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. 2026 Feb 13;41(1):70–83. doi: 10.1016/j.virs.2026.02.008

Evidence of human exposure to tick-borne viruses based on viromes of ticks and presence of specific antibodies among patients in Hainan Island, southern China

You Zhang a,c, Yuan Bai c, Jun Ni c, Junming Shi c,1, Yanfang Zhang c, Lesley Bell-Sakyi d, Xiaoli Wu c, Changhua He e,f, Fei Deng c, Feifei Yin b,⁎, Shu Shen c,g,⁎, Yaohui Fang c,⁎
PMCID: PMC13007299  PMID: 41690672

Abstract

Hainan Island, located in the South China Sea, is known as an area with diseases related to Rickettsia spp. or spirochete infection; however, the potential threat there from infection with tick-borne viruses (TBVs) remains obscure. In the present study, the dominant tick species, including Rhipicephalus sanguineus and Rhipicephalus microplus, were collected in Hainan Island, and tick viromes were investigated by metagenomic sequencing. In total, 27 viral species were identified belonging to the families Orthomyxoviridae, Flaviviridae, Nairoviridae, Phenuiviridae, Totiviridae, Chuviridae, Rhabdoviridae, and Parvoviridae, amongst which one novel virus and 13 new strains were discovered. Subsequently, individual ticks were screened for seven TBVs, Huanggang Rhabd tick virus 1 (HRTV1), Lihan tick virus (LHTV), Mivirus (MIV), Guangdong tick quaranjavirus (GTQV), Wenchang Ephemerovirus (WEPMV), Jingmen tick virus (JMTV), and brown dog tick phlebovirus (BDPTV), resulting in high prevalence rates of 16.97%, 9.59%, 10.33%, 7.38%, 7.01%, 6.27%, and 3.69%, respectively. While co-infection with multiple viruses was more frequent in R. sanguineus, R. microplus ticks generally had higher viral loads. Four febrile patients showed antibody responses to three TBVs, one each to LHTV and JMTV, and two to GTQV; the patient with antibodies to JMTV also showed neutralizing activity against this virus. This study promoted our understanding of the diversity and complexity of the TBV community in Hainan Island. The results provide serological evidence that human exposure to TBVs like JMTV may have occurred in Hainan, raising concern about potential risks from TBVs and the need to perform further surveys of TBVs among ticks, animals and humans.

Keywords: Ticks, Virome, Tick-borne viruses, Serological exposure, Hainan Island

Highlights

  • •

    Metagenomic analysis identified 27 tick-associated viruses, including one novel virus and 13 new strains in Hainan Island.

  • •

    Seven TBVs showed high prevalence, with frequent co-infection in R. sanguineus and higher viral loads in R. microplus.

  • •

    Antibodies to LHTV, GTQV, and JMTV were detected in febrile patients, indicating possible human exposure.

Introduction

Ticks are important vectors of worldwide distribution, which are capable of carrying and transmitting viruses associated with human and animal diseases (Zhou et al., 2023). Tick-borne viruses (TBVs) such as Crimean-Congo hemorrhagic fever virus (CCHFV) (WHO, 2021), tick-borne encephalitis virus (TBEV) (WHO, 2020), severe fever with thrombocytopenia syndrome virus (SFTSV) (Yu et al., 2011) and African swine fever virus (Galindo and Alonso, 2017) have caused disease epidemics in different countries, and pose an increasing threat to public health and the livestock industry (Mansfield et al., 2017).

Its vast territory gives China diverse ecological environments and creates a suitable habitat for at least 124 tick species (Jia et al., 2020). A recent study discovered over 1000 viruses carried by ticks from 31 provinces of China, revealing a huge TBV community of significant diversity. The study also suggested a role of ecological environments in shaping the geographic distributions of different tick species, which consequently influences the diversity of TBVs (Ni et al., 2023). Previous studies on viromes of tick species also showed different viral communities in regions of China (Kong et al., 2022; Ni et al., 2023). Discovery of novel TBVs based on tick viromes has promoted the identification of pathogenic TBVs that are associated with human or animal diseases, and will continue to do so. TBVs such as Tacheng tick virus 2 (Dong et al., 2021), Tamdy virus (Moming et al., 2021), Jingmen tick virus (Taniguchi, 2019), and Beiji Nairovirus (Wang, Y.C. et al., 2021) were first identified from different tick species by metagenomic sequencing and were then found to be the causative agents of human diseases, raising the concern about the increasing risk from novel emerging TBVs. However, the spillover capability for most other viruses, such as Lihan tick virus (Lopez et al., 2020) and Quaranjavirus (Sameroff et al., 2021), identified by metagenomics is still unclear. Their potential association with human or animal diseases remains to be investigated.

Hainan Island, a large tropical island located in the South China Sea, possesses a hot and humid ecological environment characterized by an average annual temperature above 20 °C, annual rainfall exceeding 1,000 mm, and humidity consistently above 70% (Guo et al., 2021). The island has a varied topography including mountains, hills, plains and fertile tropical rainforests, providing an ideal habitat for ticks that are active year-round (Zheng et al., 2023). So far, more than 20 tick species have been identified, of which Rhipicephalus spp. are the dominant population distributed throughout the island (Sun et al., 2013; Zhao, G.P. et al., 2021). Very few studies have reported the viromes of ticks from Hainan Island. One recent study characterized viromes of Rhipicephalus ticks collected from four sampling sites in Hainan and identified viral sequences belonging to 12 viral families (Wang et al., 2024). However, understanding of tick viromes, the diversity of the TBV community, and the potential threat it poses throughout Hainan Island are still very limited.

Multiple tick-borne pathogens have been identified among ticks, animals and humans in Hainan, including Borrelia, Anaplasma, Rickettsia, Ehrlichia and Coxiella (Lu et al., 2021; Zhao, H.Q. et al., 2021; Zheng et al., 2023). Since the first human rickettsia infection reported in 1996, Rickettsia spp. have been considered the most important tick-borne pathogens in Hainan (Wu, J. 2014). Recent epidemiological surveys detected Rickettsia DNA in 15.38% (18/117) of patients with unknown fever (Wu, J. 2014), and over 46.1% (378/821) of healthy farmers were Rickettsia seropositive (Jin et al., 2011). A retrospective survey showed that Lyme disease was diagnosed in 1.9%–16.67% of patients with arthritis in different counties of Hainan (Zhang, L. et al., 2021). These suggested a substantial threat from infection with tick-borne pathogens in Hainan; however, the threat from infection or exposure to TBVs remains to be characterized.

In this study, R. sanguineus and R. microplus were collected from livestock in 15 counties of Hainan Island, and their viromes were investigated. A survey of the prevalence of viruses identified at high abundance based on metagenomic data was performed among individual ticks. The association of prevalence rates of the tested viruses with tick species, sampling locations, and animal hosts was characterized, and co-infection with multiple viruses detected in individual ticks was analyzed. Antibody responses to viruses were surveyed among febrile patients, and neutralization tests were conducted with the antibody-positive serum samples. The results provided fundamental data on tick viromes and a precise prevalence rate of TBVs among individual ticks, and demonstrated serological evidence of human exposure to TBVs in Hainan. The findings improved our understanding of the TBV community and spillover risks in this tropical island, and raised the need to carry out ongoing surveillance of viruses in ticks and of human and animal infection with emerging TBVs.

