Viral DNA Research Articles

B-237 Engineered Reverse Transcriptases and Taq DNA Polymerases deliver robust yield in the presence of clinically relevant inhibitors

Abstract Background The past five years has seen explosive growth in molecular diagnostic assays that rely on sensitive and accurate detection of nucleic acids across a wide range of sample types. As the assays are required to become increasingly sensitive, the need for highly functional and purpose-built enzymatic tools has grown. Reverse transcriptase (RT) and Taq DNA Polymerase (Taq DNAP) are commonly used to detect RNA and/or DNA viruses in patient samples for point of care (POC) diagnostics. However, crude patient samples introduce inhibitors that decrease enzyme activity and increase false negative results in POC testing. In addition, there is a need for faster turn-around time for patient diagnosis which can be enabled by engineering more active and sensitive Taq variants. Herein, we present on our engineered RT and Taq DNAP enzymes that were developed to confer greater activity, thermostability, inhibitor tolerance and/or sensitivity. Methods We take a 3-pronged approach to protein engineering: rational design, computational design and directed evolution. Using these techniques, we identified and characterized many RT and Taq DNAP variants. We deeply characterized each RT variant by measuring processivity, inhibitor tolerance and thermostability. First strand synthesis was done at temperatures from 42-65°C in the presence and absence of various clinically relevant inhibitors followed by qPCR to assess cDNA yield. Next, we characterized the inhibitor tolerance of the Taq variants by performing qPCR reactions in the presence of inhibitors. To determine if the Taq variant would be an appropriate fit for fast PCR, the polymerization activity of the Taq DNAP variants were characterized using a primer extension assay. Results Reverse transcriptases termed; StellarScript, StellarScript HT and StellarScript HT+ exhibit high processivity at 42°C. StellarScript HT+ exhibited the highest inhibitor tolerance, processivity and thermostability at temperatures up to 65°C thus making StellarScript HT+ well suited to produce high yields with clinical samples. Taq Pol variants were identified that efficiently amplify DNA in the presence of inhibitors. Conclusions Based on the increased polymerase activity and inhibitor tolerance, WMG Taq variants are well suited to give robust DNA yields with clinical samples. We were successful in engineering RT and Taq DNAP variants that are highly thermostable, active and inhibitor tolerant and thus will enable breakthroughs in existing POC diagnostics.

B-350 The application of MALDI-TOF imaging to biomarker discovery in a mouse model of papillomavirus associated tumorigenesis

Abstract Background Human papillomavirus (HPV) can cause infection and dysplasia in cutaneous epithelium of immunodeficient patients. An animal model has been developed to study HPV-associated tumorigenesis using Nude mouse (Foxn1nu) and mouse papillomavirus (MmuPV1). This study applies MALDI-TOF imaging to compare the expression pattern of small molecules between viral-infected dysplastic mouse skin and mock-infected control tissue. Three sets of molecules showing distinct patterns are identified in correlation with the upregulation of an enzyme in related biosynthetic pathway. Methods Nude mice were wounded on tail and inoculated with MmPV1 suspension (1×109 viral DNA genomes per infected site) or PBS 24 hours later for treatment and control group, respectively. Tail skins were harvested 4 weeks post-infection and processed for cryosectioning. Tissue sections were analyzed using timsTOF flex (Bruker) under negative ion mode with 9-aminoacridine as the matrix. Viral infection (MmuPV1-L1 antigen) and enzyme expression (3'-phosphoadenosine 5'-phosphosulfate synthetase 1, PAPSS1) were assessed by immunofluorescence on adjacent sections. Results Three peaks demonstrated distinct distribution patterns in mouse skin. One peak at m/Z = 465 was identified as cholesterol sulfate (CS) by tandem MS and comparing with the standard. CS was enriched in keratinocytes, hair follicles, and blood vessels in normal skins, and it increased in dysplastic skins with enhanced angiogenesis. The combined peaks at m/Z = 327 & 329 were tentatively identified as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These long chain polyunsaturated fatty acids (LC-PUFAs) were present in the dermis as foci partially co-localized with CS, and they also increased in virus-infected skins. The peak at m/Z = 426 may represent a combination of ADP and APS. Although these two isobars cannot be separated by tandem MS, APS was revealed by the presence of sulfur isotopes at expected natural abundance (34S and 32S). They were present at a low level in the epidermis of normal skins but significantly increased after virus infection. APS and ADP were closely associated with the proliferating cells, and the signal intensity also increased with the degree of tissue dysplasia. As a single factor, APS & ADP demonstrated a fair differential power for MmuPV1-induced dysplastic skin and normal control (AUC of ROC curve = 0.75). The bifunctional enzyme PAPSS1 catalyzes the conversion of ATP to 3’-phosphonato-5’-adenyl sulfate (PAPS) via APS as an intermediate. Interestingly, immunofluorescence showed the expression of PAPSS1 also increased significantly with the severity of tissue dysplasia and MmuPV1 infection. Conclusions MALDI-TOF imaging reveals three sets of molecules with distinct patterns in mouse skin, and their levels are increased during MmuPV1-induced epithelium dysplasia. As a precursor of the common sulfate donor (PAPS), increased APS and ADP in the epidermis of virus-infected skins indicate an upregulation of sulfation pathway and adenine metabolism. This finding is supported by enhanced expression of enzyme PAPSS1 and an increased sulfation reaction product, CS. In addition, DHA and DPA are also increased in the dermis of virus-infected skins, implying a disturbance in LC-PUFA metabolism. This study demonstrates combined use of MALDI-TOF imaging and immunostaining in the search of novel biomarkers of HPV-associated tumorigenesis.

