Prevalence and Diversity of Severe Fever with Thrombocytopenia Syndrome Virus and Co-Infection with Babesia microti in Ticks from Central and Eastern Parts of China

TL;DR

This study found that ticks in central and eastern China commonly carry SFTSV and often co-infect with Babesia microti, especially in Haemaphysalis longicornis.

Contribution

The study reports the prevalence and diversity of SFTSV and co-infection with Babesia microti in multiple tick species across China.

Findings

SFTSV was detected in all five tick species, with Haemaphysalis longicornis being the most prevalent carrier.

Babesia microti co-infection was found in SFTSV-positive ticks, particularly in Haemaphysalis longicornis.

The highest SFTSV-positive pool rate was observed in Xinyang, Henan (20.0%).

Abstract

This study was conducted to investigate tick species that may harbour severe fever with thrombocytopenia syndrome virus (SFTSV) and Babesia microti in the provinces of Henan, Anhui, and Zhejiang, as well as in Shanghai in the central and eastern parts of China. Between March and September 2023, 721 pools of ticks were collected belonging to three genera and five species: Haemaphysalis longicornis (n = 612; 84.9%), Haemaphysalis fusca (n = 94; 13.0%), Rhipicephalus microplus (n = 10; 1.4%), Amblyomma testudinarium (n = 3; 0.4%), and Haemaphysalis wellingtoni (n = 2; 0.3%). The SFTSV-positive pool rate was 20.0%, 13.0%, 5.8%, and 4.1% in Xinyang, Henan; Songjiang, Shanghai; Lu’an, Anhui; and Zhoushan, Zhejiang, respectively. SFTSV was detected in all five tick species collected. Among the SFTSV-positive pools, H. longicornis constituted the highest proportion (83.9%, 78/93), whereas pools containing R. microplus and H. wellingtoni each represented the lowest proportion (1.1%, 1/93). Babesia microti was assayed only in these SFTSV-positive tick pools, and co-infection was found in both H. longicornis and H. wellingtoni, though it was most frequent in H. longicornis.

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1. Introduction

Ticks parasitize a wide range of hosts including mammals, reptiles, birds, and amphibians [1]. They are widely distributed across China, and inhabit diverse natural environments such as forests, grasslands, and hills. Ticks are known vectors for a variety of zoonotic pathogens, including those that cause severe fever with thrombocytopenia syndrome (SFTS), babesiosis, Lyme disease, human granulocytic anaplasmosis (HGA), and others [2]. The SFTS virus (SFTSV) has been gradually increasing in prevalence since it was first detected in China in 2009 [3]. To date, 27 provinces in China have reported human cases of SFTSV, with Zhejiang, Henan, and Anhui provinces accounting for the majority of cases. In 2022, human-to-human transmission of the SFTSV was reported in Xinyang, Henan, China [4]. SFTS has increased in the central and eastern regions of China [5]. These regions, particularly the provinces of Henan, Anhui, and Zhejiang, exhibit the highest incidence of SFTSV. Morbidity and mortality of SFTS is high and poses a serious public health threat [6]. There are considerable geographical variations in the case-fatality rate (CFR) of SFTS, which ranges from 5.3% to 16.2% in China, 27% in Japan, and 23.3% in Korea [7]. Although several virus genotypes have been identified in Eastern China, there is a paucity of systematic investigations into the viruses carried by vector ticks in the region [8,9]. Variability in SFTSV has led to the emergence of numerous recombinant strains over time. The recombinant strain of the S-fragment was the most prevalent, reaching a frequency of up to 9.3% and contributing to the widespread SFTSV epidemic [10]. Thus, the present study focused on the amplification of some fragments of the S gene of SFTSV. While human case reports and hospital-based surveillance have outlined the expanding geographic range of SFTSV [11], data on the virus’s prevalence and genetic makeup within its primary arthropod vectors—ticks—remain fragmented, particularly in hyperendemic regions of central and eastern China [12]. Systematic surveillance of tick populations is crucial to understand enzootic transmission cycles, identify potential bridge vectors, and map the genetic landscape of circulating viral strains, which can inform public health risk assessments.

