Viral DNA Research Articles

Intravitreal Dexamethasone Implants for Macular Edema Secondary to Acute Retinal Necrosis

ABSTRACT Purpose To assess the effectiveness and risk of intravitreal injection of dexamethasone implants in treating macular edema (ME) secondary to acute retinal necrosis (ARN). Methods In this retrospective, noncomparative case series study, five patients who developed secondary ME after ARN and received an intravitreal dexamethasone implant injection were enrolled. The features of secondary ME on OCT and the outcomes of dexamethasone intravitreal implanting were presented. Results The mean age of the patients was 59 years (range, 51–61 years). All patients had unilateral involvement, and all 5 eyes showed mild to moderate anterior uveitis, retinal necrosis, and vasculitis. Herpes zoster virus was detected in all eyes using PCR, and timely antiviral and anti-inflammatory treatment was performed. Aqueous humor samples were negative for herpes zoster virus DNA, and resolution of viral retinitis was noted upon the occurrence of ME. Additionally, three eyes received pars plana vitrectomy with silicone oil prior to ME development. All eyes presented with intraretinal fluid, hyper-reflective foci, and impairments of the external limiting membrane/ellipsoid zone at varying degrees on OCT images. Epiretinal membrane was exhibited in 80% of eyes, but no vitreoretinal traction was detected. Subretinal fluid was visible in 60% of eyes. ME was relieved effectively in all eyes after intravitreal dexamethasone implanting. One of these patients experienced three episodes of ME. No recurrence of retinal necrosis or corticosteroid-associated ocular hypertension was observed during the follow-up period. Conclusion Intravitreal injection of dexamethasone implants can effectively alleviate ME secondary to ARN and improve visual acuity with no adverse reactions.

Isolation and pathogenicity of a fowl adenovirus 8b (FAdV-8b) strain in Cherry Valley ducks

ABSTRACT Inclusion body hepatitis (IBH) is an economically important viral disease primarily affecting the poultry industry. In this study, we isolated a strain of FAdV-8b (strain SDYT) from naturally infected ducks and the hexon and fiber gene sequences were analysed by polymerase chain reaction (PCR) amplification. In order to study the pathogenicity of FAdV-8b in Cherry Valley ducks, we inoculated 10- and 20-day-old ducks with 0.3 ml of FAdV-4 virus (TCID50 of 105.5/0.1 ml) either orally or intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines, viral load and antibody levels were observed and recorded within 15 days after infection. Pathomorphological investigations revealed that ducks in the experimental group exhibited hepatitis. Histopathology showed multiple organ damage, including serious liver and kidney lesions. Furthermore, elevated levels of inflammatory cytokines and antibodies were noticed, due to the infection and innate immune response. At a later stage of infection, immunosuppression occurred, resulting in decreased levels of cytokines. Determination of viral load showed that the virus was present in several organs, with the highest viral DNA load found in the liver, followed by the kidney. Compared to birds infected orally, the intramuscular group exhibited the highest viral load. In summary, this study increases our understanding of the pathogenicity of FAdV-8b in ducks and establishes a model that will inform antiviral drug testing and vaccine evaluation for IBH, thereby preventing and reducing the spread of IBH in the poultry industry. RESEARCH HIGHLIGHTS A strain (SDYT) of fowl adenovirus 8b (FAdV-8b) was successfully isolated from ducks. Cherry Valley ducks were successfully infected with FAdV-8b. Different routes of infection can lead to duck mortality, more pronounced when birds are injected intramuscularly. FAdV-8b (SDYT) was distributed in various tissues and organs of ducks, causing different degrees of lesions.

Successful treatment of Keratitis caused by Mycobacterium chelonae and an overview of previous cases in Europe

Introduction and purposeMycobacterium (M.) chelonae is responsible for a half of relatively rare nontuberculous mycobacteria (NTM) keratitis. We report a case of M. chelonae keratitis in a woman following sclerocorneal suture extraction after cataract surgery.ResultsA 70-year-old woman presented with a red eye and corneal infiltration of her left eye six weeks following sclerocorneal suture extraction after an elective cataract surgery in another institute. She complained of a sharp, cutting pain and photophobia. Since initial corneal scrapes and conjunctival swabs proved no pathogen using culture and PCR methods, non-specific antibiotics and antifungal agents were administered. As keratitis was complicated by an inflammation in the anterior chamber and vitreous, samples of the vitreous fluid were sent for microbiologic examination. DNA of Epstein-Barr virus (EBV) was repeatedly detected. Since the intrastromal abscess had formed, corneal re-scrapings were performed and M. chelonae was detected using culture, MALDI-TOF MS and PCR methods. Therapy was changed to a combination of oral and topical clarithromycin, intravitreal, topical and intracameral amikacin, and oral and topical moxifloxacin. The successful therapy led to stabilization. The optical penetrating keratoplasty was performed and no signs of the infection recurrence were found.ConclusionsThe diagnosis of nontuberculous mycobacterial keratitis is difficult and often delayed. An aggressive and prolonged antimicrobial therapy should include systemic and topical antibiotics. Surgical intervention in the form of corneal transplantation may be required in the active and nonresponsive infection. In the presented case this was necessary for visual rehabilitation due to scarring.