Novel adomavirus associated with proliferative skin lesions affecting the dermal denticles of a sand tiger shark (Carcharias taurus).

A captive sand tiger shark (Carcharias taurus) presented with progressive, hard, raised, miliary skin lesions localized to the lateral trunk and peduncle. Histopathologic evaluation of biopsy samples revealed dysplastic proliferation of odontogenic epithelium with the production of collagenous material. Inclusion bodies and viral particles were not observed with light or transmission electron microscopy, respectively. However, using next generation sequencing with Illumina MiSeq and PCR followed by Sanger sequencing, the complete genome of a novel adomavirus, tentatively named sand tiger shark adomavirus (STAdoV), was obtained from the affected tissue. The genome was circular and 18.5 kilobases with bidirectionally transcribed genes, namely EO1, EO2 & 4, EO3, LO4, LO5, LO6, LO7, LO8, and SET. In situ hybridization using RNAscope® technology and a STAdoV specific probe localized viral DNA to the nuclei of proliferating epithelial cells. Adomaviruses are an emerging viral group with structural and replicative genes sharing a complex evolutionary history with adenoviruses and small circular DNA tumor viruses including papillomaviruses and polyomaviruses. Adomaviruses are described in a number of fish species in association with both necrotizing and proliferative diseases. BLAST analysis of the viral genome revealed greatest nucleotide identity (71.29%) to guitarfish adomavirus (GAdoV), another elasmobranch virus associated with proliferative (epidermal) skin lesions. Lesions in the index animal persisted for approximately 1 year during which time four conspecifics developed similar proliferations. Ultimately, lesions in all sharks regressed spontaneously without recurrence for 2 years.

Regulation of R-Loops in DNA Tumor Viruses.

R-loops are triple-stranded nucleic acid structures that occur when newly synthesized single-stranded RNA anneals to duplex DNA upon the collision of replication forks with transcription complexes. These RNA-DNA hybrids facilitate several transcriptional processes in the cell and have been described extensively in the literature. Recently, evidence has emerged that R-loops are key regulators of DNA tumor virus transcription and the replication of their lifecycle. Studies have demonstrated that R-loops on the Human Papillomavirus (HPV) genome must be resolved to maintain genome maintenance and avoid viral integration, a hallmark of HPV cancers. For Epstein-Barr virus (EBV), R-loops are formed at the oriLyt to establish lytic replication. Structural maintenance of chromosome proteins 5/6 (SMC5/6) bind to these viral R-loops to repress EBV lytic replication. Most viruses in the herpesvirales order, such as KSHV, contain R-loop-forming sequences. In this perspective, we will describe the current, although limited, literature demonstrating the importance of RNA-DNA hybrids to regulate DNA virus transcription. We will also detail potential new areas of R-loop research and how these viruses can be used as tools to study the growing field of R-loops.

