Detection of novel tick-borne pathogen, Alongshan virus, in Ixodes ricinus ticks, south-eastern Finland, 2019

The newly identified tick-borne Alongshan virus (ALSV), a segmented Jingmen virus group flavivirus, was recently associated with human disease in China. We report the detection of ALSV RNA in Ixodes ricinus ticks in south-eastern Finland. Screening of sera from patients suspected for tick-borne encephalitis for Jingmen tick virus-like virus RNA and antibodies revealed no human cases. The presence of ALSV in common European ticks warrants further investigations on its role as a human pathogen.

The newly identified tick-borne Alongshan virus (ALSV), a segmented Jingmen virus group flavivirus, was recently associated with human disease in China. We report the detection of ALSV RNA in Ixodes ricinus ticks in south-eastern Finland. Screening of sera from patients suspected for tick-borne encephalitis for Jingmen tick virus-like virus RNA and antibodies revealed no human cases. The presence of ALSV in common European ticks warrants further investigations on its role as a human pathogen.

Recent reports have associated two members of Jingmen virus group, Alongshan virus (ALSV) and
Jingmen tick virus (JMTV), to febrile disease in humans [1,2]. Here we report the presence and genetic characterisation of ALSV in Ixodes ricinus ticks in Kotka archipelago, south-eastern Finland.
These viruses share the genome organisation of four segments, two of which show similarity to the NS3 and NS5 proteins of non-segmented RNA viruses in the genus Flavivirus. The other two segments appear to originate from an unknown ancestor. Together, the viruses form a separate and diverse group tentatively called the Jingmen virus group in the family Flaviviridae [9].

Detection of Jingmen-like virus in Kotka archipelago
In 2019, while performing a metatranscriptomic analysis of ticks collected in 2011 from Haapasaari island, Kotka archipelago, south-eastern Finland, we detected a full genome of JMTV-like virus together with tickborne encephalitis virus (TBEV) genome. Thereafter, we used RT-PCR to screen 198 I. ricinus ticks collected from the Kotka archipelago in 2017 and 2018 for the presence of JMTV-like RNA. We found another positive tick from a neighbouring Kuutsalo island in the Kotka archipelago, and obtained the full genome using nextgeneration sequencing. The viruses (GenBank accession numbers MN107153 to MN107160) cluster together with ALSV (MH158415 to MH158418) from Heilongjian Province, China, and form a cluster distinct from the other members JMTV group, including the strains found in Kosovo (MH133313 to MH133324) [2,7]

Human and tick samples
The emerging reports on the association of JMTV-like viruses with human disease in China [1,2] led us to investigate sera of TBE-suspected cases for JMTV-like virus RNA or antibodies against recombinant proteins of ASLV in 2019. The sera panel included 974 serum samples from 879 individuals. These samples were originally sent for TBEV antibody testing to Helsinki University Hospital laboratory (Helsinki, Finland) from May to November 2018. All samples were tested for JMTV RNA by RT-PCR, with 304 from 283 individuals (median age: 48 years, range: 1-88 years) for antibodies to JMTV VP1a, VP1b, membrane and capsid proteins.
For the RNA detection, we could verify that the RT-PCR detects local Finnish strains of ALSV, but we had no human ALSV positive control samples available for the antibody tests. We also studied three serial samples from two patients positive in an earlier sample for JMTV RNA from Kosovo at dilutions 1:20 and 1:80 for reference. These two patients shown to be infected with Kosovo strains of JMTV (capsid/membrane 63.5-64.0% amino acid identity, glycoprotein 49.9-50.1% amino acid identity) did not exhibit clear reactivity to the ALSV recombinant protein [7]. The 90 and 108 ticks collected in 2017 and 2018, respectively, from Kuutsalo The NS3 segment amino acid sequences of all available tick-borne Jingmen tick virus-like viruses were aligned using the ClustalW algorithm. The phylogenetic tree was constructed using the Bayesian Markov chain Monte Carlo (MCMC) method, implemented in MrBayes version 3.2 [19]. The GenBank accession number of the strains sequenced in this study are MN107155 and MN107159.
island, Kotka archipelago in south-eastern Finland were tested for JMTV RNA (Table 2).

