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The most common DNA virus found in wastewaters globally is the cross-assembly phage (crAssphage). King Saud University wastewater treatment plant (KSU-WWTP); Manfoha wastewater treatment plant (MN-WWTP); and the Embassy wastewater treatment plant (EMB-WWTP) in Riyadh, Saudi Arabia were selected, and 36 untreated sewage water samples during the year 2022 were used in the current study. The meteorological impact on crAssphage prevalence was investigated. CrAssphage prevalence was recorded using PCR and Sanger sequencing. The molecular diversity of crAssphage sequences was studied for viral gene segments from the major capsid protein (MCP) and membrane protein containing the peptidoglycan-binding domain (MP-PBD). KSU-WWTP and EMB-WWTP showed a higher prevalence of crAssphage (83.3%) than MN-WWTP (75%). Phylogenetic analysis of MCP and MP-PBD segments depicted a close relationship to the Japanese isolates. The MCP gene from the current study's isolate WW/2M/SA/2022 depicted zero evolutionary divergence from 3057_98020, 2683_104905, and 4238_99953 isolates (d = 0.000) from Japan. A significant influence of temporal variations on the prevalence of crAssphage was detected in the three WWTPs. CrAssphage displayed the highest prevalence at high temperatures (33–44 °C), low relative humidity (6–14%), and moderate wind speed (16–21 Km/h). The findings provided pioneering insights into crAssphage prevalence and its genetic diversity in WWTPs in Riyadh, Saudi Arabia. [ABSTRACT FROM AUTHOR]

The promise of viral vector‐based gene therapy (GT) as a transformative paradigm for treating severely debilitating and life‐threatening diseases is slowly coming to fruition with the recent approval of several drug products. However, they have a unique mechanism of action often necessitating a tortuous clinical development plan. Expertise in such complex therapeutic modality is still fairly limited in this emerging class of adeno‐associated virus (AAV) vector‐based gene therapies. Because of the irreversible mode of action and incomplete understanding of genotype–phenotype relationship and disease progression in rare diseases careful considerations should be given to GT product's benefit–risk profile. In particular, special attention needs to be paid to safe dose selection, reliable dose exposure response (including clinically relevant endpoints), or creative approaches in study design targeting small patient populations during clinical development. We believe that quantitative tools encompassed within model‐informed drug development (MIDD) framework fits quite well in the development of such novel therapies, as they enable us to benefit from the totality of data approach in order to support dose selection as well as optimize clinical trial designs, end point selection, and patient enrichment. In this thought leadership paper, we provide our collective experiences, identify challenges, and suggest areas of improvement in applications of modeling and innovative trial design in development of AAV‐based GT products and reflect on the challenges and opportunities for incorporating MIDD tools and more in rational development of these products. [ABSTRACT FROM AUTHOR]

The study of gene therapy for malignant tumors is a rapidly developing area that shows great potential in the battle against cancer. Although it has the capacity for success, tackling the complexity and variety of tumor biology remains a substantial obstacle. Several approaches have been investigated to address this problem, including the use of viral vector-based gene delivery systems, non-viral techniques such as nanoparticles and electroporation, and genome editing technologies like CRISPR-Cas9. These treatments seek to alter the genetic material of tumor cells in order to either impede their growth or trigger cell death. Nevertheless, there exist substantial obstacles that must be surmounted prior to the regular implementation of gene therapy in clinical settings. These factors include assuring precise targeting, minimizing unintended consequences, optimizing the transport of therapeutic genes into tumor cells, and controlling immune responses to the vectors used. To overcome these problems, it will be necessary to establish interdisciplinary alliances and conduct thorough preclinical research to guarantee both safety and effectiveness. However, due to the tremendous progress in molecular biology and gene editing technology, gene therapy has significant potential as a new method for effectively treating malignant tumors. [ABSTRACT FROM AUTHOR]

Introduction: African swine fever (ASF) is an acute and highly contagious disease and its pathogen, the African swine fever virus (ASFV), threatens the global pig industry. At present, management of ASF epidemic mainly relies on biological prevention and control methods. Moreover, due to the large genome of ASFV, only half of its genes have been characterized in terms of function. Methods: Here, we evaluated a previously uncharacterized viral gene, L60L. To assess the function of this gene, we constructed a deletion strain (SY18ΔL60L) by knocking out the L60L gene of the SY18 strain. To evaluate the growth characteristics and safety of the SY18ΔL60L, experiments were conducted on primary macrophages and pigs, respectively. Results: The results revealed that the growth trend of the recombinant strain was slower than that of the parent strain in vitro. Additionally, 3/5 (60%) pigs intramuscularly immunized with a 105 50% tissue culture infectious dose (TCID50) of SY18ΔL60L survived the 21-day observation period. The surviving pigs were able to protect against the homologous lethal strain SY18 and survive. Importantly, there were no obvious clinical symptoms or viremia. Discussion: These results suggest that L60L could serve as a virulence- and replication-related gene. Moreover, the SY18ΔL60L strain represents a new recombinant live-attenuated ASFV that can be employed in the development of additional candidate vaccine strains and in the elucidation of the mechanisms associated with ASF infection. [ABSTRACT FROM AUTHOR]

Prion-like spreading of protein misfolding is a characteristic of neurodegenerative diseases, but the exact mechanisms of intercellular protein aggregate dissemination remain unresolved. Evidence accumulates that endogenous retroviruses, remnants of viral germline infections that are normally epigenetically silenced, become upregulated in neurodegenerative diseases such as amyotrophic lateral sclerosis and tauopathies. Here we uncover that activation of endogenous retroviruses affects prion-like spreading of proteopathic seeds. We show that upregulation of endogenous retroviruses drastically increases the dissemination of protein aggregates between cells in culture, a process that can be inhibited by targeting the viral envelope protein or viral protein processing. Human endogenous retrovirus envelopes of four different clades also elevate intercellular spreading of proteopathic seeds, including pathological Tau. Our data support a role of endogenous retroviruses in protein misfolding diseases and suggest that antiviral drugs could represent promising candidates for inhibiting protein aggregate spreading. Endogenous retroviruses, or genomic relics of ancient viral infection, have been associated with certain neurodegenerative diseases. Here, Liu et al. report a pathway by which reactivated viral gene products contribute to intercellular protein aggregate spreading. [ABSTRACT FROM AUTHOR]