A-270 Evaluation of a multiplex panel to determine antibody profile to monkeypox virus in vaccinated, exposed, and naïve individuals

Abstract Background Monkeypox Virus (MPXV) is a double-stranded DNA virus of the Orthopoxvirus genus that causes Mpox disease with symptoms such as swollen lymph nodes, chills, exhaustion, and blisters that appear on the face, inside the mouth, hands, feet etc. The genus orthopoxvirus (OPXV) has four species, namely, MPXV, smallpox-causing variola major virus (VARV), variola minor virus, and cowpox virus (CPXV), that cause disease in humans. In 1980, the World Health Assembly declared that smallpox had been eradicated due to a successful global vaccination program. This vaccine also provided cross-protection against other OPXV. Historically, the pox disease was rarely seen outside West and Central Africa. The first case of Mpox was identified in 1970 in a 9-year-old boy from the Democratic Republic of Congo (DRC), two years after the smallpox vaccination was discontinued. Since then, it has been reported in 11 African countries. In 2003, the first Mpox outbreak outside of Africa was in the USA and was linked to contact with infected pet prairie dogs. The recent 2022-2023 Mpox outbreak has generated renewed interest in the scientific community to understand the level of OPXV-specific immunity in the various geographic regions, among different age groups, and especially in the risk groups and its impact on future outbreaks by using integrated serosurveillance. To meet the need of the hour, we developed a novel multiplex assay for the detection of human IgG antibodies using Luminex® xMAP® technology for the detection of the human IgG profiles. Methods We have evaluated 11 different recombinant antigens from VARV, Vaccinia virus (VACV), and MPXV in a multiplex assay for detecting human IgG antibodies to OPXV using Luminex® xMAP® technology. We have covalently coupled magnetic fluorescent microspheres to three recombinant proteins from VARV, three from VACV, and five from MPXV to interrogate IgG responses. This 15-plex assay also includes four internal controls that assure the assay and reagent performance. In this preliminary evaluation, we have tested 84 de-identified serum and plasma from healthy subjects. We evaluated 24 serum samples of MPXV-exposed individuals procured commercially. We performed principal component analysis to determine which of these 11 antigens contributed the maximum differential and a result analysis algorithm was developed to predict these individuals' positive and negative antibody outcomes. Results The healthy subjects that were assumed to have been vaccinated showed positive response to A35R of MPXV; A36R of VARV, A33R of VACV, the homologous proteins in the three species and were distinct from unvaccinated younger individuals. There was some reactivity with B6R of MPXV protein. In MPXV-exposed subjects, we observed strong positive antibody responses to A35R of MPXV, A36R of VARV, A33R of VACV, and B6R of MPXV proteins in 19 out of 24 individuals. Fifteen individuals showed a moderate positive response to the A27L protein of VACV, and 8 out of 24 showed a weak positive response to the A29 protein of MPXV. Conclusions This panel is a useful tool to understand the level of OPXV-specific immunity by measuring IgG profiles for successful serosurveillance programs for public health.

Geminiviruses and Food Security: A Molecular Genetics Perspective for Sustainable Agriculture in Africa

The African continent is vulnerable to food insecurity. Increased food costs, job losses, and climate change force Africans to chronic hunger. Biotechnology can be used to mitigate this by using techniques such as CRISPR/Cas9 systems, TALENs, and ZFNs. Biotechnology can utilize geminiviruses to deliver the necessary reagents for precise genome alteration. Additionally, plants infected with geminiviruses can withstand harsher weather conditions such as drought. Therefore, this article discusses geminivirus replication and its use as beneficial plant DNA viruses. It focuses explicitly on genome editing to increase plant resistance by manipulating plants’ salicylic acid and jasmonic acid pathways.

Efficient Strategy for Synthesizing Vector-Free and Oncolytic Herpes Simplex Type 1 Viruses.

