miR-3188 inhibits hepatitis B virus transcription by targeting Bcl-2.

Transcription of the covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is subject to dual regulation by host factors and viral proteins. MicroRNAs (miRNAs) can regulate the expression of target genes at the post-transcriptional level. Systematic investigation of miRNA expression in HBV infection and the interaction between HBV and miRNAs may deepen our understanding of the transcription mechanisms of HBV cccDNA, thereby providing opportunities for intervention. miRNA sequencing and real-time quantitative PCR (qRT-PCR) were used to analyze miRNA expression after HBV infection of cultured cells. Clinical samples were analyzed for miRNAs and HBV transcription-related indicators, using qRT-PCR, enzyme-linked immunoassay (ELISA), and Western blot. miRNA mimics or inhibitors were used to study their effects on the HBV life cycle. The target genes of miR-3188 and their roles in HBV cccDNA transcription were also identified. The expression of 10 miRNAs, including miR-3188, which was significantly decreased after HBV infection, was measured in clinical samples from patients with chronic HBV infection. Overexpression of miR-3188 inhibited HBV transcription, whereas inhibition of miR-3188 expression promoted HBV transcription. Further investigation confirmed that miR-3188 inhibited HBV transcription by targeting Bcl-2. miR-3188 is a key miRNA that regulates HBV transcription by targeting the host protein Bcl-2. This observation provides insights into the regulation of cccDNA transcription and suggests new targets for anti-HBV treatment.

Origin and dispersal history of Hepatitis B virus in Eastern Eurasia.

Hepatitis B virus is a globally distributed pathogen and the history of HBV infection in humans predates 10000 years. However, long-term evolutionary history of HBV in Eastern Eurasia remains elusive. We present 34 ancient HBV genomes dating between approximately 5000 to 400 years ago sourced from 17 sites across Eastern Eurasia. Ten sequences have full coverage, and only two sequences have less than 50% coverage. Our results suggest a potential origin of genotypes B and D in Eastern Asia. We observed a higher level of HBV diversity within Eastern Eurasia compared to Western Eurasia between 5000 and 3000 years ago, characterized by the presence of five different genotypes (A, B, C, D, WENBA), underscoring the significance of human migrations and interactions in the spread of HBV. Our results suggest the possibility of a transition from non-recombinant subgenotypes (B1, B5) to recombinant subgenotypes (B2 - B4). This suggests a shift in epidemiological dynamics within Eastern Eurasia over time. Here, our study elucidates the regional origins of prevalent genotypes and shifts in viral subgenotypes over centuries.

Establishing a screening strategy for non-discriminatory reactive blood donors by constructing a predictive model of hepatitis B virus infection status from a single blood center in China.

Background: When employing the transcription-mediated amplification method for screening blood donors, there are some non-discriminatory reactive results which are screening assay reactive but HBV-DNA discriminatory assay negative. This raises concerns regarding the possibility of false positives among donors, which may lead to permanent deferral of blood donors and affect blood supply. This study aimed to elucidate the infection status of these non-discriminatory reactive blood donors and develop and validate a model to predict individualized hepatitis B status to establish an optimal screening strategy. Methods: Supplementary tests were conducted on initial non-discriminating reactive donations to determine their HBV infection status, including repeat testing, viral load, serological marker detection, and follow-up. Primary clinical variables of the donors were recorded. Based on the Akaike information criterion, a stepwise forward algorithm was used to identify the predictive factors for information and construct a predictive model. The optimal screening strategy was determined through cost-effectiveness analysis. Results: At the Blood Center of Zhejiang Province, 435 cases of initial non-discriminatory reactive donations were collected over two successive periods and sub-categorized through repeated testing into the following three groups: non-repeated positive group, non-discriminated positive group, and non-repeated HBV-DNA positive group. The HBV discriminatory rate increased after repeated testing (110/435, 25.29%). According to supplementary tests, the HBV-DNA positivity rate was 65.52% (285/435), and occult HBV infection was a significantly different among groups (χ2 = 93.22, p < 0.01). The HBV serological markers and viral load in the non-repeated positive group differed from those in the other two groups, with a lower viral load and a higher proportion of false positives. The predictive model constructed using a stepwise forward algorithm exhibited high discrimination, good fit, high calibration, and effectiveness. A cost-effectiveness analysis indicated that utilizing repeated discriminatory testing and the predictive model is an extremely beneficial screening approach for non-discriminatory reactive blood donors. Conclusion: Nearly two-third (65.52%) of the non-discriminatory reactive blood donors were HBV-DNA positive. Our innovative approach of constructing a predictive model as a supplementary screening strategy, combined with repeated discriminatory experiments, can effectively identify the infection status of non-discriminatory reactive blood donors, thereby increasing the safety of blood transfusions.

