Adeno-associated virus (AAV) is a non-enveloped DNA virus infecting a wide variety of species, tissues and cell types, which is recognized as safe and effective method for delivering therapeutic transgenes. AAV vector is the most popular viral gene delivery system in clinical delivery system with unique and multiple advantages, such as tissue tropism, transduction specificity, long-lasting gene expression, low immune responses, without host chromosome incorporation. Till now, four AAV-based gene therapy drugs have already been approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Despite the success of AAV vectors, there still have some remaining challenges to limit the further usage, such as poor packaging capacity, low organ specificity, pre-existing humoral immunity, and vector dose-dependent toxicity. In the present review, we address the different approaches to optimize AAV vector delivery system with focus on capsid engineering, packaging capacity, immune response at the clinical level. The review further investigates the potential of manipulating AAV vectors in preclinical applications and clinical translation, which emphasizes the challenges and prospects in viral vector selection, drug delivery strategies, immune reactions in cancer, neurodegenerative disease, retinal disease, SARS-CoV-2, and monkeypox. Finally, it forecasts future directions and potential challenges of artificial intelligence (AI), vaccine, and nanobody, which emphasizes the need for ethical and secure approaches in AAV application.

Respiratory specimens collected via nasopharyngeal and throat swabs are the recommended method of choice for the molecular detection of the Influenza A virus. However, they often cause discomfort to patients and require trained healthcare workers. This study aimed to validate a cost-effective nucleic acid extraction-free PCR method using SYBR-Green chemistry with saliva samples to diagnose respiratory illnesses caused by Influenza A viruses. This study enrolled symptomatic pediatric and adult patients with influenza-like illness. A SYBR-Green based nucleic acid amplification test was used with CDC-recommended primers specific for detecting Influenza A viruses. Results from saliva PCR tests were compared with those of standard TaqMan chemistry-based PCR detection from nasopharyngeal swabs. The sensitivities of saliva PCR tests with and without nucleic acid extraction were 83% and 82%, respectively. Both the methods showed a specificity of 98%. Overall, the sensitivity and specificity of saliva as a sample using the developed SYBR-Green-based PCR for Influenza A virus detection were 90% and 98%, respectively. Given the high sensitivity, specificity, positive and negative predictive values, likelihood ratios, agreement percentage, and kappa statistics, this study concluded that saliva is a potential specimen for diagnosing Influenza A virus infection by extraction free nucleic acid amplification testing.

Background It is unclear whether iron levels affect BKPyV reactivation after kidney transplantation. Our goal is to analyze the relationship between preoperative iron levels in kidney transplant recipients and postoperative BK virus reactivation. Additionally, we aim to develop and validate a personalized prediction model for BK virus reactivation. Methods We retrospectively analyzed the relationship between preoperative iron levels and BK virus reactivation in 626 kidney transplant recipients. The cohort was randomly divided into a training cohort and a validation cohort in a 2:1 ratio. Independent risk factors associated with BK virus reactivation were identified in the training cohort using COX proportional hazards regression. Based on the analysis results, a nomogram was constructed to develop individualized risk prediction models for BK viremia and BK virus nephropathy. Results Among the 626 kidney transplant recipients, 38 cases (6.1%) of BK viremia and 18 cases (2.9%) of BK virus nephropathy were diagnosed. Univariate analysis revealed that UIBC, TIBC, and TSAT were associated with the occurrence of BK viremia and BKPyVAN. In the training cohort, multivariate analysis showed that preoperative TSAT was an independent risk factor for BK viremia (1.03 [1.01-1.05], P=0.009) and BK virus nephropathy (1.05 [1.01-1.09], P=0.011). The C-index for the nomogram model predicting BK viremia and BK virus nephropathy after kidney transplantation was 0.77 and 0.82 in the training cohort, and 0.88 and 0.92 in the validation cohort, respectively. Conclusions Preoperative transferrin saturation levels in kidney transplant recipients are closely associated with the reactivation and progression of BK virus after transplantation.