Results

R. sanguineus and R. microplus ticks from Hainan island have different viromes

A total of 493 ticks collected from livestock were identified as R. sanguineus (52.7%) or R. microplus (47.3%) (Supplementary Table S1). Eleven tick pools were prepared for RNA sequencing, generating a total of 855,674,748 reads. From each pool, 388 to 800,470 viral-related reads were found, accounting for 0.0005%–0.9394% of the total reads across all pools (Supplementary Table S2). These viral-related reads were assigned to at least 27 different viral species belonging to eight viral families: Orthomyxoviridae, Flaviviridae, Nairoviridae, Phenuiviridae, Totiviridae, Chuviridae, Rhabdoviridae, and Parvoviridae (Supplementary Tables S3 and S4). Different viral communities were observed between the two tick species. The virome of R. sanguineus was predominantly composed of Chuviridae (45.3%), Phenuiviridae (19.8%) and Orthomyxoviridae (21.6%), while that of R. microplus was dominated by viruses of the Flaviviridae family (54.9%) (Fig. 1A). While viral sequences of Chuviridae, Phenuiviridae, Orthomyxoviridae, Parvoviridae, and Rhabdoviridae were found in both R. sanguineus and R. microplus, those belonging to Flaviviridae, Nairoviridae, and Totiviridae were only found in R. microplus (Fig. 1B). Clustering analysis based on the heatmap presenting the abundance of viral species in each tick pool, showed that the pools were divided into three clusters. One cluster comprised four pools of R. microplus (M5, M4, M3, and M2), the second cluster contained one pool of R. microplus (M1) and two pools of R. sanguineus (S2 and S1), and the third cluster comprised four pools of R. sanguineus (S6, S5, S4, and S3) (Fig. 1C). Rarefaction curve analysis of virus species-richness in R. sanguineus and R. microplus ticks showed that the sequencing depth of all pools was sufficient (Fig. 1D). The alpha diversity analysis showed that both Shannon and Simpson indexes were higher in R. microplus than in R. sanguineus, suggesting a higher viral diversity in the former than in the latter, although the difference was not statistically significant (Fig. 1E). Nevertheless, the beta diversity analysis based on Non-metric Multidimensional Scaling (NMDS) revealed a significant difference in viral species between R. sanguineus and R. microplus (metaMDS: stress = 0.052, PERMANOVA: P < 0.05) (Fig. 1F), while geographic distribution did not significantly affect tick virome diversity (Supplementary Fig. S1). Moreover, the linear discriminant analysis (LDA) indicated that the Phenuiviridae was associated with R. sanguineus, while Tacheng tick virus 3, JMTV, and Flaviviridae sequences were associated with R. microplus (Fig. 1G). All results indicated that the variation in viral communities was primarily driven by tick species identity (PERMANOVA, P < 0.05), rather than by the geographical locations where the ticks were collected (longitude or latitude, P > 0.05). Owing to the relatively small number of ticks collected from dogs, their data were not analyzed separately.

Fig. 1.

Fig. 1

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Characteristics of viral composition, distribution, and relative abundance in R. sanguineus and R. microplus ticks from Hainan Island. The proportion of sequenced reads associated with different viral families (A) and each viral family (B) in the R. sanguineus and R. microplus tick sequencing libraries are shown. C The heatmap of viral species identified from 11 tick libraries and the Euclidean distance matrix were used to calculate the hierarchical clustering. The viral species (below) and tick species (right) are marked on the heatmap. Tick species are represented by different colors (red: R. sanguineus, yellow: R. microplus). D Rarefaction curves of virus species richness in R. sanguineus and R. microplus ticks. The x-axis represents the sequencing depth (103 TPM), and the y-axis shows the cumulative number of virus species observed. Each curve corresponds to a different sample or group of samples. E The alpha diversity of virome distribution among R. sanguineus and R. microplus ticks calculated by Shannon index (left) and Simpson index (right). F NMDS analysis of virome distribution among R. sanguineus and R. microplus ticks. G Linear Discriminant Analysis (LDA) plot represents the differentiation of tick species based on virus species.

Novel viruses and new viral strains were identified from ticks

Complete genome sequences of three new strains [Guangdong tick quaranjavirus (GTQV), Huanggang Rhabd tick virus 1 (HRTV1), and Wuhan Tick Virus 1 (WTV1)] and one novel virus, Wenchang ephemerovirus (WEPMV), were obtained. Named according to the locations where the tick samples were collected (Table 1).

Table 1.

Novel viruses and new strains identified in ticks collected on Hainan Island in this study.

Virus name Strain Viral family Tick species Genome/Segment length (nt) Closest virus %Identity (aa)
Guangdong tick quaranjavirus (GTQV) Baisha/R.sanguineus/2018 Orthomyxoviridae R. sa
R. mi		 2369 (PB1) Guangdong tick quaranjavirus 99
2413 (PB2) 98
2367 (PA) 99
1634 (HA) 99
1736 (NP) 99
Huanggang Rhabd tick virus 1 (HRTV1) Lingshui/R.sanguineus/2018 Rhabdoviridae R. sa 6522 (RdRP) Huanggang Rhabd tick virus 1 99
1458 (NP) 98
1287 (GP) 96
Wuhan Tick Virus 1 (WTV1) Wenchang/R.sanguineus/2018 Rhabdoviridae R. sa 6573 (RdRP) Wuhan Tick Virus 1 99
1287 (NP) 98
1410 (GP) 99
Wenchang Ephemerovirus (WEPMV) Wenchang/R.sanguineus/2018 Rhabdoviridae R. sa 8357 (L) Bovine ephemeral fever virus 75
1830 (GP) 62
669 (M) 78
Jingmen tick virus (JMTV) Qiongzhong/R.microplus/2021 Flaviviridae R. mi 1355 (NS5) Jingmen tick virus-XJ364 99
Changing tick virus 2 (CPTV2) Wenchang/R.sanguineus/2018 Chuviridae R. sa 6471 (RdRP) Changping tick virus 2 92
1371 (NP) 92
2052 (GP) 92
Wuhan tick virus 2 (WTV2) Wenchang/R.sanguineus/2018 Chuviridae R. sa 6726 (RdRP) Wuhan tick virus 2 isolate DN277489 96
1236 (NP) 98
2052 (GP) 98
Wuhan tick virus 2 (WTV2) Baisha/R.sanguineus/2018 R. sa 6570 (RdRP) 97
1173 (NP) 97
2052 (GP) 97
Lihan tick virus (LHTV) Lingshui/R.sanguineus/2018 Phenuiviridae R. sa 6503 (L seg) Lihan tick virus strain LH-1 97
1888 (S seg) 97
Lihan tick virus (LHTV) Wenchang/R.sanguineus/2018 R. sa 6504 (L seg) 97
1955 (S seg) 97
Lihan tick virus (LHTV) Tunchang/R.microplus/2018 R. mi 6501 (L seg) 97
1878 (S seg) 97
Lihan tick virus (LHTV) Qiongzhong/R.microplus/2021 R. mi 1843 (S seg) 94
6162 (L seg) 96
Brown dog tick phlebovirus 1 (BDPTV1) Lingao/R.microplus/2018 Phenuiviridae R. sa 2010 (S seg) Brown dog tick phlebovirus 1 83
6556 (L seg) 95
Brown dog tick phlebovirus 2 (BDPTV2) Dingan/R.sanguineus/2018 R. sa 2010 (S seg) Brown dog tick phlebovirus 2 isolate TTP-Pool-5 83
6556 (L seg) 95
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R. sa: R.sanguineus, R. mi: R. microplus.

