A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia.

Tilapia farming is one of the most important sectors in aquaculture worldwide and of major importance to global food security. Infectious spleen and kidney necrosis virus (ISKNV) has been identified as an agent of high morbidity and mortality, threatening tilapia aquaculture. ISKNV was detected in Lake Volta, Ghana, in September 2018 and spread rapidly, with mortality rates between 60 and 90% and losses of more than 10 tonnes of fish per day. Understanding the spread and evolution of viral pathogens is important for control strategies. Here, we developed a tiled-PCR sequencing approach for the whole-genome sequencing of ISKNV, using long read sequencing to enable field-based, real-time genomic surveillance. This work represents the first use of tiled-PCR for whole genome recovery of viruses in aquaculture, with the longest genome target (>110 kb dsDNA) to date. Our protocol was applied to field samples collected from the ISKNV outbreaks from four intensive tilapia cage culture systems across Lake Volta, between October 2018 and May 2022. Despite the low mutation rate of dsDNA viruses, 20 single nucleotide polymorphisms accumulated during the sampling period. Droplet digital PCR identified a minimum requirement of template in a sample to recover 50% of an ISKNV genome at 275 femtograms (2410 viral templates per 5 µL sequencing reaction). Overall, tiled-PCR sequencing of ISKNV provides an informative tool to assist in disease control in aquaculture.

Torque teno virus (TTV): A gentle spy virus of immune status, predictive marker of seroconversion to COVID-19 vaccine in kidney and lung transplant recipients.

To date, no comprehensive marker to monitor the immune status of patients is available. Given that Torque teno virus (TTV), a known human virome component, has previously been identified as a marker of immunocompetence, it was retrospectively investigated whether TTV viral load may also represent a marker of ability to develop antibody in response to COVID-19-BNT162B2 vaccine in solid organ transplant recipients (SOT). Specifically, 273 samples from 146 kidney and 26 lung transplant recipients after successive doses of vaccine were analyzed. An inverse correlation was observed within the TTV copy number and anti-Spike IgG antibody titer with a progressive decrease in viremia the further away from the transplant date. Analyzing the data obtained after the second dose, a significant difference in TTV copy number between responsive and nonresponsive patients was observed, considering a 5 log10 TTV copies/mL threshold to discriminate between the two groups. Moreover, for 86 patients followed in their response to the second and third vaccination doses a 6 log10 TTV copies/mL threshold was used to predict responsivity to the booster dose. Although further investigation is necessary, possibly extending the analysis to other patient categories, this study suggests that TTV can be used as a good marker of vaccine response in transplant patients.

Obesity is associated with a higher Torque Teno viral load compared to leanness.

Introduction: Obesity affects a rising proportion of the population and is an important risk factor for unfavorable outcomes in viral disease including severe acute respiratory syndrome coronavirus 2- associated diseases. Torque Teno virus (TTV) is a ubiquitous and apathogenic virus which reflects the immune function of its host. The aim of this study was to investigate the association between obesity and TTV load - an indirect marker of compromised viral immune response. Methods: TTV was quantified by TTV R-GENE® PCR in a total of 89 participants of which 30 were lean (BMI <25 kg/m2) and 59 were obese (BMI >30 kg/m2). For 38 subjects, follow-up was available after bariatric surgery. Results: TTV load was higher in individuals with obesity (median 2.39, IQR: 1.69-3.33 vs. 1.88, IQR 1.08-2.43 log10 copies/mL; p = 0.027). Multivariable linear modeling revealed an independent association between TTV load and obesity. TTV was positively correlated with waist-to-hip ratio and inversely with 25OH vitamin D levels. Interleukin 6 and fasting insulin resistance were confounders of the association between TTV and obesity, while age was an effect modifier. TTV load increased by 87% (95% CI 2-243%) in the year following bariatric surgery. Discussion: A higher TTV load in obese individuals may reflect compromised immune function and thus might serve for risk stratification of unfavorable outcomes during infectious disease, including coronavirus disease 2019, in this population. Our data warrant further analysis of TTV-based risk assessment in obese individuals in the context of infectious disease-associated outcomes.

Detection of Torquetenovirus and Redondovirus DNA in Saliva Samples from SARS-CoV-2-Positive and -Negative Subjects.

OBJECTIVES: Torquetenovirus (TTV) and Redondovirus (ReDoV) are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies. A large-scale epidemiological study was performed to investigate their prevalence and loads in saliva samples according to SARS-CoV-2 status. METHODS: Saliva samples from 448 individuals (73% SARS-CoV-2 negative and 27% SARS-CoV-2 positive) aged 23-88 years were tested. SARS-CoV-2 and TTV were determined in saliva by specific qualitative and quantitative real-time PCRs, respectively. A sub-cohort of 377 subjects was additionally tested for the presence and load of ReDoV in saliva, and a different sub-cohort of 120 subjects for which paired saliva and plasma samples were available was tested for TTV and ReDoV viremia at the same timepoints as saliva. RESULTS: TTV in saliva was 72% prevalent in the entire cohort, at a mean DNA load of 4.6 log copies/mL, with no difference regardless of SARS-CoV-2 status. ReDoV was found in saliva from 61% of the entire cohort and was more prevalent in the SARS-CoV-2-negative subgroup (65% vs. 52%, respectively). In saliva, the total mean load of ReDoV was very similar to the one of TTV, with a value of 4.4 log copies/mL. The mean viral loads in subjects infected with a single virus, namely, those infected with TTV or ReDoV alone, was lower than in dually infected samples, and Tukey's multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier (p = 0.004). Differently from TTV, ReDoV was not detected in any blood samples. CONCLUSIONS: This study establishes the prevalence and mean value of TTV and ReDoV in saliva samples and demonstrates the existence of differences between these two components of the human virome.

