Researchers from the Institute of Virology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland have issued warnings about the discovery of the novel Alongshan Virus (ALSV) in ticks in Switzerland with possible implications that the virus could be now widespread not only in Switzerland but in many parts of Europe.
The study findings of the discovery of Alongshan virus in ticks in Switzerland were published in the journal Zenodo.
In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected.
Alongshan virus (ALSV) is a member of the unclassified Jingmen virus group within the family of the Flaviviridae. In contrast to the viruses of the genera flavivirus, hepacivirus, pegivirus, and pestivirus, which have non-segmented genomes, the genomes of the unclassified Jingmen viruses are segmented.
Specifically, the ALSV genome consists of four segments of positive sense ssRNA. ALSV was first detected in China in 2017, in patients with a history of tick bites and tick-borne encephalitis virus (TBEV) symptoms but negative TBEV diagnostics (1). In 2019, ALSV was also detected in Ixodes ricinus ticks in Finland, but human infection cases were not found (2).
Our study aimed to determine the virome of ticks collected in 2021 and 2022 from different environments and regions of Switzerland. For sequence analysis, ticks were divided into pools according to species, developmental stage, gender, and place of collection, and homogenized in 500 µl of phosphate-buffered saline (PBS) using the TissueLyser II (Qiagen, Germany) at 20 Hz for 2 min.
Total RNA was extracted, reverse transcribed, and amplified, and libraries were prepared for next-generation sequencing (NGS) as previously described (3). A paired-end NGS run of 2 x 100-nt read length was performed on an Illumina NovaSeq sequencing system at the Functional Genomics Center Zurich (Switzerland).
In total, 3.2 x 106 raw reads were sequenced, and quality controlled using FastQC (version 0.11.7). PCR primers, sequencing adaptors and low-quality ends were trimmed by Trimmomatic (version 0.39), and contigs were assembled using metaSPAdes (v3.14.0), with settings as described previously (4, 5). Assembled contigs were compared against the NCBI nt database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) using blastn (v2.10.1+) and visualized, manually confirmed, and corrected using the SeqMan Pro software version 17 (DNAStar [Lasergene, USA]) based on available complete genomes.
From a pool of 60 adult male Ixodes ricinus ticks collected in the Canton Grisons, Switzerland, we fully sequenced segments 1-4 of an ALSV genome, with a length of 3028, 2788, 2808 and 2744 nt, a GC content of 53%, 53%, 54%, and 53%, and a sequencing depth of 3765x, 4008x, 3399x and 1909x, respectively.
The coding sequences at amino acid levels showed high similarities to NS5-like protein (99.8%), glycoprotein VP1a (99.2%), NS3-like protein (99.9%), membrane protein (99.8%) and capsid protein (100%) of an ALSV strain from France (MN095519–MN095522), and to glycoprotein VP1b (99.6%) of ALSV strains from Finland (MN107154) and Russia (MN648776). This finding expands the list of pathogens that can potentially be transmitted by ticks in Switzerland and warrants further investigations regarding the prevalence in ticks and the impact on public health.