RNA and antibody detection
Ticks were homogenised and RNA was extracted as described previously [10]. Total nucleic acids from human serum samples were extracted using MagNa Pure LC 2.0 instrument and Total Nucleic Acid Isolation Kit (Roche, Basel, Switzerland). Viral RNA was detected with real-time or conventional reverse transcription (RT)-PCR targeting the NS5 gene. Primers and the probe were designed based on sequences available to us in August 2018, and we used JM F1312 as the forward primer (5'-TTCGGRGCMTGGCAMCTSACCT-3'),

JM1548
as the reverse primer (5'-CCKGTTDTCCATYTGGTADCCCAT-3'), and JM2 as the probe (FAM-CTCCTAAAGATGTTAAACACTGC-BHQ). Conventional RT-PCR without the probe was initially used for tick samples with SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, California, United States (US)). Patient and tick samples were screened with real-time RT-PCR using the TaqMan Fast Virus 1-Step Master Mix (Thermo Scientific, Waltham, Massachusetts, US). An in vitro transcribed RNA served as the positive control.
Synthetic gene constructs encoding JTMV glycoproteins VP1a, VP1b, membrane and capsid proteins were The NS5 segment amino acid sequences of all available tick-borne Jingmen tick virus-like viruses were aligned using the ClustalW algorithm. The phylogenetic tree was constructed using the Bayesian Markov chain Monte Carlo (MCMC) method, implemented in MrBayes version 3.2 [19]. The GenBank accession number of the strains sequenced in this study are MN107156 and MN107160.
cloned into pCAGGS/MCS [11]. The recombinant and empty plasmids were transfected into Vero E6 cells using Fugene HD according to the manufacturer's instructions. The transfected cells were fixed onto microscopic slides with acetone, serum samples were diluted 1:20 in phosphate-buffered saline and immunofluorescence assay was performed as described previously [12].

Next generation sequencing and phylogenetic analysis
Tick homogenates were treated with a mixture of micrococcal nuclease (New England BioLabs Ipswich, Massachusetts, US) and benzonase (Millipore, Burlington, Massachusetts, US) for 1 hour at 37 °C, followed by RNA extraction using TriPure Isolation reagent (Roche, Basel, Switzerland). RRNA was removed using a NEBNext rRNA Depletion Kit (New England BioLabs) according to the manufacturer's protocol. The sequencing library was prepared using a NEBNext Ultra II RNA Library Prep Kit (New England BioLabs). The library fragment sizes were measured using agarose gel The putative capsid/membrane segment amino acid sequences of all available tick-borne Jingmen tick virus-like viruses were aligned using the ClustalW algorithm. The phylogenetic tree was constructed using the Bayesian Markov chain Monte Carlo (MCMC) method, implemented in MrBayes version 3.2 [19]. The GenBank accession number of the strains sequenced in this study are MN107153 and MN107157. electrophoresis and the concentrations using Qubit dsDNA BR Assay Kit (Life Technologies, Carlsbad, California, US) and NEBNext Library Quant Kit for Illumina (New England BioLabs). Sequencing was conducted using MiSeq Reagent Kit V2 with 150 bp reads.
Complete genome sequences of all available tick-borne JMTV-like viruses were downloaded from GenBank (accessed June 2019). The amino acid sequences were aligned using the ClustalW algorithm followed by manual refinement. In addition, NS5 sequences of the representatives of all flavivirus species were retrieved from NCBI Reference Sequence Database (RefSeq) and aligned with MAFFT programme version 7 [17] using E-INS-i algorithm, followed by removal of ambiguously aligned amino acid sites using TrimAl programme [18].
The phylogenetic trees were constructed using the Bayesian Markov chain Monte Carlo (MCMC) method, implemented in MrBayes version 3.2 [19] with two The putative glycoprotein segment amino acid sequences of all available tick-borne Jingmen tick virus-like viruses were aligned using the ClustalW algorithm. The phylogenetic tree was constructed using the Bayesian Markov chain Monte Carlo (MCMC) method, implemented in MrBayes version 3.2 [19]. The GenBank accession number of the strains sequenced in this study are MN107154 and MN107158.

Figure 5
The phylogenetic tree of NS5 of all species in the family Flaviviridae  independent runs and four chains per run. The analysis was run for 5 million states and sampled every 5,000 steps.

Conclusion
Our findings show that ALSV, a newly described tickborne human pathogen, is also present in southeastern Finland. Notably, ALSV was detected in I. ricinus ticks, a tick species that is common across the European continent. Despite apparent ALSV circulation in the south-eastern archipelago of Finland, no ALSV RNA or antibodies to selected recombinant ALSV proteins were found in ca 900 Finnish patients suspected for TBEV infection in recent years. While our results suggest low human infection pressure, further research using other methods, including properly evaluated ALSV antibody tests, and focusing on other geographic areas and patient cohorts beyond meningitis or encephalitis cases is needed.