Quaranjavirus: GTQV BS18

GTQV reads were detected in six pools of R. sanguineus and R. microplus, comprising 4,094–91,620 reads and accounting for 4.37%–87.51% of the viral-related reads. The viral genome consists of five segments ranging in length from 1,634 to 2,413 nt and shares high sequence identities (98.14%–99.2% nt and 98.17%–99.48% aa) with Guangdong tick quaranjavirus, a member of the genus Quaranjavirus (family Orthomyxoviridae) initially identified in Rhipicephalus ticks from Guangdong, China (Guo et al., 2022). Based on its near-identical genomic sequence similarity to Guangdong tick quaranjavirus, this virus is classified as a new strain of this species. Phylogenetic analysis based on PB1 protein confirmed that the newly identified variant, designated Guangdong tick quaranjavirus Baisha/R.sanguineus/2018 (GTQV-BS18), clusters within the same clade as the prototype GTQV (Fig. 2A). The broad distribution of GTQV-BS18 across Hainan Island is further evidenced by its detection in R. sanguineus pools collected from Baisha, Wenchang, Haikou, Dongfang, Wuzhishan, and Sanya, as well as in one R. microplus pool from Lin'gao (Supplementary Table S3).

Fig. 2.

Fig. 2

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Phylogenetic analyses of novel viruses belonging to Orthomyxoviridae and Rhabdoviridae identified in ticks in this study. The phylogenetic trees were constructed using the protein sequences of PB1 from Orthomyxoviridae (A) and RdRp from Rhabdoviridae (B). These sequences were aligned with those from representative viruses obtained from public databases. The trees were generated using the maximum likelihood method with bootstrap values to assess the reliability of the branches. Viruses identified in our tick libraries are highlighted with red-filled circles. The scale bar represents the number of substitutions per site, and the bootstrap values (percentage of 1000 replicates) are indicated at the nodes. Bootstrap values greater than 50 are indicated.

Alpharicinrhavirus: HRTV1 LS18 and WTV1 WC18

The genomic sequences of HRTV1 and WTV1, which belong to the genus Alpharicinrhavirus, were identified in pools of R. sanguineus (Fig. 2B). They have been designated as Huanggang Rhabd tick virus 1 Linshui/R.sanguineus/2018 (HRTV1 LS18) and Wuhan Tick Virus 1 Wenchang/R.sanguineus/2018 (WTV1 WC18), respectively. HRTV1 LS18 exhibited 95.46% nucleotide and 99.11% (RNA-dependent RNA polymerase, RdRp) amino acid sequence identity with Huanggang Rhabd tick virus 1, which was initially identified in Rhipicephalus ticks from Hubei, China. Since the amino acid sequence divergence in the RdRp falls below the 10% threshold established by the ICTV for species demarcation within the genus Alpharicinrhavirus, they are classified as new strains of HRTV1 and WTV1. Similarly, WTV1 WC18 showed 96.35% nucleotide and 99.41% (RdRp) amino acid identity with Wuhan Tick Virus 1, also reported from Rhipicephalus ticks in Hubei, China. Phylogenetic analysis reveals that both currently unclassified viruses are placed within the Alpharicinrhavirus genus of the family.

Ephemerovirus: WEPMV

WEPMV, found in R. sanguineus, belongs to the genus Ephemerovirus in the family Rhabdoviridae, which contains ephemeroviruses usually found in cattle and water buffalo (Fig. 2B). It had the closest similarity (aa identities: 75.23% of RdRp, 69.91% of Glycoprotein (GP), and 78.10% of matrix protein) to bovine ephemeral fever virus (BEFV) strain EGY-2005 (Fig. 2B), which is the pathogenic agent of an acute inflammatory disease in buffalo in Egypt (Walker and Klement, 2015). The amino acid sequence divergence of RdRp exceeds 15%, surpassing the ICTV-recommended threshold for species demarcation. Therefore, we formally propose its classification as a distinct novel species (King et al., 2011).

In addition, 10 new strains of known viruses were also identified: JMTV (unclassified), LHTV (Phenuiviridae), WTV2 (Chuviridae), Mivirus (MIV, Chuviridae), Changping tick virus 2 (CPTV2, Chuviridae), and brown dog tick phlebovirus (BDPTV, Phenuiviridae) (Table 1, Supplementary Fig. S2, Supplementary Fig. S3). Of the seven identified viruses, JMTV, LHTV, WTV2, and Mivirus had been previously documented in R. microplus and R. sanguineus. BDPTV and CPTV2, which were originally identified in Amblyomma sp., Dermacentor sp., and Haemaphysalis sp., have been detected in R. microplus and R. sanguineus for the first time.

The precise prevalence and abundance of TBVs among individual ticks

The precise prevalence of seven of the novel TBVs and new strains was investigated by qRT-PCR among 271 individual ticks (165 R. sanguineus collected from sheep and cattle and 106 R. microplus collected from cattle). We found that 112 of the 271 ticks were positive for at least one of the seven tested viruses, resulting in a total infection rate of 41.32%. HRTV1 showed the highest infection rate (46/271; 16.97%), followed by MIV (28/271; 10.33%), JMTV (27/271; 9.96%), LHTV (26/271, 9.59%), GTQV (20/271, 7.38%), WEPMV (19/271, 7.01%), and BDPTV (9/271; 3.69%) (Table 2). These viruses exhibited varying levels of abundance in individual ticks, with MIV showing the highest median viral loads (1.3 × 104 copies/tick; 95% Confidence interval (CI): 2.0 × 104–8.7 × 103), followed by LHTV (3.6 × 103 copies/tick; 95% CI: 1.1 × 104–1.1 × 103), WEPMV (3.0 × 103 copies/tick; 95% CI: 1.3 × 104–7.4 × 102), GTQV (3.9 × 103 copies/tick; 95% CI: 3.9 × 104–3.91 × 102), HRTV1 (1.2 × 103 copies/tick; 95% CI: 2.72 × 103–5.72 × 102), JMTV (1.2 × 103 copies/tick; 95% CI: 2.85 × 103–4.8 × 102), and the lowest was BDPTV (3.0 × 102 copies/tick; 95% CI: 6.6 × 102–1.4 × 102) (Supplementary Fig. S4).

