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African swine fever (ASF) is an acute and severe infectious disease caused by the African Swine Fever Virus (ASFV). ASFV exhibits significant resistance and stability in the environment, which, coupled with its double-stranded DNA and large genome, predisposes it to contaminate laboratory samples. This characteristic can lead to false-positive results in swine farm settings even days after disinfection, as detectable through polymerase chain reaction (PCR) or real-time fluorescent quantitative PCR (qPCR) assays. To meet the demand for efficient clinical methods capable of discriminating between ASFV nucleic acid and ASFV virions, this study aims to ascertain the efficacy of the nuclease "BenzoNuclease" in distinguishing ASFV nucleic acid (ASFV-DNA) from ASFV virions. BenzoNuclease is a versatile nucleic acid enzyme with the capacity to degrade nearly all forms of DNA and RNA. Initially, this research established a highly sensitive general PCR detection method for ASFV. Subsequently, a positive control was constructed using the M13 bacteriophage to substitute for active ASFV, facilitating the development of an improved qPCR method. It is important to note that common disinfectants have the potential to deactivate BenzoNuclease. However, in an environment simulating actual production applications, residual disinfectants do not interfere with the enzymatic efficacy of BenzoNuclease, thus not affecting the detection capabilities of this method. Positive clinical samples from pig farms, upon testing with the improved method, revealed that three samples were positive, indicating the presence of viral particles, while the remaining samples were negative, indicating the presence of nucleic acids. This provides an additional new option for sample testing in pig farms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Dynamic therapy response is strongly associated with cancer outcomes. This study aimed to evaluate the significance of longitudinal Epstein-Barr virus (EBV) DNA and radiological tumor regression in risk stratification and response-adaptive treatment in locally-advanced nasopharyngeal carcinoma (LA-NPC). In total, 1312 patients from two centers were assigned to the training and validation cohorts. Based on the multipoint examination of EBV-DNA and tumor response, four post-induction chemotherapy, four mid-radiotherapy, and four post-radiotherapy subgroups were established. Then seven phenotypes were further generated according to different permutations and combinations. These phenotypes were subsequently congregated into four response clusters, which reflect distinct biological treatment responses. The four response clusters correlated with an evident 5-year progression-free survival in both the training and external validation cohorts (5-year: training cohort 91.1 %, 82.8 %, 30.6 %, and 10.0 %; external validation 94.4 %, 55.6 %, 40.0 %, and 12.7 %) had superior prognostic performance compared to TNM staging and nomogram model (concordance index: training cohort-0.825 vs. 0.603 vs. 0.756 and external validation-0.834 vs. 0.606 vs. 0.789). Importantly, the response clusters exhibited an excellent capability in selecting candidates who can benefit from adjuvant chemotherapy. In conclusion, risk stratification based on the dynamic assessment of both radiological and biological responses can significantly enhance prognostic insights and shed light on individualized treatment modifications in LA-NPCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Campylobacter jejuni is a leading cause of foodborne illness worldwide. The application of bacteriophages offers a promising approach to specifically target and reduce C. jejuni contamination in food products. In this study, two C. jejuni phages were characterized, and their ability to inhibit bacterial growth in combination with ethylenediaminetetraacetic acid (EDTA) was investigated. Both phages exhibited tolerance to a wide range of temperature (4-60 °C) and pH (3-9). Phage vB_CjeM-PC10 and vB_CjeM-PC22 were found to have a latent period of 30 min and 20 min and a burst size of 7 and 35 PFU/cell, respectively. Phage vB_CjeM-PC10 has a linear double-stranded DNA (dsDNA) genome of 51,148 bp with 77 ORFs and 29% GC content. Phage vB_CjeM-PC22 has a circular dsDNA genome of 32,543 bp with 56 ORFs and 28% GC content. At 42 °C, the combination of these phages (MOI = 10) and EDTA decreased the count of viable C. jejuni by 5.2 log 10 and inhibited the regrowth of resistant cells for 48 h. At 4 °C, phage vB_CjeM-PC10 alone (MOI = 1000) reduced the count of viable C. jejuni by 3 log 10 in brain heart infusion (BHI) broth and 2 log 10 on chicken skin after incubation for 48 h. Although these phages were effective against C. jejuni, they cannot be utilized directly for food safety applications because they are lysogenic. Nevertheless, these findings expand the genome library of C. jejuni phages and enrich data resources by highlighting potential strategies for controlling C. jejuni infections., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Polyomaviruses (PyVs) cause diverse diseases in a variety of mammalian hosts. During the life cycle, PyVs recruit nuclear host factors to viral genomes to facilitate replication and transcription. While host factors involved in DNA replication, DNA damage sensing and repair, and cell cycle regulation have been observed to bind PyV DNA, the complete set of viral and host proteins comprising the PyV replisome remains incompletely characterized. Here, the iPOND-MS technique (Isolation of Proteins on Nascent DNA coupled with Mass Spectrometry) was used to identify the proteome bound to murine PyV (MuPyV) DNA immediately following synthesis and 2 hours post-synthesis. Several novel MuPyV DNA interactors were identified on newly synthesized viral DNA (vDNA), including MCM complex members, DNA primase, DNA polymerase alpha, DNA ligase, and replication factor C. Though displaying partial overlap, the host and viral proteins bound to MuPyV DNA 2 hours post-synthesis lacked many of the replication proteins found on newly synthesized vDNA. These data help distinguish between the host factors critical for MuPyV DNA replication and those involved in downstream processing.IMPORTANCEPolyomaviruses are the causative agents of serious diseases in humans, including progressive multifocal leukoencephalopathy (PML), BK virus nephropathy, and Merkel cell carcinoma. The exact mechanisms by which the virus replicates, and which host cell proteins are required, are incompletely characterized. Identifying the host proteins necessary for efficient viral replication in the cell may reveal targets for downstream targets that may suppress viral replication in vivo ., Competing Interests: The authors declare no conflict of interest.

After the ejection of viral DNA into the host cytoplasm, the temperate bacteriophage (phage) lambda integrates a cascade of expressions from various regulatory genes, coupled with DNA replication, to commit to a decision between lysis and lysogeny. Higher multiplicity of infection (MOI) greatly shifts the decision toward the lysogenic pathway. However, how the phage separates the MOI from replicated viral DNA during lysis-lysogeny decision-making is unclear. To quantitatively understand the role of viral DNA replication, we constructed a reporter system facilitating the visualization of individual copies of phage DNA throughout the phage life cycle, along with the lysis-lysogeny reporters. We showed that intracellular viral DNA diverges between the lytic and lysogenic pathways from the early phase of the infection cycle, mostly due to the synchronization and success of DNA injection, as well as the competition for replication resources, rather than the replication rate. Strikingly, we observed two distinct replication patterns during lysogenization and surprisingly heterogeneous integration kinetics, which advances our understanding of temperate phage life cycles. We revealed that the weak repression function of Cro is critical for an optimal replication rate and plays a crucial role in establishing stable lysogens., Importance: Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution., Competing Interests: The authors declare no conflict of interest.

Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3' inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in the presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high-molecular-weight AAV2 DNA concatemers in the presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase., Importance: AAV is a small helper virus-dependent, non-pathogenic parvovirus. The AAV genome replication mechanism was extensively studied in the presence of AdV as the helper virus and described to proceed using RHR. Surprisingly, HSV-1 co-infection facilitates RCR of the AAV2 DNA. We directly compared AdV5 and HSV-1 supported AAV2 DNA replication and showed that AAV2 can adapt its replication mechanism to the helper virus. A detailed understanding of the AAV replication mechanism expands our knowledge of virus biology and can contribute to increase gene therapy vector production., Competing Interests: The authors declare no conflict of interest.

