Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS is still in its early stages of broader translation into clinical applications. To further support the development, implementation, optimization and standardization of mNGS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mNGS for viral diagnostics to share methodologies and experiences, and to develop application guidelines. Following the ENNGS publication Recommendations for the introduction of mNGS in clinical virology, part I: wet lab procedure in this journal, the current manuscript aims to provide practical recommendations for the bioinformatic analysis of mNGS data and reporting of results to clinicians.

Since 2010, the West Nile virus (WNV) has been established in Greece. We describe the epidemiology of diagnosed human WNV infections in Greece with a focus on the 2022 season. During the transmission period, clinicians were sending samples from suspected cases for testing. Active laboratory-based surveillance was performed with immediate notification of diagnosed cases. We collected clinical information and interviewed patients on a timely basis to identify their place of exposure. Besides serological and molecular diagnostic methods, next-generation sequencing was also performed. In 2022, 286 cases of WNV infection were diagnosed, including 278 symptomatic cases and 184 (64%) cases with neuroinvasive disease (WNND); 33 patients died. This was the third most intense season concerning the number of WNND cases, following 2018 and 2010. Most (96%) cases were recorded in two regions, in northern and central Greece. The virus strain was a variant of previous years, clustering into the Central European subclade of WNV lineage 2. The 2022 WNV season was quite intense in Greece. The prompt diagnosis and investigation of cases are considered pivotal for the timely response, while the availability of whole genome sequences enables studies on the molecular epidemiology of the disease.

In 2022, Greece was the second most seriously affected European country in terms of the West Nile virus (WNV), after Italy. Specifically, Central Macedonia was the region with the most reported human cases (81.5%). In the present study, 30,816 female Culex pipiens sensu lato mosquitoes were collected from May to September 2022 in the seven regional units of Central Macedonia; they were then grouped into 690 pools and tested for WNV, while next-generation sequencing was applied to the samples, which showed a cycle threshold of Ct < 30 in a real-time RT-PCR test. WNV was detected in 5.9% of pools, with significant differences in the detection rate among regional units and months. It is of interest that in the Thessaloniki regional unit, where most of the human cases were observed, the virus circulation started earlier, peaked earlier, and lasted longer than in the other regional units. All sequences clustered into the Central European subclade of WNV lineage 2, and the virus strain differed from the initial Greek strain of 2010 by 0.52% and 0.27% at the nucleotide and amino acid levels, respectively. Signature substitutions were present, such as S73P and T157A in the prM and E structural proteins, respectively. The screening of mosquitoes provides useful information for virus circulation in a region with a potential for early warning, while the availability of whole-genome sequences is essential for further studies, including virus evolution.

Greece is one of the countries in Europe most affected by West Nile virus (WNV), and since 2010, when the virus caused a large outbreak with 197 human neuroinvasive cases, outbreaks occur almost every year. Mosquito surveillance is an indirect sign of virus circulation; therefore, the purpose of the study was the molecular detection of WNV in 45,988 C. pipiens s.l. mosquitoes collected during 2024 in four Regions of Greece and the genetic characterization of the virus strains. WNV was detected in 41 of 1316 (3.12%) Culex spp. mosquito pools. Next-generation sequencing was applied to the WNV-positive samples that had a high viral load. All WNV sequences belong to Cluster B of the sub-lineage Europe WNV-2A presenting a temporal clustering. The WNV infection rates varied highly across the Regions, regional units and months, being higher in Thessaly and Central Macedonia Regions, especially in July and September. All mosquito pools were also tested for Usutu virus (USUV), and one pool was found positive, with sequence clustering into the EU-2 lineage. A subset of mosquitoes (737 pools) was tested for additional viruses, and bunya-like viruses were detected in 6 pools with sequences clustering into four distinct subclades. The prompt detection of pathogenic viruses is helpful for the design of control measures, while the detection of insect-specific viruses provides insights into viral diversity and evolution.

Ixodes ricinus ticks are vectors of a plethora of pathogens. The purpose of this study was to screen 398 I. ricinus ticks for a variety of pathogens. Following the pooling, homogenization, and extraction of total nucleic acids, a real-time PCR was applied for the detection of a panel of tick-borne pathogens, while additional conventional PCRs combined with Sanger sequencing were applied for the detection of viruses and typing of Rickettsia and Borrelia species. At least one pathogen was detected in 60 of the 80 (75%) tick pools. Rickettsia spp. predominated, as it was detected in 63.75% of the pools (51/80; MIR 12.81%), followed by Borrelia spp. (35 pools (45%); MIR 8.79%), while Anaplasma phagocytophilum was detected in 2 pools (2.5%, MIR 0.5%). The ticks of six Rickettsia-positive pools were tested individually (from stored half-ticks); all sequences were identical to those of R. monacensis. Similarly, the ticks of six Borrelia-positive pools were tested individually, and it was shown that four belonged to the genospecies Borrelia garinii and two to Borrelia valaisiana. Phleboviruses were detected in 3 pools (3.75%; MIR 0.75%), with sequences clustering in the Ixovirus genus, while nairoviruses were detected in 7 pools (8.75%; MIR 1.76%), with one sequence clustering in the Orthonairovirus genus, and six clustering in the Norwavirus genus. Although a small number of ticks from only one area in Greece were tested, a variety of pathogens together with recently identified viruses were detected, prompting further studies in ticks and surveillance studies in humans.