Relapsing-remitting multiple sclerosis (RRMS) is defined by elevated IgG/IgA/IgM responses targeting Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA) and deoxyuridine-triphosphatases (dUTPases) of Human herpsesvirus-6 (HHV-6) and EBV. These responses suggest that the viruses are being replicated and reactivated. An increased prevalence of chronic fatigue syndrome, depression, and anxiety is associated with signs of immune activation in RRMS. Nevertheless, there is a lack of data regarding the association between viral reactivation and neuropsychiatric symptoms of RRMS. This study investigated the IgG/IgA/IgM responses to EBNA, and EBV and HHV-6-dUTPases, in 58 remitted RRMS patients and 63 normal controls. The McDonald criteria were employed to establish the diagnosis of MS. The Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score were employed to evaluate disabilities caused by RRMS. We evaluated the scores of the Hamilton Depression (HAMD) and Anxiety (HAMA) Rating Scales, and Fibro-Fatigue (FF) scale. One latent construct was extracted from the EDSS, MSSS, FF, HAMD, and HAMA scores. We discovered that the combined effects of IgG and IgM-HHV-6-dUTPAses accounted for 63.7% of the variance in this construct. Furthermore, the total FF, HAMA, and HAMD scores were substantially associated with the IgG and IgM-HHV-6-dUTPAses, accounting for approximately 38.7% to 51.0% of the variance. The three neuropsychiatric rating scale scores were also significantly correlated with IgA reactivity directed to both dUTPases and IgG/IgA/IgM to EBNA. In conclusion, the reactivation and replication of HHV-6 and EBV significantly contributes to chronic fatigue syndrome, as well as symptoms of depression and anxiety due to RRMS.

We investigated sewage samples from different wastewater treatment plants in Belgium for the presence of measles virus (MV). Samples from Brussels North were found to contain MV genotype D8. Twelve measles cases were reported at that time in the Brussels capital region, additional infections are likely occurring under the radar.

Background: Acute-on-chronic liver failure (ACLF) is a disease characterized by systemic inflammatory response with high mortality. Nowadays, there is no prediction model for its long-term prognosis. We aimed to establish and validate a prognostic prediction model incorporating inflammation indexes to forecast the long-term prognosis of patients with hepatitis B-related ACLF (HBV-ACLF). Methods: Retrospective analysis of 986 patients’ clinical data with HBV-ACLF from Third Affiliated Hospital of Sun Yat-sen University between January 2014 and December 2018 were conducted. The patients were randomly divided into the training cohort (690 cases) and the validation cohort (296 cases) according to the ratio of 7:3. LASSO and Cox regression analysis were used to determine the independent risk factors for long-term mortality. Results: The following variables were identified: age, cirrhosis, hepatic encephalopathy, total bilirubin (TBIL), international normalized ratio (INR), Monocyte to lymphocyte ratio (MLR), and Neutrophil to platelet ratio (NPR), and a new nomogram was constructed to predict the survival rate of 1 -month, 3-month, and 12-month by weighting the scores of each variable. The C-index was 0.777 (95%CI 0.752-0.802), and the AUC was 0.829 (95%CI 0.798-0.859) in the training cohort. The predictive value of the nomogram demonstrated a superior ability to predict long-term survival rate compared to MELD score (0.767, 95% CI: 0.730-0.804, P<0.001), and COSSH-ACLF II score (0.807, 95%CI: 0.774-0.840, P=0.028). Evaluation using calibration curves and decision curve analysis (DCA) suggested its practical utility. Conclusions: The novel inflammation scoring system, including MLR and NPR, can well predict long-term mortality in HBV- ACLF patients.

not-yet-known not-yet-known not-yet-known unknown Oncolytic virotherapy (OVT) aims to disrupt the tumor microenvironment and provide a unique therapeutic approach against solid tumors. Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for treating various solid tumors and has been approved to treat melanoma and glioma in human patients. Previously, we reported the generation of an engineered HSV-1 vaccine strain VC2, which has shown exceptional promise as an oncolytic and immunotherapeutic virus. In the present work, we engineered VC2 to constitutively express the murine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) gene inserted in place of HSV-1 Glycoprotein C (gC). We tested the efficacy of VC2-GMCSF for its ability to generate anti-tumor response in the 4T1 stage four metastatic breast cancer mouse model. GM-CSF expression enhanced VC2 viral replication and infectious virus production. Tumors formed after 7 days of engraftment in the mammary fat-pad of Balb/CJ mice were treated by injecting ~10 6 plaque forming units (PFU) once. Intra-tumor treatment did not appreciably reduce average primary tumor sizes. However, metastatic foci were significantly reduced in mice lungs treated with VC2-GMCSF compared to VC2 or mock treatment. VC2-GMCSF intratumoral treatment induced a stronger intratumor T cell infiltration but not an increased cytotoxic activity. A significant T cell infiltration was observed in the metastatic areas in VC2-GMCSF treated animals, which was associated with reduced pro-tumor marker PDL1 and VEGF gene expression. These results show that constitutive expression of GM-CSF enhanced the overall efficacy of VC2 for oncolytic virotherapy. VC2-GMCSF holds promise as an oncolytic and immunotherapeutic virotherapy for breast and other cancers.