SARS-CoV-2 nonstructural protein 13 (nsp13) has been shown to selectively suppress the transcription of episomal DNA while sparing chromosomal DNA. Hepatitis B Virus (HBV) harbors covalently closed circular DNA (cccDNA), a form of viral episomal DNA found within infected hepatocyte nuclei. The persistence of cccDNA is the major cause of chronic HBV infection. In this study, we investigated the impact of SARS-CoV-2 nsp13 on HBV replication, particularly in the context of cccDNA. Our findings demonstrate that nsp13 effectively hinders HBV replication by suppressing the transcription of HBV cccDNA, both in vitro and in vivo . Additionally, we observed that SARS-CoV-2 nsp13 binds to HBV cccDNA and its NTPase and helicase activities contribute significantly to inhibiting HBV replication. Furthermore, our screening identified the interaction between nsp13 and structural maintenance of chromosomes 4, opening new avenues for future mechanistic inquiries. This study presents the evidence suggesting the potential utilization of SARS-CoV-2 nsp13 as a strategy to impede HBV replication by specifically targeting cccDNA. These findings provide a proof of concept for exploring nsp13 as a prospective approach in combating HBV infection., Importance: To effectively combat hepatitis B virus (HBV), it is imperative to develop potent antiviral medications targeting covalently closed circular DNA (cccDNA). Our investigation aimed to assess the impact of SARS-CoV-2 nsp13 on HBV replication across diverse HBV models, confirming its ability to significantly reduce several HBV replication markers. Additionally, our identification of the interaction between nsp13 and SMC4 opens the door for further mechanistic exploration. This marks a paradigm shift in our approach to HBV antiviral therapy, introducing an entirely novel perspective. Our findings propose a novel strategy for developing anti-HBV drugs that specifically target HBV cccDNA., Competing Interests: Yuchen Xia, Aixin Li, and Bei Zhang are listed as inventors on a patent related to the research presented in this paper (patent no. ZL2021106624210, titled "The application of the SARS-CoV-2 nsp13 gene").

Background: Xanthomonas vasicola pv. musacearum is responsible for the widespread Banana Xanthomonas Wilt in banana cultivation regions across the globe. Biocontrol measures for disease management remain limited amidst increasing antimicrobial resistance and unsustainable conventional agricultural practices. The purpose of this study is to explore a viable alternative or adjunct strategy through the use of bacteriophages for disease management., Results: Kintu was isolated from sewage and displayed clear and circular plaques measuring 3 mm. Based on transmission electron microscopy, Kintu displays siphovirus characteristics, including an icosahedral head and a non-contractile tail. Kintu infects 78% (22 out of 28) Ugandan Xvm strains, has an optimal multiplicity of infection of 1, a 10 min adsorption and latent period, a 35 min burst period, and a burst size of 15 particles per bacterium. Phage titers remain stable for two and half months (75 days) in SM buffer at -20 o C and - 40 o C but decrease significantly (p ≤ 0.0001) at 4 o C. Kintu is active at pH 3 and 11, maintains viability at temperatures between 25 o C and 120 o C and tolerates UV irradiation for up to 2 min and 20 s. Kintu inhibits Xvm growth at MOI ratios of 0.1, 1 and 10. The genome is a double stranded DNA molecule that consists of 48,985 base pairs and a G + C content of 51.71%. Antibiotic resistance genes or genes associated with a lysogenic life cycle are absent. There is limited sequence similarity of Kintu with other phages, making it a novel phage belonging to an unclassified genus of the class Caudoviricetes., Conclusion: Kintu is a novel bacteriophage that infects and lyses Xanthomonas vasicola pv. musacearum, the causative agent for Banana Xanthomonas Wilt. Its stability across diverse temperatures and pH conditions highlights its potential as a biocontrol agent for managing the disease., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Occult hepatitis B virus (HBV) infection (OBI) is a significant challenge for HBV prevention and control. We investigated the prevalence and surface (S) gene mutations of OBI among blood donors in Huzhou City, eastern China. The hepatitis B surface antigen (HBsAg) was routinely screened among 44,256 blood donors. HBV-DNA was detected using the Roche cobas®system. Serum samples that were HBsAg negative and HBV-DNA positive were selected, and the HBV S gene was amplified and sequenced. HBV genotype and S gene mutations were analyzed. The OBI rate in these blood donors was 0.070 % (31/44,256). Among the blood donors with OBI, only two cases (2/31, 6.5 %) were anti-HBc negative. The S gene sequences of 28 samples were successfully obtained, and we found that HBV genotype C (21/28, 70 %) was predominant among blood donors with OBI. Most S gene mutations were associated with OBI, and the high frequency mutations included N40S, G44E, Q51R/P, T113A/S,T118K/M, P120Q/S/T, and Y161F/S. Notably, amino acid substitutions at some sites differed from those reported previously, such as Y72F, G102V, P127L, Q129P, and S143T. Additionally, six novel mutations (S31I/N/R, P46L, S58C, C76Y, Y200F/C, and I208T) that may be associated with OBI were found. OBI was detected in a certain proportion of blood donors in Huzhou City. S gene mutations play an important role in OBI development. Further research is required to explore the functions of novel S gene mutants in OBI pathogenesis. The findings of this study may provide important insights to prevent HBV transmission through blood transfusions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The extraction of DNA from okra (Abelmoschus esculentus) is challenging due to its high mucilage and polysaccharide content, which can hinder both the yield and quality of DNA. In this study, an improved DNA isolation method is described incorporating a key modification being the use of solution I (1 M NaCl and 2% Sarcosyl) as a pre-treatment before applying the CTAB buffer, resulting in high-purity genomic DNA in just 1 h and 45 min., making it suitable for handling large sample sizes due to its rapid processing capabilities. This enhanced DNA extraction method was crucial for the accurate and rapid molecular detection of Okra enation leaf curl virus (OELCuV), a monopartite begomovirus that has spread across various regions of India. Transmitted by the whitefly (Bemisia tabaci), OELCuV causes leaf curling, enations, and stunted growth in okra, leading to significant yield losses. The surveys conducted during the 2020-21 and 2021-22 sowing seasons revealed disease incidence ranging from 14.03 to 67.57%. The extracted DNA via the improved DNA extraction method enhanced the speed of PCR based molecular identification of OELCuV, using virus-specific coat protein primers. The amplified CP genes were cloned and sequenced to study the CP gene based diversity among OELCuV isolates from different states of India. The CP gene nucleotide identity among the studied OELCuV isolates ranged from 95.57 to 99.27%, while comparison with previously reported Indian OELCuV CP sequences, the nucleotide identity ranged from 89.35 to 98.83%. The successful application of this optimized DNA extraction method sped up the detection process but also holds promise for broader use in the molecular study of okra and other mucilaginous crops, particularly in the rapid and reliable identification of begomoviruses. The optimized DNA extraction method significantly accelerated the detection of OELCuV, demonstrating its efficiency and reliability. This method shows strong potential for broader applications in the molecular study of okra and other mucilaginous crops, making it a valuable tool for future research and disease management., Competing Interests: Declarations Conflict of interest The authors declare no conflict of interest. Ethical approval We adhered to all relevant guidelines and legislation concerning virus infected okra leaf sample collection and their usage in this study. The plant leaf of Okra (Abelmoschus esculentus), in the present study is not endangered. Ethical clearance and permission for collecting virus-infected okra samples were obtained from ACSEN HyVeg, India, and JK Agri Genetics Ltd, India., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Human papillomavirus 16 (HPV16) infection is the leading cause of cervical cancer. The current mainstream method for detecting HPV16 is quantitative real-time PCR (qPCR). However, due to its time-consuming nature and reliance on expensive qPCR instruments, there is growing interest in more convenient, rapid and private approaches such as recombinase polymerase amplification (RPA). In this study, we innovated an effective method for HPV16 detection by integrating a RPA system with lateral flow strip (LFS) detection. Primers with optimal efficiency and specificity were designed, and false positive signals caused by dimers were eliminated by reducing the probe concentration. The RPA-LFS method demonstrates high sensitivity and specificity, capable of detecting 230 copies per reaction of HPV16 within 25 min without cross-reactivity to other subtypes. It exhibits good tolerance, remaining unaffected by 1.0% miconazole, 0.5% tioconazole and 1.0% hemoglobin. The results of clinical samples detected by this method were consistent with those of qPCR. The method provides a practical reference for HPV16 diagnosis and can be valuable in home and resource-limited settings, contributing to the reduction of cervical cancer incidence.