Babesia microti, a protozoan parasite transmitted by hard ticks, is also a significant zoonotic threat [13]. It is closely associated with rodents and is transmitted by a wide range of tick species. The first human case of Babesia microti was identified in California, USA, in 1969, which subsequently led to the documentation of infections in North America, Europe, and Japan. In 2012, the first human case in China was diagnosed in Zhejiang Province [14]. Since then, instances of human infections with Babesia microti has been reported in Yunnan, Henan, Jiangsu, and other locations across China [15], including two additional cases in Heilongjiang and Zhejiang provinces [16].

SFTS and babesiosis have very similar clinical symptoms, including fever and thrombocytopenia. A retrospective survey of SFTS patients in Henan revealed that co-infection with Babesia microti occurred in 9.8% of cases [17]. This suggests the existence of vectors that facilitate co-infection of SFTSV with Babesia microti, although further research is required to substantiate this hypothesis. The objective of this study was to detect SFTSV in tick vectors from the central and eastern regions of Henan, Anhui, Zhejiang, and Shanghai. Furthermore, this study investigated the potential co-infection of SFTSV with Babesia microti, to provide a foundation for the development of effective control strategies for emerging tick-borne diseases.

2. Materials and Methods

2.1. Tick Collection

Ticks were collected from March to September 2023 using the standard drag and flag methods in the central and eastern parts of China. Collection sites included grasslands and bushes across (a diameter of 10–50 m site) each county in Xinyang (Henan Province, 32.149° N, 114.091° E), Lu’an (Anhui Province, 31.730° N, 115.929° E), Zhoushan (Zhejiang Province, 29.987° N, 122.203° E), and Songjiang (Shanghai, 31.034° N, 121.223° E). We conducted the collection at each site for 30 min and it lasted for three days. The number of tick samples collected from each site did not exceed 50. The distribution of tick species by region is detailed in Table S1.

2.2. Identification of Tick Species and Developmental Stages

Based on microscopic observations and molecular biology methods, as described in [18], ticks were classified into different species and developmental stages [19]. For nymphs that were difficult to identify morphologically, molecular identification was performed by amplifying and sequencing a fragment of the internal transcribed spacer (ITS) gene. PCR amplification was conducted using universal primers ITS-F (5′-CGAGACTTGGTGTGAATTGCA-3′) and ITS-R (5′-TCCCATACACCACCACATTTCCCG-3′) [20]. The PCR conditions were: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s; with a final extension at 72 °C for 7 min. The resulting sequences were compared with reference sequences in the GenBank database using the BLAST algorithm on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 10 January 2026). The sequences obtained from ITS amplification are included in Table S2.

2.3. DNA and RNA Extraction

A total of 721 tick pools were constituted for nucleic acid extraction and pathogen screening, based on developmental stage: larvae (up to 15 individuals per pool), nymphs (up to 3 individuals per pool), and adults (processed individually, i.e., one per pool). For each pool, individual ticks were washed with 75% ethanol (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) and ground separately. The homogenates from all individuals within the same pool were then combined for nucleic acid extraction using a DNA/RNA rapid nucleic acid extraction kit with the magnetic bead method (Shanghai Barbarian Biotechnology Co., Shanghai, China). cDNA was synthesized from the extracted RNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China), which includes a genome cleanup module for complete removal of residual genomic DNA. DNA and cDNA were stored at –80 °C.

2.4. Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Primers (SFTSV-F-TGTCAGAGTGGTCCAGGATT, SFTSV-R-ACCTGTCTCCTTCAGCTTCT, and SFTSV-S-TGGAGTTTGGTGAGCAGCAGC) were designed for the S-fragment of SFTSV [21]. The amplification conditions were: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min, for 45 cycles. The 20 μL reaction system consisted of 10 μL of Premix Ex TaqTM (2×) (Vazyme, Nanjing, China), 0.8 μL of primer-probe mixture, 2 μL of template, and 7.2 μL of ddH_2_O.

