Fabrizio Carletti

West Nile virus (WNV) is a flavivirus whose cycle involves mosquitoes and avian hosts that occasionally may infect horses and humans as “dead‐end hosts.” Human infection is mostly asymptomatic, sometimes leading to febrile or neuroinvasive disease. In 2025, Italy experienced its largest epidemic with an unprecedented geographic spread (79 provinces; 52% of cases in central Italy). Here we report whole‑genome sequencing and phylogeographic analysis of 39 WNV lineage 2 samples collected in Lazio and Campania regions between July and September 2025 from humans and animal samples. All 2025 strains belonged to a Southern Italian clade that diversified around 2020, with evidence of earlier circulation in 2024. All the 2025 genomes shared a substitution that lies in envelope protein domain III (K399R), potentially influencing interactions with the viral membrane. The same mutation was previously detected in Eastern European strains, leading to the hypothesis of introductions in Italy via migratory birds from these regions. Phylogeography identified strong viral dispersal routes within Central Italy supporting a pattern of short‑range spread and local overwintering. Overall, results highlight the roles of ecological conditions and cross‑border transmission dynamics in shaping the 2025 Italian epidemic, underscoring the need for integrated genomic/ecological surveillance.

Bundibugyo virus (BDBV) remains one of the least genomically characterized filoviruses, partly due to the absence of primer schemes specifically optimized for its full genome amplification. Existing amplicon-based protocols developed for the Zaire Ebola virus or other orthoebolaviruses provide incomplete coverage when applied to BDBV, limiting genomic surveillance during outbreaks. In this study, we present a newly designed multiplex PCR primer scheme tailored to the genomic features of BDBV and optimized for sequencing on the Ion Torrent platform. The panel comprises 37 overlapping primer pairs spanning the entire ~19 kb BDBV genome, designed to minimize amplicon dropouts and ensure uniform coverage across regions with variable GC content and sequence divergence. Validation using BDBV RNA, derived from a diluted aliquot of a clinical specimen collected from a patient who tested positive for BDBV during the 2026 outbreak, demonstrated >99% genome recovery, high coverage uniformity, and robust performance even in low-titer samples. This represents, to the best of our knowledge, the first experimentally validated primer scheme specifically developed for BDBV. The protocol provides a reliable and accessible tool for genomic surveillance, outbreak investigation, and evolutionary studies, supporting improved diagnostic and public health responses to BDBV disease.

Arboviral infections represent a growing challenge for global public health due to vector expansion, globalization, and climate change. Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) often co-circulate and present overlapping clinical manifestations, making rapid and accurate etiological diagnosis difficult. In this context, multiplex real-time RT-PCR assays can support clinical management and epidemiological surveillance. In this study, analytical and preliminary retrospective comparisons of the Novaplex^TM Tropical Fever Virus Assay, a multiplex RT-PCR system designed for the simultaneous detection of DENV, ZIKV, CHIKV, and WNV, were performed. Analytical sensitivity was assessed using serial viral-stock dilutions and Probit regression analysis to estimate the limit of detection (LOD) at a 95% probability. The LOD was 4.41 Log copies/mL for WNV2, 3.67 for DENV2, 2.57 for ZIKV, and 3.43 for CHIKV in PBS. In spiked plasma or urine, LODs ranged from 2.04 to 4.79 Log copies/mL. No cross-reactivity with Usutu virus was observed under the conditions tested. In a retrospective comparison of 34 clinical specimens with singleplex assays, agreement was the highest for WNV, substantial for CHIKV, and limited for DENV. These findings support Novaplex as a complementary tool for arboviral diagnosis and surveillance, although further clinical studies are warranted.