Evaluation of analytical performance of the STANDARDTM M10 MPX/OPX assay for the simultaneous DNA detection and clade attribution of Monkeypox virus.

Monkeypox virus (MPXV) infection confirmation needs reliable polymerase chain reaction (PCR) assays; in addition, viral clade attribution is a key factor in containment measures, considering a more severe syndrome in clade I and the possibility of simultaneous circulation. This study evaluates the performance of all-in-one STANDARD M10 MPX/OPX (SD BIOSENSOR, South Korea - M10). Frozen samples from 205 subjects were selected and stratified according to routine test results (RealStar® Orthopoxvirus PCR Kit 1.0, Altona DIAGNOTICS, Germany - RS; RS-1): in detail, 100 negative skin lesions (SL) and 200 positive samples at the variable stage of infection were analysed. Positive samples were retested with RS (RS-2). Positive and Negative Percent Agreements (PPA, NPA) were calculated. The median (IQR) Ct values of RS and M10 (OPXV target) assays were highly similar. The PPA of M10 compared to RS-1 was 89.5% considering system interpretation, and 96.0% when the operator classified results as positive if any target was detected; NPA was 100%. Comparing the RS-2 run and M10, an overall concordance of 95.3% between assays was found; however, considering operator interpretation, M10 returned more positive results than RS-2. The occurrence of False-Negative results was likely associated with the influence of thawing on low viral concentration; no False-Positive tests were observed. All samples collected at the time of Mpox diagnosis were positive and M10 correctly attributed the clade (West-Africa/II). The M10 MPX/OPX assay demonstrated high reliability in confirming MPXV infection and clade attribution.

Biomarkers for progressive multifocal leukoencephalopathy: emerging data for use of JC virus DNA copy number in clinical trials.

Progressive multifocal leukoencephalopathy is a rare but devastating demyelinating disease caused by the JC virus (JCV), for which no therapeutics are approved. To make progress towards addressing this unmet medical need, innovations in clinical trial design are needed. Quantitative JCV DNA in CSF has the potential to serve as a valuable biomarker of progressive multifocal leukoencephalopathy disease and treatment response in clinical trials to expedite therapeutic development, as do neuroimaging and other fluid biomarkers such as neurofilament light chain. Specifically, JCV DNA in CSF could be used in clinical trials as an entry criterion, stratification factor, or predictor of clinical outcomes. Insights from the investigation of candidate biomarkers for progressive multifocal leukoencephalopathy might inform approaches to biomarker development for other rare diseases.

Characterization of mutations in hepatitis B virus DNA isolated from Japanese HBsAg-positive blood donors in 2021 and 2022.

Missense mutations in certain small envelope proteins diminish the efficacy of antibodies. Consequently, tracking the incidence and types of vaccine-escape mutations (VEMs) was crucial both before and after the introduction of universal hepatitis B vaccination in Japan in 2016. In this study, we isolated hepatitis B virus (HBV) DNA from 58 of 169 hepatitis B surface antigen (HBsAg)-positive blood samples from Japanese blood donors and determined the nucleotide sequence encoding the small envelope protein. DNA from six (10%) of the samples had VEMs, but no missense mutations, such as G145R, were detected. Complete HBV genome sequences were obtained from 29 of the 58 samples; the viral genotype was A1 in one (3%), A2 in three (10%), B1 in nine (31%), B2 in five (17%), B4 in one (3%), and C2 in 10 (34%) samples. Tenofovir-resistance mutations were detected in two (7%) samples. In addition, several core promoter mutations, such as 1762A>T and 1764G>A, and a precore nonsense mutation, 1986G>A, which are risk factors for HBV-related chronic liver disease, were detected. These findings provide a baseline for future research and highlight the importance of ongoing monitoring of VEMs and drug resistance mutations in HBV DNA from HBsAg-positive blood donors without HBV antibodies.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

HIV-1 uncoating requires long double-stranded reverse transcription products.