Background: Human Immunodeficiency Virus (HIV), Human T Lymphotropic Virus (HTLV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) coinfection may lead to disease progression or worsen its clinical presentation. Viral coinfections screening during blood donation is critical. Methods: To identify risk factors for coinfection among blood donors, we assessed the blood donations at the Fundação de Hematologia e Hemoterapia da Bahia, from 2008 – 2017. We compared single infection versus coinfection, defined as individuals with two or more blood-borne viruses. A multivariable logistic regression model was performed to evaluate independent associations between characteristics of donors with single infection and multiple infection using “non-infection” category as reference. Results: Among 777,446 collected blood donations, 27,458 (3.5%) were reactive, most (n=26,677, 97.6%) for a single infection and 681 (2.4%) for coinfection. The most frequent coinfections were HBV-HIV (30.6%), HBV-HCV (30.4%), and HBV-HTLV (24.4%). Male sex, lower education, being single, and being a first-time donor were independently associated with both single and coinfection. Nevertheless, the adjusted odds for risk factors of coinfection were notably higher than for single infection. Conclusions: Single and coinfection shared identical risks, but they were significantly higher for coinfection. Preventive strategies addressing the identified risks can decrease transmission of blood-borne viruses via blood transfusion.

Introduction BK polyomavirus-associated nephropathy (BKPyVAN) is a well-known complication of kidney transplantation (KTx).The mainstay of prevention is reduction of immunosuppression upon detection of BK polyomavirus (BKPyV) viremia, which precedes BKPyVAN. However, this reduction may inadvertently increase the risk of alloimmunity particularly in patients with a high BKPyV load, where significant immunosuppression reduction is often necessary. This single-center, retrospective cohort study assesses the risk of de novo donor-specific antibodies (dnDSA) development and biopsy-proven acute rejection (BPAR) following high and low BKPyV viremia. Methods All patients who underwent KTx at Leiden University Medical Center between 2011 and 2020 were included. Patients were grouped according to high (maximum BKPyV serum load >10E4 copies/ml), low (maximum serum BKPyV load <10E4 copies/ml) and absent BKPyV viremia, and analyzed for the development of dnDSA and BPAR, using Cox regression. Results Of 1076 KTx recipients included, 108(10%) developed a BKPyV viremia with a maximum serum load below 10E4 copies/ml, whereas 121(11.2%) developed a BKPyV viremia exceeding 10E4 copies/ml. The risk of dnDSA development was higher in patients with a high BKPyV viremia, compared to patients without viremia (adjusted Hazard Ratio of 1.9(95% CI1.1-3.2, p=0.017). No significant difference in dnDSA risk was observed between patients with low and absent BKPyV viremia. Risk of BPAR did not differ between groups. Conclusion Our study shows that higher BKPyV loads in KTx patients are associated with a higher risk for dnDSA development, highlighting the importance of exploring additional strategies for the prevention and treatment of BKPyV infections in KTx recipients.

Introduction: The study aims to assess awareness, attitude, apprehension, action expected from policy makers, and acceptability towards HPV vaccination amongst health care providers (HCP), medical students, paramedical staff and administrative personnel. Methods: A cross-sectional study was conducted with 501 participants, including HCP, medical students, paramedical and administrative staff. Their awareness, attitudes, apprehensions, actions expected from policymakers, and acceptability towards HPV vaccine were assessed through a Google Form survey. Results: Of the 501 participants, 92.4% were aware of HPV vaccines, 24.6% had a thorough understanding of HPV, but only 5.8% had received the vaccine. Most participants held a positive attitude toward cervical cancer prevention and HPV vaccination, although 46.7% expressed apprehensions. Initially, 278 participants (55.8%) were willing to receive the HPV vaccine, while 116 (44.2%) were not. After viewing the educational video, willingness increased to 394 participants (78.6%), and those unwilling decreased to 107 (21.4%). Factors such as age, gender, marital status, education, and professional role did not significantly predict knowledge or apprehension regarding HPV and the HPV vaccine. However, professional role and younger adult (20-39 years) groups were statistically significant predictors of a positive attitude as well as stronger expectations for action from policymakers towards HPV vaccine. Conclusion: The study reveals significant gaps in HPV vaccine awareness and uptake among medical professionals, paramedics, and administrative staff. It highlights the need for broad educational programs to address these knowledge gaps, emphasizing HPV’s health impacts and preventive measures. Multimedia educational interventions effectively boost vaccine acceptance among the groups and the general public.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt’s lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels. The USP7 inhibitor GNE6776 altered EBNA1 protein interactions, including disrupting its ability to bind to USP7. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. GNE6776 selectively inhibited EBV + gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. Transcriptomic studies revealed that USP7 inhibition differentially affected EBV + cancer cells compared to EBV - cells with a significant effect on chromosome segregation and mitotic cell division pathways. Our findings indicate that USP7 inhibition perturbs EBNA1 stability and function and can be exploited to target EBV + cancer cells selectively.

