using gross pathology, histopathology, immunohistochemistry, ultrastructural pathology, PCR, and sequence analysis, we investigated the concurrent infection of JSRV and MVV in 319 Dorper rams slaughtered in a private slaughterhouse in Inner Mongolia, in 2022. Results: Of the 319 rams included, 3 showed concurrent JSRV and MVV infection. Gross lung pathology showed diffuse enlargement, consolidation, and greyish-white miliary nodules on the lung surface; the trachea was filled with a white foamy fluid; hilar and mediastinal lymph nodes were significantly enlarged. Histopathology results revealed typical OPA and MVD lesions in the lung tissue. Immunohistochemical results were positive for JSRV envelope protein (Env) in the tumor cells and MVV CA in alveolar macrophages. Transmission electron microscopy showed several virions and autophagosomes in the lung tissue, severely damaged mitochondria, and the induced mitophagy. Nucleotide sequences obtained for JSRV env and MVV gag showed the highest homology with the Inner Mongolian strains of JSRV env (JQ837489) and MVV gag (MW248464). Conclusions and Relevance: Our study confirmed that OPA and MVD co-occurrence and identified the pathological changes in Inner Mongolia, China, thereby providing references for the identification of concurrent JSRV and MVV infections. [ABSTRACT FROM AUTHOR]

Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno‐associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non‐viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non‐viral vectors. [ABSTRACT FROM AUTHOR]

Respiratory Syncytial Virus (RSV) is a significant cause of lower respiratory tract infections (LRTI) across all demographics, with increasing mortality and morbidity among high-risk groups such as infants under two years old, the elderly, and immunocompromised individuals. Although newly approved vaccines and treatments have substantially reduced RSV hospitalizations, accessibility remains limited, and response to treatment varies. This underscores the importance of comprehensive studies on host–RSV interactions. tRNA-derived RNA fragments (tRFs) are recently discovered non-coding RNAs, notable for their regulatory roles in diseases, including viral infections. Our prior work demonstrated that RSV infection induces tRFs, primarily derived from the 5′-end of a limited subset of tRNAs (tRF5), to promote RSV replication by partially targeting the mRNA of antiviral genes. This study found that tRFs could also use their bound proteins to regulate replication. Our proteomics data identified that PABPC1 (poly(A)-binding protein cytoplasmic 1) is associated with tRF5-GluCTC, an RSV-induced tRF. Western blot experimentally confirmed the presence of PABPC1 in the tRF5-GluCTC complex. In addition, tRF5-GluCTC is in the anti-PABPC1-precipitated immune complex. This study also discovered that suppressing PABPC1 with its specific siRNA increased RSV (-) genome copies without impacting viral gene transcription, but led to less infectious progeny viruses, suggesting the importance of PABPC1 in virus assembly, which was supported by its interaction with the RSV matrix protein. Additionally, PABPC1 knockdown decreased the production of the cytokines MIP-1α, MIP-1β, MCP-1, and TNF-α. This is the first observation suggesting that tRFs may regulate viral infection via their bound proteins. [ABSTRACT FROM AUTHOR]

A newly isolated rhabdovirus-like agent, named SHRV-In, obtained from snakehead fish in India was further characterized in this study. The virus demonstrated growth in various cell lines including those derived from snakehead and seabass species. It exhibited sensitivity to acidic conditions (pH 3.0) and exposure to 56 °C. Five structural proteins of 20 to 68 kDa were found in the purified virus. Experimental infection trials conducted on catfish (Pangasius pangasius) resulted in mortality 72 h post-infection, whereas no mortality was observed in rohu (Labeo rohita) and common carp (Cyprinus carpio) fingerlings. Molecular characterization of the virus involved sequencing of the N, P, M, and G gene fragments after PCR amplification using specific primers. Sequence analysis of all four viral genes showed high similarity with the previously isolated snakehead rhabdovirus (SHRV) from Thailand. Phylogenetic analysis and gene sequence alignment placed the SHRV-In isolate within the Novirhabdovirus group, which includes viruses like VHSV (viral hemorrhagic septicemia virus), IHNV (infectious hematopoietic necrosis virus), and SHRV. However, our repeated trials failed to amplify the NV gene in this isolate SHRV-In. [ABSTRACT FROM AUTHOR]

