Adenovirus DNA Research Articles

Stepwise virus assembly in the cell nucleus revealed by spatiotemporal click chemistry of DNA replication.

Biomolecular assemblies are fundamental to life and viral disease. The spatiotemporal coordination of viral replication and assembly is largely unknown. Here, we developed a dual-color click chemistry procedure for imaging adenovirus DNA (vDNA) replication in the cell nucleus. Late- but not early-replicated vDNA was packaged into virions. Early-replicated vDNA segregated from the viral replication compartment (VRC). Single object tracking, superresolution microscopy, fluorescence recovery after photobleaching, and correlative light-electron microscopy revealed a stepwise assembly program involving vDNA and capsid intermediates. Depending on replication and the scaffolding protein 52K, late-replicated vDNA with rapidly exchanging green fluorescent protein-tagged capsid linchpin protein V and incomplete virions emerged from the VRC periphery. These nanogel-like puncta exhibited restricted movements and were located with the capsid proteins hexon, VI, and virions in the nuclear periphery, suggestive of sites for virion formation. Our findings identify VRC dynamics and assembly intermediates, essential for stepwise productive adenovirus morphogenesis.

Discovery of the first sea turtle adenovirus and turtle associated circoviruses

Turtles are an evolutionarily unique and morphologically distinctive order of reptiles, and many species are globally endangered. Although a high diversity of adenoviruses in scaled reptiles is well-documented, turtle adenoviruses remain largely understudied. To investigate their molecular diversity, we focused on the identification and characterisation of adenoviruses in turtle-derived organ, swab and egg samples. Since reptile circoviruses have been scarcely reported and no turtle circoviruses have been documented to date, we also screened our samples for circoviruses. Host−virus coevolution is a common feature of these viral families, so we aimed to investigate possible signs of this as well. Two screening projects were conducted: one on Brazilian samples collected from animals in their natural habitat, and the other on Hungarian pet shop samples. Nested PCR systems were used for the detection of adeno- and circoviruses and purified PCR products were Sanger sequenced. Phylogenetic trees for the viruses were reconstructed based on the adenoviral DNA polymerase and hexon genes, circoviral Rep genes, and for the turtle hosts based on mitochondrial cytochrome b amino acid sequences. During the screening, testadeno-, siadeno-, and circovirus strains were detected. The circovirus strains were classified into the genus Circovirus, exhibiting significant evolutionary divergence but forming a monophyletic clade within a group of fish circoviruses. The phylogenetic tree of turtles reflected their taxonomic relationships, showing a deep bifurcation between suborders and distinct monophyletic clades corresponding to families. A similar clustering pattern was observed among the testadenovirus strains in their phylogenetic tree. As a result, this screening of turtle samples revealed at least three new testadenoviruses, including the first sea turtle adenovirus, evidence of coevolution between testadenoviruses and their hosts, and the first turtle associated circoviruses. These findings underscore the need for further research on viruses in turtles, and more broadly in reptiles, to better understand their viral diversity and the evolutionary processes shaping host–virus interactions.

Evaluation of the AltoStar AM16 system for the quantitation of AdV, CMV and HHV-6 DNA from clinical specimens

The vulnerability of immunocompromised patients to common or opportunistic viral infections is particularly high. The quantitation of viral load in clinical specimens is important for the diagnosis and management of the infection and reactivation in this patient population, particularly transplant recipients. As the new regulation “IVDR” regarding in vitro diagnosis methods is about to come into effect in France, diagnostic laboratories have to implement methods and systems compatible with this new regulation. Technical performance of the AltoStar® Adenovirus (AdV), Cytomegalovirus (CMV) and human Herpesvirus-6 (HHV-6) DNA PCR Kits 1.5 was assessed on the AltoStar Automation system AM16 using reference kits in 146 clinical samples. Overall agreement in clinical specimens was 87.5 % (28/32), 96.8 % (62/64), 100 % (22/22), 100 % (28/28) and 92.8 % (26/28) for AdV, CMV (WB samples and other matrices), HHV-6 A&B respectively. Quantitative results were highly correlated and estimated to be equivalent within a 0.057–0.648 log-amount difference.We found that altona kits on The AltoStar AM16 system are suitable for clinical monitoring of AdV, CMV and HHV-6 in immunocompromised hosts.

