2.1 Tick collection and identification
In April, 2021, ticks were collected from cattle or by flagging a white cotton cloth over vegetation in three sites of Jilin Province, northeastern China, including Huadian (126°73'E, 42°97'N), Dunhua (128°13'E, 43°21'N), and Jiaohe (127°33'E, 43°72'N) (Figure 1). All the ticks were preliminarily identified by the morphological classification combined with polymerase chain reaction (PCR) assay targeting the 16S rRNA of the ticks as described elsewhere[6].
2.2 DNA and RNA extraction
Every 10 ticks were pooled according to the collection sites and species for molecular detection (Table 1). Pools of ticks in tubes were washed with 75% alcohol and rinsed twice with double distilled water. 500-μL Dulbecco’s modified Eagle’s minimum (DMEM) and two stainless steel beads (3 mm diameter) were added to the tubes, and crushed using the Tissuelyser (Jingxin, Shanghai, China) at 70Hz for 2min. The lysates were centrifuged at 12 000 rpm for 10min at 4°C, and the supernatant was collected for DNA and RNA extraction. Total DNA of ticks was extracted using a Universal Genomic DNA Kit (Cwbio, Beijing, China) to identify tick species, and viral RNA was extracted using a TIANamp Virus RNA kit (Tiangen, Beijing, China) in accordance with the manufacturer’s instructions. The extracted virus RNA was reversed using the PrimeScript 1st Strand cDNA Synthesis kit (TaKaRa, Kyoto, Japan) and used for LCMV detection and whole genome amplification.
2.3 Detection of LCMV and whole genome amplification
The prevalence of LCMV in ticks were detected by real-time RT-PCR and primers were designed based on the S sequences of strain DH46 (MG554177), JX14 (MG554175), JX4 (MG554174), and JX31 (MG554176). The following components: 10.0 μL of Probe Premix Ex Taq (TaKaRa), 7.4 μL of double distilled water, 0.4 μL of forward primer (LCMV-NP F:5′-TGATGAGTCYTTCACGTCCCA-3′) and reverse primer (LCMV-NP R:5′-GACAACACAGCAGCTTGACC-3′) for each, 0.8 μL of probe (LCMV-NP P: FAM-5′- TCACCACACCAGTTGCACCCT-3′-BHQ1) and 1 μL of cDNA template were added to a total reaction volume of 20 μL. The PCR reaction parameters were 95℃ for 30s, followed by 50 circles at 95℃ for 5s, 60℃ for 30s. The positive pools were used for whole genome amplification by nested PCR assay with the primers listed in (Table S1 and S2). All the PCR products were separated by using 1% agarose gel electrophoresis for sanger sequencing, and the acquired sequences were assembled using DNAMAN software.
2.4 Phylogenetic analysis
Nucleotide sequences of the whole genome of LCMV were aligned using ClustalW and calculated for identities by MegAlign program available within DNAstar V7.1. The phylogenetic relationships were estimated using the maximum-likelihood method with the Tamura-Nei model conducted in MEGA software version 7.0. A bootstrapping analysis of 1000 replicates was conducted, and the bootstrap values more than 60 were shown in the phylogenetic trees.
2.5 Recombination analysis
To find the potential recombination events in the evolutionary history of LCMV, the full length of L and S segment sequences of LCMV strains were concatenated in order, and then analyzed using RDP, geneconv, bootscan, maxchi, chimera, siscan, and 3seq prediction methods in RDP4 software[7]. The reorganization event should meet the following two conditions: ⅰ, the event should be verified by at least two methods with P < 0.05, ⅱ, and the RDP recombination consensus score should be > 0.6[8] . Additionally, if the recombination event met the first condition, but the RDP recombination consensus score was between 0.4 and 0.6, a possible recombination event can be considered. Otherwise, the event was considered untenable. Moreover, the aligned LCMV sequences were also analyzed using Simplot version 3.5.1 to confirm the recombination events[9].
2.6 Statistical analyses
The prevalence of LCMV infection in ticks was calculated by using the program PooledInfRate Excel Add-In version 4.0[10]. Maximum likelihood estimation (MLE) method with 95% confidence intervals (CIs) and one-sample analysis for unequal pool sizes were used to analyze the infection rates for pooled ticks. Significant difference (p < 0.05) between the tick species or collection regions were performed by Fisher's exact test using SAS statistical software, version 9.3 (SAS Institute, Inc., Cary, NC).