Glutamine cyclase, an enzyme involved in posttranslational modifications, is encoded by the glutaminyl-peptide cyclotransferase (QPCT) gene. Gene microarray analysis revealed that the QPCT gene was highly expressed in HepG2.2.15 cells compared with that in HepG2 cells. The serum expression level of the QPCT gene was detected by ELISA and was significantly greater in HBV-infected patients than in healthy controls. The mRNA and protein expression levels of the QPCT gene were markedly greater in the HBV-expressing cell lines (HepG2.2.15, and HepG2 and Huh7 cells transfected with the pBlu-HBV plasmid) than in the HepG2 and Huh7 cells. The levels of HBV pgRNA and HBV-DNA copy number, as well as the levels of HBeAg and HBsAg, also increased in the HepG2 and Huh7 cell lines cotransfected with the QPCT gene expression plasmid and the HBV 1.3-fold plasmid. Our study indicated that HBV can promote the expression of the QPCT gene, which in turn promotes the expression and replication of HBV., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Parvoviruses have evolved to possess a linear single-stranded DNA (ssDNA) genome ranging from 4 to 6.3 kb. Adeno-associated virus (AAV), a member of the Parvoviridae family, contains approximately 5 kb of linear ssDNA within its capsid. This ssDNA features two 145-base inverted terminal repeats (ITRs) positioned at each end. ITRs have a T-shaped hairpin secondary structure, which plays a crucial role in viral replication. To investigate the impact of ITRs on ssDNA stability, we conducted a DNA denaturation-reannealing assay in 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate buffer at pH7.9. Conventional double-stranded DNA (dsDNA) fragments retain a reannealing capability of over 50% for sizes under 8.8 kb, gradually losing this capability as sizes increase; however, dsDNA fragments in rAAV ranging from 0.7 to 6.3 kb did not exhibit a reannealing profile. This suggests that the presence of ITRs at both ends hinders annealing between the complementary strands. These results indicate that ITR structures preferentially induce an ssDNA conformation less than 6.3 kb in size, and that the stability of AAV ssDNA contributes to the viral life cycle, including processes such as infection, replication, and packaging. Considering the size of parvovirus genomes, it appears that their genomes require reversible flexibility in complementary DNA strands; simultaneously, this adaptability needs to be regulated by specific palindromic ITRs at both ends., Competing Interests: Declarations Competing interests The author declares no competing interests., (© 2024. The Author(s).)

High-risk human papillomavirus infection (HR-HPV) is necessary but not the only factor needed to develop cervical cancer. It is essential to estimate cervical cancer development risk in the population of high-risk HPV-positive women and to avoid unnecessary examinations and treatment in low-risk individuals. The study aimed to identify associations between different personal factors, vaginal microflora, sexually transmitted, high-risk HPV infection, and various degrees of cervical precancerous lesions. A study was performed in 2016-2020. The study group consisted of 112 patients with abnormal cervical cytology results referred for colposcopic examination. 120 women who came for a routine gynecological check-up were included in the control group. Material from the cervix and upper vaginal fornix was taken for pH measurement, wet mount microscopy, testing the six most common high-risk HPV DNA types (16/18, 31, 33, 45, 58), HPV E6/E7 mRNA, and 7 genital infections-C. trachomatis, N. gonorrhea, T. vaginalis, M. hominis, M. genitalium, U. urealyticum, U. parvum. Results showed that women with all grades of cervical intraepithelial neoplasia (CIN) more often were smokers, had increased vaginal pH levels, and had positive HR-HPV DNA and HR HPV E6/E7 mRNA expression. Abnormal vaginal microflora, especially types associated with aerobic vaginitis, and M. hominis were significantly more often found in women with CIN2+. The presence of C.trachomatis, U. parvum, and U.urealyticum did not differ between the groups. The most important factors independently associated with CIN2+ were positive high-risk HPV E6/E7 mRNA expression (OR 59.4, 95% CI 14.84-237.51), and positive high-risk HPV DNA (OR 3.9, 95% CI 1.16-13.23). Higher education level was associated with reduced risk of CIN2+ (OR 0.2, 95% CI 0.07-0.71). In conclusion, this study reports HR-HPV DNA of the most common six types and E6/E7 mRNA positivity as the most significant factors associated with CIN2+ lesions and higher education related to lower risk of high-grade cervical lesions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Plisko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Atypical squamous cells of undetermined significance (ASC-US) often present diagnostic challenges with cytology-based results, leading to potential underdiagnosis or overdiagnosis. An effective triage method is essential for managing these cases to reduce unnecessary referrals and treatment., Methods: A total of 322 women diagnosed with ASC-US were tested for HPV-DNA and the PAX1 and JAM3 methylation (PAX1 m /JAM3 m ) test in the study., Results: Methylation levels of PAX1 and JAM3 were significantly elevated in cervical lesions classified as CIN2 or more severe lesions (CIN2+). The methylation assay demonstrated a sensitivity of 83.8% and a specificity of 95.8%, outperforming HPV-DNA testing in differentiating high-grade cervical lesions among women with ASC-US. Moreover, PAX1 m /JAM3 m testing significantly reduced the colposcopy referral rate for further diagnostic procedures in high-risk HPV-positive women by 79.5%., Conclusions: PAX1 m /JAM3 m testing shows promise as a reliable supplemental method to HPV-DNA testing for the triage of women with cytologic ASC-US. In addition, the molecular triage based on the CISCER assay or single PAX1 or JAM3 methylation, had better effects in the women with non-HPV16/18 group. This approach could potentially minimize overtreatment and unnecessary referrals in clinical practice, enhancing patient management and resource utilization., Competing Interests: Declarations Ethical approval The name of the Approval Committee or the Internal Review Board (IRB). This research has been approved by the Ethics Committee of Zhejiang Provincial People’s Hospital. Human Ethics and Consent to participate declarations All participants have provided written informed consents according to the Declaration of Helsinki, and which have been approved by the Ethics Committee of Zhejiang Provincial People’s Hospital (No.2021SJ020). Consent for publication Not applicable. Clinical trial number Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: The World Health Organization has set ambitious goals to eliminate cervical cancer, necessitating evidence on increasing coverage and access to screening and treatment in high-burden areas. We implemented a pilot program to assess the feasibility of obtaining self-collected specimens for high-risk human papillomavirus (hr-HPV) testing in Nzulezo stilt village, a hard-to-reach community in Ghana, and inviting only hr-HPV-positive women to a central location for colposcopy and possible treatment. Subsequently, this study aimed to investigate the prevalence of hr-HPV infection and cervical lesions among the women and to explore factors potentially associated with hr-HPV infection among them., Methods: This pilot community-based cross-sectional study utilized data from screening sessions held from 2 to 20 November 2021 with specimens collected by participants using Evalyn brushes. HPV DNA testing was performed using the Sansure MA-6000 platform, while visual inspection utilized the Enhanced Visual Assessment (EVA) mobile colposcope. Univariate and multivariable nominal logistic regression was employed to explore factors associated with hr-HPV positivity., Results: Among 100 women screened (mean age, 43.6 ± 14.5 years), the overall hr-HPV prevalence rate was 39.0% (95% CI, 29.4-49.3). The prevalence rates of hr-HPV genotypes were stratified as follows: HPV16-8.0% (95% CI, 3.5-15.2), HPV18-5.0% (95% CI, 1.6-11.2), and other genotype(s) - 31.0% (95% CI, 22.1-41.0). Single-genotype infections with HPV16 and HPV18 were found in 4.0% (95% CI, 1.1-9.9) and 3.0% (95% CI, 0.6-8.5) of women, respectively. Mixed infections were observed in 1.0% (95% CI, 0.0-5.4) for HPV16 + 18, 3.0% (95% CI, 0.6-8.5) for HPV16 + other type(s), and 1.0% (95% CI, 0.0-5.4) for HPV18 + other type(s). The prevalence of cervical lesions among hr-HPV-positive women screened via colposcopy was 11.4% (95% CI, 3.2-26.7). In the multivariable model, reliance on other sources for medical bill payment was associated with hr-HPV infection (aOR, 0.20; 95% CI, 0.04-0.93), whereas age was not (aOR, 1.02; 95% CI, 0.99-1.05)., Conclusions: A high hr-HPV infection prevalence was recorded among the women. Utilizing technologies such as self-sampling, HPV DNA testing, and mobile colposcopy enables screening and treatment in remote and hard-to-reach communities where access to cervical cancer screening and treatment would otherwise be limited. Further research is warranted to assess the value and scalability of this approach in similar remote areas and its potential implementation in future programs., (© 2024. The Author(s).)