Synthesizing viral genomes plays an important role in fundamental virology research and in the development of vaccines and antiviral drugs. Herpes simplex virus type 1 (HSV-1) is a large DNA virus widely used in oncolytic virotherapy. Although de novo synthesis of the HSV-1 genome has been previously reported, the synthetic procedure is still far from efficient, and the synthesized genome contains a vector sequence that may affect its replication and application. In the present study, we developed an efficient vector-free strategy for synthesis and rescue of synthetic HSV-1. In contrast to the conventional method of transfecting mammalian cells with a completely synthesized genome containing a vector, overlapping HSV-1 fragments synthesized by transformation-associated recombination (TAR) in yeast were linearized and cotransfected into mammalian cells to rescue the synthetic virus. Using this strategy, a synthetic virus, F-Syn, comprising the complete genome of the HSV-1 F strain, was generated. The growth curve and electron microscopy of F-Syn confirmed that its replication dynamics and morphogenesis are similar to those of the parental virus. In addition, by combining TAR with in vitro CRISPR/Cas9 editing, an oncolytic virus, F-Syn-O, with deleted viral genes ICP6, ICP34.5, and ICP47 was generated. The antitumor effect of F-Syn-O was tested in vitro. F-Syn-O established a successful infection and induced dose-dependent cytotoxic effects in various human tumor cell lines. These strategies will facilitate convenient and systemic manipulation of HSV-1 genomes and could be further applied to the design and construction of oncolytic herpesviruses.

Inhibiting KSHV replication by targeting the essential activities of KSHV processivity protein, PF-8.

Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are no cures for KSHV infection. KSHV establishes biphasic lifelong infections. During the lytic phase, new genomes are replicated by seven viral DNA replication proteins. The processivity factor's (PF-8) functions to tether DNA polymerase to DNA, so new viral genomes are efficiently synthesized. PF-8 self-associates, interacts with KSHV DNA replication proteins and the viral DNA. Inhibition of viral DNA replication would diminish the infection within a host and reduce transmission to new individuals. In this review we summarize PF-8 molecular and structural studies, detail the essential protein-protein and nucleic acid interactions needed for efficient lytic DNA replication, identify future areas for investigation and propose PF-8 as a promising antiviral target. Additionally, we discuss similarities that the processivity factor from Epstein-Barr virus shares with PF-8, which could promote a pan-herpesvirus antiviral therapeutic targeting strategy.

Molecular characterization and functional analysis of ZAP-like gene in common carp (Cyprinus carpio)

The zinc finger antiviral protein (ZAP) is a host antiviral factor that could restrict the replication of various RNA and DNA viruses. To date, the antiviral properties of ZAP gene have been demonstrated in multiple mammals and a few of bird species, while no data is available regarding the immune role of ZAP in fish. In this study, one ZAP-like gene (CcZAPL) was identified form common carp and its antiviral role was investigated. Expression analysis showed that CcZAPL was widely expressed in multiple fish tissues, with highest level in the head kidney, and confocal microscopy analysis showed the sublocation of CcZAPL mainly in the nucleus of Epithelioma papulosum cyprini (EPC) cells. After in vivo stimulation by Spring viraemia of carp virus (SVCV), CcZAPL was induced in gene expression, and in EPC cells overexpression of CcZAPL led to significantly decreased virus load of SVCV and diminished cytopathic effect (CPE). Moreover, after SVCV infection in vitro, expressions of cytokines including IFN, ISG15, PKR, Mx and TNF-α were observed to be up-regulated in CcZAPL-overexpressed EPC cells. Our findings indicated that CcZAPL played a positive role in the control of SVCV, which will allow us to gain new insights into the immune role of ZAP in fish antiviral immunity.

Получение и оценка биологической активности экзогенной мРНК, кодирующей белок MxA человека

Human MxA protein induced by type I and III interferons is an important innate immunity mediator, it shows antiviral activity against a broad spectrum of RNA and DNA viruses. According to the latest data, the MxA protein overexpression increases chemotherapy sensitivity and represents one of the favorable prognostic factors in patients with breast cancer. The exogenous mRNA capable of intracellular MxA protein production not only has the potential for treatment of viral respiratory infection, but also can become an important fundamental research tool. The study aimed to construct and produce the exogenous mRNA encoding the functional human cytoplasmic MxA protein by in vitro transcription (IVT); to study its translational properties; to assess and identify the patterns of the expression of some interferon system genes in response to introduction of this exogenous mRNA into cells. As a result of the study, the exogenous mRNAs capable of effective translation (up to 20 ng/mL of protein from 100 ng of mRNA per well of the 96-well plate) in the eukaryotic cell systems were successfully constructed and produced by IVT (in the amount of up to 200 µg); diffuse distribution of the MxA protein in the MDCK cells was confirmed; significant changes in the expression of the interferon-stimulated genes, such as OAS1, PKR (EIF2AK2), MDA5, RIG-I, were revealed. Our further research will be focused on assessing the developed exogenous mRNAs’ therapeutic potential against influenza A and B viruses, respiratory syncytial virus, and coronavirus SARS-CoV-2.