Table 2.

Prevalence of viruses identified in this study among individual ticks from Hainan Island.

Location Tick species Host species No. of ticks tested HRTV1 WEPMV LHTV BDPTV JMTV MIV GTQV
Baisha R. sa Bovine 35 0 0 0 0 0 4 (11.42%) 9 (25.71%)
Chengmai R. sa Bovine 17 1 (5.88%) 6 (35.29%) 0 0 0 0 0
Haikou R. mi Bovine 40 14 (35%) 0 6 (15%) 3 (7.5%) 5 (12.5%) 0 0
Lingao R. mi Bovine 24 0 0 0 1 (4.16%) 0 0 2 (8.33%)
Lingshui R. sa Ovine 25 8 (32.00%) 12 (48.00%) 5 (20%) 1 (4%) 0 2 (8.00%) 0
Qionghai R. sa Bovine 2 0 0 0 0 0 0 0
Qiongzhong R. mi Bovine 42 17 (40.47%) 0 8 (19.04%) 3 (7.14%) 2 (4.76%) 0 4 (14.7%)
Tunchang R. sa Bovine 17 6 (35.29%) 0 7 (41.17%) 0 0 0 0
Wanning R. sa Ovine 30 0 1 (3.33%) 0 1 (3.33%) 0 1 (3.33%) 0
Wenchang R. sa Ovine 34 0 0 0 0 10 (29.41%) 20 (58.82%) 3 (8.82%)
R. sa Bovine 5 0 0 0 0 0 1 (20.00%) 2 (40.00%)
Total 271 46 (16.97%) 19 (7.01%) 26 (9.59%) 9 (3.69%) 17 (6.27%) 28 (10.33%) 20 (7.38%)
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R. sa: R.sanguineus, R. mi: R.microplus.

The prevalence rates and abundance of each virus were analyzed in association with tick species and animal hosts. The positive rates of HRTV1, LHTV, BDPTV and JMTV in R. sanguineus were generally higher than those in R. microplus, while GTQV appeared with a higher prevalence rate in R. microplus than in R. sanguineus (Fig. 3A). Except for MIV and WEPMV which were absent in R. sanguineus, the other five tested viruses had RNA loads in R. microplus ticks significantly higher than in R. sanguineus ticks (P< 0.05) (Fig. 3B). Moreover, the rates of HRTV1, LHTV, GTQV, and BDPTV were higher in ticks collected from bovine than ovine hosts, while those of MIV, WEPMV and JMTV were much higher in ticks from ovine than bovine hosts (Fig. 3C). The viral loads of HRTV1, LHTV, BDPTV, MIV, and JMTV were significantly higher in individual ticks from sheep than in those from cattle, while ticks from cattle exhibited higher loads of GTQV than those from sheep. The load of WEPMV was higher in ticks from sheep than from cattle, but this was not significant (Fig. 3D).

Fig. 3.

Fig. 3

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Comparative analysis of the correlation of tick species, host and geographical factors with distribution of emerging TBVs in Hainan Island. The positive detection rates of different viruses among ticks from different tick species (A), host species (B) are shown. Red circles represent R. sanguineus, yellow circles represent R. microplus, blue circles represent cattle-derived ticks, and navyblue circles represent sheep-derived ticks. The numerical values of virus positivity are annotated on the corresponding circles. Comparison of viral copies carried by tick individuals from various tick species (C) and host species (D) are shown. Statistical differences in viral loads were assessed using the Wilcoxon rank-sum test, and significance levels are indicated as follows: ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

Co-infection with viruses in individual ticks

We noted that 42 of the 112 viral RNA-positive ticks (36.6%) were detected positive for two or more of the tested viruses, which suggested that these ticks were co-infected with different viruses (Table 3). The seven tested viruses were all involved in co-infections. HRTV1 was involved in 21 of the 42 co-infections (50%), while BDPTV was involved only in five co-infections (11.90%). Ticks with viral co-infection were found in eight counties, namely Wenchang (12/42, 28.6%), Lingshui (10/42, 23.8%), Qiongzhong (7/42, 16.7%), Haikou (5/42, 11.9%), Tunchang (4/42, 9.5%), Basha (2/42, 4.8%), Lingao (1/42, 2.38%), and Wanning (1/42, 2.38%).Thirty-two of the 42 co-infected ticks (76.2%) were positive for two viruses, eight ticks (19.0%) were positive for three viruses, and two ticks, one each from Haikou and Lingshui, were positive for four viruses (4.8%). HRTV1, MIV, and LHTV, which exhibited a relatively high prevalence in individual ticks, were also the most likely to be involved in co-infections. We also identified several significant virus-virus interactions (Supplementary Fig. S5, Supplementary Table S5). Most notably, a strong positive association was observed between MIV and JMTV (Phi = 0.34, Q = 0.0047), indicating a synergistic co-infection pattern. Conversely, a strong negative association between MIV and HRTV1 (Phi = −0.44, q = 1.80 × 10−5) as well as MIV and LHTV (Phi = −0.32, q = 2.12 × 10−4) suggests potential competitive exclusion.

Table 3.

Prevalence of co-infections identified in this study among individual ticks from Hainan Island.

Locations Tick species Host species No. of co-infected ticks/total HRTV1 (n (%)∗) WEPMV (n (%)∗) LHTV (n (%)∗) BDPTV (n (%)∗) JMTV (n (%)∗) MIV (n (%)∗) GTQV (n (%)∗) No. of co-infecting viruses
2 3 4
Baisha R. sa Bovine 2/35 0 0 0 0 0 2 (4.76%) 2 (4.76%) 2 (4.76%) 0 0
Chengmai R. sa Bovine 0/17 0 0 0 0 0 0 0 0 0 0
Haikou R. mi Bovine 5/40 5 (11.90%) 0 5 (11.90%) 1 (2.38%) 2 (4.76%) 0 0 3 (7.14%) 1 (2.38%) 1 (2.38%)
Lingao R. mi Bovine 1/24 0 0 0 1 (2.38%) 0 0 1 (2.38%) 1 (4.16%) 0 0
Lingshui R. sa Ovine 10/25 6 (14.29%) 9 (21.43%) 5 (11.90%) 1 (2.38%) 1 (2.38%) 2 (4.76%) 0 7 (28.0%) 2 (8.00%) 1 (4.00%)
Qionghai R. sa Bovine 0/2 0 0 0 0 0 0 0 0 0 0
Qiongzhong R. mi Bovine 7/42 6 (14.29%) 0 4 (9.52%) 2 (4.76%) 2 (4.76%) 0 4 (9.52%) 3 (7.14%) 4 (9.52%) 0
Tunchang R. sa Bovine 4/17 4 (9.52%) 0 4 (9.52 %) 0 0 0 0 4 (23.52%) 0 0
Wanning R. sa Ovine 1/30 0 0 0 0 0 0 0 1 (3.33%) 0 0
Wenchang R. sa Ovine 11/34 0 0 0 0 10 (23.80%) 11 (26.19%) 2 (4.76%) 10 (25.64%) 1 (2.56%) 0
R. sa Bovine 1/5 0 0 0 0 0 1 (2.38%) 1 (2.38%) 1 (2.56%) 0 0
Total 42 21 (50%) 9 (21.43%) 18 (42.86%) 5 (11.90%) 15 (35.71%) 16 (38.10%) 10 (23.81%) 32 (76.19%) 8 (19.05%) 2 (4.76%)
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R. sa: R.sanguineus, R. mi: R.microplus ∗ % of total ticks collected at this site.