Oncolytic viruses are emerging as promising cancer therapeutic agents, with several poxviruses, including vaccinia virus (VACV) and myxoma virus, showing significant potential in preclinical and clinical trials. Modified vaccinia virus Ankara (MVA), a laboratory-derived VACV strain approved by the FDA for mpox and smallpox vaccination, has been shown to be incapable of replicating in human cells unless zinc finger antiviral protein (ZAP) is repressed. Notably, ZAP deficiency is prevalent in various cancer types. We hypothesized that MVA could selectively target and replicate in ZAP-deficient cancer cells. Our study examined MVA’s replication across multiple cancer cell lines with varying ZAP expression levels, revealing that MVA replicates more efficiently in cells with lower ZAP expression. Additionally, we assessed MVA’s oncolytic potential using a xenograft mouse model, where cancer cells were transplanted into immunodeficient mice. The data demonstrated that MVA significantly reduced tumors with lower ZAP expression without causing morbidity in nude mice. These findings suggest that MVA holds promise for further development as a targeted therapy for ZAP-deficient cancers.

Background: Inter-genogroup reassortment of Rotavirus A (RVA) strains has highlighted the spread of unusual RVA strains worldwide. We previously reported the equine-like G3 RVA as predominant strains in Indonesia in 2015-2016. However, since July 2017, typical human genotypes G1 and G3 have replaced these strains completely. To understand how dynamic changes in RVA occur in Indonesia, we performed a detailed epidemiological study. Main body: A total of 356 stool specimens were collected from hospitalized children in Sidoarjo, Indonesia between 2018 and 2022. Whole-genome sequencing was performed for all 26 RVA-positive samples using next-generation sequencing. Twenty-four samples were determined to be the unusual RVA G9P[4], while 2 were G9P[6]. Detailed analysis revealed that seven G9P[4] strains had the typical DS-1-like backbone, while the other strains exhibited a double-reassortant profile (G9-N1) on the DS-1-like backbone. The Bayesian evolutionary analyses suggested that the Indonesian G9P[4] strains share a common ancestor with previously reported G9P[4] strains in the VP7 and VP4 genes. Conclusions: G9P[4] DS-1-like strains were identified as the predominant genotype in Indonesia in 2021 for the first time. These results suggest that the G9P[4] strains were generated from the previous G9P[4] strains that had undergone further intra-reassortments with the other circulating strains.

SARS-CoV-2 continues to mutate, leading to breakthrough infections. The development of new vaccine strategies to combat variant strains is crucial. Protein cyclization can enhance thermal stability and may improve immunogenicity. Here, we designed a cyclic tandem dimeric receptor-binding domain protein (cirRBD2) using the split intein Cth-Ter. Cyclization does not affect the antigen epitopes of the RBD but results in better thermal stability than that of its linear counterpart (linRBD2). Compared with those immunized with linRBD2, mice immunized with two doses of 5 μg of cirRBD2 produced significantly greater levels of broad-spectrum neutralizing antibodies than those immunized with linRBD2 and generated a considerable cellular immune response. In the VEEV-VRP-hACE2-transduced mouse model, two doses of 5 μg of cirRBD2 provided protection against infection with BA.5, XBB.1.9, and partial against EG.5 which has more mutations. This study developed a novel circular RBD dimer subunit vaccine for SARS-CoV-2 that exhibits broad-spectrum neutralizing activity against various variants. A similar strategy can be applied to develop vaccines for other pathogens, especially for thermally stable vaccines.