Quantitative Virus-Associated RNA Detection to Monitor Oncolytic Adenovirus Replication.

Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It is, however, difficult to obtain evidence for intratumoral oncolytic adenovirus replication if direct access to the tumor is not possible. Detection of systemic adenovirus DNA, which is sometimes used as a proxy, has limited value because it does not distinguish between the product of intratumoral replication and injected virus that did not replicate. Therefore, we investigated if detection of virus-associated RNA (VA RNA) by RT-qPCR on liquid biopsies could be used as an alternative. We found that VA RNA is expressed in adenovirus-infected cells in a replication-dependent manner and is secreted by these cells in association with extracellular vesicles. This allowed VA RNA detection in the peripheral blood of a preclinical in vivo model carrying adenovirus-injected human tumors and on liquid biopsies from a human clinical trial. Our results confirm that VA RNA detection in liquid biopsies can be used for minimally invasive assessment of oncolytic adenovirus replication in solid tumors in vivo.

PCR-based study on viral pathogens circulation among cervids in the Moscow region

A molecular survey of selected viruses in free-ranging cervids was conducted in 15 different districts of Moscow region. Samples were collected from 178 game animals including 144 moose (Alces alces), 19 roe deer (Capreolus capreolus) and 15 deer without species information. Nasal swabs and tissue samples including parts of the nasal septum, upper tracheal rings, lung, heart, liver, kidneys and pooled organ samples were tested using polymerase chain reaction (PCR). Samples were studied for pestiviruses, herpesviruses, coronaviruses, group A rotaviruses, adenoviruses, hepatitis e and parainfluenza type 3 virus. None of the samples were positive for Bovine Coronavirus and SARS-COV-2, hepatitis E virus and parainfluenza type 3 virus. PCR results were positive for bovine herpesviruses (5.05%), pestiviruses (0.56%), rotaviruses (1.68%). DNA of a new adenovirus, presumably causing a mild course of animal respiratory disease, was detected in samples of 6 animals (3.37%). In conclusion, the conducted studies have shown that game animals of the Moscow region can be a natural reservoir of cattle viruses, and this must be taken into account when planning and organizing measures for the control and eradication of such notifiable diseases as bovine viral diarrhoea and infectious bovine rhinotracheitis. Monitoring studies and general disease surveillance of wild animal populations provide additional information on the epidemiology of infectious diseases in the region and allow timely measures to be taken to protect wild animals, domestic animals and the public.

Case Report: Acquired hemophilia A following COVID-19 vaccine

Background: In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified and has subsequently been responsible for immense morbidity and mortality. This resulted in the development of vaccines in record time, potentially posing safety issues. Several cases of acquired hemophilia A (AHA) following coronavirus disease 2019 (COVID-19) vaccines have been described in the literature. AHA is a rare autoimmune disease due to the production of IgG autoantibodies to coagulation FVIII that burdens high morbidity and mortality. It is presumed mainly on identifying an increased activated partial thromboplastin time (aPTT), even without bleeding and is confirmed by determining a FVIII inhibitor. To the best of our knowledge, this is the first report of AHA after inactivated virus COVID-19 treated with low dose rituximab (RTX). Lower RTX doses may lead to shorter infusion duration, lower risk of adverse events and lower costs. Case presentation: We report a case of AHA occurring in a 69-year-old man 60 days after an inactivated Sinovac-coronavac COVID-19 vaccine. Diagnosis of AHA was based on clinical presentation and confirmed with prolonged activated partial thromboplastin time, Factor VIII deficiency and the presence of an FVIII inhibitor. Follow-up showed a significant clinical and biological improvement, aPTT was normalized as of 18 days of treatment with corticosteroids along with low dose rituximab. Conclusions: In the literature, the reported cases of AHA following COVID-19 vaccination occurred after Adenoviral Vector DNA- and SARS-CoV-2 mRNA-based COVID-19 vaccines. Here, AHA was presented following inactivated virus COVID-19 vaccine. AHA treated with low dose rituximab can limit occurrence of side effects and the cost of treatment.