This is the first description of the complete genome sequence of a newly characterized monopartite begomovirus isolated from an asymptomatic uncultivated plant, Melochia tomentosa, collected in Burkina Faso. The sequence was obtained through rolling-circle amplification, cloning, and Sanger sequencing. The provisional species name "Begomovirus melochiae" and common virus name "melochia associated virus" (MeAV) are proposed. The MeAV genome was found to share the most nucleotide sequence similarity with three African monopartite begomoviruses: tomato curly stunt virus (74%), pepper yellow vein Mali virus (73%), and tomato leaf curl Cameroon virus (73%). Phylogenetic analysis confirmed its relationship to Old World monopartite begomoviruses. The discovery of MeAV in an uncultivated and asymptomatic plant provides a further example of the high diversity of begomoviruses in sub-Saharan African ecosystems., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Hepatitis B virus (HBV) infection is a major public health issue and is responsible for considerable morbidity and mortality globally. In Pakistan, the prevalence of chronic HBV infection varies from 2% to 4%, with an estimated exposure rate of approximately 34%. The major objective of this study was to determine the prevalence of HBV genotypes and the pattern of escape mutations in the HBV S gene in two major provinces of Pakistan: Punjab and Khyber Pakhtunkhwa. A total of 146 serum/plasma samples collected from hepatitis B patients were sequenced by the Sanger method. Phylogenetic analysis indicated the intermixed circulation of closely related strains of HBV genotype D and subgenotypes HBV/D1, HBV/D2, and HBV/A2 in both provinces, and various escape mutations (Y100C, P120S/T, G119R, C121S/Y, R122K, T126I, A128V, Q129H/R, G130R, T131N/I, M133T/I, Y134N, C137W, S143L, and D144E) were identified. These findings provide important insights into the current prevalence of HBV genotypes in Pakistan and will help in the establishment of more-efficient disease interventions and patient management strategies., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Cervical cancer is the 2nd most common female cancer among Sri Lankan females and is almost associated with sexually transmitted cervicovaginal human papillomavirus (HPV) infection. The study objectives were to determine the prevalence of vaginal and cervical HPV infection among 35year old ever-married women and assess the validity of primary healthcare provider-collected vaginal HPV/DNA specimens as a cervical cancer screening tool to improve the coverage of the programme., Method: A descriptive cross-sectional study was carried out from the 1st of September 2018 to the 31st of January 2019. Ever-married women 35 years of age in Kalutara district were the study population. Two women from each Public Health Midwife area (n = 413) were selected randomly from the relevant area eligible families register/s. HPV/DNA cervical specimen and vaginal specimen collection (n = 621) were carried out. Specimens were screened by the Cobas 4800 HPV/DNA automated Polymerase Chain Reaction (PCR) machine. Participants' profiles were recorded by the research assistants using an interviewer-administered questionnaire., Results: The overall prevalence of vaginal and cervical HPV infection was 7.08% (95% CI; 5.2-9.4%) and 6.12% (95% CI; 4.26-8.3%) respectively. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy, and the kappa coefficient of the vaginal HPV/DNA screening method vs. cervical HPV/DNA screening method were 100%, 98.9%, 86.4%, 100%, 99% and 0.92 respectively., Conclusions: Vaginal HPV/DNA specimen screening method can be used as a cervical cancer screening tool due to its high validity. Pilots of the feasibility should be set up before the regional or national rollout of vaginal sampling strategies., (© 2024. The Author(s).)

The objective of this study was to establish a more sensitive and specific diagnostic method for detecting plasma BK polyomavirus (BKPyV) DNA load in patients after renal transplantation using droplet digital polymerase chain reaction (ddPCR) and to validate the methodology. The linear range, lower limit of detection, accuracy, precision, and specificity of the detection system were evaluated by using the WHO BKPyV standard (7.2 log 10 IU/mL) as a reference, in accordance with the relevant documents of the Clinical and Laboratory Standards Institute. Plasma samples were collected from 74 renal transplantation patients with urinary BKPyV-DNA levels exceeding 7 log 10 copies/mL. Quantitative PCR (qPCR) and ddPCR were performed, and their diagnostic efficacy for BKPyV-DNA in the diagnosis of BK polyomavirus-associated nephropathy was evaluated using a receiver operating characteristic (ROC) curve. The coefficients of variation for the repeated detection of BKPyV standard DNA were 2.55 and 4.71 at concentrations of 6.2 and 3.2 log 10 IU/mL, respectively. The linear range was 2.2-6.2 log 10 IU/mL, and the lowest detection limit was 100 IU/mL. By utilizing histopathological examination of renal biopsy as the gold standard for BKPyV diagnosis, the area under the ROC curve of 74 post-transplantation plasma samples detected by the ddPCR system was found to be 0.875 (95% CI: 0.797-0.953, P < 0.01). The optimal threshold was 512.86 copies/mL, with a sensitivity of 90.0% and a specificity of 67.6%. In comparison, the area under the ROC curve for qPCR was 0.668 (95% CI: 0.583-0.752, P < 0.01), with an optimal threshold of 11,481.54 copies/mL, a sensitivity of 35.0%, and a specificity of 100.0%. Pairwise comparison (Delong test) of the ROC curves of the two systems showed a significant difference in the area under the curve, with a difference of 0.207 and a P -value <0.01. The BKPyV nucleic acid detection system, based on ddPCR, is appropriate for the regular monitoring of the BK polyomavirus, specifically in plasma samples containing low viral DNA loads while it provides the benefits of both absolute quantification and high sensitivity.IMPORTANCEIt was previously believed that droplet digital polymerase chain reaction had limitations, including high cost, limited throughput, and cumbersome operation, which hindered its widespread application in clinical practice. However, the current fully automated digital PCR platform, combined with streamlined operations, can detect 96 samples at once, and the entire process can be completed within an hour, laying a solid foundation for its extensive use., Competing Interests: The authors declare no conflict of interest.

Cytomegalovirus (CMV) colitis is a serious concern worsening the prognosis of patients with ulcerative colitis (UC). We aimed to assess risk factors and prognostic impact of CMV colitis in patients with moderate-to-severe UC flare. We conducted a retrospective, observational, single-center study. Consecutive adult patients hospitalized for moderate-to-severe UC from January 2020 to June 2023 were included. The primary endpoint was a diagnosis of CMV-colitis according to immunohistochemistry on tissue biopsies. The secondary endpoint was the need for colectomy within 30 days. Overall, 135 patients were included. CMV colitis was diagnosed in n = 37 (27.4%): n = 19 (51.4%) endoscopically, the remaining on surgical specimens. Of them, n = 23 (62.2%) had positive CMV-DNAemia with a median value of 1,008 cp/mL (interquartile range 318-2,980). Differences between the two groups (CMV colitis vs non-CMV) included age (60 vs 41 years, P = 0.004), Charlson Comorbidity Index (1 vs 0, P = 0.003), steroid refractoriness (86.5% vs 62.2%, P = 0.007), and positive CMV-DNAemia (62.2% vs 10.1%, P < 0.001). At multivariable analysis, steroid-refractory disease, Charlson Comorbidity Index, and CMV-DNAemia were associated with CMV colitis. Overall, n = 54 (39.7%) patients underwent colectomy, and this was significantly more common in patients with CMV colitis vs non-CMV group (54.1% vs 34.4%, P = 0.049). Kaplan-Meier showed that antiviral therapy seems to have a relevant impact on colectomy ( P < 0.001). CMV-DNA blood detection is independently associated with CMV-positive refractory UC. Since CMV colitis may increase the risk of colectomy and antiviral treatment seems to reduce such risk, prospective studies are needed to confirm the role of CMV-DNA blood detection to early diagnose CMV colitis., Importance: Cytomegalovirus (CMV) colonic reactivation worsens the prognosis of patients with active ulcerative colitis. Blood CMV-DNA reactivation is strongly associated with CMV colitis. Prompt diagnosis and treatment of CMV colitis can avoid surgery in most cases., Competing Interests: P.G. received honoraria from Janssen, Abbvie, Pfizer, Celgene, Takeda, Ferring, MSD, Amgen, and Alfa-Sigma, and he participated in a company sponsored speaker's bureau of Abbvie, Janssen, Takeda, FGerring, MSD, Sofar, and Chiesi. F.R. received honoraria from Janssen, Abbvie, Pfizer, Takeda, Ferring, and MSD, and he participated in a company sponsored speaker's bureau of Abbvie, Jansen, Takeda, Ferring, MSD, Sofar, and Chiesi. The other authors have no conflicts of interest to declare.