Serologic evidence of human exposure to tick-borne viruses

When tested by Luminescence immunoprecipitation system (LIPS) assay, serum samples from four of the 170 fever of unknown origin (FUO) patients had antibody specific for one of three viruses, including one sample positive for LHTV (Patient 1), one for JMTV (Patient 2), and two for GTQV (Patients 3 and 4) (Fig. 4A), which were confirmed by Western blotting (Fig. 4B) showing specific antibody responses to the respective viral antigens.

Fig. 4.

Fig. 4

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Serological evidence of exposure to JMTV, LHTV and GTQV among FUO patients from Hainan Island. A Serological evidence of JMTV, LHTV and GTQV exposure based on LIPS assay. The luciferase activity (LU/mL) of different viruses in serum samples of FUO patients and healthy people are represented by circles of different colors. The threshold for LIPS detection is shown by a dotted line. B Western blot assay was performed to verify the specificity of LIPS serological screening results. Positive control validation was used to verify the success and size of viral protein expression in cell lysis after transfection. Protein markers are shown on the left, and the patient's number is marked above. Immunofluorescence assays (IFA) and microneutralization tests (MNT) were conducted to detect JMTV antibodies. C JMTV-infected BME/CTVM23 cells were fixed, Patient 2 serum sample was employed as primary antibody, the positive control utilized α-YGTV-CP as the primary antibody, and uninfected BME/CTVM23 cells were the negative control. The JMTV antigens were labeled with green fluorescence, cell nuclei were stained blue. D MNT were conducted to assess serum neutralizing activity. The serum sample was heat-inactivated, diluted 1:16, and mixed with JMTV at a concentration of 100 TCID50 for the MNT. BME/CTVM23 cells infected with 100 TCID50 of JMTV were used as the negative control. Three replicates (Wells 1, 2, and 3) were set up for both the MNT and negative control. The MNT results were evaluated using immunofluorescence assay (IFA) to visualize viral infection within the cells. The scale bar in all images represents 100 μm.

Patient 1 was a 48-year-old man from Haikou who had been suffering from hypertension for 10 years. Patient 2 was a 33-year-old woman living in Haikou and having no chronic diseases. Patient 3 was a 44-year-old man from Wanning, and Patient 4 was a 63-year-old man from Ding'an with chronic hepatitis B infection. All four patients had been admitted to the hospital for having fever and common clinical manifestations such as headache and chills, and they had significantly elevated WBC counts and increased levels of C-reaction protein (CRP) in blood routine tests (Supplementary Table S6). All four patients denied a history of tick bites. Patient 4 was diagnosed with Rickettsia infection based on a colloidal gold assay which was used to detect Rickettsia tsutsugamushi antibody (Wantai BioPharm, Beijing, China). The four hospitalized patients were treated with doxycycline or penicillin, and eventually discharged after an average hospital stay of 7 days. Furthermore, the antibody response to JMTV from the serum sample of Patient 2 was verified by IFAs, which suggested the presence of IgM and IgG antibody responses to JMTV (Fig. 4C). Neutralization activity against JMTV was also identified. It showed that JMTV infection was inhibited in BME/CTVM23 cells after incubation with the diluted serum sample (Fig. 4D). These results suggested that Patient 2 may have had an acute infection with JMTV.

Discussion

Hainan Island, the only tropical island located in the South China Sea, may have experienced and still faces an increasing threat from endemic diseases such as tick-borne spotted fever and Lyme disease caused by tick-borne pathogens (Wu, J. 2014; Zhao, G.P. et al., 2021). The virome analyses of pooled ticks identified a diverse community of TBVs, including at least 27 viral species belonging to eight viral families. Of these families, the Flaviviridae, Orthomyxoviridae, Phenuiviridae, Rhabdoviridae, and Chuviridae presented a relatively high viral abundance, which was similar to the findings of previous studies on tick viromes from other locations in China (Guo et al., 2022; Ni et al., 2023; Shi et al., 2021). One recent study has preliminarily addressed tick-borne viromes across four counties in Hainan (Wang et al., 2024). Nevertheless, our study broadened the surveillance to 15 of the total 19 counties in Hainan, and one novel virus (WEPMV) was identified, suggesting the presence of more diverse viruses there.

GTQV was identified in both R. microplus and R. sanguineus ticks and was found to be closely related to Wellfleet Bay virus, which has been associated with extensive die-offs of common eider ducks in Massachusetts (Allison et al., 2015), and Cygnet River virus, which has been identified as the causative pathogen of a severe duck diarrheal disease outbreak in Australia (Kessell et al., 2012). Further, in our study, GTQV was found in R. sanguineus, a three-host tick capable of infesting a wide range of domestic and wild hosts including dogs, cats, rodents, cattle, horses, goats, humans and, occasionally, birds (Dantas-Torres, 2010; Szabo et al., 2012). The genus Quaranjavirus is known to be transmitted to birds mainly by argasid ticks, and have been isolated from ticks and seabirds in Egypt (Al-Khalifa et al., 2007), South Africa (Sang et al., 2006), and Nigeria (Kemp et al., 1975). The large population of land-based migratory birds and small ruminants in Hainan Island, together with agricultural practices and fodder, may have led to the transmission of GTQV from birds to mammals such as cattle and sheep through ticks. BEFV is a member of the family Rhabdoviridae and is an economically significant arbovirus that causes ephemeral fever in cattle and water buffalo (Murphy et al., 1972; Trinidad et al., 2014). Here, WEPMV, which is closely related to BEFV, was identified in fully engorged R. microplus ticks from cattle. Accordingly, the potential for WEPMV to be transmitted between ticks and cattle, and its association with bovine ephemeral fever, warrant further investigation among livestock on the island. In accordance with previous reports (Dincer et al., 2019; Wu et al., 2023), JMTV was predominantly identified in R. microplus ticks, suggesting a pivotal role for R. microplus in vectoring JMTV across a vast geographical area. Given that JMTV has been linked to human febrile illness (Taniguchi, 2019), it is imperative to examine its epidemiological characteristics in Hainan Island.

Although a lot of potential factors, such as tick engorgement status, host age and sex, and sampling time, may affect the viral populations that we detected among these ticks, the results of clustering analyses of tick viromes indicated that the distribution of the viruses in Hainan Island was correlated with tick species more than with geographic locations. The prevalence of seven viruses of high abundance in the tick pools was investigated among individual ticks. The results demonstrated that HRTV1 exhibited the highest prevalence rate, exceeding 15%, while GTQV demonstrated the lowest prevalence, at approximately 4%. These findings were unexpected, as the rates found in our study were approximately two to eight times higher than the previously recognized minimum infection rates of JMTV (1.4 %) and other phleboviruses (0.67%–3.28%) in pooled ticks, as reported in previous studies in the mainland of China (Pang et al., 2022; Shao et al., 2020; Wang, A. et al., 2021). The results may be influenced by the sensitivity of the detection methods employed and may also be contingent on the sample sizes, sampling locations, and tick species under consideration. Nevertheless, the results also indicate that the infection rates of viruses in individual ticks could be higher than previously recognized.