Genomic and biogeographic characterisation of the novel prasinovirus Mantoniella tinhauana virus 1.

Mamiellophyceae are a ubiquitous class of unicellular green algae in the global ocean. Their ecological importance is highlighted in studies focused on the prominent genera Micromonas, Ostreococcus, and Bathycoccus. Mamiellophyceae are susceptible to prasinoviruses, double-stranded DNA viruses belonging to the nucleocytoplasmic large DNA virus group. Our study represents the first isolation of a prasinovirus in the South China Sea and the only one to infect the globally distributed genus Mantoniella. We conducted a comparative analysis with previously identified viral relatives, encompassing morphological characteristics, host specificity, marker-based phylogenetic placement, and whole-genome sequence comparisons. Although it shares morphological and genetic similarities with established prasinoviruses, this novel virus showed distinct genetic traits, confining its infection to the species Mantoniella tinhauana. We also explored the global biogeography of this prasinovirus and its host by mapping metagenomic data and analysing their relationship with various environmental parameters. Our results demonstrate a pronounced link between the virus and its host, both found predominantly in higher latitudes in the surface ocean. By gaining an increased understanding of the relationships between viruses, hosts, and environments, researchers can better make predictions and potentially implement measures to mitigate the consequences of climate change on oceanic processes.

Liquid biopsy with plasma Epstein-Barr virus DNA characterizes biological relapse for the prediction of cancer recurrence in non-disseminated nasopharyngeal carcinoma

PurposeTo investigate whether a bounce in plasma Epstein-Barr virus (EBV) DNA during posttreatment surveillance of nasopharyngeal carcinoma (NPC) informs the risk of clinical recurrence and its implication for early therapeutic intervention. Methods950 non-disseminated NPC patients with completed remission in 3 months after treatment were retrospectively screened. Detectable EBV DNA with no evidence of clinical relapse during follow-up was deemed as DNA bounce. The diagnostic and prognostic performance of EBV DNA bounce was assessed for subsequent failures. ResultsTumor recurrence occurred in 6.6 %, 10.1 % and 65.8 % in the group with persistently negative EBV DNA, single positive test and ≥ 2 positive tests, respectively. EBV DNA bounce over twice was associated with worse disease-free survival (DFS), locoregional recurrence-free survival (LRRFS), and distant metastasis-free survival (DMFS) than the other two groups. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for the prediction of recurrence were 0.56, 0.95, 0.66, 0.93 and 0.90 using two positive tests, which were hence deemed as biological relapse. Serial cutoffs (EBV DNA 1 ≥ 40 copies/ml or EBV DNA 2 ≥100 copies/ml) further defined a high-risk subgroup with an eventual recurrence rate of 77.9 % and 3-year DFS of merely 20.5 %. Prophylactic medical intervention with capecitabine or S1 significantly improved the 3-year DFS when compared to those with observation. ConclusionsThe earliest two positive tests of EBV DNA represent a biomarker of biological relapse that allows early detection of clinical recurrence in EBV-related NPC. For high-risk biological relapse, preemptive intervention provides potential survival benefits.

Kidney Rejection Prediction Model Combining Blood Gene Expression, Donor-Derived Cell-Free DNA, Urine Chemokines, and Torque Teno Virus

Kidney Rejection Prediction Model Combining Blood Gene Expression, Donor-Derived Cell-Free DNA, Urine Chemokines, and Torque Teno Virus

Hepatitis B and/or C virus reactivation in patients receiving chemotherapy: An observational study