Identifying distinct noncanonical structures in pathogenic genomes is crucial for developing new diagnostic tools. This study uncovers stable G-quadruplex (GQ) structures in conserved DNA sequences unique to the monkeypox virus (MPV). Furthermore, we developed a method for the detection of target GQ using a fluorogenic probe.

Background: Primary screening for high-risk human papillomavirus (hrHPV) with cytological triage for women with non-16/18 hrHPV-positive status has become popular in China. However, cytology relies on the subjective judgment of pathologists, leading to inconsistent clinical performance., Methods: A total of 657 hrHPV-positive women aged 25-64 years were enrolled in this cross-sectional study. All participants underwent colposcopic biopsy after cytology triage, with cytology residual specimens undergoing DNA methylation testing. CIN2+ and CIN3+ sensitivity and specificity were compared between the different triage strategies (n=487): PAX1 methylation (PAX1 m ) , Glycophorin C methylation (GYPC m ), cytology, and combinations between them or with HPV16/18., Results: The area under the receiver operating characteristic curves (AUCs) for PAX1 m and GYPC m in detecting CIN2 or worse (CIN2+) were 0.867 (95% confidence interval [CI]: 0.796-0.937) and 0.873 (95% CI: 0.808-0.938), respectively. The sensitivities of PAX1 m and GYPC m were consistent with those of cytology for both CIN2+ and CIN3+ detection. The relative specificities of PAX1 m and GYPC m for CIN2+ detection compared to cytology were 2.83 (95% CI: 2.33-2.45) and 3.09 (95% CI: 2.40-3.98), respectively. The relative specificities of combining HPV 16/18 with PAX1 m and GYPC m for CIN2+ detection compared to cytology were 3.38 (95% CI: 2.96-3.86) and 3.67 (95% CI: 3.15-4.27), respectively. Compared to low levels of DNA methylation, high levels of PAX1 m and GYPC m resulted in odd ratios (ORs) of 57.66 (95% CI: 13.57-409.12, p < 0.001) and 23.87 (95% CI: 6.49-115.42, p < 0.001) for CIN3+, adjusted for HPV 16/18 and cytology results., Conclusions: PAX1 m and GYPC m demonstrated superior ability to identify cervical precancerous lesions and cervical cancer, with AUC values exceeding 0.85. For detecting CIN2+/CIN3+ in women with hrHPV-positive status, DNA methylation (combined with HPV 16/18) showed higher specificity than cytology (combined with HPV 16/18) and is a potential molecular biomarker for detecting cervical (pre)cancer., (© 2024. The Author(s).)

High-risk human papillomavirus (HR-HPV) testing, utilizing both DNA and RNA methods, offers enhanced sensitivity compared to cytology for detecting high-grade cervical intraepithelial neoplasia (CIN2+). Meanwhile, HR-HPV E6 and E7 mRNAs are more likely to differentiate the transient infection from the persistent than DNA. Aptima HPV can not only detect HPV mRNA but also HPV DNA though it is much more efficient at detecting HPV RNA than DNA. Currently, there are few studies on the distribution of HPV E6 and E7 mRNA and DNA in the same individual. It is interesting to compare the clinical performance of the Onclarity and Aptima HPV assays and to assess variations in viral load across different histological grades at both DNA and mRNA levels. The analysis included 1607 women (902 from a cervical cancer screening population and 705 inpatients and outpatients), with cervical cytological samples tested using the Aptima and Onclarity HPV assays. Both assays demonstrated high agreement for HPV types 18/45 and 16. Signal-to-cutoff (S/CO) values and Ct values for various HR-HPV types increased with histological severity from normal tissue to cancer. Notably, HPV18 Ct values exceeded those for HPV16 and 45 in women with ≥ CIN1 lesions but decreased sharply in cancer cases. Across the screening population, both assays showed similar sensitivity and predictive values for detecting CIN2+ lesions. The area under the curve (AUC) for CIN2+ and CIN3+ detection in the study population was robust (CIN2+: 0.880, 0.863; CIN3+: 0.881, 0.863). The DNA level for various HR-HPV types increased with histological severity from normal tissue to cancer, which might impact the performance of Aptima HPV assay. Both assays showed similar sensitivity and predictive values for detecting CIN2+ lesions., (© 2024 Wiley Periodicals LLC.)

Background: Human papillomavirus (HPV), the most common pathogen causing sexually transmitted diseases worldwide, is also an oncogenic virus. Due to the inadequacy of serologic tests in the diagnosis of HPV, NAATs (nucleic acid amplification tests), such as PCR (polymerase chain reaction), represent the gold standard today. Endocervical brush sampling in women has been successfully used for HPV genotyping for many years. The aim of this study was to determine the diagnostic efficacy for PCR HPV genotyping of multisite samples taken simultaneously with a swab from the external genitalia in addition to endocervical brush sampling in women applying for Hr-HPV screening., Methods: This study included 105 asymptomatic patients who came to the Gynecology Polyclinic of the University of Health Sciences Tepecik Training and Research Hospital between February 2023 and June 2023 for control purposes. Both the samples taken with a brush from the cervical area and the samples taken with a swab from the external genital area were sent to a screening laboratory for testing for Hr-HPV DNA. The samples were analyzed by real time PCR., Results: The success rate of positive detection of swab samples of HPV type 16 was significantly higher, with a difference of 17.6% (p < 0.0001). For HPV type 18, the swab sample had a significantly higher positive detection rate, with a difference of 60.0% (p = 0.002)., Conclusions: The results of this study show that HPV genotyping from the external genital area can be performed as an alternative to cervical sampling in sexually active women to increase the reach of a screening program.

Two Ralstonia phages, FLC1-1B and FLC4-3B, were isolated from leaf litter compost, using Ralstonia pseudosolanacearum, which is a causal agent of bacterial wilt disease, as a host. The genomic DNA sequences of FLC1-1B and FLC4-3B were determined and found to be 290,008 bp and 291,257 bp in length, respectively, and they were therefore classified as jumbo phages. However, they did not show high similarity to any jumbo phage genomic sequence in the NCBI nt database. The closest hit in a BLAST search was the jumbo phage ripduovirus RP12, with only 35% coverage and 77% sequence identity, whereas the FLC1-1B and FLC4-3B sequences were 99.0% identical. Based on these findings, FLC1-1B and FLC4-3B should be classified as members of a new genus in the order Caudoviricetes. FLC4-3B was found to suppress wilt disease in tomato plants, suggesting that it has potential as a biocontrol agent for managing R. pseudosolanacearum infections., (© 2024. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Human papillomavirus (HPV) has emerged as a significant contributor to cancer incidence globally, particularly in the context of oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP). This study aimed to develop and validate droplet digital PCR (ddPCR) assays for the detection of circulating tumor HPV DNA (ctHPV-DNA) in plasma, focusing on high-risk HPV genotypes associated with these cancers., Methods: ddPCR assays for HPV16, 18, 33, 35, 56, and 59 were developed and tested using gBlocks, HPV cell-free DNA, fragmented tumor HPV+ DNA, and plasma samples from patients with HPV+ OPSCC (n = 110) and HNCUP (n = 9)., Results: Assays demonstrated robust technical sensitivity across all tested HPV genotypes. Clinical application of the assays on a cohort of patients with HPV+ OPSCC and HNCUP revealed high sensitivity (91.6%) and wide variability in ctHPV-DNA levels. Analyses revealed correlations between ctHPV-DNA levels and TNM stage and tumor viral load. The association between ctHPV-DNA and tumor viral load persisted even after adjusting for TNM stage. At posttreatment, 72.5% of samples had reached undetectable ctHPV-DNA levels. Having detectable ctHPV-DNA posttreatment was associated with a higher ctHPV-DNA level at diagnosis and higher viral load at diagnosis., Conclusion: The findings underscore the potential of ctHPV-DNA as a biomarker for monitoring HPV+ cancers and offer insights into tumor dynamics. Implementation of these assays in clinical practice could enhance no-invasive treatment monitoring and recurrence detection in HPV-associated cancers., Clinical Trials: NCT05904327., (© 2024. The Author(s).)

