Vardeu, M. | bioRxiv Science

bioRxiv2026

Extensive and heterogeneous viral circular RNA production during Zika virus infection

Circular RNAs (circRNAs) are covalently closed RNA molecules with regulatory functions. Although virus-derived circRNAs (vcircRNAs) are well characterized in DNA viruses, their occurrence in RNA viruses remains poorly understood. Recent reports in coronaviruses and hepatitis C virus suggest that vcircRNA production extends beyond nuclear-replicating viruses. Here, we show that Zika virus (ZIKV), a vector-borne orthoflavivirus, produces vcircRNAs during infection of neural stem cells. Using a custom computational pipeline optimized for viral back-splice junction detection, we identified at 72 hours post infection a core set of 113 putative vcircRNAs shared across three biological replicates and distributed throughout the viral genome. Eight candidates spanning different genomic regions and abundance levels were experimentally validated by rolling-circle reverse transcription and Nanopore sequencing. These findings provide the first evidence of vcircRNA production in an orthoflavivirus, suggesting that circular RNA biogenesis may represent a previously underappreciated feature of RNA virus biology.

microbiology↗

bioRxiv2026

First detection and characterization of Alongshan virus in Ixodes ricinus ticks from Italy, 2021-2022

Alongshan virus (ALSV) is an emerging tick-borne segmented RNA virus belonging to the Jingmenvirus group and has been reported in humans, ticks, and vertebrates across Asia and Europe. Despite its potential public health relevance, its distribution and genetic diversity remain poorly characterized in several European regions where tick-borne pathogens are endemic. In this study, we developed a specific TaqMan-based real-time RT-PCR assay targeting a conserved region of ALSV segment 2 and used it to investigate the presence of ALSV RNA in Ixodes ricinus ticks collected in northeastern Italy. The assay showed high linearity over a broad dynamic range and no cross-reactivity with related flaviviruses. A total of 212 archival tick samples collected between March 2021 and November 2022 were screened, and 28 samples (13.2%) tested positive for ALSV RNA. Positive ticks were detected in the provinces of Belluno and Vicenza and included individual adult males and nymph pools. A subset of positive samples was further characterized by nested PCR and Sanger sequencing of all four genomic segments. Phylogenetic analyses showed that Italian ALSV sequences clustered within the broader European ALSV diversity and were closely related to strains from Central and Northern Europe, without forming a distinct country-specific lineage. Sequence comparisons suggested purifying selection and revealed differences in predicted structural proteins between European and Chinese strains. These findings provide the first molecular evidence of ALSV circulation in Italy and support further studies to clarify its epidemiology, host range, genetic diversity, and potential clinical relevance. IMPORTANCEAlongshan virus (ALSV) is an emerging tick-borne virus identified in febrile patients in China and subsequently detected in ticks in Russian Federation and several European countries. Although severe disease has not yet been reported in humans, surveillance and elucidation of the virus distribution are essential to assess its pathogenicity and potential public health impact. We developed a specific real-time RT-PCR protocol and detected ALSV in Ixodes ricinus ticks collected in northeastern Italy. Sequence analyses suggested multiple introductions and revealed differences in structural proteins between European and Chinese strains, suggesting potential adaptation and differences in pathogenicity. Since the clinical signs of ALSV infection in humans may overlap with those of tick-borne encephalitis (TBE), differential diagnostic procedures should be developed to improve patient management, particularly in TBE-endemic regions such as northeastern of Italy.

microbiology↗