Evaluation of a custom designed hybridisation assay for whole genome sequencing of human adenoviruses direct from clinical samples

Background: Human Adenoviruses are a common cause of disease and can cause significant morbidity and mortality in immunocompromised patients. Nosocomial transmission events can occur with whole genome sequencing playing a crucial role. This study evaluates the performance of a custom designed SureSelectXT target enrichment assay based on 14 adenovirus genomes for sequencing direct from clinical samples.Methods: Modifications were made to the SureSelectXT low input protocol to enhance performance for viral targets. Consensus sequences were generated using an in-house designed three stage bioinformatics pipeline. We assessed, percentage of on target reads, average depth of coverage and percentage genome coverage to determine assay performance across a range of sample matrices.Results: Whole genome sequences were successfully generated for 91.6 % of samples assessed. Adenovirus DNA concentration was a good indicator of enrichment success. Highly specific enrichment was observed with only 6 % of samples showing < 50 % on target reads. Respiratory and faecal samples performed well where bloods showed higher levels of non-specific enrichment likely confounded by low adenovirus DNA concentrations. Protocol performance did not appear impacted by Adenovirus type or species.Conclusion: Overall performance of this modified SureSelectXT protocol appears in line with previously published works although there are some confounding factors requiring further investigation. The use of a small RNA bait set has the potential to reduce associated costs which can be prohibitive.

Development of a Nanoplate-Based Digital PCR Test Method for Quantitative Detection of Human Adenovirus DNA.

Digital polymerase chain reaction (dPCR) assay is an advanced PCR technique that allows for the simultaneous detection and absolute quantification of diverse pathogens.Commercially validated kits available for detecting all subtypes of human adenovirus (HAdV) are limited. This study aimed to demonstrate the development of an in-house nanoplate-based dPCR assay with high sensitivity, even at low copy numbers. In this methodological study, the standardized HAdV DNA was prepared by amplifying the specific hexon gene region with real-time PCR and purifying the HAdV DNA using magnetic beads from HAdV-positive extractions. Dilutions were tested in triplicate during three independent runs to determine the dynamic range, the limit of detection (LoD), the limit of quantification (LoQ), precision, and reproducibility. The primer and probe sequences used in the study were selected based on a literature review to ensure the detection of all HAdV serotypes in a single run. The selected primers were verified using the US National Center for Biotechnology Information (NBCI) nBLAST tools, and the target sequence was determined using the BioEdit software. The DNA concentration of the stock solution was measured using a Qubit fluorometer. The estimated copy number of the stock solution per milliliter was calculated based on the length of the amplified base sequence and fluorometer measurement. The dynamic range of the test was determined to be from 770.4 to 0.9476 cp/μl, with the LoD and LoQ values both being 0.9476 cp/μl. The coefficient of determination (r 2) value of the test was 0.9986. The results demonstrated that the dPCR method could be an ideal tool for the diagnosis and absolute quantification of human adenoviruses, especially in low copy numbers. In order to determine the reproducibility of the test and validate the method for field use, it needs to be developed and adapted in various laboratories and supported by clinical studies.

Construction of recombinant adenovirus-5 vector to prevent replication-competent adenovirus occurrence

Human adenovirus-5 (hAd5) is an important gene delivery vector, which has been widely used in various fields of biomedicine, such as gene therapy, cancer therapy, and vaccine development. However, replication-competent adenovirus (RCA) generated when adenoviral vectors are prepared in HEK293 cells has remained a concern. In this study, the human adenovirus-5 was modified to shorten the length of homologous sequence between the adenovirus and HEK293 genomic DNA, thereby reducing the production of RCA. The recombinant hAd5 was amplified and serially passaged 12 times in HEK293 cells. The amounts of RCA at passage 2, 4, 6, 8, 10, and 12 were detected by quantitative real-time PCR. The results demonstrated that the modification of adenoviral vector could effectively reduce the production of RCA during serial passages in HEK293 cells.

AAV production in stable packaging cells requires expression of adenovirus 22/33K protein to allow episomal amplification of integrated rep/cap genes

Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis-acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. ‘TESSA’ is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.

Markers of antroponotic viral infections in vervet monkeys arrived from their natural habitat (Tanzania).