Can torque teno virus be a predictor of SARS-CoV-2 disease progression in cancer patients?

INTRODUCTION: Cancer patients with SARS-CoV-2 infection can experience a broad range of clinical manifestations and outcomes. Previous studies have demonstrated an association between torque teno virus (TTV) load and deficiencies of the immune system. The impact of SARS-CoV-2 and TTV viral loads in cancer patients is unknown. METHODS: In this retrospective study, 157 cancer patients and 191 noncancer controls were analysed for SARS-CoV-2 RNA and TTV DNA presence. RESULTS: SARS-CoV-2 RNA was detected in 66.2% of cancer patients and in 68.6% of noncancer control subjects. In SARS-CoV-2-positive patients, TTV was detectable in 79.8% of cancer patients, while in controls, TTV was detected in 71.7% of subjects. No statistically significant correlation was found between TTV and SARS-CoV-2 loads in cancer patients. However, the 100-day survival rate in cancer patients who died from COVID-19 was significantly lower in the TTV-positive group than in the TTV-negative group (P = 0.0475). In the cancer TTV-positive group, those who died also had a higher load of TTV than those who did not die (P = 0.0097). CONCLUSIONS: Our findings indicated that the presence of TTV in nasopharyngeal swabs from cancer patients was related to a higher number of deaths from COVID-19 and to a higher TTV DNA load.

Monitoring of Torque Teno virus DNAemia in critically ill COVID-19 patients: May it help to predict clinical outcomes?

BACKGROUND: Torque teno virus (TTV) DNA load in plasma directly associates with the net state of immunosuppression and inflammation in different clinical settings, including transplantation and chronic inflammatory diseases. OBJECTIVES: We investigated whether plasma TTV DNA load may predict the occurrence of certain infectious events and overall mortality in critically ill COVID-19 patients. PATIENTS AND METHODS: 50 patients (median age, 65.5 years) were recruited. TTV DNA load was quantitated in serial plasma specimens by real-time PCR. Serum levels of interleukin-6, C-reactive protein, ferritin, lactate dehydrogenase, Gamma-Glutamyl Transferase (GGT), alanine transaminase (ALT) and aspartate transaminase (AST) and absolute lymphocyte counts (ALC) in paired specimens were available. Nosocomial bloodstream infections and ventilator-associated pneumonia and overall mortality were the clinical outcomes. RESULTS: TTV DNA was detected in 38 patients (76%). A weak inverse correlation (Rho=-0.28; P = 0.004) was observed between TTV DNA loads and ALC. No direct correlation was found between TTV DNA load and serum levels of any of the above biomarkers. Patients with detectable TTV DNA had an increased risk of subsequently developing infectious events (HR 9.28; 95% CI, 1.29-69.5; P = 0.03). A trend (P = 0.05) towards higher TTV DNA area under a curve between days 7 and 17 after ICU admission (AUC7-17) was observed in patients who died, as compared to survivors. CONCLUSION: Our findings suggested that plasma TTV DNA load monitoring may be helpful for predicting the occurrence of severe nosocomial infections and mortality in critically ill COVID-19 patients.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Kinetics of torquetenovirus DNA load in a recent kidney transplant recipient with mild SARS-CoV-2 infection and a failed antibody response.

Kidney transplant (KT) recipients are at an increased risk for severe COVID-19 because of their immunosuppressed state. A 42-year-old KT patient was diagnosed with COVID-19 three months after KT. Despite lymphopenia and several risk factors, he had a mild disease course. Nasopharyngeal real-time reverse transcriptase polymerase chain reaction for SARS-CoV-2 became negative 48 days after detection. SARS-CoV-2 IgG antibodies became negative after day 40. TTV DNA load increased with the onset COVID-19 and reduced after its resolution. This is the first report where TTV DNA load was measured during the course of COVID-19.

Ginkgolic acid inhibits fusion of enveloped viruses.

Ginkgolic acids (GA) are alkylphenol constituents of the leaves and fruits of Ginkgo biloba. GA has shown pleiotropic effects in vitro, including: antitumor effects through inhibition of lipogenesis; decreased expression of invasion associated proteins through AMPK activation; and potential rescue of amyloid-ß (Aß) induced synaptic impairment. GA was also reported to have activity against Escherichia coli and Staphylococcus aureus. Several mechanisms for this activity have been suggested including: SUMOylation inhibition; blocking formation of the E1-SUMO intermediate; inhibition of fatty acid synthase; non-specific SIRT inhibition; and activation of protein phosphatase type-2C. Here we report that GA inhibits Herpes simplex virus type 1 (HSV-1) by inhibition of both fusion and viral protein synthesis. Additionally, we report that GA inhibits human cytomegalovirus (HCMV) genome replication and Zika virus (ZIKV) infection of normal human astrocytes (NHA). We show a broad spectrum of fusion inhibition by GA of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, we show inhibition of a non-enveloped adenovirus. Our experiments suggest that GA inhibits virion entry by blocking the initial fusion event. Data showing inhibition of HSV-1 and CMV replication, when GA is administered post-infection, suggest a possible secondary mechanism targeting protein and DNA synthesis. Thus, in light of the strong effect of GA on viral infection, even after the infection begins, it may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)).