Real-time RT-PCR of the 18S rRNA gene sequence was used to identify B. microti in SFTSV-positive tick pools. The probe primers (Bm18Sf-AACAGGCATTCGCCTTGAAT and Bm18Sr-CCAACTGCTCCTATTAACCATTACTCT) were designed for the 18S rRNA fragment of B. microti [22]. The amplification process and reaction system were the same as those for the SFTSV.

Positive controls for SFTSV were cDNA samples of SFTSV cultured in vitro in the laboratory. Positive controls for B. microti were blood DNA samples from infected mice. The negative controls were uninfected tick DNA samples, both provided by our laboratory.

2.5. Nested PCR and Sequencing

For SFTSV-positive samples (e.g., Cq value < 35), partial S-fragment sequences were amplified using nested PCR [23]. The first round of amplification primers used were SFTSV-1-F (5′-CATCATTGTCTTTGCCCTGA-3′) and SFTSV-1-R (5′-AGAAGACAGAGTTCACAGCA-3′). The second round of amplification primers were SFTSV-2-F (5′-AAYAAGATCGTCAAGGCATCA-3′) and SFTSV-2-R (5′-TAGTCTTGGTGAAGGCATCTT-3′). The amplification process was 95 °C for 3 min; 94 °C for 20 s; 55 °C for 40 s; 72 °C for 30 s, 35 cycles; and 72 °C for 5 min. The 50 μL reaction system consisted of 25 μL of Premix Ex TaqTM (2×) (Vazyme, Nanjing, China), 4 μL of primer mixture, 2 μL of template, and 19 μL of ddH_2_O. The PCR products were analyzed through electrophoresis on 1% agarose gel. The identity of the PCR product at high quantities (>0.1 µg/µL) was further confirmed through automated sequencing conducted by Beijing Tsingke Biotech Co., Ltd. (Beijing, China).

2.6. Phylogenetic Analysis

The partial S-fragment sequences obtained from the 93 SFTSV-positive samples (GenBank accession numbers PQ268222–PQ268228) were analyzed alongside reference sequences downloaded from GenBank. These reference sequences were selected to represent the major known lineages and genotypes of SFTSV documented in China and neighboring countries. Multiple sequence alignment was performed using the MUSCLE algorithm implemented in MEGA11 (Mega Limited, Auckland, New Zealand). The evolutionary history was inferred using the Maximum Likelihood method. The Tamura 3-parameter model was selected as the best-fit nucleotide substitution model based on the lowest Akaike Information Criterion (AIC) score. A discrete Gamma distribution was not used (Uniform Rates). Initial tree(s) for the heuristic search were obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances, and the topology with the highest log likelihood (−6542.84) was selected. A heuristic search was then performed using the Nearest-Neighbor-Interchange (NNI) method. The analysis involved 18 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option), resulting in a final dataset of 1785 positions. Bootstrap support was assessed with 1000 replicates; clusters with bootstrap values ≥70% were considered well-supported. Evolutionary analyses were conducted in MEGA11. Genotypes were defined as monophyletic clades with both strong bootstrap support (≥70%) and within-group pairwise nucleotide identity of ≥97%.

3. Results

3.1. Geographical Distribution of Tick Species

A total of 721 tick pools belonging to three genera and five species were collected (Table 1). The most abundant species in our samples was Haemaphysalis longicornis, present in 84.9% of pools, followed by Haemaphysalis fusca (13.0%), Rhipicephalus microplus (1.4%), Amblyomma testudinarium (0.4%), and Haemaphysalis wellingtoni (0.3%). The majority of pools contained nymphs (69.5%), followed by adults (24.1%) and larvae (6.4%).