Introduction: Zoonotic transition of Influenza A viruses is the cause of epidemics with high rates of morbidity and mortality. Predicting which viral strains are likely to transition from their genetic sequence could help in the prevention and response against these zoonotic strains. We hypothesized that features predictive of viral hosts could be leveraged to identify biomarkers of zoonotic viral transition. Methods: We trained deep learning models to predict viral hosts based on the virus mRNA or protein sequences. Our multi-host dataset contained 848,630 unique nucleotide sequences obtained from the NCBI Influenza Virus and Influenza Research Databases. Each sequence, representing one gene from one viral strain, was classified into one of the three host categories: Avian, Human, and Swine. Trained models were analyzed using various neural network interpretation methods to identify interesting candidates for zoonotic transition biomarkers. Results: Using mRNA sequences as input led to higher prediction accuracies than amino acids, suggesting that the codon sequence contains information relevant to viral hosts that is lost during protein translation. UMAP visualization of the latent space of our classifiers showed that viral sequences clustered according to their host of origin. Interestingly, sequences from pandemic zoonotic viral strains localized at the margins between hosts, while zoonotic sequences incapable of Human-to-Human transmission localized with non-zoonotic viruses from the same host. In addition, host prediction for pandemic zoonotic sequences had low prediction accuracy, which was not the case for the other zoonotic strains. This supports our hypothesis that ambiguously predicted viral sequences bear features associated with cross-species infectivity. Finally, we compared misclassified sequences to well-classified ones to extract interesting candidates for zoonotic transition biomarkers. While features varied significantly between pairs of species and viral genes, several codons were conserved in Swine-to-Human and Avian-to-Human misclassified sequences, and in particular in the NA, HA, and NP genes, suggesting their importance for zoonosis in Humans. Discussion: Analysis of viral sequences using neural network interpretation approaches revealed important genetic differences between zoonotic viruses with pandemic potential, compared to non-zoonotic viral strains or zoonotic viruses incapable of Human-to-Human transmission. [ABSTRACT FROM AUTHOR]

Herein, we introduce a novel assay for multiple-gene recognition by ligation–double transcription mediated fluorometric profiling. We demonstrated the capability of the system to identify potential multi-gene classifiers for diagnostic use by employing a combination of a ligation–double transcription approach with a selective fluorophore probe–RNA hybridization/graphene oxide quenching system. The system is efficient and requires only 45 minutes for total experimentation and offers high sensitivity (369.6, 408, and 407.8 copies per mL for the O, E, and N genes of SARS-CoV-2, respectively) and specificity (selective to until two mismatches). Our system is expected to expedite the precise diagnosis of RNA-virus-related diseases with multiple gene classifiers. By focusing on distinct viral genes, our method allowed for the detection of various RNA viruses in a variety of sample pools. [ABSTRACT FROM AUTHOR]

Compared to free-living viruses (< 0.22 m) in the ocean, planktonic viruses in the "cellular fraction" (0.22 ~ 3.0 μm) are now far less well understood, and the differences between them remain largely unexplored. Here, we revealed that even in the same seawater samples, the "cellular fraction" comprised significantly distinct virus communities from the free virioplankton, with only 13.87% overlap in viral contigs at the species level. Compared to the viral genomes deposited in NCBI RefSeq database, 99% of the assembled viral genomes in the "cellular fraction" represented novel genera. Notably, the assembled (near-) complete viral genomes within the "cellular fraction" were significantly larger than that in the "viral fraction," and the "cellular fraction" contained three times more species of giant viruses or jumbo phages with genomes > 200 kb than the "viral fraction." The longest complete genomes of jumbo phage (~ 252 kb) and giant virus (~ 716 kb) were both detected only in the "cellular fraction." Moreover, a relatively higher proportion of proviruses were predicted within the "cellular fraction" than "viral fraction." Besides the substantial divergence in viral community structure, the different fractions also contained their unique viral auxiliary metabolic genes; e.g., those potentially participating in inorganic carbon fixation in deep sea were detected only in the "cellular-fraction" viromes. In addition, there was a considerable divergence in the community structure of both "cellular fraction" and "viral fraction" viromes between the surface and deep-sea habitats, suggesting that they might have similar environmental adaptation properties. The findings deepen our understanding of the complexity of viral community structure and function in the ocean. [ABSTRACT FROM AUTHOR]

Soil Sinorhizobium phage AP-16-3, a strain phylogenetically close to Rhizobium phage 16-3, was isolated in a mountainous region of Dagestan, belonging to the origin of cultivated plants in the Caucasus, according to Vavilov N.I. The genome of phage AP-16-3 is 61 kbp in size and contains 62 ORFs, of which 42 ORFs have homologues in the genome of Rhizobium phage 16-3, which was studied in the 1960s–1980s. A search for Rhizobium phage 16-3-related sequences was performed in the genomes of modern strains of root nodule bacteria belonging to different species, genera, and families. A total of 43 prophages of interest were identified out of 437 prophages found in the genomes of 42 strains, of which 31 belonged to Sinorhizobium meliloti species. However, almost all of the mentioned prophages contained single ORFs, and only two prophages contained 51 and 39 ORFs homologous to phages related to 16-3. These prophages were detected in S. meliloti NV1.1.1 and Rh. leguminosarum OyaliB strains belonging to different genera; however, the similarity level of these two prophages did not exceed 14.7%. Analysis of the orphan genes in these prophages showed that they encoded predominantly virion structural elements, but also enzymes and an extensive group of hypothetical proteins belonging to the L, S, and E regions of viral genes of phage 16-3. The data obtained indicate that temperate phages related to 16-3 had high infectivity against nodule bacteria and participated in intragenomic recombination events involving other phages, and in horizontal gene transfer between rhizobia of different genera. According to the data obtained, it is assumed that the repetitive lysogenic cycle of temperate bacteriophages promotes the dissolution of the phage genetic material in the host bacterial genome, and radical updating of phage and host bacterial genomes takes place. [ABSTRACT FROM AUTHOR]

Treatment options for herpesvirus infections that target the interactions between the virus and the host have been identified as promising. Our previous studies have shown that transcription factors p53 and Fos are essential host determinants of gallid alpha herpesvirus 1 (ILTV) infection. The impact of p53 and Fos on ILTV replication has 'not been fully understood yet. Using the sole ILTV-permissive chicken cell line LMH as a model, we examined the effects of hosts p53 and Fos on all phases of ILTV replication, including viral gene transcription, viral genome replication, and infectious virion generation. We achieved this by manipulating the expression of p53 and Fos in LMH cells. Our results demonstrate that the overexpression of either p53 or Fos can promote viral gene transcription at all stages of the temporal cascade of ILTV gene expression, viral genome replication, and infectious virion production, as assessed through absolute quantitative real-time PCR, ILTV-specific RT-qPCR assays, and TCID50 assays. These findings are consistent with our previous analyses of the effects of Fos and p53 knockdowns on virus production and also suggest that both p53 and Fos may be dispensable for ILTV replication. Based on the synergistic effect of regulating ILTV, we further found that there is an interaction between p53 and Fos. Interestingly, we found that p53 also has targeted sites upstream of ICP4, and these sites are very close to the Fos sites. In conclusion, our research offers an in-depth understanding of how hosts p53 and Fos affect ILTV replication. Understanding the processes by which p53 and Fos regulate ILTV infection will be improved by this knowledge, potentially paving the way for the development of novel therapeutics targeting virus–host interactions as a means of treating herpesvirus infections. [ABSTRACT FROM AUTHOR]