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly significant infectious disease that poses a substantial threat to the global pig industry. In recent years, the NADC30-like strain has gradually emerged as prevalent in China, causing a profound impact on the country's pig farming industry. Therefore, it is important to conduct an in-depth study on the characteristics and gene functions of the NADC30-like strain. An infectious cDNA clone is an indispensable tool for investigating the functions of viral genes. In this current study, we successfully isolated a NADC30-like strain and constructed its full-length infectious cDNA clone. The utilization of this clone will facilitate our investigation into the viral replication, pathogenesis, and immune response associated with the PRRSV NADC30-like strain. [ABSTRACT FROM AUTHOR]

Viral infection is frequently assayed by ongoing expression of viral genes. These assays fail to identify cells that have been exposed to the virus but limit or inhibit viral replication. To address this limitation, we used a dual-labeling vesicular stomatitis virus (DL-VSV), which has a deletion of the viral glycoprotein gene, to allow evaluation of primary infection outcomes. This virus encodes Cre, which can stably mark any cell with even a minimal level of viral gene expression. Additionally, the virus encodes GFP, which distinguishes cells with higher levels of viral gene expression, typically due to genome replication. Stereotactic injections of DL-VSV into the murine brain showed that different cell types had very different responses to the virus. Almost all neurons hosted high levels of viral gene expression, while glial cells varied in their responses. Astrocytes (Sox9+) were predominantly productively infected, while oligodendrocytes (Sox10+) were largely abortively infected. Microglial cells (Iba1+) were primarily uninfected. Furthermore, we monitored the early innate immune response to viral infection and identified unique patterns of interferon (IFN) induction. Shortly after infection, microglia were the main producers of IFNb, whereas later, oligodendrocytes were the main producers. IFNb+ cells were primarily abortively infected regardless of cell type. Last, we investigated whether IFN signaling had any impact on the outcome of primary infection and did not observe significant changes, suggesting that intrinsic factors are likely responsible for determining the outcome of primary infection. [ABSTRACT FROM AUTHOR]

Cervical cancer remains a significant health challenge for women worldwide, with a disproportionate impact on developing regions like sub-Saharan Africa. Taking advantage of recent advancements in developing suitable preclinical models to study cell proliferation, differentiation, and gene expression, we used RNA sequencing to compare the transcriptomic profiles of SiHa cervical cancer cells grown in 3D versus 2D culture systems. Pathway analysis of 3D cultures revealed upregulation of immune activation, angiogenesis, and tissue remodeling pathways. The high expression of cytokines, chemokines, matrix metalloproteinases, and immediate early genes, suggests that 3D cultures replicate the tumor microenvironment better than 2D monolayer cultures. HPV gene expression analysis further demonstrated higher expression levels of HPV16 E1, E2, E6, and E7 genes in 3D versus 2D cultures. Further, by using a set of linear models, we identified 79 significantly differentially expressed genes in 3D culture compared to 2D culture conditions, independent of HPV16 viral gene effects. We subsequently validated five of these genes at the protein level in both the SiHa cell line and a newly developed, patient-derived cervical cancer cell line. In addition, correlation analysis identified 26 human genes positively correlated with viral genes across 2D and 3D culture conditions. The top five 3D versus 2D differentially expressed and HPV-correlated genes were validated via qRT-PCR in our patient derived cell line. Altogether, these findings suggest that 3D cultures provide superior model systems to explore mechanisms of immune evasion, cancer progression and antiviral therapeutics. [ABSTRACT FROM AUTHOR]