Tick-borne encephalitis virus (TBEV) can cause life-threatening CNS infection. Changes in cerebrospinal fluid (CSF) metabolites may reflect critical aspects of host responses and end-organ damage in neuro infection and neuroinflammation. In this study, we applied an untargeted metabolomics screen of CSF samples to investigate the metabolites profile and explore biomarkers for TBEV infection. By analyzing CSF samples from 77 patients with TBEV infection and 23 without TBEV infection, tryptophan metabolism and Citrate cycle were found to be the top important metabolic pathways in differentiating the control and case groups; acetoacetate, 5’-deoxy-5’-(methylthio)-adenosine, 3-methyl-2-oxobutanoic acid,etc. were identified to be metabolic biomarkers（| log 2 FC|>1,VIP>1,FDR<0.05）in CSF and clearly separated the TBEV infection from the non-infected samples. Moreover, four metabolites were identified to be associated with fatal outcome, including kynurenic acid, 5-hydroxyindole-3-acetic acid, DL-tryptophan, indole-3-acrylic acid, demonstrating the potential predictive biomarkers for severe TBEV infection. This study explored the metabolic profile of TBEV infection both in CSF samples and identified candidate biomarkers for TBEV infection, which might be useful in target screening for differential diagnosis and therapeutic inter-vention.

SARS-CoV-2, a positive-strand RNA virus, utilizes both genomic replication and subgenomic mRNA transcription. Whole genome sequencing (WGS) from clinical samples can estimate viral gene expression levels. We conducted WGS on 529 SARS-CoV-2 positive clinical samples from Assam and northeastern India to track viral emergence and assess gene expression patterns. Our results reveal differential expression across structural, non-structural, and accessory genes, with notable upregulation of the M gene, especially in the Omicron variant, followed by E and ORF6. The mean Transcript Per Million (TPM) expression levels of the M gene were significantly higher in Omicron variants (175611±46921), peaking in the KP.1/KP.2 sublineage (220493±34917), compared to the Delta variant (129717±33773). The relative fold change of M gene expression between Delta and Omicron 2024 subvariants showed a 1.6-fold change. Variant-wise gene expression analysis suggests a correlation between gene expression and viral mutation, impacting replication and transmission. As anticipated, the expression levels of genes surge with the increase in the virus mutation. The Chi-square trend for average substitution count vs. average TPM of the M gene was highly significant (72.78, p<0.0001). The M gene’s high expression and low mutation rate make it an ideal target for designing a real-time RT-PCR kit assay. These findings highlight the need for continuous surveillance and understanding of viral gene expression dynamics for effective COVID-19 management. Further studies are necessary to elucidate the significance of these observations in viral pathogenesis and transmission dynamics.

Human papillomavirus infection is a prevalent viral condition affecting the female reproductive system, with high-risk HPV (hrHPV) posing significant risks. The vaginal microbiome plays a crucial role in the progression of cervical intraepithelial neoplasia (CIN). This study investigates the correlation between cervicovaginal microbiota and HPV status in cervicovaginal samples collected from 173 randomly selected participants enrolled in a clinical trial assessing self-sampling for HPV typing. Employing linear discriminant analysis effect size and receiver operating characteristic methodologies, microbial diversity across different cervical lesion categories was examined, identifying potential bacterial species as markers for cervical abnormalities. Among the cases analyzed, the positive predictive value of HPV infection was 74.2%, with Fannyhessea vaginae showing a relative abundance exceeding 0.00123%. This research also investigated the relationship between HPV alpha 7 and alpha 9 and various bacterial species. The findings indicated a potential association between HPV alpha 7 and Streptococcus, particularly Streptococcus pyogenes. Further observational studies are warranted to establish the association between vaginal microbiota dysbiosis and the carcinogenic potential of (hr)HPV-induced cervical cancer. The proposed microbiological biomarkers hold promise for reducing unnecessary invasive procedures and facilitating CIN risk assessment.

Human papillomavirus (HPV) is the most common sexually transmitted viral infection worldwide, which has been suggested to affect male fertility. However, reported evidence is scarce and inconclusive. Moreover, the putative effects of coinfections remain unveiled. In the present study, we analyzed male urogenital infection by HPV and coinfection with several other uropathogens, sperm quality and biomarkers of semen inflammation in a cohort of 205 men attending a urology clinic. A considerable prevalence (19%) of HPV male urogenital infection was found. Interestingly, HPV was detected coinfecting with at least one other uropathogen in most cases (74.4%). Notably, the most frequently detected coinfection was C. trachomatis (CT, 52% of cases). Remarkably, patients solely infected with HPV showed no significant alterations in conventional sperm parameters as well as reduced concentrations of IL-6 and leukocytes in semen. However, patients coinfected with HPV and CT showed significantly impaired sperm concentration and motility and increased levels of IL-6 in semen. These results indicate that HPV infection alone does not significantly alter sperm quality. However, the coinfection with CT decreases sperm quality and induces semen inflammation, suggesting that concurrent HPV and CT infection in semen represents a distinct clinical entity with particular characteristics.