Various human viruses have been identified in wild monkeys and in captive primates. Cases of transmission of viruses from wild monkeys to humans and vice versa are known. The aim of this study was to identify markers of anthroponotic viral infections in vervet monkeys (Chlorocebus pygerythrus) arrived from their natural habitat (Tanzania). Fecal samples (n = 56) and blood serum samples (n = 75) obtained from 75 animals, respectively, on days 10 and 23 after admission to the primate center, were tested for the markers of anthroponotic viral infections (Ebola virus, Marburg virus, lymphocytic choriomeningitis, hepatitis C virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), parainfluenza types 1 and 3, intestinal adenoviruses, rotaviruses) by enzyme immunoassay (ELISA) and polymerase chain reaction (PCR). Among the examined animals, markers of 6 out of 11 tested viral infections were identified. Detection rates of IgG antibodies to HSV-1,2 (15.9%) and CMV (15.9%) were two times as low as IgG antibodies to EBV (31.8%). Among the markers of respiratory viral infections, IgG antibodies to parainfluenza virus type 1 were found (6.8%). 14.3% of the animals had rotavirus antigen, and 94% had simian adenovirus DNA. Markers of hemorrhagic fevers Ebola, Marburg, LCM, hepatitis C, and type 3 parainfluenza were not detected. When importing monkeys from different regions of the world, an expanded screening for viral infections is needed considering the epidemiological situation both in the country of importation and in the country of destination.

Evidence of vertical transmission of fowl adenovirus 8b in ducks

Fowl adenovirus mainly causes hydropericardium hepatitis syndrome (HHS), inclusion body hepatitis (IBH) and gizzard erosion (GE), etc. In 2015, the first outbreak of HHS was reported in broiler chickens in central China, followed by an outbreak in waterfowl. The first outbreak of HHS in broiler flocks in central China in 2015, followed by outbreaks in waterfowl, has severely restricted the healthy development of the poultry industry. During the investigation, fowl adenovirus was detected in ducklings from a total of seven hatcheries in Shandong, Inner Mongolia and Jiangsu provinces. In addition, the DNA of fowl adenovirus was detected in breeding ducks and their progeny. To test the hypothesis that FAdV can be transmitted vertically, sixty 250-day-old Cherry Valley breeder ducks were divided equally into three groups for experimental infection. FAdV-8b SDLY isolate (duck/Shandong/SDLY/2021, SDLY) preserved in our laboratory was injected intramuscularly into group A and inoculated orally into group B. FAdV-8b DNA was detected in the yolk membranes, embryos and allantoic fluid of duck embryos in the FAdV-infected group after inoculation. In addition, the FAdV-8b hexon gene isolated from yolk membranes, embryos, allantoic fluid and duck eggs was close to 100% nucleotide homology to the FAdV-8b hexon gene isolated from laying duck ovaries, indicating that fowl adenovirus can be transmitted vertically in ducks. These findings provide evidence for the possible vertical transmission of fowl adenovirus from breeder ducks to ducklings.

B-239 Long-read Nanopore Sequencing is a Practicable Method of Targeted or Untargeted Microbial Contamination Detection

Abstract Background Sequencing-based methods for microbe detection are poised to replace both in vivo and PCR-based detection methods due to advantages in speed, sensitivity, specificity, and multiplexing. Here we describe pilot experiments to assess the practicability of nanopore sequencing for microbe detection. Methods Quantitative genomic Human Adenovirus 5 or Mycoplasma hominis DNA was spiked into remnant human serum and nucleic acids were extracted. 16S rRNA PCR amplification and barcoding was performed for Mycoplasma hominis identification. Sequencing was performed with a R9.4.1 flowcell on the MinION sequencer. Basecalling and genome alignment was performed with Guppy v.6.4.2. Results The performance of untargeted sequencing was evaluated by spiking quantitative Adenovirus DNA at 104 genome copies per microliter. Illustrative data from a single run is presented. Replicates yielded similar results. 1 520 991 reads between 31 bases and 970 kilobases were generated. 162 reads between 700 bases and 11.3 kilobases aligned to the Adenovirus genome (Image 1). To evaluate the performance of PCR amplified sequencing, we spiked Mycoplasma genomic DNA into serum or water. The entire 16S rRNA gene (1.5 kb) was PCR amplified and genomic DNA was spiked in at 104, 102, 10, and 0 (control) copies per microliter. A 10% signal: noise ratio cutoff yielded 100% specificity between spiked samples and negative controls. Conclusion Short read sequencing techniques are adequate for microbial detection but are capital intensive and prone to false positive results. We demonstrate that nanopore sequencing can generate long reads (&amp;gt;1.5 kb) with high-confidence microbe genome alignment. Our methods were sensitive to 104 and 10 copies per microliter utilizing untargeted and targeted methods, respectively. Further method optimization and validation will need to be performed, but these results demonstrate that nanopore sequencing is a practicable approach for microbe detection.