Introduction: Goose astrovirus (GAstV) is a newly emerging pathogen that is currently widespread among geese, causing visceral gout and leading to substantial gosling mortalities, posing a severe threat to the waterfowl industry. GAstV II is the predominant epidemic strain, characterized by its high morbidity and mortality rate. Consequently, there is an urgent necessity to develop an effective diagnostic approach to control the dissemination of GAstV II, particularly in clinical farms with limited laboratory resources. Methods: In this study, a novel multi-enzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combined assay was developed. Different primers designed specific targeting a highly conserved region within the viral RdRp gene for the detection of GAstV II. Primers optimized and MIRA-LFD assay analyzed its performance regarding limits of detection, specificity, and efficiency of detection. Results: The developed MIRA amplification is conducted at a constant temperature and accomplished within 10 minutes. Subsequent naked-eye observation of the LFD strips merely takes 5 minutes. The established MIRALFD method exhibits high specificity, with no cross-reaction with other pathogens and attains a detection sensitivity of 1 copy/ml, which is consistent with the reverse transcription quantitative PCR (RT-qPCR) assay. Further evaluation with clinical samples indicates that the accuracy of this MIRA-LFD method correlates well with RT-qPCR for the detection of GAstV II. Conclusion: In summary, the convenience, sensitivity, and rapidity of this newly developed detection method offer a significant advantage for on-site diagnosis of GAstV II. [ABSTRACT FROM AUTHOR]

This review investigates links between post-acute sequelae of SARS-CoV-2 infection (PASC), post-infection viral persistence, mitochondrial involvement and aberrant innate immune response and cellular metabolism during SARS-CoV-2 infection. Advancement of proteomic and metabolomic studies now allows deeper investigation of alterations to cellular metabolism, autophagic processes and mitochondrial dysfunction caused by SARS-CoV-2 infection, while computational biology and machine learning have advanced methodologies of predicting virus–host gene and protein interactions. Particular focus is given to the interaction between viral genes and proteins with mitochondrial function and that of the innate immune system. Finally, the authors hypothesise that viral persistence may be a function of mitochondrial involvement in the sequestration of viral genetic material. While further work is necessary to understand the mechanisms definitively, a number of studies now point to the resolution of questions regarding the pathogenesis of PASC. [ABSTRACT FROM AUTHOR]

Background Human cytomegalovirus (HCMV) is the most common and serious opportunistic infection after solid organ and hematopoietic stem cell transplantation. In this study, we used whole-genome HCMV data to investigate viral factors associated with the clinical outcome. Methods We sequenced HCMV samples from 16 immunocompromised pediatric patients with persistent viremia. Eight of the 16 patients died of complications due to HCMV infection. We also sequenced samples from 35 infected solid organ adult recipients, of whom 1 died with HCMV infection. Results We showed that samples from both groups have fixed variants at resistance sites and mixed infections. Next-generation sequencing also revealed nonfixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with nonfixed variants in these patients. These genes formed a viral signature that discriminated patients with HCMV infection who died from those who survived with high accuracy (area under the curve = 0.96). Lymphocyte numbers for a subset of patients showed no recovery posttransplant in the patients who died. Conclusions We hypothesize that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T-cell function, potentially identifying early those patients requiring nonpharmacological interventions. [ABSTRACT FROM AUTHOR]

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies. [ABSTRACT FROM AUTHOR]