The Tomato leaf curl Palampur virus (ToLCPMV) is a bipartite begomovirus that poses a substantial risk to agriculture by infecting a variety of crops, including cucurbitaceous group. This study examines the manifestation of encapsidation and synergism by ToLCPMV in bitter gourd (Momordica charantia) and focuses on its epidemiological approaches and implications of managing this virus in tomatoes growing areas. Through the utilization of molecular and biological techniques, we have successfully ascertained the epidemiology of this highly destructive virus, highlighting the vital roles played by its two genetic components. An analysis was conducted to identify the mechanism by which the virus clusters its DNA into virions, known as the encapsidation process. Additionally, the impact of synergism with other viral or environmental factors over the degree of infection was examined. The evolutionary rate differences among sites were modeled deploying a discrete Gamma distribution with 5 categories and a [+G] parameter. The results of this study provide important and unique information about synergism, encapsidiation and host-virus interactions. Sequencing study revealed that the bipartite ToLCPMV is linked to the occurrence of leaf curl disease in bitter gourd. The DNA-A and DNA-B of the ToLCPMV isolates infecting bitter gourd (SP1-4) showed 89 %, 93 %, 95 %, and 98 % similarity respectively. Mean evolutionary rates in these categories were 0.19, 0.47, 0.79, 1.24, 2.31 substitutions per site. Unexpectedly, the DNA-A sequences of ToLCPMV that infect this particular host seemed to be an amalgamation of sequences that are closely associated with tomato leaf curl New Delhi virus (ToLCNDV). Additionally, reiterate cropping of tomatoes with vegetables expanded the virus's host geographic region. This understanding will create some specific ways to regulate the dissemination of ToLCPMV and minimize its adverse impacts in tomato growing regions. Through the implementation of these strategies, the ability of crops to withstand and recover from adverse conditions can be enhanced, so encouraging the adoption of sustainable farming practices in affected regions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: CXC chemokine receptor 7 (CXCR7) plays pivotal roles in different virus infections. However, no research focused on the role of CXCR7 in hepatitis B virus (HBV)-infected patients. The primary aim of this study is to elucidate the role of CXCR7 in predicting the treatment response of chronic hepatitis B (CHB) patients undergoing pegylated interferon-alpha (PegIFNα) therapy., Methods: Two cohorts with a total of 945 Chinese CHB patients (Cohort 1, n = 238; Cohort 2, n = 707) were enrolled in this retrospective study, all the patients were positive for hepatitis B e antigen (HBeAg) and received PegIFNα treatment for 48 weeks and followed-up for 24 weeks post-treatment. Nineteen tag single-nucleotide polymorphisms (SNPs) were selected within and surrounding the CXCR7 gene region. The associations of CXCR7 SNPs and polygenic score (PGS) with PegIFNα treatment response were investigated in the two cohorts., Results: Among the 19 candidate SNPs of CXCR7, rs2952665 (A > G) was significantly associated with combined response (CR, defined as HBeAg seroconversion and HBV DNA level <3.3log 10 IU/mL, P = 0.002) and hepatitis B surface antigen (HBsAg) decline (P = 0.015) in the two cohorts at week 72. Furthermore, a PGS comprising CXCR7&#95;rs2952665 and five additional SNPs, which were previously recognized as biomarkers of PegIFNα treatment response, demonstrated a robust correlation with both CR (P = 1.38 × 10 -12 ) and HBsAg decline (P = 0.003) in all the patients., Conclusion: This research illustrated that CXCR7&#95;rs2952665 is a promising predictor of the PegIFNα therapy efficiency in Chinese HBeAg-positive CHB patients. A PGS consisting of CXCR7&#95;rs2952665 and five previously reported SNPs predicts treatment response to PegIFNα better., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: This study aimed to identify the specific vaccine strain associated with herpes zoster (HZ) in children following a series of diagnosed cases and to explore whether differences in single nucleotide polymorphisms (SNPs) among various vaccine strains are linked to an increased incidence of herpes zoster after vaccination., Methods: From February 2021 to March 2024, children <12 years old suspected of vaccine-related varicella-like rash or HZ were included. Varicella zoster virus DNA isolated from the patients were sequenced to differentiate vaccine type versus wild-type. 3D protein structures of pORF62 were simulated using open reading frame 62 sequences extracted from whole genome sequencing of vOka, MAV/06, Oka/SK vaccines, and pOka reference., Results: A total of 27 children with a median age of 2.1 (interquartile range, 1.5-3.4) years old presented with vaccine-related varicella-like rash (n = 4/27, 14.8%) or HZ (n = 23/27, 85.2%). One patient with varicella-like rash and 34.8% (n = 8/23) with HZ had disseminated skin involvement. All were immunized with the Oka/SK strain varicella vaccine. Genotyping showed 88.2% (n = 15/17) had SNPs specific to the Oka/SK strain, and two had SNPs considered pOka type contained within the Oka/SK vaccine. Despite accumulations of SNPs in ORF 62 of Oka/SK, the translated amino acid sequence and 3D protein structure were identical to wild-type pOka's pORF62. In vOKA and MAV/06, changes in amino acids occurred at two positions, S628G and R958G, within pORF62. The predicted 3D protein structure of vOka and MAV/06's pORF62 showed that the α helical structure within region I undergoes conformational change, potentially increasing difficulties in interactions with infection-related proteins and thereby decreasing virulence. pORF62 in pOka and Oka/SK exhibited more stable structure complex of the α helical structure., Discussion: Lack of structural alternations in region I of pORF62 due to the absence of critical genetic polymorphisms in open reading frame 62 could be associated with the heightened incidence of adverse events., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: To analyze the trend of intraocular viral load after antiviral treatment in patients with varicella-zoster virus-induced acute retinal necrosis and to explore the effect of viral genotypes on clinical manifestations., Methods: In this case series, viral load was detected using polymerase chain reaction from aqueous humor during treatment; viral load curves were fitted, and the time required to reach the inflection point between plateau phase and logarithmic reduction phase was estimated. Variations in viral genomes were detected by metagenomic sequencing., Results: Twenty eyes of 20 patients were included. The median (interquartile range) initial viral load was 5.9 × 10 7 (1.1 × 10 7 -1.1 × 10 8 ) copies/mL. The average duration of retinitis was 5 ± 3 weeks. The average time required to reach the inflection point was 4.2 ± 1.6 days. Time required to reach the inflection point was correlated with the duration of retinitis ( P = 0.025). Patients with varicella-zoster virus carrying the p.S715* variation in ribonucleotide reductase ( RNR ) subunit 1 gene had lower initial viral loads (median 1.3 × 10 7 copies/mL) than those without (median 1.1 × 10 8 copies/mL; adjusted P = 0.030)., Conclusions: The inflection of viral load curve is helpful to estimate the length of plateau phase and the duration of retinitis during antiviral treatment in patients with acute retinal necrosis. Loss-of-function variation in RNR gene might be correlated with lower virulence of varicella-zoster virus., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Opthalmic Communications Society, Inc.)

Dense packaging of genomic DNA is crucial for organismal survival, as DNA length always far exceeds the dimensions of the cells that contain it. Organisms, therefore, use sophisticated machineries to package their genomes. These systems range across kingdoms from a single ultra-powerful rotary motor that spools the DNA into a bacteriophage head, to hundreds of thousands of relatively weak molecular motors that coordinate the compaction of mitotic chromosomes in eukaryotic cells. Recent technological advances, such as DNA proximity-based sequencing approaches, polymer modelling and in vitro reconstitution of DNA loop extrusion, have shed light on the biological mechanisms driving DNA organization in different systems. Here, we discuss DNA packaging in bacteriophage, bacteria and eukaryotic cells, which, despite their extreme variation in size, structure and genomic content, all rely on the action of molecular motors to package their genomes., (© 2024. Springer Nature Limited.)