HIV-1 cores, which contain the viral genome and replication machinery, must disassemble (uncoat) during viral replication. However, the viral and host factors that trigger uncoating remain unidentified. Recent studies show that infectious cores enter the nucleus and uncoat near the site of integration. Here, we show that efficient uncoating of nuclear cores requires synthesis of a double-stranded DNA (dsDNA) genome >3.5 kb and that the efficiency of uncoating correlates with genome size. Core disruption by capsid inhibitors releases viral DNA, some of which integrates. However, most of the viral DNA is degraded, indicating that the intact core safeguards viral DNA. Atomic force microscopy and core content estimation reveal that synthesis of full-length genomic dsDNA induces substantial internal strain on the core to promote uncoating. We conclude that HIV-1 cores protect viral DNA from degradation by host factors and that synthesis of long double-stranded reverse transcription products is required to trigger efficient HIV-1 uncoating.

Characterization of the brain virome in human immunodeficiency virus infection and substance use disorder.

Viruses can infect the brain in individuals with and without HIV-infection: however, the brain virome is poorly characterized. Metabolic alterations have been identified which predispose people to substance use disorder (SUD), but whether these could be triggered by viral infection of the brain is unknown. We used a target-enrichment, deep sequencing platform and bioinformatic pipeline named "ViroFind", for the unbiased characterization of DNA and RNA viruses in brain samples obtained from the National Neuro-AIDS Tissue Consortium. We analyzed fresh frozen post-mortem prefrontal cortex from 72 individuals without known viral infection of the brain, including 16 HIV+/SUD+, 20 HIV+/SUD-, 16 HIV-/SUD+, and 20 HIV-/SUD-. The average age was 52.3 y and 62.5% were males. We identified sequences from 26 viruses belonging to 11 viral taxa. These included viruses with and without known pathogenic potential or tropism to the nervous system, with sequence coverage ranging from 0.03 to 99.73% of the viral genomes. In SUD+ people, HIV-infection was associated with a higher total number of viruses, and HIV+/SUD+ compared to HIV-/SUD+ individuals had an increased frequency of Adenovirus (68.8 vs 0%; p<0.001) and Epstein-Barr virus (EBV) (43.8 vs 6.3%; p=0.037) as well as an increase in Torque Teno virus (TTV) burden. Conversely, in HIV+ people, SUD was associated with an increase in frequency of Hepatitis C virus, (25 in HIV+/SUD+ vs 0% in HIV+/SUD-; p=0.031). Finally, HIV+/SUD- compared to HIV-/SUD- individuals had an increased frequency of EBV (50 vs 0%; p<0.001) and an increase in TTV viral burden, but a decreased Adenovirus viral burden. These data demonstrate an unexpectedly high variety in the human brain virome, identifying targets for future research into the impact of these taxa on the central nervous system. ViroFind could become a valuable tool for monitoring viral dynamics in various compartments, monitoring outbreaks, and informing vaccine development.

A quantitative assay for the assessment of cutaneous human papillomaviruses and polyomaviruses over time: A proof-of-concept in two patients with atopic dermatitis and psoriasis.

The human skin virome, unlike commensal bacteria, is an under investigated component of the human skin microbiome. We developed a sensitive, quantitative assay to detect cutaneous human resident papillomaviruses (HPV) and polyomaviruses (HPyV) and we first used it to describe these viral populations at the skin surface of two patients with atopic dermatitis (AD) and psoriasis (PSO). We performed skin swabs on lesional and non-lesional skin in one AD and one PSO patient at M0, M1 and M3. After extraction, DNA was amplified using an original multiplex PCR technique before high throughput sequencing (HTS) of the amplicons (named AmpliSeq-HTS). Quantitative results were ultimately compared with monoplex quantitative PCRs (qPCRs) for previously detected viruses and were significantly correlated (R2 = 0.95, ρ = 0.75). Fifteen and 13 HPV types (mainly gamma and beta-HPVs) or HPyV species (mainly Merkel Cell Polyomavirus (MCPyV)) were detected on the skin of the AD and PSO patients, respectively. In both patients, the composition of the viral flora was variable across body sites but remained stable over time in non-lesional skin samples, mostly colonized with gamma-papillomaviruses. In lesional skin samples, beta-papillomaviruses and MCPyV were the major components of a viral flora more prone to vary over time especially with treatment and subsequent clinical improvement. We believe this method might be further used in extensive studies to further enhance the concept of an individual cutaneous viral fingerprint and the putative role of its alterations through various skin diseases and their treatments.