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 divides 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. Author summary: Enteroviruses are frequent causes of diseases that range from mild common colds to severe conditions such as poliomyelitis, acute flaccid myelitis and viral myocarditis. Enterovirus particles are tiny, even by virus standards, and they can only package a small amount of viral genetic material. With such a small genetic 'toolbox', how can these viruses so vigorously hijack the interior of a cell and turn it into a viral factory? One reason is that the viral genes often encode multifunctional proteins. Here, we studied one such multifunctional viral protein, called 2C. This protein is found in all enteroviruses and in related viruses, and the virus cannot replicate without it. We developed a new way of studying 2C that allowed us to work with the complete 2C protein 'in its element', namely bound to a membrane. We found that 2C not only binds one membrane, but can gather several membranes together–a function that may be important to building the viral factory. We also found that 2C can bind RNA (the chemical form of the virus' genetic material) to membranes and can cleave RNA. Our findings reinforce the view of 2C as an organizer of the viral factory. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is a formidable threat to the stability of the global grape and wine industries. It is the primary etiological agent of grapevine leafroll disease (GLD) and significantly impairs vine health, fruit quality, and yield. GLRaV-3 is a member of the genus Ampelovirus, Closteroviridae family. Viral genes within the 3′ proximal unique gene blocks (UGB) remain highly variable and poorly understood. The UGBs of Closteroviridae viruses include diverse open reading frames (ORFs) that have been shown to contribute to viral functions such as the suppression of the host RNA silencing defense response and systemic viral spread. This study investigates the role of GLRaV-3 ORF8, ORF9, and ORF10, which encode the proteins p21, p20A, and p20B, respectively. These genes represent largely unexplored facets of the GLRaV-3 genome. Here, we visualize the subcellular localization of wildtype and mutagenized GLRaV-3 ORFs 8, 9, and 10, transiently expressed in Nicotiana benthamiana. Our results indicate that p21 localizes to the cytosol, p20A associates with microtubules, and p20B is trafficked into the nucleus to carry out the suppression of host RNA silencing. The findings presented herein provide a foundation for future research aimed at the characterization of the functions of these ORFs. In the long run, it would also facilitate the development of innovative strategies to understand GLRaV-3, mitigate its spread, and impacts on grapevines and the global wine industry. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) poses a global threat worldwide causing pandemics, epidemics, and seasonal outbreaks. Annual modification of vaccines is costly due to continual shifts in circulating genotypes, leading to inadequate coverage in low- and middle-income countries like India. Additionally, IAVs are evolving resistance to approved antivirals, necessitating a search for alternative treatments. In this study, the antiviral role of the FDA-approved antibiotic minocycline against IAV strains was evaluated in vitro and in vivo by quantifying viral gene expression by qRT-PCR, viral protein levels by Western blotting, and viral titers. Our findings demonstrate that minocycline at a non-toxic dose effectively inhibits IAV replication, regardless of viral strain or cell line. Its antiviral mechanism operates independently of interferon signaling by targeting the MEK/ERK signaling pathway, which is crucial for the export of viral ribonucleoproteins (vRNPs). Minocycline prevents the assembly and release of infectious viral particles by causing the accumulation of vRNPs within the nucleus. Moreover, minocycline also inhibits IAV-induced late-stage apoptosis, further suppressing viral propagation. The antiviral activity of minocycline against IAVs could offer a promising solution amidst the challenges posed by influenza and the limitations of current treatments. [ABSTRACT FROM AUTHOR]

Computational models of homologous protein groups are essential in sequence bioinformatics. Due to the diversity and rapid evolution of viruses, the grouping of protein sequences from virus genomes is particularly challenging. The low sequence similarities of homologous genes in viruses require specific approaches for sequence- and structure-based clustering. Furthermore, the annotation of virus genomes in public databases is not as consistent and up to date as for many cellular genomes. To tackle these problems, we have developed VOGDB, which is a database of virus orthologous groups. VOGDB is a multi-layer database that progressively groups viral genes into groups connected by increasingly remote similarity. The first layer is based on pair-wise sequence similarities, the second layer is based on the sequence profile alignments, and the third layer uses predicted protein structures to find the most remote similarity. VOGDB groups allow for more sensitive homology searches of novel genes and increase the chance of predicting annotations or inferring phylogeny. VOGD B uses all virus genomes from RefSeq and partially reannotates them. VOGDB is updated with every RefSeq release. The unique feature of VOGDB is the inclusion of both prokaryotic and eukaryotic viruses in the same clustering process, which makes it possible to explore old evolutionary relationships of the two groups. VOGDB is freely available at vogdb.org under the CC BY 4.0 license. [ABSTRACT FROM AUTHOR]