The prevalence rates of the seven tested TBVs were compared between the two tick species and also the two animal hosts. Our findings indicated that R. sanguineus and R. microplus ticks in Hainan Island exhibited disparate capabilities in harboring viruses. Specifically, the higher viral positive rate (R. sanguineus: 6.3%–29.8%; R. microplus: 2.3%–16.3%) and a greater number of co-infection events (R. sanguineus: 29; R. microplus: 13) were observed in R. sanguineus. This was expected, as R. sanguineus is a three-host tick, which has chances to be exposed to multiple viruses, while R. microplus is a one-host tick that always feeds on one same host. In contrast, R. microplus exhibited a higher viral abundance (R. sanguineus: ∼101−4 viral copies per tick; R. microplus: ∼101−6 viral copies per tick). The host species may also be a determining factor in the viral communities present in ticks. Ticks collected from sheep were more likely to harbor MIV, WEPMV, and JMTV, while ticks from bovine had a higher likelihood of carrying HRTV1, LHTV, GTQV, and BDPTV. Of these viruses, WEPMV, which is closely related to the bovine disease-associated BEFV (Li et al., 2015), was predominantly detected in ticks collected from sheep in this study. A previous study reported a seropositive rate of 14% for BEFV among cattle in Hainan in 2015 (Li et al., 2015). This highlights the necessity for further investigation into the potential cross-reaction between WEPMV and BEFV, as well as the importance of conducting additional surveys of both viruses among livestock in Hainan. While the impact of variations in individual ticks (such as size and engorgement) on viral detection cannot be overlooked, our study conducted across a wide area of Hainan Island highlights the significant role that ticks and host species play in the distribution of TBVs. Although our study revealed the presence of multiple viruses in ticks, the specific transmission routes (horizontal or vertical) remain to be determined. Future studies are needed to investigate the transmission dynamics of these viruses to better understand their epidemiology and to evaluate their spillover potential.

The viral abundance, represented by the viral RNA loads in individual ticks, indicated the potential for persistent virus maintenance within tick populations. This persistence offers insight into virus–tick interaction dynamics and possible spillover risk to hosts. R. sanguineus exhibited significantly higher viral loads of HRTV1, LHTV, BDPTV, MIV, and JMTV compared to R. microplus, suggesting differences in viral replication efficiency or host adaptation between these tick species. Moreover, the sheep-derived ticks harbored higher viral loads as for HRTV1, LHTV, BDPTV, MIV, and JMTV, whereas cattle-associated ticks showed higher GTQV RNA loads, suggesting a role of hosts in viral abundance of ticks. These data provide a basis for further assessment of virus spillover risks in Hainan Island. Subsequently, we found that 36.6% of virus-positive ticks were co-infected with multiple viruses, revealing complex viral ecology in this tropical region. Co-infection of multiple viruses within a single tick could make a gene pool, which may facilitate viral recombination and thus affect viral evolution, potentially leading to the generation of pathogenic variants. Co-infection may also alter transmission dynamics via viral interactions (synergistic or competitive), because many viruses could be tick-restricted viruses and are persistently existing in ticks. Moreover, the co-infection of TBVs like SFTSV with rickettsia has been reported in patients previously, suggesting the possibility of infection by multiple pathogens after a tick bite, although no cases with infection with two different TBVs have been identified so far. Nevertheless, our findings highlight the need to characterize co-infection of viruses and to investigate the mechanisms underlying co-infections, which may benefit the strategies for specific virus surveillance and control among ticks in the future.

The serological response to seven of the TBVs was initially evaluated among a group of FUO patients. Four patients exhibited positive antibody responses to three of the viruses, LHTV, JMTV and GTQV, resulting in a positive rate of 2.35% among the 170 tested patients. Further identification of serologic responses to JMTV in one serum sample indicated that this patient might have an acute infection with JMTV. However, we tried but failed to detect JMTV RNA in serum samples of this patient. Alternatively, the patient may have been in the acute to recovering phase, as neutralizing antibodies were identified. Unlike its presence in ticks of globally wide distribution, there have been only a few human cases confirmed with JMTV infection, because they exhibited antibody response and positive viral RNA at the eschar site of the tick bite (Jia et al., 2019). Since we only identified one patient with neutralizing antibody to JMTV, the link between JMTV infection and clinical manifestation still needs to be clarified once more cases are identified.

Epidemiological features of the seven tested TBVs are summarized collectively and presented on a map of Hainan Island (Fig. 5). This shows that five viruses were present in ticks from Lingshui and Qiongzhong, suggesting that multiple viruses may be prevalent in these regions. GTQV, LHTV, and JMTV, which may be potential pathogens and to which humans may have been exposed, were widely distributed throughout the island (Table 2 and Fig. 5). In Haikou, both LHTV and JMTV were detected in ticks, and antibodies specifically reacting with antigens of these two viruses were identified in serum samples of febrile patients, indicating spillover and spread of tick-borne viruses to humans in the area. Consequently, further research on the transmission and impact of these viruses is required, along with the implementation of enhanced monitoring and control measures in these regions to reduce the risk of virus transmission.

Fig. 5.

Fig. 5

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Overview of the ecological distribution characteristics of seven TBVs found in Hainan Island based on screening individual ticks. The positive detection rates of HRTV1, LHTV, BDPTV, JMTV, MIV, WEPMV, and GTQV are shown as bar plots. The locations tick and host species are indicated by different icons (red: R. sanguineus, yellow: R. microplus, hexagon: ovine, circle: bovine, square: canine). The blue shaded area represents PCR evidence of TBV prevalence in the region, while the diagonally striped area represents NGS evidence of TBV prevalence in the region. The star icon indicates that the corresponding virus antibody was detected in fever patients in a region. Bar charts created using R v4.3.3 package ggplot2 (version 3.5.1) were added to present the prevalence rates of viruses in respective locations. The map has been reviewed and approved by Hainan Administration of Surveying Mapping and Geoinformation, with approval number: Qiong S (2026) No. 036.

There are limitations of this study. It is a relatively small sample size that was used to investigate the prevalence of viral infection in individual ticks. While this approach allowed for precise rates to be determined, it may have resulted in an underrepresentation of the true distribution of these viruses. Regarding the human serological survey, the cohort size (n = 170) and the low number of seropositive individuals (n = 4) are limited. Therefore, the estimated seroprevalence should be considered preliminary, and broader generalizations regarding population-level exposure should be made with caution. Although our study provides insights into the co-infection patterns of seven selected viruses, the complexity of viral co-infections in ticks is likely greater due to the potential presence of other undetected viruses. Unfortunately, the lack of isolated laboratory strains of LHTV and GTQV prevented an investigation into the potential presence of neutralizing antibodies to these viruses in the patients. Nevertheless, the serological evidence gave rise to concerns regarding the medical significance of LHTV and GTQV.