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is estimated to affect 0.4%–2.5% of the global population. Most cases are unexplained; however, some patients describe an antecedent viral infection or response to antiviral medications. We report here a multicenter study for the presence of viral nucleic acid in blood, feces, and saliva of patients with ME/CFS using polymerase chain reaction and high‐throughput sequencing. We found no consistent group‐specific differences other than a lower prevalence of anelloviruses in cases compared to healthy controls. Our findings suggest that future investigations into viral infections in ME/CFS should focus on adaptive immune responses rather than surveillance for viral gene products. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes. Author summary: The goal of this study was to determine what role PCNA associated with HSV-1 replication forks plays during viral infection. Using two commercially available PCNA inhibitors, we provide evidence that targeting specific functions of PCNA block HSV-1 DNA replication and subsequent steps in the infectious cycle. Although it was previously thought that viral factors alone facilitate the process of HSV-1 DNA replication, results presented here contradict this long-standing view. With the continued emergence of antiviral resistant HSV-1 variants, these results provide insight into how cellular proteins can be targeted for antiviral treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: PRV can infect most mammals, mainly affecting the nervous and reproductive systems of infected animals, and can cause the death of piglets and other susceptible animals. Identifying effective antiviral agents against PRV to prevent a latent infection is essential. Piceatannol is a bioactive polyphenol substance. It is mainly found in grapes, mushrooms, blueberries, passion fruit and other edible fruits and vegetables. In this study, we evaluated the inhibitory effect of piceatannol on PRV replication in vivo and in vitro and investigated the mechanism of action of piceatannol against PRV. Piceatannol could exert an anti-PRV effect by reducing the transcription level of viral genes, reducing PRV-induced apoptosis and elevating the levels of IL-4, TNF-α and IFN-γ in the serum of mice. Animal experiments showed that piceatannol could delay the onset of disease, reduce the viral load in the brain and kidney and reduce the pathological changes in the tissues and organs of the mice to improve the survival rate of the mice (14.3%). Therefore, the anti-PRV activity of piceatannol in vivo and in vitro was systematically evaluated in this study to provide scientific data for developing a new alternative measure for controlling PRV infection. Pseudorabies virus (PRV) belongs to the family Herpesviridae. PRV has a wide host range and can cause cytopathic effects (CPEs) in PK-15 cells. Therefore, PRV was used as a model to study the antiviral activity of piceatannol. The results showed that piceatannol could restrain PRV multiplication in PK-15 cells in a dose-dependent manner. The 50% inhibitory concentration (IC50) was 0.0307 mg/mL, and the selectivity index (SI, CC50/IC50) was 3.68. Piceatannol could exert an anti-PRV effect by reducing the transcription level of viral genes, inhibiting PRV-induced apoptosis and elevating the levels of IL-4, TNF-α and IFN-γ in the serum of mice. Animal experiments showed that piceatannol could delay the onset of disease, reduce the viral load in the brain and kidney and reduce the pathological changes in the tissues and organs of the mice to improve the survival rate of the mice (14.3%). Therefore, the anti-PRV activity of piceatannol in vivo and in vitro was systematically evaluated in this study to provide scientific data for developing a new alternative measure for controlling PRV infection. [ABSTRACT FROM AUTHOR]

Background: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. Results: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. Conclusions: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies. [ABSTRACT FROM AUTHOR]

Introduction: Planktothrix agardhii is a microcystin-producing cyanobacterium found in Sandusky Bay, a shallow and turbid embayment of Lake Erie. Previous work in other systems has indicated that cyanophages are an important natural control factor of harmful algal blooms. Currently, there are few cyanophages that are known to infect P. agardhii, with the best-known being PaV-LD, a tail-less cyanophage isolated from Lake Donghu, China. Presented here is a molecular characterization of Planktothrix specific cyanophages in Sandusky Bay. Methods and Results: Putative Planktothrix-specific viral sequences from metagenomic data from the bay in 2013, 2018, and 2019 were identified by two approaches: homology to known phage PaV-LD, or through matching CRISPR spacer sequences with Planktothrix host genomes. Several contigs were identified as having viral signatures, either related to PaV-LD or potentially novel sequences. Transcriptomic data from 2015, 2018, and 2019 were also employed for the further identification of cyanophages, as well as gene expression of select viral sequences. Finally, viral quantification was tested using qPCR in 2015–2019 for PaV-LD like cyanophages to identify the relationship between presence and gene expression of these cyanophages. Notably, while PaV-LD like cyanophages were in high abundance over the course of multiple years (qPCR), transcriptomic analysis revealed only low levels of viral gene expression. Discussion: This work aims to provide a broader understanding of Planktothrix cyanophage diversity with the goals of teasing apart the role of cyanophages in the control and regulation of harmful algal blooms and designing monitoring methodology for potential toxin-releasing lysis events. [ABSTRACT FROM AUTHOR]

Most acute gastroenteritis (AGE) outbreaks and sporadic cases in developing countries are attributable to infection by human norovirus (HuNoV), the enteric virus mainly transmitted via fecal-contaminated water. However, it has been challenging to study HuNoV due to the lack of suitable systems to cultivate and replicate the virus, hindering the development of treatments and vaccines. Researchers have been using virus-like particles (VLPs) and infectious viral clones to overcome this challenge as alternatives to fresh virus isolates in various in vitro and ex vivo models. VLPs are multiprotein structures that mimic the wild-type virus but cannot replicate in host cells due to the lack of genetic materials for replication, limiting downstream analysis of the virus life cycle and pathogenesis. The development of in vitro cloning systems has shown promise for HuNoV replication studies. This review discusses the approaches for constructing HuNoV-VLPs and infectious viral clones, the techniques involved, and the challenges faced. It also highlights the relationship between viral genes and their protein products and provides a perspective on technical considerations for producing efficient HuNoV-VLPs and infectious viral clones, which could substitute for native human noroviruses in future studies. [ABSTRACT FROM AUTHOR]