Effect of S-region mutations on HBsAg in HBsAg-negative HBV-infected patients.

BACKGROUND: Occult HBV infection (OBI) is a special form of hepatitis B virus (HBV) infection that may cause Liver cirrhosis and hepatocellular carcinoma, causing significant harm to patients. Given the insidious nature of OBI, it is usually not easy to be detected. Most of the samples currently studied are concentrated on blood donors, however, patients in this special state have not been fully studied. This project aimed to study the effect of HBV S region mutations on HBsAg in patients with clinical OBI. METHODS: Collect 107 HBsAg-/HBV DNA + blood samples from Beijing Youan Hospital, Capital Medical University from August 2022 to April 2023. Next, the successfully extracted and amplified HBV DNA S regions were sequenced. Construct mutant plasmids to verify the cell function of the high-frequency mutation sites and explore the possible molecular mechanism. RESULTS: Sixty-eight HBsAg-negative samples were sequenced, revealing high-frequency amino acid substitution sites in the HBV S protein, including immune escape mutations (i.e., sY100C、sK122R、sI126T、sT131P、and sS114T) and TMD (Transmembrane domain) region substitutions (i.e., sT5A、sG10D、sF20S、and sS3N). We constructed a portion of the mutant plasmids and found that sT5A, sF20S, sG10D, sS3N, sI68T, and sI126T single point mutations or combined mutations may decrease HBsAg expression or change the antigenicity of HBsAg leading to detection failure. CONCLUSIONS: HBsAg-negative patients may show various mutations and amino acid replacement sites at high frequency in the HBV S-region, and these mutations may lead to undetectable Hepatitis B surface antigen (HBsAg), HBsAg antigenic changes or secretion inhibition.

Nonlinear relationship between viral load and TCT in single/multiple HPV52 infection.

PURPOSE: To determine the correlation between HPV (human papillomavirus) 52 viral load, multiple infections and ThinPrep cytology test (TCT), to inform clinical management of HPV52-positive women after cervical cancer screening. METHODS: A total of 1,882 female patients who had positive quantitative HPV tests at Yuebei People's Hospital from January 2020 to December 2022, of whom 533 tested positive for HPV52. We excluded patients who combined HPV16 and/or HPV 18 positivity and whom HPV52 viral load could not be calculated. The final enrollment was 488 patients, including 400 NILM, 48 ASC-US, 28 LSIL and 12 HSIL. The HPV test is a quantitative multiplexed fluorescent PCR assay that provides both HPV genotyping and viral load. RESULTS: In our study, there were differences in the median distribution of viral loads among various cytological class categories. The risk of TCT results (LSIL or worse) was increased with the increase of HPV52 viral load, for every LOG unit increase in HPV52 viral load, the risk increased by 26.6%. More importantly, we found a nonlinear relationship between HPV52 viral load and TCT results (LSIL or worse) in both single and multiple infections. When the viral load reaches a threshold, the risk of abnormal cytological results increases significantly. CONCLUSION: HPV52 viral load is an independent risk factor for TCT results (LSIL or worse). The relationship between HPV52 viral load and TCT results (LSIL or worse) is not linear. Viral load may be used as a triage indicator for HPV52-positive patients, thus improving the post-screening clinical management of HPV52-positive women.

High rate of high-risk human papillomavirus among benign and breast cancer patients in Ethiopia.

INTRODUCTION: There have been numerous studies that showed the presence of human papillomavirus (HPV) in breast cancer; nonetheless, there is ongoing debate regarding their association. Given few studies in Ethiopia, we aimed to investigate the magnitude of HPV infection in Ethiopian breast cancer patients. METHODS: A total of 120 formalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained, and basic demographic, clinical, and histological data were collected from medical records. DNA was extracted from archived FFPE breast tissue specimens using GeneRead DNA FFPE Kit. The AnyplexTM II HPV28 Detection Kit (Seegene, Korea) was used to detect HPV by following the manufacturer's instructions. The SPSS Version 25 was used to enter and analyze data. RESULTS: Among the 120 study participants; HPV (both high-risk and low-risk) was detected in 20.6% of breast cancer and 29.6% of non-malignant breast tumors. The most common genotype was the high-risk HPV 16 genotype. The frequency of HPV was nearly 10-fold higher in estrogen receptor-positive than ER-negative breast cancer. The percentage of HPV in the luminal (luminal A and luminal B) breast cancer subtypes was also much higher than in the non-luminal subtypes (HER-2 enriched and triple-negative breast cancer). CONCLUSION: This study did not find a significant difference in HPV expression between breast cancer and non-malignant breast tumors; however, the higher percentage of HPV in ER-positive compared to ER-negative breast cancer warrants further attention.