Conclusion

Our study revealed some fundamental information about tick viromes in Hainan Island, and demonstrated the prevalence of novel TBVs and new TBV strains at high rates in individual ticks. The sero-prevalence study suggested TBV exposure in humans and underscored the possible public health significance of JMTV, LHTV, and GTQV. Overall, the results suggested the areas with potential risks of TBV spread and spillover in Hainan. Extensive surveillance of the potential zoonotic TBVs, including JMTV, LHTV, and GTQV, is suggested to be performed subsequently, which would benefit designing the strategies for controlling TBV spreading and for the first aid to emerging tick-borne viral diseases in Hainan.

Materials and methods

Collection of ticks from domestic animals and serum samples from patients

Ticks were collected from the body surface of cattle, sheep and dogs from 15 of the 19 counties in Hainan, China, in 2018 and 2021 (Supplementary Table S1). The collection process involved carefully examining the animals' skin and removing ticks using fine-tipped forceps to minimize damage to the specimens. Tick species were first identified morphologically by an experienced technician based on key morphological characteristics such as body size, color, and the presence of specific anatomical structures. The identification was conducted using a stereomicroscope and referenced to established taxonomic keys and literature (Xiao et al., 2024). For molecular confirmation, selected ticks with morphological features representative of R. sanguineus or R. microplus were subjected to DNA extraction. DNA was extracted from individual ticks using a commercial DNA extraction kit (TAKARA, Japan), following the manufacturer's protocol. The extracted DNA was then used as a template for PCR amplification targeting the mitochondrial cytochrome C oxidase subunit-I (cox1) gene, using primers and conditions as previously described (Zhang et al., 2021, Zhang et al., 2021).

Serum samples from 170 patients diagnosed with fever of unknown origin (FUO) (≥ 38.3 °C for at least one week without definitive diagnosis) were collected between 2017 and 2019 from the Affiliated Hospital of Hainan Medical University, Hainan Province. A team of trained health personnel provided all participants with comprehensive information about the research process. Sociodemographic data, including age, gender, occupation, and history of animal contact, were obtained through structured interviews. Serum samples from twenty healthy individuals archived in the National Virus Resource Center (NVRC, Wuhan, China) were used as controls. None of the samples included in this study had previously been included in another study.

Library preparation, RNA-seq sequencing and bioinformatic analyses

Ticks were washed three times with sterile RNA- and DNA-free phosphate buffered saline (PBS) and grouped (10–40 individuals per pool) according to tick species, sampling location, and hosts from which they were collected. Ticks were homogenized, and total RNA was prepared using Trizol (Life Invitrogen, USA) as previously described (Zhang, Y. et al., 2021). For each pool, 1 μg of RNA was used for library preparation and subjected to RNA-Seq sequencing using a HiSeq 3000 sequencer according to the manufacturer's instructions (Illumina, San Diego, USA) as previously described (Zhang, Y. et al., 2021). Raw sequencing data in FASTQ format were first subjected to quality control using FastQC (v0.20.0) to assess sequence quality. For host read removal, raw paired-end reads were aligned to the Ixodoidea genome database (downloaded from GenBank) using Bowtie2 (v2.3.3.1). The reference database included the following tick species with their corresponding GenBank assembly accessions: Ixodes persulcatus (GCA_013358835.2), Haemaphysalis longicornis (GCA_013339765.2), Dermacentor silvarum (GCF_013339745.2), Hyalomma asiaticum (GCA_013339685.2), Rhipicephalus sanguineus (GCA_013339695.2), and Rhipicephalus microplus (GCA_013339725.2) (Jia et al., 2020). Unaligned read pairs were retained as host-filtered reads. Filtered reads were de novo assembled into contigs using Trinity (v2.5.1) with a maximum memory of 300 GB and 48 threads. For gene expression quantification, the cleaned reads were mapped back to the assembled contigs using RSEM via the align_and_estimate_abundance.pl script provided by Trinity, yielding transcript per million (TPM) values. To functionally annotate the assembled contigs, BLASTn searches were performed against the NCBI nucleotide (nt) database with parameters-evalue 1e-5-max_target_seqs 1, and DIAMOND BLASTx searches were run against the NCBI non-redundant protein (nr) database with equivalent stringency. Both BLASTn and BLASTx results were exported in custom format including query ID, subject ID, identity, alignment length, e-value, bitscore, title, and taxonomy ID (staxids). The annotation results were merged with TPM values to generate a unified annotation table for downstream analysis. To identify viral sequences, taxonomy IDs from BLAST results were extracted and annotated using TaxonKit (v0.10.1). Taxonomic lineages were screened to retain only those classified under the “Viruses” (TaxID: 10239; corresponding to 11,273 species across 3,818 genera per ICTV, 2024), thereby including all established DNA and RNA viral lineages while excluding subviral agents. Contigs annotated as viruses in either BLASTn or BLASTx were retained as putative viral contigs, along with their TPM values. The rarefaction curve was generated using the RarefactionCurve.r script, and alpha diversity was analyzed using the AlphaDiversity.r script. NMDS and LEfSe analyses were performed using the online platform available at https://www.bic.ac.cn/BIC/#/(Chen et al., 2024) (Supplementary Fig. S6).

Reference sequences for phylogenetic tree construction were selected based on the NCBI Taxonomy database. Taxonomic information, including viral families, genera, and species, was retrieved to understand the phylogenetic relationships of the viruses. Representative viral genera and species were chosen to cover those annotated in our study and to reflect diversity in geographic distribution, host range, and pathogenicity. Sequences were retrieved and filtered using the NCBI Taxonomy database and BLAST, with quality assessment ensuring genomic integrity. PhyloSuite (Zhang et al., 2020) was used to conduct, manage and streamline the analyses with the help of several plug-in programs: Sequence was aligned with MAFFT (Katoh and Standley, 2013) using ‘--auto’ strategy and normal alignment mode. Gap sites were removed with trimAl (Capella-Gutierrez et al., 2009) using “-automated1” command. ModelFinder (Kalyaanamoorthy et al., 2017) was used to select the best-fit model using BIC criterion. Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al., 2015) under the rtREV + I + G4+F model for 10000 ultrafast (Minh et al., 2013) bootstraps. According to the taxonomy disciplines announced by the International Committee on Taxonomy of Viruses (ICTV), the criteria used to define novel virus species vary among different viral families and genera. These species demarcation criteria, as described in the relevant ICTV Reports, are based on comparative analyses of nucleotide and amino acid sequences, particularly those of the RNA-dependent RNA polymerase (RdRp) (King et al., 2011).