NR2E3 is a nuclear hormone receptor gene required for the correct development of the retinal rod photoreceptors. Expression of NR2E3 protein in rod cell precursors suppresses cone-specific gene expression and, in concert with other transcription factors including NRL, activates the expression of rod-specific genes. Pathogenic variants involving NR2E3 cause a spectrum of retinopathies, including enhanced S-cone syndrome, Goldmann–Favre syndrome, retinitis pigmentosa, and clumped pigmentary retinal degeneration, with limited evidence of genotype–phenotype correlations. A common feature of NR2E3-related disease is an abnormally high number of cone photoreceptors that are sensitive to short wavelength light, the S-cones. This characteristic has been supported by mouse studies, which have also revealed that loss of Nr2e3 function causes photoreceptors to develop as cells that are intermediate between rods and cones. While there is currently no available cure for NR2E3-related retinopathies, there are a number of emerging therapeutic strategies under investigation, including the use of viral gene therapy and gene editing, that have shown promise for the future treatment of patients with NR2E3 variants and other inherited retinal diseases. This review provides a detailed overview of the current understanding of the role of NR2E3 in normal development and disease, and the associated clinical phenotypes, animal models, and therapeutic studies. [ABSTRACT FROM AUTHOR]

Liver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the cellular E3 ligase LXR-inducible degrader of low-density lipoprotein receptor (IDOL) targets the human cytomegalovirus (HMCV) UL136p33 protein for turnover. UL136 encodes multiple proteins that differentially impact latency and reactivation. UL136p33 is a determinant of reactivation. UL136p33 is targeted for rapid turnover by the proteasome, and its stabilization by mutation of lysine residues to arginine results in a failure to quiet replication for latency. We show that IDOL targets UL136p33 for turnover but not the stabilized variant. IDOL is highly expressed in undifferentiated hematopoietic cells where HCMV establishes latency but is sharply downregulated upon differentiation, a stimulus for reactivation. We hypothesize that IDOL maintains low levels of UL136p33 for the establishment of latency. Consistent with this hypothesis, knockdown of IDOL impacts viral gene expression in wild-type (WT) HCMV infection but not in infection where UL136p33 has been stabilized. Furthermore, the induction of LXR signaling restricts WT HCMV reactivation from latency but does not affect the replication of a recombinant virus expressing a stabilized variant of UL136p33. This work establishes the UL136p33-IDOL interaction as a key regulator of the bistable switch between latency and reactivation. It further suggests a model whereby a key viral determinant of HCMV reactivation is regulated by a host E3 ligase and acts as a sensor at the tipping point between the decision to maintain the latent state or exit latency for reactivation. [ABSTRACT FROM AUTHOR]

The molecular diversity of Epstein–Barr virus (EBV) is exceptionally complex and based on the characterization of sequences coding for several viral genes. The aim of this study was to analyze the distribution of EBV types 1 and 2 and to characterize LMP1 variants in a cohort of 73 patients with infectious mononucleosis (IM), as well as to investigate a possible association between viral diversity and relevant clinical parameters. Population-based sequencing of EBNA-2 gene showed the presence of EBV type 1 in all IM patients. Analysis of LMP1 gene found a restricted repertoire of LMP1 variants with the predominance of wild-type B95-8, China1, Mediterranean and North Carolina variants with the presence of more than one LMP1 variant in 16.4% of patients. Co-infections with different LMP1 variants were associated with significantly higher levels of C-reactive protein and lower levels of maximal neutrophil counts and minimal platelet count. The results of this study have shown a narrow repertoire of LMP1 variants and an exclusive presence of EBV type 1 in a cohort of IM from Croatia, suggesting a characteristic local molecular pattern of this virus. The clinical importance of distinct immunobiological features of IM patients with LMP1 variant co-infections needs to be investigated further. [ABSTRACT FROM AUTHOR]

HSV-1 hijacks the cellular vesicular secretion system and promotes the secretion of extracellular vesicles (EVs) from infected cells. This is believed to facilitate the maturation, secretion, intracellular transportation and immune evasion of the virus. Intriguingly, previous studies have shown that noninfectious EVs from HSV-1-infected cells exert antiviral effects on HSV-1 and have identified host restrictive factors, such as STING, CD63, and Sp100 packed in these lipid bilayerenclosed vesicles. Octamer-binding transcription factor-1 (Oct-1) is shown here to be a pro-viral cargo in non-virion-containing EVs during HSV-1 infection and serves to facilitate virus dissemination. Specifically, during HSV-1 infection, the nuclear localized transcription factor Oct-1 displayed punctate cytosolic staining that frequently colocalized with VP16 and was increasingly secreted into the extracellular space. HSV-1 grown in cells bereft of Oct-1 (Oct-1 KO) was significantly less efficient at transcribing viral genes during the next round of infection. In fact, HSV-1 promoted increased exportation of Oct-1 in non-virioncontaining EVs, but not the other VP16-induced complex (VIC) component HCF- 1, and EV-associated Oct-1 was promptly imported into the nucleus of recipient cells to facilitate the next round of HSV-1 infection. Interestingly, we also found that EVs from HSV-1-infected cells primed cells for infection by another RNA virus, vesicular stomatitis virus. In summary, this investigation reports one of the first pro-viral host proteins packed into EVs during HSV-1 infection and underlines the heterogenetic nature and complexity of these noninfectious double-lipid particles. [ABSTRACT FROM AUTHOR]

Tailed bacteriophages (order, Caudovirales) account for the majority of all phages. However, the long flexible tail of siphophages hinders comprehensive investigation of the mechanism of viral gene delivery. Here, we report the atomic capsid and in-situ structures of the tail machine of the marine siphophage, vB_DshS-R4C (R4C), which infects Roseobacter. The R4C virion, comprising 12 distinct structural protein components, has a unique five-fold vertex of the icosahedral capsid that allows genome delivery. The specific position and interaction pattern of the tail tube proteins determine the atypical long rigid tail of R4C, and further provide negative charge distribution within the tail tube. A ratchet mechanism assists in DNA transmission, which is initiated by an absorption device that structurally resembles the phage-like particle, RcGTA. Overall, these results provide in-depth knowledge into the intact structure and underlining DNA delivery mechanism for the ecologically important siphophages. Tailed bacteriophages account for the majority of all phages. Here, the authors employ cryo-EM and structure prediction techniques to investigate the atomic structure of the R4C phage capsid and the in- situ structure of its unique long rigid tail. [ABSTRACT FROM AUTHOR]