NFIB facilitates replication licensing by acting as a genome organizer

The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G1 and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.

Outbreak of adenovirus D8 in a neonatal intensive care unit involving multiple simultaneous transmission pathways

Adenovirus (ADV) outbreaks in neonatal intensive care units (NICU) can lead to durable transmission and serious adverse outcomes. This study describes the investigation and control of an ADV-D8 outbreak in an NICU, associated with ophthalmologic equipment used during retinopathy of prematurity (ROP) screening. Cases were observed in neonates, parents and nurses. The outbreak investigation was performed including sampling patients, parents and health care workers as well as the environment for molecular detection of ADV DNA. The investigation was also conducted in the guest house where some parents were temporary residents. A retrospective cohort study focused on neonates hospitalized during the epidemic period to assess the risk associated with ROP examination. Fifteen cases were identified in neonates; all but one presented with conjunctivitis. Two healthcare workers and 18 parents acquired conjunctivitis. ADV DNA was identified on the RetCam and on the freezer shared by parents. All ADV-positive samples were typed as ADV-D8. ADV infections occurred more frequently in neonates who had ROP examinations (37.8% (14/37) vs (0.9% (1/110); P<0.001) (relative risk 41.6; (5.7-305.8)). The RetCam was disinfected between two examinations using a disinfectant that was virucidal on ADV after a 30-min contact. This outbreak was significantly associated with ROP examination with a RetCam that had a disinfection protocol ill-adapted to rapid patient turnover. In addition, nosocomial transmission via the parents to neonates and parent-to-parent transmission is likely to have played a role in the dissemination of cases. No further cases were observed after the new disinfection procedure was enforced.

Monitoring of cytomegalovirus, Epstein-Barr virus and adenovirus infections in hematopoietic stem cell transplant recipients

Purpose: Haematopoietic stem cell transplant (HSCT) recipients with iatrogenic immunosuppression are high-risk patients for viral infections. The aim of this study was to investigate the incidence of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) infections in HSCT recipients. &#x0D; Materials and Methods: We prospectively monitored 35 patients aged 0-17 years who had allogeneic (n=30) and autologous (n=5) HSCT by quantitative real-time polymerase chain reaction tests for CMV, EBV, and ADV. The monitoring was performed one week before HSCT and weekly for the first 100 days, once a month up to one year after HSCT. In addition, seropositivity for viruses was analysed by Enzyme-Linked Immuno Sorbent Assay a week before transplantation.&#x0D; Results: Before transplantation, all 35 (100%) patients who underwent HSCT were CMV IgG positive, 30 (85.7% - 95% CI: 74.1%-97.3%) HSCT recipients were found to be EBV IgG positive. CMV infection was found in 24 (80% - 95% CI: 65.7%-94.3%), ADV infection in 11 (36.7% - 95% CI: 19.4%-53.9%) and EBV infection in 8 (26.7% - 95% CI: 10.8%-42.5%) allogeneic HSCT patients. In this group, CMV DNA viral load in 8 (26.7%) patients, of which one (3.3%) coinfected with EBV DNA and one (3.3%) with ADV DNA, was higher than 1000 copies/mL which was required for pre-emptive treatment. Among 5 autologous HSCT recipients, CMV DNA was detected in 2 patients, EBV DNA in 5 and ADV DNA in 2. Pre-emptive treatment was given to 11 (%31.4 - 95% CI: 16%-46.8%; 6 CMV, 2 EBV, 1 ADV, 1 CMV-EBV and 1 CMV-ADV infection) of 35 patients. Thus, the development of viral disease was prevented in 7 (63.6% - 95% CI: 35.2%-92.1%). Of the total 35 patients, only 2 (5.7% - 95% CI: 0.0%-13.4%) died due to viral infection. &#x0D; Conclusion: Early diagnosis of viral infections by prospective monitoring of viral loads in HSCT patients would be effective in preventing morbidity and mortality by ensuring timely initiation of pre-emptive therapy.