Multiscale modeling of HBV infection integrating intra- and intercellular viral propagation to analyze extracellular viral markers.

Chronic infection with hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. Thus, quantifying and understanding the dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, such study requires repeated liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because liver biopsy is potentially morbid and not common during hepatitis B treatment. We here aimed to develop a noninvasive method for quantifying cccDNA in the liver using surrogate markers in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations, integrates experimental data from in vitro and in vivo investigations. By applying this model, we roughly predicted the amount and dynamics of intrahepatic cccDNA within a certain range using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The noninvasive quantification of cccDNA using our proposed method holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

Structural and sequence analysis of the RPO30 gene of sheeppox and goatpox viruses from India.

Sheeppox and goatpox are transboundary viral diseases of sheep and goats that cause significant economic losses to small and marginal farmers worldwide, including India. Members of the genus Capripoxvirus (CaPV), namely Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV), are antigenically similar, and species differentiation can only be accomplished using molecular approaches. The present study aimed to understand the molecular epidemiology and host specificity of SPPV and GTPV circulating in India through sequencing and structural analysis of the RNA polymerase subunit-30 kDa (RPO30) gene. A total of 29 field isolates from sheep (n = 19) and goats (n = 10) belonging to different geographical regions of India during the period: Year 2015 to 2023, were analyzed based on the sequence and structure of the full-length RPO30 gene/protein. Phylogenetically, all the CaPV isolates were separated into three major clusters: SPPV, GTPV, and LSDV. Multiple sequence alignment revealed a highly conserved RPO30 gene, with a stretch of 21 nucleotide deletion in all SPPV isolates. Additionally, the RPO30 gene of the Indian SPPV and GTPV isolates possessed several species-specific conserved signature residues/motifs that could act as genotyping markers. Secondary structure analysis of the RPO30 protein showed four α-helices, two loops, and three turns, similar to that of the E4L protein of vaccinia virus (VACV). All the isolates in the present study exhibited host preferences across different states of India. Therefore, in order to protect vulnerable small ruminants from poxviral infections, it is recommended to take into consideration a homologous vaccination strategy.

A high-throughput DNA analysis method based on isothermal amplification on a suspension microarray for detecting mpox virus and viruses with comparable symptoms.

BACKGROUND: Mpox is a zoonotic disease caused by mpox virus (MPXV) infection. Since May 2022, there has been a marked increase in human mpox cases in different regions. Rash, fever, and sore throat are typical signs of mpox. However, other viruses, such as the B virus (BV), herpes simplex virus types 1 (HSV-1), herpes simplex virus types 2 (HSV-2), and varicella zoster virus (VZV), can also infect people and cause comparable symptoms. Therefore, clinical symptoms and signs alone make distinguishing MPXV from these viruses difficult. RESULTS: In this study, we combined suspension microarray technology with recombinase-aided amplification technology (RAA) to establish a high-throughput, sensitive, and quantitative method for detecting MPXV and other viruses that can cause similar symptoms. The experimental results confirmed that the technique has outstanding sensitivity, with a minimum detection limit (LOD) of 0.1 fM and a linear range of 0.3 fM to 20 pM, spanning five orders of magnitude. The approach also exhibits exquisite selectivity, as the amplified signal can only be detected when the target virus nucleic acid is present. Additionally, serum recoveries ranging from 80.52% to 119.09% suggest that the detection outcomes are generally considered reliable. Moreover, the time required for detection using this high-throughput method is very short. After DNA extraction, the detection signal amplified by isothermal amplification on the bead array can be obtained in just 1 h. SIGNIFICANCE AND NOVELTY: Our research introduces a new technique that utilizes suspension microarray technology and isothermal amplification to create a high-throughput nucleic acid assay. This innovative method offers multiple benefits compared to current techniques, such as being cost-effective, time-efficient, highly sensitive, and having high throughput capabilities. Furthermore, the assay is applicable not only for detecting MPXV and viruses with similar symptoms, but also for clinical diagnostics, food safety, and environmental monitoring, rendering it an effective tool for screening harmful microorganisms.