Simple Summary: African swine fever (ASF) causes serious economic losses to the global pig industry. Unfortunately, there is currently no effective and safe vaccine, although its development is actively continuing. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. However, deletion of the same gene in different ASFV isolates can lead to different results. In our work, we have shown that, unlike some other isolates, the deletion of the CD2 gene from the genome of the virulent Congo strain does not lead to its weakening, although it affects the onset of ASF infection in domestic pigs. African swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background. [ABSTRACT FROM AUTHOR]

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2–12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions. Author summary: There is heterogeneity in the rates of clinical disease progression in antiretroval therapy-naïve people living with HIV (PLWH). In heterosexual HIV-1 transmission, only a single or very few viral strains, called transmitted/founder (T/F) viruses establish infection in a newly infected host. The long terminal repeat (LTR) is the viral promoter that drives viral gene transcription and is important for the HIV-1 life cycle. In this study we investigated the impact of HIV-1 subtype C T/F virus LTR genetic variation on transcriptional activity, clinical disease outcomes and response to cell activation. Our data show inter-patient T/F LTR genetic variation and limited intrahost evolution by 12 months post infection. T/F LTR variants exhibit differential basal LTR transcriptional activity, which is significanly increased in the presence of the Transactivator of transcription (Tat) protein. Furthermore, we show that T/F LTR transcription activity significanly correlates positively with viral load and viral load set point but negatively with CD4 T cell count. Lastly, we show that T/F LTR variants exhibit differential responses to cell activators PMA, TNF-α, Prostratin and SAHA. Taken together our data suggest that T/F viruses LTR genetic variation and functional heterogeneity are important determinants of clinical outcomes and virus reactivation potential. [ABSTRACT FROM AUTHOR]

Background: Cuban studies show high levels of viral resistance in treated and untreated patients. In 2017, dolutegravir (DTG) was introduced as a rescue therapy in Cuba, and in 2018 as a first-line regimen. The aim of this study was to determine the primary and secondary resistance of HIV-1 to DTG in Cuban patients with multiple resistances. Methods: The sample consisted of 29 patients, of which 23 had not been treated with DTG. The viral RNA isolated from plasma was amplified and the fragment of the pol gene encoding viral integrase (3,854-5,981 bp) was sequenced. The viral subtype of the 29 viruses was determined. Results: No primary resistance mutations to DTG were detected in any patient. The polymorphic positions found with greater frequency in the treated patients were: 112 and 119 (both with 73.9%), 125 (69.5%), and 201 (78.2%). Virological suppression was achieved in 18 out of 23 patients (78.2%) and in 14 out of 23 (60.8%) at 3 and 6 months after the start of treatment, respectively. Of the six patients who had received DTG prior to this study, four (66.7%) developed resistance to integrase inhibitors. The subtypes found were: B (41%), CRF19_cpx (24%), CRF18_cpx and C (both 14%), and G (7%). Conclusion: Primary resistance to DTG was not detected, while secondary resistance was high in those patients who received a single daily dose of the antiviral. It is necessary to delve into the role of some polymorphisms, more frequent in certain subtypes, and their contribution to primary resistance. [ABSTRACT FROM AUTHOR]

Tomato yellow leaf curl virus (TYLCV) is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China. In this study, we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibited that of viral DNA and virion-sense strand mRNAs. However, two other resistant lines did not have such virus inhibition pattern. Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes, as compared to the susceptible line, suggesting a di-versity of tomato TYLCV-resistance mechanisms. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) latency in CD34+ progenitor cells is the outcome of a complex and continued interaction of virus and host that is initiated during very early stages of infection and reflects pro- and anti-viral activity. We hypothesized that a key event during early infection could involve changes to host miRNAs, allowing for rapid modulation of the host proteome. Here, we identify 72 significantly upregulated miRNAs and three that were downregulated by 6hpi of infection of CD34+ cells which were then subject to multiple in silico analyses to identify potential genes and pathways important for viral infection. The analyses focused on the upregulated miRNAs and were used to predict potential gene hubs or common mRNA targets of multiple miRNAs. Constitutive deletion of one target, the transcriptional regulator JDP2, resulted in a defect in latent infection of myeloid cells; interestingly, transient knockdown in differentiated dendritic cells resulted in increased viral lytic IE gene expression, arguing for subtle differences in the role of JDP2 during latency establishment and reactivation of HCMV. Finally, in silico predictions identified clusters of genes with related functions (such as calcium signaling, ubiquitination, and chromatin modification), suggesting potential importance in latency and reactivation. Consistent with this hypothesis, we demonstrate that viral IE gene expression is sensitive to calcium channel inhibition in reactivating dendritic cells. In conclusion, we demonstrate HCMV alters the miRNAome rapidly upon infection and that in silico interrogation of these changes reveals new insight into mechanisms controlling viral gene expression during HCMV latency and, intriguingly, reactivation. [ABSTRACT FROM AUTHOR]

For effective control of several viral diseases, live virus vaccines are given preference over killed or inactivated vaccines for their higher immunogenic potentials. Live virus vaccine strains are developed by attenuation by passaging in nonnatural host species, tissues, embryonated eggs, etc., or by transferring viral genes/genomes to some other vector virus. The attenuated viruses fail to infect the species of their choice; though can elicit immunity when used as vaccines. The vector viruses are modified by the addition of a section of the genome of the disease-producing viruses in their genome to offer the desired immunity in vaccinated humans or animals. During mass vaccinations, the attenuated live viruses or vector viruses spread in the biosphere. It is suspected that many erstwhile low-profile disease agents genetically related to the viruses used in live vaccine production are causing more severe disease symptoms, changing tissue or organ of predilection, and even changing or broadening host species specificity. Three representative viral diseases affecting three species (human, goat, and cattle) are discussed as examples to enlighten the possible linkage between the spread of the attenuated or vector viruses in the biosphere and the increased virulence of related disease-producing viruses. [ABSTRACT FROM AUTHOR]

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses. The cGAS-STING signalling pathway is critical in mediating host antiviral immunity. Here, Yang et al screen vaccinia viral genes to identify and then characterise that the viral protein E5 is a major inhibitor of cGAS by mediating cGAS ubiquitination and degradation. [ABSTRACT FROM AUTHOR]