Human adenovirus DNA polymerase is evolutionarily and functionally associated with human telomerase reverse transcriptase based on in silico molecular characterization that implicate abacavir and zidovudine.

Human adenoviruses (HAdVs) are non-enveloped, small double stranded DNA (dsDNA) viruses that cause asymptomatic infections, clinical syndromes and significant susceptibility to infections in immunocompromised people. The aim of the present study was to identify critical host proteins and HAdV hypothetical proteins that could be developed as potential host-viral targets for antiHAdV therapy. Here, the function of selected hypothetical proteins of HAdV based on phylogenetic relationship with the therapeutic targets of antiretroviral drugs of human immunodeficiency virus (HIV) was predicted computationally, and characterized the molecular dynamics and binding affinity of DNA polymerase of HAdV. Thirty-eight hypothetical proteins (HPs) of human adenovirus (HAdV) were used in this study. The results showed that HAdV DNA polymerase (P03261) is related to Human TERT (O14746) and HLA-B (P01889) genes. The protein-protein interaction of human five molecular targets (PNP, TERT, CCR5, HLA-B, and NR1I2) of ARVDs are well-coordinated/networked with CD4, AHR, FKBP4, NR3C1, HSP90AA1, and STUB1 proteins in the anti-HIV infection mechanism. The results showed that the free energy score of abacavir and zidovudine binding to HAdV DNA polymerase are -5.8 and -5.4kcalmol-1 respectively. Also, the control drug, cidofovir and ganciclovir have less binding affinity for DNA polymerase of HAdV when compare to that of abacavir and zidovudine. Similarity was observed in the binding of abacavir and zidovudine to HAdV DNA polymerase (ASP742, ALA743, LEU772, ARG773 and VAL776). In conclusion, combination of abacavir and zidovudine was predicted to be potential therapy for controlling HAdV infection targeting HAdV DNA polymerase.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

Kuwanon X from Mulberry Leaves Exhibits Antiviral Activity Against Human Adenoviruses

Aim: This study aimed to investigate the anti-adenoviral activity of stilbene derivatives from mulberry leaves. Materials &amp; methods: The anti-adenoviral activity was tested against adenoviruses -3 and -7 on human airway epithelial cell models. Cytotoxicity was assessed by LDH assay. Adenoviral DNA was quantified by qPCR. Results: All five tested stilbene derivatives from mulberry leaves exhibited anti-adenoviral activity, with Kuwanon X showing the highest inhibitory effect. Kuwanon X showed no apparent cytotoxicity for a wide range of concentrations. The mechanistic study revealed that Kuwanon X did not affect viral entry and nuclear translocation of the adenoviral genome but reduced viral DNA production. Conclusion: Stilbene derivatives like Kuwanon X from mulberry leaves are good candidates for antiviral treatment against AdV.

Droplet Digital PCR Development for Adenovirus Load Monitoring in Children after Hematopoietic Stem Cell Transplantation

Human adenovirus (AdV) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with life-threatening clinical manifestations. Although real-tme quantitative PCR (qPCR) has been widely used to measure AdV loads, it has not been standardized for AdV. Droplet digital PCR (ddPCR) is a novel pathogen detection technology that enables the absolute quantification of viral loads. ddPCR would enable a more accurate AdV DNA detection compared with qPCR. In this study, ddPCR was developed for AdV DNA and its performance characteristics compared with those of qPCR. AdV DNAemia incidence during the first 12 weeks after allogenic HSCT was then retrospectively examined by qPCR and ddPCR in 97 HSCT procedures using the preserved 545 DNA samples. ddPCR exhibited better reproducibility and sensitivity, as well as equivalent quantifiability, compared with qPCR. AdV DNA among HSCT patients was detected in 11 (2.0%) and 49 (9.0%) of 545 samples by qPCR and ddPCR, respectively. AdV DNA levels >1000 copies/mL were observed in five cases by qPCR and/or ddPCR. However, two patients developed fulminant hepatitis and died; other patients remained asymptomatic with subsequently undetectable AdV DNA. In conclusion, ddPCR was more sensitive and reproducible in detecting AdV DNA among pediatric HSCT recipients than qPCR. ddPCR offers the potential to provide a more accurate DNAemia detection, determine cutoff values for treatment initiation, and enable antiviral efficacy assessment.