Cerebrospinal Fluid and Peripheral Blood Lymphomonocyte Single-Cell Transcriptomics in a Subject with Multiple Sclerosis Acutely Infected with HIV.

Signatures of neurodegeneration in clinical samples from a subject with multiple sclerosis (MS) acutely infected with HIV were investigated with single-cell transcriptomics using 10X Chromium technology. Sequencing was carried out on NovaSeq-TM, and the analysis was performed with Cell Ranger software (v 7.1.0) associated with a specifically established bioinformatic pipeline. A total of 1446 single-cell transcriptomes in cerebrospinal fluid (CSF) and 4647 in peripheral blood mononuclear cells (PBMCs) were obtained. In the CSF, many T-cell lymphocytes with an enriched amount of plasma cells and plasmacytoid dendritic (pDC) cells, as compared to the PBMCs, were detected. An unsupervised cluster analysis, putting together our patient transcriptomes with those of a publicly available MS scRNA-seq dataset, showed up-regulated microglial neurodegenerative gene expression in four clusters, two of which included our subject's transcriptomes. A few HIV-1 transcripts were found only in the CD4 central memory T-cells of the CSF compartment, mapping to the gag-pol, vpu, and env regions. Our data, which describe the signs of neurodegenerative gene expression in a very peculiar clinical situation, did not distinguish the cause between multiple sclerosis and HIV infection, but they can give a glimpse of the high degree of resolution that may be obtained by the single-cell transcriptomic approach.

Genetic and structural characterization of dengue virus involved in the 2023 autochthonous outbreaks in central Italy.

Dengue virus (DENV) has been expanding its range to temperate areas that are not usually affected, where the spread of vectors has been facilitated by global trade and climate change. In Europe, there have been many cases of DENV imported from other regions in the past few years, leading to local outbreaks of DENV among people without travel history. Here we describe the epidemiological and molecular investigations of three transmission events locally acquired DENV infections caused by serotypes 1, 2 and 3, respectively, in the Latium Region from August to November 2023. Next-generation or Sanger sequencing was used to obtain the whole genomes, or the complete E-gene of the viruses, respectively. The structure of the DENV-1 and DENV-3 sequences was analysed to identify amino acid changes that were not found in the closest related sequences. The major cluster was supported by DENV-1 (originated in South America), with 42 autochthonous infections almost occurring in the eastern area of Rome, probably due to a single introduction followed by local sustained transmission. Seven DENV-1 subclusters have been identified by mutational and phylogenetic analysis. Structural analysis indicated changes whose meaning can be explained by the adaptation of the virus to human hosts and vectors and their interactions with antibodies and cell receptors.

Association Between TTV Viremia, Chronic Inflammation, and Ischemic Heart Disease Risk: Insights From MARK-AGE and Report-Age Projects.

The implication of Torquetenovirus (TTV) in ischemic heart disease (IHD) has not been thoroughly explored. This study investigated the association between TTV viremia, pro-inflammatory cytokines, and IHD risk in an aging population. This cross-sectional study included 900 non-IHD subjects and 86 individuals with IHD (aged 55-75 years) selected from the MARK-AGE project. Results were verified in another independent Report-Age cohort, including 94 inpatients with chronic IHD and 111 inpatients with non-IHD (aged 65-96 years). Multivariable logistic regression in the MARK-AGE cohort revealed that male sex, TTV viremia ≥4log, Cu/Zn ratio, diabetes, hypertension, and smoking were significant IHD predictors. Notably, TTV viremia ≥4log independently increased the IHD risk (odds ratio [OR]: 2.51, 95% confidence interval [CI]: 1.42-4.43), confirmed in the Report-Age cohort (OR: 4.90, 95% CI: 2.32-10.39). In a RASIG subgroup, individuals with TTV viremia ≥4 log, both with and without IHD, exhibited increased plasma pro-inflammatory cytokine levels (IFN-γ, IL-1ß, IL-6, IL-10, IL-12p70, TNF-α) compared to those with TTV viremia <4 log. No significant difference in cytokine production was observed between IHD patients and non-IHD with TTV viremia ≥4 log. A positive correlation between TTV viremia and DNA methylation estimator of leukocyte telomere length was observed in Report-Age patients. Additionally, IHD Report-Age patients with TTV viremia ≥4 log displayed higher NLR and SIRI index than those with TTV viremia <4 log. In conclusion, a high TTV viremia is associated with an elevated IHD risk in the older population, potentially arising from an augmented pro-inflammatory response and immunosenescence.

Subsequent Waves of Convergent Evolution in SARS-CoV-2 Genes and Proteins.