Introduction: Coronaviruses (CoVs) are naturally found in bats and can occasionally cause infection and transmission in humans and othermammals. Our study aimed to build a deep learning (DL) method to predict the adaptation of bat CoVs to other mammals. Methods: The CoV genome was represented with a method of dinucleotide composition representation (DCR) for the twomain viral genes, ORF1ab and Spike. DCR features were first analyzed for their distribution among adaptive hosts and then trained with a DL classifier of convolutional neural networks (CNN) to predict the adaptation of bat CoVs. Results and discussion: The results demonstrated inter-host separation and intra-host clustering of DCR-represented CoVs for six host types: Artiodactyla, Carnivora, Chiroptera, Primates, Rodentia/Lagomorpha, and Suiformes. The DCR-based CNN with five host labels (without Chiroptera) predicted a dominant adaptation of bat CoVs to Artiodactyla hosts, then to Carnivora and Rodentia/Lagomorpha mammals, and later to primates. Moreover, a linear asymptotic adaptation of all CoVs (except Suiformes) from Artiodactyla to Carnivora and Rodentia/Lagomorpha and then to Primates indicates an asymptotic bats-other mammals-human adaptation. Conclusion: Genomic dinucleotides represented as DCR indicate a host-specific separation, and clustering predicts a linear asymptotic adaptation shift of bat CoVs from other mammals to humans via deep learning. [ABSTRACT FROM AUTHOR]

Identifying the spatial patterns of genetic structure of influenza A viruses is a key factor for understanding their spread and evolutionary dynamics. In this study, we used phylogenetic and Bayesian clustering analyses of genetic sequences of the A/H1N1pdm09 virus with district-level locations in mainland China to investigate the spatial genetic structure of the A/H1N1pdm09 virus across human population landscapes. Positive correlation between geographic and genetic distances indicates high degrees of genetic similarity among viruses within small geographic regions but broad-scale genetic differentiation, implying that local viral circulation was a more important driver in the formation of the spatial genetic structure of the A/H1N1pdm09 virus than even, countrywide viral mixing and gene flow. Geographic heterogeneity in the distribution of genetic subpopulations of A/H1N1pdm09 virus in mainland China indicates both local to local transmission as well as broad-range viral migration. This combination of both local and global structure suggests that both small-scale and large-scale population circulation in China is responsible for viral genetic structure. Our study provides implications for understanding the evolution and spread of A/H1N1pdm09 virus across the population landscape of mainland China, which can inform disease control strategies for future pandemics. [ABSTRACT FROM AUTHOR]

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model. Author summary: We conducted a comprehensive characterization and reannotation of murine cytomegalovirus (MCMV) gene expression during lytic infection in murine fibroblasts using an integrative multi-omics approach. This unveiled hundreds of novel transcripts that explained the expression of close to 300 so far unknown viral open reading frames (ORFs). Interestingly, small viral ORFs (sORFs) were amongst the most highly expressed viral gene products and thus presumably encode for important viral microproteins of unknown function. We show that sORFs located upstream of larger ORFs tune the expression of the downstream ORFs by repressing their translation. We classified viral transcription start sites (TiSS) based on their expression kinetics obtained by the combination of metabolic RNA labelling with transcription start sites profiling. This not only identified a so far unknown viral immediate-early transcript (ie4, m166.5 RNA) but also revealed a novel class of viral late transcripts that are expressed later than canonical true late genes and lack TATA box-like motifs. We exemplify for the m145 locus how so far unknown viral TiSS give rise to abundantly expressed truncated viral proteins. In summary, we provide a state-of-the-art annotation of an important model virus, which will be instrumental for future studies on CMV biology, immunology and pathogenesis. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies. [ABSTRACT FROM AUTHOR]

Infantile hemangioma (IH) is the most prevalent benign vascular tumor in infants, with distinct disease stages and durations. Despite the fact that the majority of IHs can regress spontaneously, a small percentage can cause disfigurement or even be fatal. The mechanisms underlying the development of IH have not been fully elucidated. Establishing stable and reliable IH models provides a standardized experimental platform for elucidating its pathogenesis, thereby facilitating the development of new drugs and the identification of effective treatments. Common IH models include the cell suspension implantation model, the viral gene transfer model, the tissue block transplantation model, and the most recent three-dimensional (3D) microtumor model. This article summarizes the research progress and clinical utility of various IH models, as well as the benefits and drawbacks of each. Researchers should select distinct IH models based on their individual research objectives to achieve their anticipated experimental objectives, thereby increasing the clinical relevance of their findings. [ABSTRACT FROM AUTHOR]

Upon viral entry, components of ND10 nuclear bodies converge with incoming DNA to repress viral expression. The infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) contains a RING-type E3 ubiquitin ligase that targets the ND10 organizer, PML, for proteasomal degradation. Consequently, ND10 components are dispersed and viral genes are activated. Previously, we reported that ICP0 E3 differentiates two similar substrates, PML isoforms I and II, and demonstrated that SUMO-interaction has profound regulatory effects on PML II degradation. In the present study, we investigated elements that regulate the PML I degradation and found that: (i) two regions of ICP0 flanking the RING redundantly facilitate the degradation of PML I; (ii) downstream of the RING, the SUMO-interaction motif located at residues 362–364 (SIM362–364) targets the SUMOylated PML I in the same manner as that of PML II; (iii) upstream of the RING, the N-terminal residues 1–83 mediate PML I degradation regardless of its SUMOylation status or subcellular localization; (iv) the reposition of residues 1–83 to downstream of the RING does not affect its function in PML I degradation; and (v) the deletion of 1–83 allows the resurgence of PML I and reformation of ND10-like structures late in HSV-1 infection. Taken together, we identified a novel substrate recognition specific for PML I, by which ICP0 E3 enforces a continuous PML I degradation throughout the infection to prevent the ND10 reformation. [ABSTRACT FROM AUTHOR]

Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and "off-target", continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed. [ABSTRACT FROM AUTHOR]

The ability to specifically, safely, and efficiently transfer therapeutic payloads to the striated musculature via a minimally invasive delivery route remains one of the most important but also most ambitious aims in human gene therapy. Over the past two decades, a flurry of groups have harnessed recombinant adeno-associated viruses (AAVs) for this purpose, carrying cargoes that were packaged either in one of the various wild-type capsids or in a synthetic protein shell derived by molecular bioengineering. In this study, we provide an overview over the most commonly used techniques for the enrichment of muscle-specific (myotropic) AAV capsids, typically starting off with the genetic diversification of one or more extant wild-type sequences, followed by the stratification of the ensuing capsid libraries in different muscle types in small or large animals. These techniques include the shuffling of multiple parental capsid genes, peptide display in exposed capsid loops, mutagenesis of individual capsid residues, creation of chimeras between two viral parents, or combinations thereof. Moreover, we highlight alternative experimental or bioinformatic strategies such as ancestral reconstruction or rational design, all of which have already been employed successfully to derive synthetic AAV capsids or vectors with unprecedented in vivo efficiency and/or specificity in the musculature. Most recently, these efforts have culminated in the isolation of unique clades of myotropic vectors called AAVMYO or MyoAAV that have in common the display of the amino acid motif RGD (arginine–glycine–aspartate) on the capsid surface and that exhibit the highest transduction rate in striated muscles of mice or nonhuman primates reported to date. Finally, we note essential looming improvements that will facilitate and accelerate clinical translation of these latest generations of myotropic AAVs, including the identification and utilization of capsid selection or validation schemes that promise optimal translation in humans, and continued efforts to enhance patient safety by minimizing hepatic off-targeting. [ABSTRACT FROM AUTHOR]