Wastewater-based surveillance has emerged as a powerful approach for population-level monitoring of pathogen circulation in a timely and non-invasive manner. In this study, shotgun metagenomic sequencing was applied to wastewater samples collected from a hospital (HP), a university campus (UN), and a wastewater treatment plant (WTP). Viral sequences were taxonomically classified using Kraken2. Specifically, HP samples showed the highest viral richness, followed by WTP and UN samples (HP vs. UN, p = 0.0003; WTP vs. UN, p = 0.0018). Using Jaccard distance, significant differences were observed between WTP and UN (R² = 0.181, p < 0.001), WTP and HP (R² = 0.159, p < 0.001), and UN and HP (R² = 0.223, p < 0.001), and similarly, for Sørensen–Dice dissimilarity: WTP vs. UN (R² = 0.238, p < 0.001), WTP vs. HP (R² = 0.212, p < 0.001), and UN vs. HP (R² = 0.307, p < 0.001). Human-associated viral families were detected across all sources, predominantly Poxviridae, Orthoherpesviridae, Polyomaviridae and Circoviridae. Furthermore, the taxonomic composition of indirectly associated viruses, mainly Anelloviridae and Crassvirales, was examined. Overall, these findings support the potential of wastewater metagenomics as a reliable tool for monitoring viral diversity within environmental and public health contexts, although further research is needed to establish its operational utility for routine surveillance applications within a One Health framework.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe hemorrhagic disease and is endemic in Africa, the Middle East, and Asia, with increasing relevance in Eastern and Southern Europe due to climate change. Rapid detection methods are essential for outbreak control and timely therapeutic intervention. In this study, we present a lateral flow device (LFD) for direct detection of CCHFV RNA. Recombinase Polymerase Amplification (RPA) is used to amplify viral RNA, enhancing test sensitivity. CCHFV-specific oligonucleotide probes enable optical detection of amplification products. Two approaches were evaluated: (i) external RPA performed in a tube followed by LFD detection (total time 55 minutes), and (ii) integrated RPA conducted directly on the LFD (total time 80 minutes), reducing pipetting steps and simplifying handling. Using inactivated CCHFV cultures, limits of detection (LODs) for the external RPA system ranged from 125 to 12,500 copies/µl, whereas the integrated system showed an LOD of 125,000 copies/µl. Both approaches eliminate the need for bulky equipment such as thermal cyclers required for PCR, reducing analysis time and operational complexity. While the external RPA system achieves PCR-like sensitivity, the integrated system offers advantages in ease of use and reduced contamination risk due to encased on-device amplification. Overall, the presented LFD-based detection system represents a promising alternative to PCR, particularly in resource-limited settings where rapid decision-making is critical for controlling viral outbreaks.

West Nile virus (WNV) diagnosis relies on nucleic acid amplifications, but these techniques do not discriminate between infectious and non-infectious viral particles. This limitation can be bypassed by using a genome-binding dye (PMAxx) that is unable to cross membranes and can only bind to the genomes of non-intact (i.e., non-infectious) viral particles. This study evaluated a workflow combining PMAxx treatment with digital PCR to improve the molecular discrimination of intact WNV particles. Fifty-five samples (35 plasma, 20 urine) from 41 patients with WNV fever (WNF) or WNV neuroinvasive disease (WNND) were analyzed. Samples were tested with/without PMAxx treatment. Overall, PMAxx treatment resulted in a significant reduction in detectable viral RNA (median reduction: 1.0 Log copies/mL; p < 0.0001), indicating that a substantial fraction of RNA detected by standard methods originated from non-infectious particles. This reduction was more visible in urine (1.8 Log copies/mL) than in plasma (0.4 Log copies/mL), suggesting a higher proportion of degraded viral particles or free RNA in urine. Stratification by clinical presentation showed significant reductions in both WNF and WNND patients, with no significant differences between groups. This approach may represent a valuable adjunct for improving diagnostic interpretation and epidemiological assessment of WNV infection, particularly in matrices characterized by prolonged RNA persistence.

Borealpox virus (Alaskapox virus, BRPV), a neglected zoonotic Orthopoxvirus (OPXV), has been reported only in Alaska, usually causing mild infections. A recent fatal case in the Kenai Peninsula has raised concerns about its public health impact. Like other OPXVs, BRPV diagnosis relies on molecular tools, making species-specific assays essential. In this study, we developed and validated two BRPV-specific molecular assays: a TaqMan-based real-time PCR (rtPCR) and a droplet digital PCR (ddPCR) using the QIAcuity platform. Both target the viral CC-chemokines inhibitor (vCCI) gene, showing high sensitivity (limit of detection ~0.3–0.5 copies/μL), excellent specificity (no cross-reactivity with other OPXVs or rash-causing viruses), and strong reproducibility. While rtPCR is ideal for routine diagnostics, ddPCR offers absolute quantification without standard curves, enhancing the detection of low viral loads. Although the lack of clinical BRPV-positive samples limits full validation, both assays show strong potential for improving BRPV detection, helping to distinguish it from other OPXVs and supporting the early identification of emerging orthopoxvirus threats.