The number of tick species was higher in the Xinyang area. Among these, Am. testudinarium and R. microplus were found only in Lu’an. H. wellingtoni was found only in Xinyang.

3.2. Characterization of the Geographic Distribution of SFTSV

SFTSV was detected in ticks from all four regions (Table 2). SFTSV was present in 93 of 721 pools. The positive pool rate ranged from 20.0% to 4.1% and was greatest in Xinyang followed by Songjiang, and similar among Lu’an and Zhoushan.

3.3. Distribution Profile by Life Stage and Species

SFTSV was detected in all five collected tick species (Table 3). The species-specific positive pool rate varied, with the highest rates observed in Am. testudinarium (66.7%, 2/3) and H. wellingtoni (50.0%, 1/2), followed by H. longicornis (12.7%, 78/612), H. fusca (11.7%, 11/94), and R. microplus (10.0%, 1/10).

When analyzed by developmental stage, the stage-specific positive pool rate was highest in adults (16.7%, 37/221), followed by nymphs (12.1%, 50/413) and larvae (6.9%, 6/87) (Table 3).

However, in terms of the composition of the 93 SFTSV-positive pools, nymphs constituted the majority (53.8%, 50/93), followed by adults (37.6%, 35/93) and larvae (6.5%, 6/93), reflecting the predominance of nymphs in our field collections.

3.4. Phylogenetic Analysis of SFTSV

Partial S-fragments were amplified and sequenced from all 93 SFTSV-positive tick samples. Phylogenetic analysis revealed that the obtained sequences clustered into seven distinct genotypes, designated as Shanghai-A (PQ268222), Shanghai-B (PQ268223), Shanghai-C (PQ268224), Zhejiang-A (PQ268225), Zhejiang-B (PQ268226), Anhui-A (PQ268227), and Henan-A (PQ268228) (Figure 1). The Shanghai-A genotype formed a tight cluster with the Qingdao strain (OQ513650.1), indicating a close phylogenetic relationship. The Shanghai-B, Anhui-A, and Zhejiang-B genotypes grouped together on a well-supported branch with the reference Liaoning strain (KC570436.1, Huaiyangshan virus) and other human-derived SFTSV strains (e.g., AB817996, KX672015), suggesting they belong to a major circulating lineage. The Zhejiang-A and Shanghai-C genotypes formed a separate, distinct cluster, which was evolutionarily adjacent to another human isolate (HQ179745.1). The Henan-A genotype was positioned on a relatively long, independent branch, indicating it is genetically distinct from the other genotypes identified in this study and from the reference Henan strain (MN510197.1).

3.5. Co-Detection of Babesia microti in SFTSV-Positive Tick Pools

Babesia microti was assayed specifically in the 93 SFTSV-positive tick pools. Babesia microti infections were detected in 18.3% of SFTSV-infected ticks (17/93) (Table 4). Among these, co-infection was detected in 18.3% (17/93) of the pools (Table 4). Among these co-infected pools, 20.5% (16/78) contained H. longicornis and 100% (1/1) contained H. wellingtoni. Nymphs comprised 22% (11/50) of the co-infected ticks.

4. Discussion

Currently, few studies have reported the association of SFTSV with ticks in China [24,25]. Most documented cases of SFTSV infection have been identified within Haemaphysalis spp. Nevertheless, a few reports have emerged concerning infection of Ixodes spp., Dermacentor spp., and Amblyomma spp. [26,27]. This is the first report of the discovery of SFTSV in Am. testudinarium in central and eastern China. In this study, the overall positive pool rate for SFTSV was 12.9%. SFTSV infection was found in all four regions, and the highest positivity rate was found in Henan.