Beginning in 2022, following widespread infection and vaccination among the global population, the SARS-CoV-2 virus mainly evolved to evade immunity derived from vaccines and past infections. This review covers the convergent evolution of structural, nonstructural, and accessory proteins in SARS-CoV-2, with a specific look at common mutations found in long-lasting infections that hint at the virus potentially reverting to an enteric sarbecovirus type.

Torquetenovirus Viremia Quantification Using Real-Time PCR Developed on a Fully Automated, Random-Access Platform.

Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing-Bablok linear regression and Bland-Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

The Emerging Tool of the Human Anellovirome.

The blood virome is dominated by the Anelloviridae family, which emerges early in life; the anellome, which represents the variety of anelloviruses within an individual, stabilizes by adulthood [...].

Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Online dashboards for SARS-CoV-2 wastewater-based epidemiology.

Aim: Wastewater-based epidemiology (WBE) is increasingly used to monitor pandemics. In this manuscript, we review methods and limitations of WBE, as well as their online dashboards. Materials & methods: Online dashboards were retrieved using PubMed and search engines, and annotated for timeliness, availability of English version, details on SARS-CoV-2 sublineages, normalization by population and PPMoV load, availability of case/hospitalization count charts and of raw data for export. Results: We retrieved 51 web portals, half of them from Europe. Africa is represented from South Africa only, and only seven portals are available from Asia. Conclusion: WBS provides near-real-time cost-effective monitoring of analytes across space and time in populations. However, tremendous heterogeneity still persists in the SARS-CoV-2 WBE literature.

Monitoring the amount of a virus in the sewage system provides a way to work out the circulation of the virus among a population at a given time. Standard procedures are needed to produce data that can be compared across countries. Timely sharing of wastewater surveillance data across publicly accessible web portals is important to inform researchers and plan public health policies. This study shows that we are still far away from standardization and timely and transparent reporting.

Amyotrophic lateral sclerosis stratification: unveiling patterns with virome, inflammation, and metabolism molecules.

Amyotrophic lateral sclerosis (ALS) is an untreatable and clinically heterogeneous condition primarily affecting motor neurons. The ongoing quest for reliable biomarkers that mirror the disease status and progression has led to investigations that extend beyond motor neurons' pathology, encompassing broader systemic factors such as metabolism, immunity, and the microbiome. Our study contributes to this effort by examining the potential role of microbiome-related components, including viral elements, such as torque tenovirus (TTV), and various inflammatory factors, in ALS. In our analysis of serum samples from 100 ALS patients and 34 healthy controls (HC), we evaluated 14 cytokines, TTV DNA load, and 18 free fatty acids (FFA). We found that the evaluated variables are effective in differentiating ALS patients from healthy controls. In addition, our research identifies four unique patient clusters, each characterized by distinct biological profiles. Intriguingly, no correlations were found with site of onset, sex, progression rate, phenotype, or C9ORF72 expansion. A remarkable aspect of our findings is the discovery of a gender-specific relationship between levels of 2-ethylhexanoic acid and patient survival. In addition to contributing to the growing body of evidence suggesting altered peripheral immune responses in ALS, our exploratory research underscores metabolic diversity challenging conventional clinical classifications. If our exploratory findings are validated by further research, they could significantly impact disease understanding and patient care customization. Identifying groups based on biological profiles might aid in clustering patients with varying responses to treatments.

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.

SARS-CoV-2 BA.2.86 ("Pirola"): Is it Pi or Just Another Omicron Sublineage?

The SARS-CoV-2 sublineage BA [...].

Torquetenovirus Loads in Peripheral Blood Predict Both the Humoral and Cell-Mediated Responses to SARS-CoV-2 Elicited by the mRNA Vaccine in Liver Transplant Recipients.

Three years into the COVID-19 pandemic, mass vaccination campaigns have largely controlled the disease burden but have not prevented virus circulation. Unfortunately, many immunocompromised patients have failed to mount protective immune responses after repeated vaccinations, and liver transplant recipients are no exception. Across different solid organ transplant populations, the plasma levels of Torquetenovirus (TTV), an orphan and ubiquitous human virus under control of the immune system, have been shown to predict the antibody response after COVID-19 vaccinations. We show here a single-institution experience with TTV viremia in 134 liver transplant recipients at their first or third dose. We found that TTV viremia before the first and third vaccine doses predicts serum anti-SARS-CoV-2 Spike receptor-binding domain (RBD) IgG levels measured 2-4 weeks after the second or third dose. Pre-vaccine TTV loads were also associated with peripheral blood anti-SARS-CoV-2 cell-mediated immunity but not with serum SARS-CoV-2 neutralizing antibody titers.

Outbreaks of autochthonous Dengue in Lazio region, Italy, August to September 2023: preliminary investigation.