Background: Viral hepatitis reactivation occurs during various chemotherapy treatments. Patients with high serum titer levels of Hepatitis B virus (HBV) DNA before chemotherapy and those receiving intensive chemotherapy for hematological malignancies are particularly at risk. Aims and Objectives: This study aimed to determine the incidence, predictors, and clinical significance of HBV and hepatitis C virus reactivations during chemotherapy. Materials and Methods: This prospective observational study was conducted at a tertiary care center. Hepatitis B and C virus status was identified before the initiation of cancer treatment. Liver function was monitored in all patients before each cycle of chemotherapy. Patients with deranged liver function were subjected to repeated viral antigen tests and DNA/RNA titer levels. Results: A total of 110 patients were identified as having hepatitis virus reactivation out of 1190 patients. The sites of malignancy were breast, 39.1% (43 patients); colorectal, 11.8% (13 patients); upper gastrointestinal, 10% (11 patients); ovary, 9.1% (10 patients); hematological malignancies, 8.2% (9 patients); lung, 5.6% (6 patients); genitourinary, 4.5% (5 patients); head-and-neck, 4.5% (5 patients); biliary tract, 3.6% (4 patients); brain tumor, 1.8% (2 patients); and others, 1.8% (2 patients). Most of the patients with reactivation received 5-FU/Capecitabine-based chemotherapy 37.3%, (41 patients), taxane plus platinum (10.9%, 12 patients), taxane only (10%, 11 patients), trastuzumab (8.2%, 9 patients), platinum-based (6.4%, 7 patients), tyrosine kinase inhibitors (6.4%, 7 patients), monoclonal antibodies (MABs) (3.6%, 4 patients), anthracyclines (2.7%, 3 patients), immunomodulators (2.7%, 3 patients), and other agents (11.8%, 13 patients). Conclusion: We recommend that Hepatitis B and C virus Screening must me performed before chemotherapy and that non-reactive patients should be vaccinated.

Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products

The CRISPR-Cas12a system has emerged as a promising tool for molecular diagnostics due to its indiscriminate trans-ssDNase activity. However, the sensitivity of Cas12a-based diagnostics remains insufficient for clinical use without a pre-amplification step such as loop-mediated isothermal amplification, and therefore the trans-cleavage activity of Cas12a needs to be enhanced. Here, we present a novel strategy to enhance the trans-cleavage activity of Cas12a by reducing the steric hindrance from cis-cleavage products. We have designed Cas12a variants with alanine mutations in the target strand loading (TSL) domain, resulting in reduced affinity for target strand (TS) overhangs to the catalytic site and significantly increased trans-cleavage efficiency by up to 5.8-fold. In addition, we used a novel salt dilution method to exploit the enhanced trans-cleavage activity of Cas12a under low ionic strength conditions (7-fold), significantly improving the sensitivity of our Cas12a-based detection system. To demonstrate the clinical potential of our Cas12a-based detection system, we validated its ability to detect small amounts of hepatitis B virus (HBV) DNA model using the combination of the KE1096AA Cas12a mutant and the salt dilution method, which enables the detection of DNA at atto-molar concentrations. Our strategy to enhance the trans-cleavage activity of Cas12a paves the way for the development of more sensitive and efficient Cas12a-based diagnostics.

Ancient viral DNA in the human genome linked to neurodegenerative diseases

BackgroundHuman endogenous retroviruses (HERVs) are sequences in the human genome that originated from infections with ancient retroviruses during our evolution. Previous studies have linked HERVs to neurodegenerative diseases, but defining their role in aetiology has been challenging. Here, we used a retrotranscriptome-wide association study (rTWAS) approach to assess the relationships between genetic risk for neurodegenerative diseases and HERV expression in the brain, calculated with genomic precision. MethodsWe analysed genetic association statistics pertaining to Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson’s disease, using HERV expression models calculated from 792 cortical samples. Robust risk factors were considered those that survived multiple testing correction in the primary analysis, which were also significant in conditional and joint analyses, and that had a posterior inclusion probability above 0.5 in fine-mapping analyses. ResultsThe primary analysis identified 12 HERV expression signatures associated with neurodegenerative disease susceptibility. We found one HERV expression signature robustly associated with amyotrophic lateral sclerosis on chromosome 12q14 (MER61_12q14.2) and one robustly associated with multiple sclerosis on chromosome 1p36 (ERVLE_1p36.32a). A co-expression analysis suggested that these HERVs are involved in homophilic cell adhesion via plasma membrane adhesion molecules. ConclusionsWe found HERV expression profiles robustly associated with amyotrophic lateral sclerosis and multiple sclerosis susceptibility, highlighting novel risk mechanisms underlying neurodegenerative disease, and offering potential new targets for therapeutic intervention.