Muscle-directed gene therapy with adeno-associated viral (AAV) vectors is undergoing clinical development for treating neuromuscular disorders and for systemic delivery of therapeutic proteins. Although these approaches show considerable therapeutic benefits, they are also prone to induce potent immune responses against vector or transgene products owing to the immunogenic nature of the intramuscular delivery route, or the high doses required for systemic delivery to muscle. Major immunological concerns include antibody formation against viral capsid, complement activation, and cytotoxic T cell responses against capsid or transgene products. They can negate therapy and even lead to life-threatening immunotoxicities. Herein we review clinical observations and provide an outlook for how the field addresses these problems through a combination of vector engineering and immune modulation. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) is causally involved in the development of head and neck squamous cell carcinoma (HNSCC). The integration of HPV drives tumorigenesis through expression of oncogenic viral genes as well as genomic alterations in surrounding regions. To elucidate involvement of epigenetic dysregulation in tumorigenesis, we here performed integrated analyses of the epigenome, transcriptome and interactome using ChIP‐seq, RNA‐seq and Hi‐C and 4C‐seq for HPV(+) HNSCCs. We analyzed clinical HNSCC using The Cancer Genome Atlas data and found that genes neighboring HPV integration sites were significantly upregulated and were correlated with oncogenic phenotypes in HPV(+) HNSCCs. While we found four HPV integration sites in HPV(+) HNSCC cell line UPCI‐SCC‐090 through target enrichment sequencing, 4C‐seq revealed 0.5 to 40 Mb of HPV‐interacting regions (HPVIRs) where host genomic regions interacted with integrated HPV genomes. While 9% of the HPVIRs were amplified and activated epigenetically forming super‐enhancers, the remaining non‐amplified regions were found to show a significant increase in H3K27ac levels and an upregulation of genes associated with GO terms, for example, Signaling by WNT and Cell Cycle. Among those genes, ITPR3 was significantly upregulated, involving UPCI‐SCC‐090‐specific super‐enhancer formation around the ITPR3 promoter and in the 80‐kb‐downstream region. The knockdown of ITPR3 by siRNA or CRISPR deletions of the distant enhancer region led to a significant suppression of cell proliferation. The epigenetic activation of HPVIRs was also confirmed in other cell lines, UM‐SCC‐47 and UM‐SCC‐104. These data indicate that epigenetic activation in HPVIRs contributes, at least partially, to genesis of HPV(+) HNSCC. [ABSTRACT FROM AUTHOR]

Infectious bursal disease virus (IBDV) causes a highly contagious disease associated with immunosuppression in young chickens. Production of either egg-based or primary cell-based high-quality vaccines requires time-consuming and costly procedures. To determine a suitable cell line for IBDV replication, L929 cell line was a candidate for the growth kinetics processing of the virus. The L929 cells were proliferated in monolayer, and doubling time was calculated. Replication kinetics an IBDV isolate at the multiplicity of infection 0.1 PFU/cell were determined using virus titration. To adapt IBDV on L929 cells, seven consecutive passages were performed. Virus titer and levels of apoptosis were quantitatively analyzed at each passage. The viral VP2 gene was amplified and sequenced in three passages. An average doubling time of 21 h was estimated for monolayers of L929 cells. Although during early passages, virus growth did not produce a clear cytopathic effect (CPE), an increase in IBDV titers was observed. Serial passages led to the evidence of marked CPEs and an increase in the virus titer in the third passage. During the fourth to seventh passages, consistent CPEs characterized by the formation of granulated and round cells were evident within 24 to 48 hours post-inoculation. The titer of the virus was increased in the third passage onwards to peak in the fourth and constant at 5.9 TCID50 until the end passage. The IBDV replication in connection with DNA fragmentation and FITC, revealed the characteristic picture of apoptosis in a time-dependent manner. We found that the IBDV could easily be adapted to L929 cells, increasing virus yields by about two orders of magnitude. These results indicated that the cell line may be useful in the production of efficient virus particles. [ABSTRACT FROM AUTHOR]

Cucumber mosaic virus (CMV) is the one of notorious virus known for its ubiquitous nature and causes substantial yield loss worldwide. The resistance against the Cucumber mosaic virus (CMV) was envisaged in Nicotiana tabacum transgenic lines by introducing viral gene fragments. The chimeric hairpin RNA constructs incorporating 401 bp of coat protein, 411 bp of replicase protein and 361 bp of 2b gene were developed respectively and transformed into N. tabacum. The regenerated transgenic lines introduced with inverted repeats of CMV gene fragments exhibited enhanced resistance against CMV. The preliminary molecular screening and qPCR confirmed the integration of transgene in the transgenic lines. The spectrum of resistance in transgenic lines was evaluated by challenge inoculation with CMV and the resistance was determined through DAC-ELISA. The complete resistance was achieved in the hpRNA-CP transformant with a very low titre (0.029) of CMV followed by hpRNA-REP (0.099) with no symptoms. [ABSTRACT FROM AUTHOR]

Phototransduction is mediated by distinct types of G protein cascades in different animal taxa: bilateral invertebrates typically utilise the Gαq pathway whereas vertebrates typically utilise the Gαt(i/o) pathway. By contrast, photoreceptors in jellyfish (Cnidaria) utilise the Gαs intracellular pathway, similar to olfactory transduction in mammals1. How this habitually slow pathway has adapted to support dynamic vision in jellyfish remains unknown. Here we study a light-sensing protein (rhodopsin) from the box jellyfish Carybdea rastonii and uncover a mechanism that dramatically speeds up phototransduction: an uninterrupted G protein-coupled receptor – G protein complex. Unlike known G protein-coupled receptors (GPCRs), this rhodopsin constitutively binds a single downstream Gαs partner to enable G-protein activation and inactivation within tens of milliseconds. We use this GPCR in a viral gene therapy to restore light responses in blind mice. Jellyfish see light in a similar way that vertebrates smell odours. Here, Michiel van Wyk and Sonja Kleinlogel show how jellyfish has adapted this slow signal detection pathway to support sophisticated vision, and use a similar method to restore vision in blind mice. [ABSTRACT FROM AUTHOR]