Between August and September 2023, three distinct autochthonous dengue virus transmission events occurred in Lazio, Italy, with the main event in Rome. The events involved three different dengue serotypes. No link with previous imported cases was identified. Here we describe the epidemiological and phylogenetic analysis of the first autochthonous cases and the implemented control actions. The multiple transmission events call for a strengthening of the vector control strategies and future research to better characterise the risk in countries like Italy.

Viral load of Torquetenovirus correlates with Sano's score and levels of total bilirubin and aspartate aminotransferase in Kawasaki disease.

Cause of Kawasaki disease (KD) is unknown. KD is often resistant to treatment with intravenous immunoglobulin (IVIG). Sano's score, which is derived from total bilirubin (TBIL), aspartate aminotransferase (AST) and C-reactive protein (CRP), is predictive of IVIG resistance in Japan. A recent study reported that Torquetenovirus (TTV), especially TTV7, was present at a high viral load in the patients with KD. We used PCR to quantify TTV load and amplicon next generation sequencing to detect individual TTV species. We used serum samples that were collected between 2002 and 2005 from 57 Japanese KD patients before IVIG treatment. Correlations between TTV load and Sano's score, the biomarkers that constitute this score, and IVIG resistance were examined. TTV load was positively correlated with Sano's score (P = 0.0248), TBIL (P = 0.0004), and AST (P = 0.0385), but not with CRP (P = 0.6178). TTV load was marginally correlated with IVIG resistance (P = 0.1544). Presence of TTV7 was correlated with total TTV load significantly (P = 0.0231). The correlations between biomarkers for KD and TTV load suggested that TTV may play a role in the pathophysiology of KD. We hypothesize that TTV7 may be associated with a higher total viral load in KD.

Inhibiting immunoregulatory amidase NAAA blocks ZIKV maturation in Human Neural Stem Cells.

Recent evidence suggests that lipids play a crucial role in viral infections beyond their traditional functions of supplying envelope and energy, and creating protected niches for viral replication. In the case of Zika virus (ZIKV), it alters host lipids by enhancing lipogenesis and suppressing ß-oxidation to generate viral factories at the endoplasmic reticulum (ER) interface. This discovery prompted us to hypothesize that interference with lipogenesis could serve as a dual antiviral and anti-inflammatory strategy to combat the replication of positive sense single-stranded RNA (ssRNA+) viruses. To test this hypothesis, we examined the impact of inhibiting N-Acylethanolamine acid amidase (NAAA) on ZIKV-infected human Neural Stem Cells. NAAA is responsible for the hydrolysis of palmitoylethanolamide (PEA) in lysosomes and endolysosomes. Inhibition of NAAA results in PEA accumulation, which activates peroxisome proliferator-activated receptor-α (PPAR-α), directing ß-oxidation and preventing inflammation. Our findings indicate that inhibiting NAAA through gene-editing or drugs moderately reduces ZIKV replication by approximately one log10 in Human Neural Stem Cells, while also releasing immature virions that have lost their infectivity. This inhibition impairs furin-mediated prM cleavage, ultimately blocking ZIKV maturation. In summary, our study highlights NAAA as a host target for ZIKV infection.

TTV and other anelloviruses: The astonishingly wide spread of a viral infection.

The broad family of viruses known as anelloviruses (AV) infects both humans and numerous animal species. They have a tiny, covalently closed single-stranded DNA genome and the astonishing capacity to infect a very high percentage of healthy and ill people with chronic infections that could last a lifetime. AV, and particularly the prototype Torquetenovirus, have established a successful interaction with the host's immune system and the rate at which they replicate is a gauge to measure overall immune function, even though many aspects of their life cycle and pathogenesis are still poorly understood.

Identification of Torquetenovirus Species in Patients with Kawasaki Disease Using a Newly Developed Species-Specific PCR Method.

A next-generation sequencing (NGS) study identified a very high viral load of Torquetenovirus (TTV) in KD patients. We aimed to evaluate the feasibility of a newly developed quantitative species-specific TTV-PCR (ssTTV-PCR) method to identify the etiology of KD. We applied ssTTV-PCR to samples collected from 11 KD patients and 22 matched control subjects who participated in our previous prospective study. We used the NGS dataset from the previous study to validate ssTTV-PCR. The TTV loads in whole blood and nasopharyngeal aspirates correlated highly (Spearman's R = 0.8931, p < 0.0001, n = 33), supporting the validity of ssTTV-PCR. The ssTTV-PCR and NGS results were largely consistent. However, inconsistencies occurred when ssTTV-PCR was more sensitive than NGS, when the PCR primer sequences mismatched the viral sequences in the participants, and when the NGS quality score was low. Interpretation of NGS requires complex procedures. ssTTV-PCR is more sensitive than NGS but may fail to detect a fast-evolving TTV species. It would be prudent to update primer sets using NGS data. With this precaution, ssTTV-PCR can be used reliably in a future large-scale etiological study for KD.