AXIN1 boosts antiviral response through IRF3 stabilization and induced phase separation

Axis inhibition protein 1 (AXIN1), a scaffold protein interacting with various critical molecules, plays a vital role in determining cell fate. However, its impact on the antiviral innate immune response remains largely unknown. Here, we identify that AXIN1 acts as an effective regulator of antiviral innate immunity against both DNA and RNA virus infections. In the resting state, AXIN1 maintains the stability of the transcription factor interferon regulatory factor 3 (IRF3) by preventing p62-mediated autophagic degradation of IRF3. This is achieved by recruiting ubiquitin-specific peptidase 35 (USP35), which removes lysine (K) 48-linked ubiquitination at IRF3 K366. Upon virus infection, AXIN1 undergoes a phase separation triggered by phosphorylated TANK-binding kinase 1 (TBK1). This leads to increased phosphorylation of IRF3 and a boost in IFN-I production. Moreover, KYA1797K, a small molecule that binds to the AXIN1 RGS domain, enhances the AXIN1-IRF3 interaction and promotes the elimination of various highly pathogenic viruses. Clinically, patients with HBV-associated hepatocellular carcinoma (HCC) who show reduced AXIN1 expression in pericarcinoma tissues have low overall and disease-free survival rates, as well as higher HBV levels in their blood. Overall, our findings reveal how AXIN1 regulates IRF3 signaling and phase separation-mediated antiviral immune responses, underscoring the potential of the AXIN1 agonist KYA1797K as an effective antiviral agent.

Foxa deficiency restricts hepatitis B virus biosynthesis through epigenic silencing.

The current absence of curative therapies capable of resolving chronic hepatis B virus (HBV) infection is a major clinical problem associated with considerable morbidity and mortality. The small viral genome limits molecular targets for drug development, suggesting that the identification of cellular factors essential for HBV biosynthesis may represent alternative targets for therapeutic intervention. Genetic Foxa deficiency in the neonatal liver of HBV transgenic mice leads to the transcriptional silencing of viral DNA by CpG methylation without affecting viability or displaying an obvious phenotype. Therefore, limiting liver Foxa activity therapeutically may lead to the methylation of viral covalently closed circular DNA (cccDNA), resulting in its transcriptional silencing and ultimately the resolution of chronic HBV infection.

Verteporfin is an effective inhibitor of HCMV replication

Human cytomegalovirus (HCMV), a double-stranded DNA virus from the Betaherpesvirinae subfamily, constitutes significant risks to newborns and immunocompromised individuals, potentially leading to severe neurodevelopmental disorders. The purpose of this study was to identify FDA-approved drugs that can inhibit HCMV replication through a drug repositioning approach. Using an HCMV progeny assay, verteporfin, a medication used as a photosensitizer in photodynamic therapy, was found to inhibit HCMV production in a dose-dependent manner, significantly reducing replication at concentrations as low as 0.5 µM, approximately 1/20th of the concentration used in anti-cancer research. Further analysis revealed that verteporfin did not interfere with HCMV host cell entry or nuclear transport but reduced viral mRNA and protein levels throughout the HCMV life cycle from the immediate-early stages. These results suggest that verteporfin has the potential to be rapidly and safely developed as a repurposed drug to inhibit HCMV infection.

Vaccination for Mpox (Monkeypox) Infection in Humans: From Basic Science to Real-World Effectiveness

Human mpox (previously known as monkeypox) is a multi-system disease caused by an orthopox DNA virus [...]

Distribution of HPV genotypes in Mashhad, Iran: insights from a 2022–2023 study

BackgroundHuman papillomavirus (HPV), is one of the main causes of cervical cancer and also one of the most common sexually transmitted infections (STIs). HPV is responsible for almost all cases of cervical cancer and plays a principal role in causing other cancers including oropharynx, penis, larynx, oral cavity, anus, vulva, and vagina. The study aims to investigate the prevalence and distribution of HPV genotypes among patients referred to private laboratories in Mashhad, located in the northeast of Iran.Methods and materials428 samples including 382 females (89.3%) and 46 males (10.7%) between January 10, 2022, and February 11, 2023, in Mashhad, Iran were evaluated to detect HPV and determine its genotypes. Cervical swabs and urine samples were collected from females and males, respectively. Viral DNA was extracted by using a CedExtra purification kit (cedbio, Iran) and viral genotypes were identified with a High + Low Papillomastrip kit (Operon, Spain). Mann Whitney U test and Chi-square test were accomplished for statistical analysis.ResultFrom the total of 428 participants analyzed, the HPV test result was positive for 129 patients (30.1%) and negative for 299 people (69.9%). Among the participants, 115 female (30.1%) and 14 male (30.4%) were positive for HPV infection. The prevalence of HPV infection among the referring people was about 30%. The most common genotype identified was HPV-6 (10.3%), followed by HPV-16 (8.7%) and HPV-51 (7.7%), the second and third most common genotypes, respectively. Additionally, HPV-39 was detected at a frequency of 6.70%. HPV-11, HPV-61, HPV-91, and HPV-44 with a frequency of 1% were among the least genotypes identified among the patients.ConclusionIn line with the results of this study, the prevalence of HPV genotypes in both genders is 30%. The results likely reflect differences in the prevalence of high-risk HPV genotypes, that are less common. Also, HPV-6 and HPV-16 genotypes that are covered by the vaccine had a significant prevalence. On the other hand, with the prevalence of HPV-51 and HPV-39 genotypes in infected people who are not covered by the Gardasil (quadrivalent) vaccine, there is a risk of related cancers in the future.