Two families, nine genera, and 130 species of ticks have been identified in China [28]. Three genera and five species were collected in this study. The most abundant species was H. longicornis, while R. microplus and H. fusca were the dominant species in the Central Plain of China. We found that Am. testudinarium and H. wellingtoni were present. SFTSV was detected in all five tick species. A recent report indicated that H. longicornis is considered the primary vector for SFTSV [29]. The species distribution among the 93 SFTSV-positive pools was as follows: H. longicornis accounted for 83.9% (78/93), H. fusca for 11.8% (11/93), Am. testudinarium for 2.2% (2/93), and both H. wellingtoni and R. microplus for 1.1% (1/93) each. Calculating the positive pool rate for each species yielded 12.7% (78/612) for H. longicornis, 11.7% (11/94) for H. fusca, 50.0% (1/2) for H. wellingtoni, and 10.0% (1/10) for R. microplus. SFTSV was detected across all tick developmental stages. Notably, the stage-specific positive pool rate was highest in adults (16.7%), suggesting a potential higher risk of transmission per feeding event. However, nymphs constituted the majority (53.8%) of all SFTSV-positive pools, likely reflecting their high field abundance and underscoring their significant role in enzootic virus maintenance.

We identified seven distinct SFTSV genotypes among the positive tick samples. Phylogenetic analysis revealed complex relationships between these genotypes and known circulating strains. The Shanghai-A genotype showed a close evolutionary relationship with the Qingdao strain (OQ513650.1). In contrast, the Shanghai-B genotype clustered within a major clade containing the Liaoning human isolate (KC570436.1) along with our Anhui-A and Zhejiang-B genotypes. Meanwhile, the Shanghai-C and Zhejiang-A genotypes formed a separate cluster adjacent to another human isolate (HQ179745.1). The Henan-A genotype appeared on a relatively independent branch. Interestingly, we isolated three different genotypes (Shanghai-A, B, and C) in the Shanghai area, which belong to separate phylogenetic lineages. This suggests that multiple SFTSV lineages can co-circulate within the same geographic region, reflecting a high degree of viral genetic diversity and possibly complex transmission dynamics. The close phylogenetic relationship of several genotypes identified in this study (e.g., Shanghai-B, Anhui-A, Zhejiang-B) with known human-pathogenic strains underscores the potential public health risk associated with tick bites in these regions. H. longicornis, possibly carried by migratory birds, may have played an important role in the rapid spread of SFTSV [29].

This study confirmed that among the five tick species collected in central and eastern China (Henan, Anhui, Zhejiang, and Shanghai), H. longicornis was the species most frequently found to harbor SFTSV and Babesia microti, was the most common species in which co-detection with Babesia microti occurred within SFTSV-positive pools. SFTSV was detected in all examined tick species, highlighting their broad potential as vectors. Significant regional variations in pathogen-positive pool rates were observed, with Xinyang, Henan exhibiting the highest prevalence. These findings reinforce the important role of H. longicornis in SFTSV ecology and potential involvement in Babesia microti transmission in the region, as evidenced by co-detection in a subset of SFTSV-positive pools, while emphasizing the risk of tick-borne pathogen co-infection. This holds significant implications for local public health prevention and control strategies.

5. Conclusions

In summary, our study demonstrates a significant prevalence and genetic diversity of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) in ticks collected from central and eastern China. H. longicornis was the most common tick species found to carry SFTSV in our survey. Co-infection with both SFTSV and Babesia microti was also most frequently observed as a co-detection within SFTSV-positive pools of this species. SFTSV was detected across all five collected tick species and all developmental stages, indicating a broad potential reservoir for viral transmission.

Notably, co-infection of SFTSV and Babesia microti in ticks, particularly in H. longicornis, raises substantial public health concerns, as it poses a risk for concurrent human infections—which can complicate clinical diagnosis and treatment. The geographical variation in SFTSV prevalence, with the highest rate observed in Xinyang, Henan, underscores the need for region-specific surveillance and control strategies.

These findings highlight the ongoing emergence and spread of SFTSV in China and emphasize the critical role of tick monitoring in preventing tick-borne diseases. Enhanced public awareness, targeted vector control, and continued pathogen surveillance are urgently needed to mitigate the health threat posed by SFTSV and co-circulating tick-borne pathogens.