The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment. Author summary: HCMV has been implicated in glioma pathogenesis, but the molecular characterization of the infection is completely lacking, hindering a thorough understanding of how HCMV contributes to glioma progression. We identified the SOX2-PML axis as a key regulator of HCMV infection in glioma cells and demonstrated in a patient-derived xenograft mouse model that the SOX2-PML axis not only controls HCMV infection but also contributes to the oncomodulatory role of the virus. Consistent with this finding, high levels of SOX2 and IE1 in gliomas predict a worse prognosis. To our knowledge, this is the first report of molecular determinants of HCMV infection in glioma cells, and the SOX2-IE1 pair may serve as a valuable prognostic factor to stratify glioma patients for targeted molecular therapies. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate. [ABSTRACT FROM AUTHOR]

Viruses are intracellular parasites that have evolved to effectively manipulate the cells they infect. As a result of the viral infection, multiple cellular processes are altered, suppressed, or redirected, partially due to the viral co-option of the host's molecular machinery. RNA biology plays a central role in virus–host interactions, since it is at the basis of viral gene expression, splicing of viral transcripts, anti-viral RNA silencing, and—at least in the case of RNA viruses—genome replication, and therefore is heavily targeted by viruses. The plant DNA geminiviruses, causal agents of devasting diseases in crops worldwide, are no exception, and RNA processing is tightly entrenched in their infection cycle. In this review, we will discuss the relevance of the manipulation of RNA biology by geminiviruses for a successful viral infection and the underlying molecular mechanisms, and suggest some of the multiple remaining open questions in this field. [ABSTRACT FROM AUTHOR]

Background: Reverse genetics systems have been crucial for studying specific viral genes and their relevance in the virus lifecycle, and become important tools for the rational attenuation of viruses and thereby for vaccine design. Recent rapid progress has been made in the establishment of reverse genetics systems for functional analysis of SARS-CoV-2, a coronavirus that causes the ongoing COVID-19 pandemic that has resulted in detrimental public health and economic burden. Among the different reverse genetics approaches, circular polymerase extension reaction (CPER) has become one of the leading methodologies to generate recombinant SARS-CoV-2 infectious clones. Although CPER has greatly facilitated SARS-CoV-2 analysis, it still has certain intrinsic limitations that impede the efficiency and robustness of virus rescue. Results: We developed an optimized CPER methodology which, through the use of a modified linker plasmid and by performing DNA nick ligation and direct transfection of permissive cells, overcomes certain intrinsic limitations of the 'traditional' CPER approaches for SARS-CoV-2, allowing for efficient virus rescue. Conclusions: The herein described optimized CPER system may facilitate research studies to assess the contribution of SARS-CoV-2 genes and individual motifs or residues to virus replication, pathogenesis and immune escape, and may also be adapted to other viruses. [ABSTRACT FROM AUTHOR]

Background: High-risk HPV is clearly associated with cervical cancer. Integration of HPV DNA into the host genome is considered a key event in driving cervical carcinogenesis. However, the mechanism on how HR-HPV integration influences the host genome structure has remained enigmatic. Methods: In our study, 25 DNA samples including 11 from fresh-frozen cervical carcinomas and 14 from fresh-frozen high-grade squamous intraepithelial lesion (HSILs) were detected using the method of HPV capture combined with next generation sequencing. Results: We calculated the frequency in each viral gene or region and found that breakpoints were prone to occur in L1 and L2 instead of E2 in the cervical cancer (P = 0.0004 and P = 5.15 × 10−40) and HSIL group (P = 2.1 × 10−32 and P = 7.06 × 10−13). The results revealed that HPV16 showed a strong tendency toward intronic region (P = 5.02 × 10−64) but a subtle tendency toward intergenic region (P = 0.04). The most frequent integration site was in the MACROD2 gene (introns 2, 4, 5, 6, 8 and 9), which in MACROD2 functional domain. Conclusion: Our results revealed that MACROD2 is HPV hot spot integration site in cervical lesions, and its deficiency alter DNA repair and sensitivity to DNA damage thought impaired PARP1 activity resulting in chromosome instability. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The begomovirus–betasatellite complex constantly threatens crops in Asia. However, the quantitative relationship between begomoviruses and betasatellites remains largely unknown. The quantities of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) and their ratio varied significantly in initial infection, and thereafter, the ratio tended to become constant. The TbCSB/TbCSV ratio in agrobacteria inoculum significantly affected that in plants in the initial infection but not thereafter. Null-mutation of βC1 that encodes a multifunctional protein important for pathogenesis in TbCSB significantly reduced the TbCSB/TbCSV ratio in plants. Viral inoculum plants with higher TbCSB/TbCSV ratios promoted whitefly transmission of the virus. The expression of AV1 encoded by TbCSV, βC1 encoded by TbCSB and the βC1/AV1 ratio varied significantly in the initial infection and thereafter the ratio tended to become constant. Additionally, the temporal dynamics of the ratio between another begomovirus and its betasatellite was similar to that of TbCSV and was positively regulated by βC1. These results indicate that the ratio between monopartite begomoviruses and betasatellites tend to become constant as infection progresses, and is modulated by βC1, but a higher betasatellite/begomovirus ratio in virally inoculated plants promotes virus transmission by whiteflies. Our findings provide novel insights into the association between begomoviruses and betasatellites. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is an acute infectious disease of domestic pigs and wild boars caused by the African swine fever virus (ASFV), with up to a 100% case fatality rate. The development of a vaccine for ASFV is hampered by the fact that the function of many genes in the ASFV genome still needs to be discovered. In this study, the previously unreported E111R gene was analyzed and identified as an early-expressed gene that is highly conserved across the different genotypes of ASFV. To further explore the function of the E111R gene, a recombinant strain, SY18ΔE111R, was constructed by deleting the E111R gene of the lethal ASFV SY18 strain. In vitro, the replication kinetics of SY18ΔE111R with deletion of the E111R gene were consistent with those of the parental strain. In vivo, high-dose SY18ΔE111R (105.0 TCID50), administered intramuscularly to pigs, caused the same clinical signs and viremia as the parental strain (102.0 TCID50), with all pigs dying on days 8–11. After being infected with a low dose of SY18ΔE111R (102.0 TCID50) intramuscularly, pigs showed a later onset of disease and 60% mortality, changing from acute to subacute infection. In summary, deletion of the E111R gene has a negligible effect on the lethality of ASFV and does not affect the viruses' ability to replicate, suggesting that E111R could not be the priority target of ASFV live-attenuated vaccine candidates. [ABSTRACT FROM AUTHOR]