Bovine papillomavirus gene expression and inflammatory pathway activation vary between equine sarcoid tumour subtypes

Equine sarcoids are common non-metastasising skin tumours in horses, associated with bovine papillomavirus (BPV) infection. Six subtypes are recognised (occult, verrucose, nodular, fibroblastic, mixed and malevolent lesions), with variable clinical behaviour. The pathophysiology underlying varying tumour phenotype is poorly understood, and previous data on associations with viral load have been conflicting. To better understand this clinical variation, we investigated associations between tumour subtype and viral load, viral early protein gene expression, and expression of 10 host genes by quantitative polymerase chain reaction in 27 sarcoids and 5 normal skin samples. Viral DNA copy number did not differ between subtypes but was significantly higher in animals with fewer tumours. Expression of BPV E2 and E6 was higher in occult lesions compared to fibroblastic or nodular lesions, while E5 expression was higher in previously-treated lesions. Of the host genes, only IL6 and IL1B differed between subtypes, with higher expression in fibroblastic lesions, while IL10 and CCL5 were elevated compared to skin in all lesion types, and elevations in TNF and TGFB1 were significant for occult lesions only. Expression of TLR9, ATR, VEGFA and PTGS2 in sarcoids was not significantly different from normal skin, suggesting differences between BPV and human papillomavirus tumorigenesis. Results for BPV viral load and gene expression differed from previous reports and are insufficient to explain the spectrum of tumour phenotypes. Activation of both pro-inflammatory and anti-inflammatory immune pathways in sarcoids could influence tumour growth and effective immune responses, and the contribution of specific infiltrating immune cells requires further investigation.

Strain-resolved de-novo metagenomic assembly of viral genomes and microbial 16S rRNAs

BackgroundMetagenomics is a powerful approach to study environmental and human-associated microbial communities and, in particular, the role of viruses in shaping them. Viral genomes are challenging to assemble from metagenomic samples due to their genomic diversity caused by high mutation rates. In the standard de Bruijn graph assemblers, this genomic diversity leads to complex k-mer assembly graphs with a plethora of loops and bulges that are challenging to resolve into strains or haplotypes because variants more than the k-mer size apart cannot be phased. In contrast, overlap assemblers can phase variants as long as they are covered by a single read.ResultsHere, we present PenguiN, a software for strain resolved assembly of viral DNA and RNA genomes and bacterial 16S rRNA from shotgun metagenomics. Its exhaustive detection of all read overlaps in linear time combined with a Bayesian model to select strain-resolved extensions allow it to assemble longer viral contigs, less fragmented genomes, and more strains than existing assembly tools, on both real and simulated datasets. We show a 3–40-fold increase in complete viral genomes and a 6-fold increase in bacterial 16S rRNA genes.ConclusionPenguiN is the first overlap-based assembler for viral genome and 16S rRNA assembly from large and complex metagenomic datasets, which we hope will facilitate studying the key roles of viruses in microbial communities.C22dm7AjjRZTtiuc33W1snVideo

MALIGNANT NEOPLASMS OF THE REPRODUCTIVE SYSTEM IN MEN AND THEIR CONNECTION WITH THE HUMAN PAPILLOMAVIRUS. (REVIEW)

The issue of malignant tumor occurrence associated with viral infections is a particularly significant one. Approximately 15-20% of tumors have some connection with various viral infections. An extensive amount of data has been gathered on neoplasms of the female reproductive system linked to the human papillomavirus (HPV). However, the connection between papillomavirus infection and malignant neoplasms in the male reproductive system requires further investigation. The aim of this review is to examine the association between malignant neoplasms of the male reproductive system and HPV, as well as to consider the issue of male vaccination. A research team conducted a literature review on the topic using databases such as the National Library of Medicine, Web of Science, and CyberLeninka. Data was collected on three types of malignant tumors in the male reproductive system: penile cancer, testicular cancer, and prostate cancer. In the context of the relationship between HPV and penile and prostate cancers, data on the presence of viral DNA in tumor tissue samples have been isolated. A study found that HPV DNA was present in semen samples from 34.9% of patients with testicular cancer, compared to only 2.4% in a control group. However, understanding of the HPV lifecycle raises questions about the link between HPV and testicular cancer. HPV multiplies in dividing epithelial cells, but testicular tissue is of mesodermal origin, and may not provide a suitable environment for HPV reproduction. Based on these findings, it is possible to draw the following conclusions: there are pathogenic reasons for the association between HPV and male reproductive system neoplasms. Further systematic review and meta-analysis are needed to fully understand this complex relationship.

LAMP-based electrochemical platform for monitoring HPV genome integration at the mRNA level associated with higher risk of cervical cancer progression.

Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.