Cutaneous plaques and squamous cell carcinoma (SCC) are common in captive North American snow leopards (SLs) ( Panthera uncia ). Our objective was to determine whether these lesions are potentially associated with papillomavirus(es). Polymerase chain reaction (PCR) was performed on 3 cutaneous plaques using degenerate primers for papillomaviruses. A putatively novel papillomavirus was identified that shared 76% sequence identity to Felis catus papillomavirus 2 . Specific PCR for this virus was performed on 5 cutaneous SCC samples and 7 normal skin samples, which were all positive. In situ hybridization for this putatively novel virus was performed, which revealed strong hybridization signals within hyperplastic cells in cutaneous plaques (n = 3) and within neoplastic cells in cutaneous SCC samples (n = 5). No hybridization signals were identified within normal skin. Ultimately, identification of a causal viral agent in the development of plaques and SCC in SLs will help guide therapeutic intervention and lay the foundation for development of prophylactic vaccines., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Like the coronavirus disease 2019, the hepatitis B virus is also wreaking havoc worldwide, which has infected over 2 billion people globally. Using an experimental animal model, our previous research observed that the hepatitis B virus genes integrated into human spermatozoa can replicate and express after being transmitted to embryos. However, as of now, this phenomenon has not been confirmed in clinical data from patients., Objectives: To explore the integration of the hepatitis B virus into patients' sperm genome and its potential clinical risks., Materials and Methods: Forty-eight patients with chronic hepatitis B virus infection were categorized into two groups: Test Group-1 comprised 23 patients without integration of hepatitis B virus DNA within the sperm genome. Test Group-2 comprised 25 patients with integration of hepatitis B virus DNA within the sperm genome. Forty-eight healthy male donors were included as control. The standard semen parameter analysis, real-time polymerase chain reaction, quantitative real-time polymerase chain reaction, sperm chromatin structure assay, fluorescence in situ hybridization, and immunofluorescence assays were utilized., Results: The difference in the median copy number of hepatitis B virus DNA per mL of sera between Test Group-1 and Group-2 was not statistically significant. In Test Group-2, the integration rate of hepatitis B virus DNA was 0.109%, which showed a significant correlation with the median copy number of hepatitis B virus DNA in motile spermatozoa (1.18 × 10 3 /mL). Abnormal semen parameters were found in almost all these 25 patients. The integrated hepatitis B virus S, C, X, and P genes were detected to be introduced into sperm-derived embryos through fertilization and retained their function in replication, transcription, and translation., Conclusion: Our findings suggest that hepatitis B virus infection can lead to sperm quality deterioration and reduced fertilization capacity. Furthermore, viral integration causes instability in the sperm genome, increasing the potential risk of termination, miscarriage, and stillbirth. This study identified an unconventional mode of hepatitis B virus transmission through genes rather than virions. The presence of viral sequences in the embryonic genome poses a risk of liver inflammation and cancer., (© 2024 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Cervical cancer is one of the most common gynecological malignancies, with the vast majority of which being caused by persistent infection with Human Papillomavirus (HPV) 16 and 18. The current available HPV detection methods are sensitive and genotyped but are restricted by expensive instruments and skilled personnel. The development of an easy-to-use, rapid, and cost-friendly analysis method for HPV is of great need. Herein, hollow palladium-ruthenium nanocages modified with two oligonucleotides (PdRu capture probes) were constructed for genotyping and simultaneous detection of target nucleic acids HPV16 and HPV18 by dual lateral flow assay (DLFA). PdRu capture probes were endowed with bi-functions for the first time, which could be used to output signals and hybridize target nucleic acids. Under optimized conditions, the PdRu based-DLFA with detection limits of 0.93 nM and 0.19 nM, respectively, exhibited convenient operation, and high sensitivity. Meanwhile, the DLFA achieved excellent rapid detection within 20 min, which was attributed to capture probes that can be directly bound to amplification-free target nucleic acids. Therefore, the development of PdRu-based DLFA can be utilized for rapid, sensitive, and simultaneous genotyping detection of HPV16 and HPV18, showing great application for nucleic acid detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Chronic Hepatitis B Virus (HBV) infection remains a significant public health concern, particularly in Africa, where the burden is substantial. HBV is an enveloped virus, classified into ten phylogenetically distinct genotypes (A-J). Tests to determine HBV genotypes are based on full-genome sequencing or reverse hybridization. In practice, both approaches have limitations. Whereas diagnostic sequencing, generally using the Sanger approach, tends to focus only on the S-gene and yields little or no information on intra-patient HBV genetic diversity, reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV diagnostic sequencing protocol suitable for clinical virology that yields both complete genome sequences and extensive intra-patient HBV diversity data. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit (Oxford nanopore Technologies, Oxford, OX4 4DQ, UK), ONT GridION sequencing, genotyping using genome detective software v1.132/1.133, a recombination analysis using jpHMM (26 October 2011 version) and RDP5.61 software, and drug resistance profiling using Geno2pheno v2.0 software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 residual diagnostic samples from HBV-infected patients in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.

Background: Hepatitis B virus (HBV) infection is a persistent global public health problem, and curing for chronic hepatitis B (CHB) through the application of existing antiviral drugs is beset by numerous challenges. The viral protein HBx is a critical regulatory factor in the life cycle of HBV. Targeting HBx is a promising possibility for the development of novel therapeutic strategies., Methods: The Nano-Glo ® HiBiT Lysis Detection System was used to screen the herbal monomer compound library for compounds that inhibit HBx expression. Western blotting was used to examine proteins expression. Southern blotting or Northern blotting were used to detect HBV DNA or HBV RNA. ELISA was performed to detect the HBsAg level. The effect of asiatic acid on HBV in vivo was investigated by using recombinant cccDNA mouse model., Results: Asiatic acid, an extract of Centella asiatica, significantly reduced the HBx level. Mechanistic studies demonstrated that asiatic acid may promote the degradation of HBx in an autophagy pathway-dependent manner. Subsequently, asiatic acid was found to reduce the amount of HBx bound to covalently closed circular DNA (cccDNA) microchromosomes, and repressive chromatin modifications then occurred, ultimately inhibiting cccDNA transcriptional activity. Moreover, in HBV-infected cells and a mouse model of persistent HBV infection, asiatic acid exhibited potent anti-HBV activity, as evidenced by decreased levels of HBV RNAs, HBV DNA and HBsAg., Conclusions: Asiatic acid was identified as a compound that targets HBx, revealing its potential for application as an anti-HBV agent., (© 2024. The Author(s).)

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.

Background: The gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent diversity of genome types (dsDNA, ssDNA, dsRNA, and ssRNA) and topologies (linear, circular, or fragments), with subsequently biases associated with current sequencing library preparation methods. To overcome these problems and improve reproducibility and comparability across studies, universal or standardized virome sequencing library construction methods are highly needed in the gut virome study., Results: We repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA + Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches., Conclusion: We introduce and improve a fast, simple, and efficient ligation-based single-stranded DNA library preparation for gut virome study. This method is compatible with Illumina sequencing platforms and only requires ligation reagents within 3-h library preparation, which is similar or even better than the advanced library preparation method (xGen). We hope this method can be further optimized, validated, and widely used to make gut virome study more comparable and reproducible. Video Abstract., (© 2024. The Author(s).)

Objective: To analyze the genotypes of VZV in Jiangsu province to identify vaccine strains and wild strains, providing a molecular biological background for the effective prevention and control of varicella., Method: Stratified sampling was used to collect herpes fluid or throat swab from patients diagnosed with varicella. ORF22 was carried out, and the restriction enzyme site of ORF38, ORF54 and ORF62 were detected., Results: All 207 virus strains were Clade 2 type by sequencing the PCR products of ORF22. The sequencing results showed that five SNP sites changed compared to the Dumas reference strain(Clade 1). From A to G at 37,902, from T to c at 38,055, from A to C at 38,081, and from G to A at 38,177, from G to A at 39,394. The prevalent VZV genotypes in Jiangsu is consistent with the P-Oka. The restriction enzyme site analysis of PCR amplification products from ORF38 (PstI), ORF54 (BglI), ORF62 (SmaI) showed that all 207 virus strains were wild-type. There were two different types of the wild strains, and 183 strains (88.4%) were PstI (+), BglI (+), SmaI (-). The wild strains between different regions showed no significant differences (χ 2  = 0.05, P = 0.982)., Conclusions: The prevalent VZV genotypes are Clade 2 and the prevalent virus strains are wild strains in Jiangsu Province, the primary wild strain observed is mainly PstI (+), BglI (+), SmaI (-)., (© 2024. The Author(s).)