Co-infection of human papillomavirus and herpes simplex virus-2 with cervical dysplasia among women in Kaduna State, Nigeria

Background: The epidemiology of human papillomavirus (HPV) and co-infections with herpes simplex virus type 2 (HSV-2) remains poorly characterized in Africa. High risk HPV (hrHPV) infection is the primary cause of 99.7% all cervical cancer especially in the presence of genital ulcer disease (GUD) which is usually caused by HSV-2. Herpes simplex virus-2 might interact directly with hrHPV to increase the risk of cervical cancer. Co-infection of HPV and HSV-2 in asymptomatic women could provide a clue to early diagnosis of cervical cancer and this could aid in the development of new strategies for effective management and improvement of the quality of life of women who are infected. If the synergy between HPV and HSV-2 can be prevented, there may be significant reduction of the incidence of cervical cancer. Methodology: This was a descriptive cross-sectional study of 515 randomly selected apparently healthy women of reproductive age whose cervical samples were screened for HPV and HSV-2 in Kaduna State, Nigeria. The cervical samples were collected by liquid-based cytology (LBC) for detection of cervical epithelial cell abnormalities (CEA) and molecular detection of HPV and HSV-2 using conventional polymerase chain reaction (PCR) assay. Extracted viral DNAs from the samples were amplified by convectional PCR using specific hrHPV (HPV 16,18, 31 and 45) primer sets and a broad spectrum MY09/11 and GP5+/6+ primers for a wider range of HPV genotypes while HSV-2 DNA was detected by florescence-based PCR assay with HSV-2 gG primers and probes. Results: PCR assay revealed that 14.7% (n=76) of the 515 selected women harbored HPV, HSV-2 or both. The prevalence of HPV, hrHPV, HSV-2 and HPV/HSV-2 co-infection among in the study participants are 11.8% (n=61), 9.3% (n=48), 5.6% 4% (n=29) and 2.3% (n=12) respectively. The frequency of HPV detection in HSV-2 infected participants (41.4%, 12/29) was significantly higher (OR=6.295, p<0.0001) than in HSV-2 negative participants (10.1%, 49/437), but only co-infections of HPV-31 (p=0.004) and HPV-18 (p=0.040) with HSV-2 were significantly associated. Cervical cytology reports on the smears revealed prevalence of cervical epithelial abnormalities (CEA) of 16.7% (n=86), with cervical dysplasia prevalence of 6.4% (n=33), consisting of high grade squamous intraepithelial lesion (HGSIL) of 1.6% (n=8), low grade squamous intraepithelial lesion (LGSIL) of 4.1% (n=21) and atypical squamous cells of uncertain significance (ASCUS) of 0.8% (n=4). The frequency of HPV detection in women with cervical dysplasia (93.9%, 31/33) was significantly higher (OR=14.22, p<0.0001) than in women without cervical dysplasia (6.2%, 30/482), and all HPV genotypes were significantly associated (p<0.05) with cervical dysplasia except HPV 16 (p=0.051). Also, the frequency of HPV/HSV-2 co-infection among women with cervical dysplasia (15.2%, 5/33) was significantly higher than among women without cervical dysplasia (1.5%, 7/482) (OR=12.12, p<0.0001). Comparing women with cervical inflammatory and atrophic changes (n=53), women with cervical dysplasia (HGSIL, LGSIL and ASC-US) had significantly higher frequency of HPV infection (χ2=42.829, p=0.000), HSV-2 infection (χ2=25.140, p=0.001) and HPV/HSV-2 co-infections (χ2=66.602, p=0.000). Conclusion: Co-infection rate of hrHPV and HSV-2 among women in Kaduna State is 2.3%. Demographic factors significantly influencing the rate of co-infections included age and marital status. In spite of the screening method using the traditional Pap smear, GUD and cervical cancer continues to be a major public health problem, thus more sensitive and specific methods such as liquid based cytology technique and polymerase chain reaction for early detection of cervical dysplasia and hrHPV or HSV-2 in asymptomatic women should be adopted for routine use

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.

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.

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