In 2023–2024, measles cases progressively increased in Europe. Multiple outbreaks were reported and viral strains with three-nucleotide mutations potentially compromising diagnostic testing were identified. We analyzed the first cases of measles reported in Lazio (Central Italy) from September 2023 to March 2024, combining molecular characterization and phylogenetic analysis with epidemiological investigation to identify transmission chains and evaluate the sensitivity of PCR tests adopted on circulating viral strains. We tested samples collected through routine measles and rubella surveillance for IgM, IgG and Real-Time PCR. We sequenced positive samples with higher viral titers using an amplicon-based whole-genome next-generation sequencing (WG-NGS) approach and performed mutational and phylogenetic analysis. Furthermore, we tested the sensitivity of the PCR molecular diagnostic assay adopted in our laboratory to identify the mutated strains. Of the 39 suspected cases, 28 were confirmed. Endemic cases were 82%; of these, 78% were sporadic at epidemiological investigation. From 21 high-titer samples, we obtained 14 strains belonging to the D8 genotype. Phylogenetic analysis identified four distinct clusters: three associating 50% of sporadic cases, and one confirming the epidemiological investigation. Several mutational patterns were identified, one of which had three nucleotide mutations potentially affecting the diagnostic test. However, our routine diagnostic PCR tests are able to detect mutated strains at different dilutions. Our results demonstrate that WG-NGS can be used to distinguish transmission chains and identify infection clusters to improve surveillance activity and enable the implementation of more targeted control measures, highlighting the importance of integrated epidemiological and genomic surveillance.

Wastewater surveillance has proven to be a cost-effective, non-invasive method for monitoring the spread and evolution of SARS-CoV-2, yet its value during today’s low-incidence phase is still being defined. Between August 2023 and July 2024, 42 composite wastewater samples were collected in Perugia, Italy and analyzed using RT-qPCR and whole-genome sequencing to identify circulating SARS-CoV-2 lineages. In parallel, clinical samples (respiratory tract samples) were collected and analyzed, allowing for direct comparisons to confirm the robustness of the wastewater findings. The sewage viral loads ranged from 8.9 × 10⁵ to 4.9 × 10⁷ genome copies inhabitant⁻¹ day⁻¹, outlining two modest community waves (September–December 2023 and May–July 2024). Sequencing resolved 403 Omicron lineages and revealed three successive subvariant phases: (i) XBB.* dominance (August–October 2023), when late-Omicron XBB subvariants (mainly EG.5.* and XBB.1.5) accounted for almost all genomes; (ii) a BA.2.86/JN surge (November 2023–March 2024), during which the BA.2.86 subvariant, driven mainly by its JN descendants (especially JN.1), rapidly displaced XBB.* and peaked at 89% in February 2024; and (iii) KP.* takeover (April–July 2024), with JN.1-derived KP subvariants rising steadily and KP.3 reaching 81% by July 2024, thereby becoming the dominant lineage. Comparisons of data from wastewater and clinical surveillance demonstrated how the former presented a much higher diversity of circulating viral lineages. Importantly, some subvariants (including BA.2.86*) were detected in wastewater weeks to months prior to clinical identification, and for longer periods. Taken together, the obtained data validated wastewater surveillance as an effective early warning system, especially during periods of low infection prevalence and/or limited molecular testing efforts. This methodology can thus complement clinical surveillance by offering valuable insights into viral dynamics at the community level and enhancing pandemic preparedness.

Mpox, caused by the Monkeypox virus (MPV), is a global public health threat. Virus isolation is the gold standard to confirm MPV infection, but this process can face many challenges. As an alternative, a new method was developed in in vitro settings using 50 µM of propidium monoazide (PMAxx, a DNA-binding agent) coupled with digital droplet PCR (ddPCR). Frozen clinical samples analyzed by PMAxx-ddPCR had a median of 0.8 copies/µL, while untreated samples had a median of 29.8 copies/µL. Since a substantial percentage of reduction was observed in these samples (>80%), it was verified whether this reduction could be due to the freezing process. This hypothesis was confirmed both in vitro and using clinical samples. A gradual increase in the mean percentage of reduction was observed after freezing–thawing cycles of MPV-isolate (59.5−81.4%). Moreover, a different percentage of reduction was observed before (68.2%) and after freezing (97.4%) the specimens, suggesting that the freezing process could reduce the number of complete viral particles. Our study shows strong evidence of the usefulness of PMAxx in clinical settings. PMAxx ensures the detection of intact MPV particles, which improves the accuracy of MPV load measurements. This method not only increases the reliability of MPV diagnosis but also overcomes virus isolation limitations.