Veterinaria Italiana

Molecular Evidence of Epizootic Haemorrhagic Disease Virus and Bluetongue Virus Circulation in Wild Ruminants in Namibia.

Wed, 04 Mar 2026 00:00:00 +0100

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-borne orbiviruses affecting domestic and wild ruminants. Information on their circulation in Namibian wildlife is limited. This study investigated the molecular detection and serotype distribution of BTV and EHDV in wild ruminants from a commercial game farm in the Khomas Region, Namibia, where wildlife and livestock coexist.

Between June and September 2019, spleen samples from 62 clinically healthy animals (kudu, oryx, and red hartebeest) were analysed by real-time RT-PCR using pan-BTV and pan-EHDV assays, followed by serotype-specific tests for selected BTV types. Two animals (3.23%) tested positive for EHDV. BTV RNA was detected in 24/62 animals (38.71%), with Ct values ranging from 28.3 to 38.4. BTV-3 and BTV-4 were the most frequently identified serotypes, while one sample was positive for BTV-1; six BTV-positive samples remained untyped. High Ct values and low RNA loads likely limited sequencing success.

Although restricted to a single farm and a limited serotype panel, this study provides preliminary molecular evidence of BTV and EHDV circulation in Namibian wild ruminants, highlighting the need for broader epidemiological investigations at the wildlife–livestock interface.

Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas–based rRNA depletion system

Wed, 04 Mar 2026 00:00:00 +0100

Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9–based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H–based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.

Development and validation of a real time RT-qPCR assay for detection of the emerging Bluetongue virus serotype 5 from field samples

Wed, 11 Mar 2026 00:00:00 +0100

Bluetongue (BT) is a WOAH-notifiable economically important disease of ruminants caused by Bluetongue virus (BTV), transmitted by Culicoides spp. biting midges. Over the past two years, Italy has experienced a marked re-emergence of BT, with thousands of outbreaks reported due to the simultaneous circulation of several BTV strains belonging to serotypes 3, 4, and 8. Moreover, in September 2025, BTV-5 was detected in Sardinia, marking its first occurrence in Europe. Following the first identification by Whole Genome Sequencing, the development of a reliable real-time RT-qPCR-based assay capable of typing the novel BTV-5 ITA 2025 strain was essential, as currently available molecular typing methods targeting BTV segment 2, which encodes the outer capsid protein VP2, are unable to detect this newly emerging strain. Therefore, in this study we developed, optimised, and validated a real-time RT-PCR assay for the detection and typing of BTV-5 ITA 2025 in field samples. The assay is characterised by high sensitivity and specificity, as well as good reproducibility, and can be effectively applied for BTV-5 ITA 2025 diagnosis in the current epidemiological context, supporting surveillance and control strategies.