Detection of viruses in individual ticks

The investigation of virus prevalence in individual ticks was performed by absolute quantification using virus-specific qRT-PCR. Total RNA was extracted from each tick using Trizol reagent. Virus-specific primers (Supplementary Table S7), designed based on conserved regions of the complete viral genomes, were used for amplification. To enable accurate quantification, RNA standards were synthesized in vitro for each virus based on the identified sequences and serially diluted (102–108 copies/μL) to generate external standard curves. The measured Ct values were converted to viral RNA copy numbers by interpolation from these curves (Zhang, Y. et al., 2021).

Luciferase immunoprecipitation system assays (LIPS)

The nucleoprotein (NP) ORFs of viruses identified by RNA-sequencing from tick pools, including Lihan tick virus (LHTV, 1431 nt), Mivirus (MIV, 1417 nt), brown dog tick phlebovirus (BDPTV, 1272 nt), Guangdong tick quaranjavirus (GTQV, 1572 nt), Huanggang Rhabd tick virus 1 (HRTV1, 1272 nt), Wenchang ephemerovirus (WEPMV, 1220 nt), and Jingmen tick virus (JMTV, 765 nt) were synthesized by Beijing Genomics Institution (Beijing, China) and confirmed by Sanger sequencing. Each of the above viral protein fragments was individually cloned into the pREN2 plasmid fused with Renilla luciferase (Ruc) as previously described (Zhang, Y. et al., 2021). The positive controls and positive thresholds for LIPS testing were set up and the assays were carried out as previously described (Chen et al., 2024; Zhang, Y. et al., 2021). Positive serum samples having an antibody response specific for any of the viral antigens were further verified by Western blot using HEK293T cells transfected with plasmids expressing the respective viral protein and using Rabbit monoclonal Anti-human IgG (AB125909, Abcam, Cambridge, UK) as the secondary antibody.

Immunofluorescence assays (IFA) and microneutralization tests

The tick cell line BME/CTVM23, derived from R. microplus, was acquired from the Tick Cell Biobank at the University of Liverpool and was maintained as previously described (Alberdi et al., 2012). JMTV strain CQ/2022 used in this study was deposited in NVRC (Wuhan, China; no. IVCAS6.9297) and was propagated in BME/CTVM23 cells as described previously (Jia et al., 2019). For immunofluorescence assay (IFA), JMTV-infected cells were fixed, permeabilized, and incubated with human serum samples (1:50 dilution in PBS), followed by goat anti-human IgG H&L-FITC (Abcam, Cambridge, UK) or goat anti-human IgM H&L-FITC (Abcam) to detect antibody responses. JMTV antigen was detected using a cross-reactive rabbit polyclonal antibody against Yanggou tick virus capsid protein (α-YGTV-CP, NVRC no. CSTR:16533.09.IVCAS11.0034) as the primary antibody, and goat anti-rabbit IgG H&L-FITC (Abcam) as the secondary antibody. Uninfected cells served as negative controls, and nuclei were stained with Hoechst 33258 (Beyotime, Shanghai, China).

For microneutralization assays, heat-inactivated serum samples (1:16 dilution) were mixed with an equal volume of JMTV suspension (100 TCID50) and incubated at 37 °C for 1.5 h. The mixture was then added to BME/CTVM23 cells (50%–60% confluence in 96-well plates) and incubated at 27 °C for 2 h. After replacing the medium, cells were cultured at 27 °C for 3 days. JMTV infection was visualized by IFA, and images were acquired using a fluorescence microscope (EVOS M7000; Invitrogen, Carlsbad, CA, USA). All tests were performed in triplicate.

Data analysis

The characteristics of the distribution and load of each virus species among tick populations were analyzed using the R Studio ggplot2 package v3.3.3. Comparative analysis of viral loads between different tick species, host species and locations was measured using the Wilcoxon test. In all analyses, values of P of 0.05 were considered statistically significant.

Data availability

The raw sequencing datasets for the current study are available in the NCBI SRA under the Bioproject with accession code PRJNA1139691 (www.ncbi.nlm.nih.gov/bioproject/1139691) and also in the Science Data Bank under accession code 10.57760/sciencedb.11378. Viral sequences analyzed in this study have been deposited in GenBank with accession numbers PQ151130–PQ151136. The analysis scripts are publicly available at GitHub: https://github.com/Yaohui-Fang/Tick-Virome-Metagenomic-Analysis-Pipeline.

Ethics statement

The study was approved by the Ethics Committee of Hainan Medical University, which complied with the principles of the Declaration of Helsinki (Ethical approval number: HYLL-2020-061). and the Institutional Review Board of the Wuhan Institute of Virology, CAS (Approval Number: WIVH33202102). Informed consent was obtained from all participants, either in written or verbal form, according to their level of literacy.

Author contributions

You Zhang: methodology, formal analysis, writing-original draft, writing-review & editing. Yuan Bai: methodology. Jun Ni: methodology. Junming Shi: methodology. Yanfang Zhang: methodology. Lesley Bell-Sakyi: methodology, writing-review & editing. Xiaoli Wu: methodology. Changhua He: methodology. Fei Deng: conceptualization, project administration, writing-review & editing. Feifei Yin: conceptualization, project administration, writing-review & editing. Shu Shen: conceptualization, project administration, writing-review & editing. Yaohui Fang: methodology, formal analysis, writing-review & editing.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Prof. Fei Deng is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article.

Acknowledgments

This work was supported by the National Key R&D Program of China (2022YFC2305100 to YFZ), the National Natural Science Foundation of China (U22A20363 to JMS), the Hainan Provincial Natural Science Foundation of China (2023-823QN352 to YZ), Nanhai Junior Talent Program of Hainan Provincial Health Commission (HNXX-WJW-2023013 to YZ), Hainan Provincial International Science and Technology Cooperation Research and Development Project (GHYF2024021 to FFY), the Youth Project of the Wuhan Institute of Virology, Chinese Academy of Sciences (2023QNTJ-03 to SS), the International Partnership Program of Chinese Academy of Sciences (088GJHZ2022022FN to SS and LBS), the Wellcome Trust (223743/Z/21/Z to LBS). We would like to thank Miss. Jiayin Jin in the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences for her supporting to the sample management and preservation.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virs.2026.02.008.

Contributor Information

Feifei Yin, Email: yinfeifeiff@163.com.

Shu Shen, Email: shenshu@wh.iov.cn.

Yaohui Fang, Email: fyh@wh.iov.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Material
mmc1.docx (14.5MB, docx)

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Supplementary Figure S1

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Supplementary Figure S3.

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Supplementary Figure S4.

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Supplementary Figure S5.

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Supplementary Figure S6.

Supplementary Figure S6

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material
mmc1.docx (14.5MB, docx)

Data Availability Statement

The raw sequencing datasets for the current study are available in the NCBI SRA under the Bioproject with accession code PRJNA1139691 (www.ncbi.nlm.nih.gov/bioproject/1139691) and also in the Science Data Bank under accession code 10.57760/sciencedb.11378. Viral sequences analyzed in this study have been deposited in GenBank with accession numbers PQ151130–PQ151136. The analysis scripts are publicly available at GitHub: https://github.com/Yaohui-Fang/Tick-Virome-Metagenomic-Analysis-Pipeline.

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