Background: Human Papillomavirus (HPV) DNA screening was a crucial element in the fight against cervical cancer and had been adopted in many countries, including China. However, the onset of the COVID-19 pandemic in March 2020 disrupted this program significantly., Methods: The aim of this study is to investigate the prevalence and distribution of HPV genotypes among the population undergoing cervical cancer screening during the pandemic period. From January 2017 to December 2022, Peking Union Medical College Hospital gathered 45,496 cervical swabs from individuals undergoing cervical cancer screening. These samples were analyzed to detect fifteen high-risk HPV (HR-HPV) DNA types and a combination of two low-risk HPV (LR-HPV) types., Results: The study revealed an overall infection rate of 11.24% (5,114/45,496), with 11.06% (5,032/45,496) of individuals infected with HR-HPV. The number of HPV screening patients and the infection rates of HPV, HR-HPV, LR-HPV, multiple genotype HPV (M-HPV), and single genotype HPV (S-HPV) during the pandemic were lower than those observed before the pandemic. Moreover, the age group with the highest percentage of infected individuals was under 45-49 years, with HPV52, HPV58, HPV16, and HPV51 being the most prevalent genotypes. Notably, HPV66 emerged as the fifth most commonly detected genotype during the pandemic. Additionally, among the eleven age groups examined, women under 25 exhibited the highest detection rate, with HPV52 and HPV16 infection rates exceeding those observed in the pre-pandemic period., Conclusions: The findings of this study offer significant insights for shaping HPV prevention strategies and enhancing cervical cancer screening initiatives in China following the epidemic., (© 2024. The Author(s).)

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (ΦT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology.IMPORTANCEFunctional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes., Competing Interests: The authors declare no conflict of interest.

During infection, the giant phiKZ phage forms a specialized structure at the center of the host cell called the phage nucleus. This structure is crucial for safeguarding viral DNA against bacterial nucleases and for segregating the transcriptional activities of late genes. Here, we describe a morphological entity, the early phage infection (EPI) vesicle, which appears to be responsible for earlier gene segregation at the beginning of the infection process. Using cryo-electron microscopy, electron tomography (ET), and fluorescence microscopy with membrane-specific dyes, we demonstrated that the EPI vesicle is enclosed in a lipid bilayer originating, apparently, from the inner membrane of the bacterial cell. Our investigations further disclose that the phiKZ EPI vesicle contains both viral DNA and viral RNA polymerase (vRNAP). We have observed that the EPI vesicle migrates from the cell pole to the center of the bacterial cell together with ChmA, the primary protein of the phage nucleus. The phage DNA is transported into the phage nucleus after phage maturation, but the EPI vesicle remains outside. We hypothesized that the EPI vesicle acts as a membrane transport agent, efficiently delivering phage DNA to the phage nucleus while protecting it from the nucleases of the bacterium., Importance: Our study shed light on the processes of phage phiKZ early infection stage, expanding our understanding of possible strategies for the development of phage infection. We show that phiKZ virion content during injection is packed inside special membrane structures called early phage infection (EPI) membrane vesicles originating from the bacterial inner cell membrane. We demonstrated the EPI vesicle fulfilled the role of the safety transport unit for the phage genome to the phage nucleus, where the phage DNA would be replicated and protected from bacterial immune systems., Competing Interests: The authors declare no conflict of interest.

Recombinase polymerase amplification (RPA) has emerged as a rapid, efficient, and highly sensitive method for nucleic acid amplification, thus becoming a focal point of research in the field of virus detection. This paper provides an overview of RPA, emphasizing its unique double-stranded DNA synthesis mechanism, rapid amplification efficiency, and capability to operate at room temperature, among other advantages. In addition, strategies and case studies of RPA in combination with other technologies are detailed to explore the advantages and potential of these integrated approaches for virus detection. Finally, the development prospect of RPA technology is prospected.

Background: Given the strong association between high-risk HPV genotypes, such as HPV 16 and 18, and cervical cancer, this study aimed to compare the distribution of common HPV genotypes in the southwest Iranian population with those included in the available vaccines., Methods: Based on the sample quality, DNA was extracted from the biological samples of 8036 individuals included in the study using three different methods (automated instrument, column, and precipitation), and a total of 21 different HPV genotypes were detected using real-time PCR., Results: The majority of participants were women (> 99%), with a positive rate of HPV infection of 29.9%, in which high-risk genotypes were dominant in 84.9% cases. The highest rate of HPV infections was observed in the age ≤ 30 years (35.9%). HPV 6 and 16 were the most frequent low- and high-risk genotypes, respectively. Multi HPV infections were observed in 35% of positive samples and the highest cross infections were observed between HPV6 and 16. Co-infection with HPV 16 and 18 was observed in 21 positive samples (1%). Although vaccination is essential to reduce the outcome of HPV infections, such as cervical cancer, other frequently occurring high-risk genotypes are not included in the 9-valent vaccine., Conclusion: Since the association between cervical cancer and other high-risk HPV types rather than 16 and 18 has been less studied, investigating their pathogenicity in cervical cancer is recommended. Furthermore, the new generation of HPV vaccines should contain other frequently occurring high-risk genotypes beyond those currently covered in approved vaccines., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The current knowledge of Staphylococcus warneri phages is limited, with few genomes sequenced and characterized. In this study, a prophage, vB&#95;G30&#95;01, isolated from Staphylococcus warneri G30 was characterized and evaluated for its lysogenic host range. The phage was studied using transmission electron microscopy and a host range. The phage genome was sequenced and characterized in depth, including phylogenetic and taxonomic analyses. The linear dsDNA genome of vB&#95;G30&#95;01 contains 67 predicted open reading frames (ORFs), classifying it within Bronfenbrennervirinae. With a total of 10 ORFs involved in DNA replication-related and transcriptional regulator functions, vB&#95;G30&#95;01 may play a role in the genetics and transcription of a host. Additionally, vB&#95;G30&#95;01 possesses a complete set of genes related to host lysogeny and lysis, implying that vB&#95;G30&#95;01 may influence the survival and adaptation of its host. Furthermore, a comparative genomic analysis reveals that vB&#95;G30&#95;01 shares high genomic similarity with other Staphylococcus phages and is relatively closely related to those of Exiguobacterium and Bacillus , which, in combination with the cross-infection assay, suggests possible cross-species infection capabilities. This study enhances the understanding of Staphylococcus warneri prophages, providing insights into phage-host interactions and potential horizontal gene transfer.

This article investigates the role of local fauna in Western Kazakhstan as potential reservoirs of the camelpox virus (CMLV). The study emphasizes analyzing possible sources and transmission pathways of the virus using polymerase chain reaction (PCR) and serological methods, including virus neutralization tests and enzyme-linked immunosorbent assays (ELISA). Samples were collected from both young and adult camels, as well as rodents, ticks and blood-sucking insects in the Mangystau and Atyrau regions. The PCR results revealed the absence of viral DNA in rodents, ticks and blood-sucking insects; also, the ELISA test did not detect specific antibodies in rodents. These findings suggest that these groups of fauna likely do not play a significant role in the maintenance and spread of CMLV. Consequently, the primary sources of transmission are likely other factors, potentially including the camels themselves. The study's results indicate the need to reassess current hypotheses regarding infection reservoirs and to explore alternative sources to enhance strategies for the control and prevention of the camelpox virus.

Torque teno virus (TTV) is a ssDNA orphan virus belonging to the Anelloviridae family, but some recent studies suggested its possible involvement in central nervous system (CNS) pathology. We analyzed serum and cerebrospinal fluid samples (CSF) from 109 patients with encephalitis for TTV infection using serological and molecular testing, virus quantitative measurement, and next-generation sequencing-based (NGS) phylogenetic analysis. TTV noncoding region (UTR) and/or open reading frame 1 (ORF-1) sequences were detected in serum of 86 (79%) patients and in nine (8%) patients in CSF. Five of the latter patients were coinfected with various entero - and herpesviruses . Anti-TTV-IgG were detected in 80 (73.4%) sera and in two (1.8%) CSF samples, while anti-TTV-IgM were present in three (2.8%) sera and in none of the CSFs. Phylogenic analysis of CSF-derived TTV ORF-1 sequences revealed the presence of three unique variants in one patient. TTV was quantified in five CSF-serum pairs: in two patients viral loads were similar, and in three serum TTV loads were approximately one log higher. Our results suggest at least an occasional replication of TTV in CNS. However, whether TTV could be the cause of encephalitis requires further studies.