Juveniles, young adults, and infants with hepatitis B virus infection: A genomic study.

Integration of hepatitis B virus (HBV) DNA into the human genome is recognized as an oncogenic factor and a barrier to hepatitis B cure. In the study, biopsy liver tissues were collected from adolescents and young adults with acute HBV infection younger than or equal to 35 years of age and from HBV-infected infant patients younger than or equal to 6 months of age. A high-throughput sequencing method was used to detect HBV DNA integration. Totally, 12 adolescents, young adults, and 6 infants were included. Among the 12 patients with acute HBV infection, immunohistochemical staining of intrahepatic hepatitis B surface antigen for all displayed negative results, and no HBV DNA integrants in the hepatocyte DNA were confirmed. All infant patients had elevated levels of alanine aminotransferase and high levels of serum HBV DNA. Numerous gene sites of hepatocyte DNA were integrated by HBV DNA for each infant patient, ranging from 120 to 430 integration sites. The fragile histidine triad gene was the high-frequency integrated site in the intragenic region for infant patients. In conclusion, hepatocyte DNA is integrated by HBV DNA in babies with active hepatitis B but seems seldom affected among adolescents and young adults with acute HBV infection. Infantile hepatitis B should be taken seriously considering abundant HBV DNA integration events.

PCR testing for herpesviruses in aqueous humor samples from patients with and without clinical corneal endothelial graft rejection.

To compare prevalence of positive PCR tests for herpesviruses between patients with and without a history of clinical corneal endothelial allograft rejection (AGR). Retrospective cross-sectional study with two-group comparison. A total of 307 aqueous humor (AH) samples from 235 Patients and 244 eyes who underwent penetrating keratoplasty or Descemet membrane endothelial keratoplasty or had a diagnostic AH aspiration due to clinical AGR between 2019 and 2023 were tested for DNA of herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). PCR test results were compared between the two groups (with/without AGR). Another sub-analysis examined the results of patients without a history of herpetic keratitis. A total of 8% of eyes with clinical AGR (9/108) had a positive PCR result for one of the herpesviruses (HSV:3, CMV:3, EBV:2, VZV:1). All patients in the group without AGR had negative PCR results for all previous viruses (0/136). The difference was statistically significant (p < 0.001). The sub-analysis of eyes without a history of herpetic keratitis also revealed significantly more positive herpes PCR results (7/87) in eyes with AGR than in eyes without AGR (0/42, p = 0.005). Clinical AGR after keratoplasty shows a significant correlation to viral replication. Herpetic infection and AGR could occur simultaneously and act synergistically. Timely differentiation between active herpetic infection and/or AGR is pivotal for proper treatment and graft preservation.

Torque Teno Virus (TTV) in Renal Transplant Recipients: Species Diversity and Variability.

Torque Teno Virus (TTV) is a nonpathogenic and ubiquitous ssDNA virus, a member of the Anelloviridae family. TTV has been postulated as a biomarker in transplant patients. This study aimed to determine the TTV species diversity and variability in renal transplant recipients and to associate species diversity with the corresponding TTV viral load. From 27 recipients, 30 plasma samples were selected. Viral load was determined using two real-time PCR assays, followed by RCA-NGS and ORF1 phylogenetic analysis. The TTV diversity was determined in all samples. Variability was determined in three patients with two sequential samples (pre- and post-transplantation). Most of the samples presented multiple TTV species, up to 15 different species were detected. In the pre-transplant samples (n = 12), the most prevalent species were TTV3 (75%) and TTV13 (75%), and the median number of species per sample was 5 (IQR: 4-7.5). TTV3 was also the most prevalent (56%) in the post-transplant samples (n = 18), and the median number of species was 2 (IQR: 1.8-5.5). No significant correlation between the number of species and viral load was found. The number and type of TTV species showed total variability over time. We report high TTV species diversity in Argentinian recipients, especially in pre-transplant period, with total intra-host variability. However, we found no significant correlation between this high diversity and TTV viral load.