ABSTRACT
Wetland virus (WELV) is a recently identified segmented orthonairovirus associated with human febrile illness in northeastern China. To elucidate its ecological drivers and transmission patterns, we conducted multi-host surveillance in seven eco-climate regions across China through an integrated approach combining molecular and serological profiling. Over 33,000 questing and feeding ticks representing 11 species were systematically screened WELV-RNA alongside 1,493 wild small mammals, while 2,578 domestic animals and 3,921 patients presenting with outdoor field or tick exposure history underwent both WELV specific molecular and serological evaluation. WELV-RNA detection via real-time RT–PCR revealed low but widespread infection, including 0.54% (8/1,493) in wild rodents (Apodemus chevrieri, Rattus norvegicus), 1.12% (29/2,578) in domestic animals, and 0.86% (275/33,091) in ticks, including five newly confirmed vector species. Serological analysis via enzyme-linked immunosorbent assay (ELISA) demonstrated 2.44% (63/2,578) IgG seroprevalence in livestock. Clinical surveillance identified acute WELV infections in 1.81% (71/3,921) of febrile patients, predominantly as monoinfections (77.46%, 55/71). Coinfected patients exhibited elevated disease severity markers, with hospitalization rates doubling compared to single-infection cases (87.50% vs. 36.36%). Spatiotemporal analysis showed spring-summer predominance of WELV for both ticks and human patients. Phylogenetic analyses revealed remarkable genetic conservation patterns of WELV strains across diverse hosts and tick vectors; and clustering across geographical regions. These findings establish WELV as an emerging tick-borne virus with trans-regional spread across heterogeneous landscapes in China. The intersecting transmission cycles among wildlife reservoirs, domestic amplifier hosts, and expanding tick vectors underscore the imperative for integrated surveillance strategies to mitigate this growing public health threat.
KEYWORDS: Wetland virus, nairovirus, ticks, human infection, tick-borne virus
Introduction
Tick-borne viruses have emerged as a growing global health challenge, with their geographic range expanding with climate change and anthropogenic disturbances [1,2]. Among these, tick-borne segmented nairoviruses have gained increasing attention due to their growing public health significance [3,4]. Over the past decade, more than 40 novel viruses have been identified within the Nairoviridae family, resulting in the classification of seven highly divergent genera and a substantial number of unclassified nairoviruses (https://talk.ictvonline.org/). Of particular note is the genus Orthonairovirus in the family Nairoviridae, containing established threats like Crimean-Congo haemorrhagic fever virus (CCHFV) – responsible for case fatality rates up to 40% in endemic regions [5], alongside emerging viruses such as Nairobi sheep disease virus (NSDV), Tamdy virus (TAMV), Yezo virus (YEZV) [6], Dugbe virus (DUGV) [7], Tacheng tick borne 1 virus (TcTV-1) [8], Songling virus (SGLV) [9], causing human diseases that range from mild to severe clinical outcomes. This taxonomic diversity mirrors complex host-vector relationships, with mammalian reservoirs spanning wildlife (e.g. Bactrian camels for TAMV) to domestic animals (e.g. sheep for NSDV) [10,11].
Our recent discovery of Wetland virus (WELV) in Northeastern China expands the pathogenic landscape of the Orthonairovirus genus [12]. Phylogenetic analysis on the whole genome sequences confirmed WELV as a distinct member of the Orthonairovirus genus, forming a unique clade within Hazara orthonairovirus genetic group. Clinical surveillance revealed multi-organ pathogenicity of WELV, causing respiratory, haemorrhagic, and neurologic manifestations. Vector competence studies identified Haemaphysalis concinna as the principal tick vector. Seroprevalence in livestock suggests stable enzootic cycles in northeastern China [12]. Although the initial findings offer significant insights into the epidemiological and clinical features of this novel virus, critical knowledge gaps persist in its eco-epidemiological profile. The current distribution of human cases remains confined to northeastern China, raising questions about WELV's spillover potential across the wide range of Ha. concinna – a tick species with expanding geographic range across Eurasian warm temperate zones [13]. This biogeographic mismatch suggests either diagnostic under-recognition in endemic regions or ecological constraints on viral dispersal, paralleling the early emergence patterns of YEZV in Japan, where initial case clustering in Hokkaido belied the broader distribution potential of its Ixodes vectors [6]. Furthermore, the role of mammalian reservoirs in perpetuating enzootic cycles remains undefined, despite serological evidence in livestock from our preliminary study.
In this study, we implemented a nationwide surveillance framework across China's seven eco-climatic regions. By integrating longitudinal sampling of ticks, wildlife, and domestic animals with enhanced clinical screening at six sentinel hospitals, we are designed to explore the transmission dynamics, genetic evolution and clinical features that are related to the infection of WELV.
Methods
Sample collection for ticks, wildlife and domestic animals
An extensive collection of questing and engorged ticks, wild small animals and domestic animals had been conducted between June 2011 and November 2024 among seven eco-climate regions in China: northeastern China, northern China, central China, southwestern China, southern China, Qinghai-Tibet, and Inner Mongolia-Xinjiang [14] (Figure 1, Table S1). Free ticks were collected by dragging a flannel flag over vegetation, while engorged ticks were collected from domestic animals. Wild small animals were captured using standardized snap trapping protocols, with tissues sample collected following aseptic dissection. Taxonomic identification of tick species and captured wildlife was performed through morphological characteristics followed by molecular confirmation. Specifically, mitochondrial 16S ribosomal DNA (16S rDNA) sequencing was employed for tick species verification, while mitochondrial cytochrome b (mt-cyt b) gene sequencing was utilized for wild animal identification (Table S2). Serum samples collected from domestic animals (canines, ovines, and bovine) underwent comprehensive testing including both molecular detection and serological analysis for WELV. All collected samples were stored at −80°C until processing. This study strictly adhered to national ethical standards for animal research, according to the Guidelines for Laboratory Animal Use and Care and the Rules for the Implementation of Laboratory Animal Medicine (1998) issued by the Ministry of Health. All field collection procedures and experimental protocols received prior approval from the institutional animal ethics committee.
Figure 1.
Detection of Wetland virus in ticks, wild and domestic animals, and patients in the mainland of China. (A) Distribution of WELV in seven eco-climate regions in China. The circle, triangle, square, and rhombus represent the ticks, patients, wild and domestic animals, respectively. The colour indicates positive rate. (B) Positive rate of WELV-RNA detected in seven eco-climate regions and four seasons. Comparisons among eco-climate regions or seasons were carried out using Chi-squared test, Chi-squared test (Yate's correction) or Fisher's Exact test where it is appropriate. * P < 0.05.
Surveillance of WELV-infected patients
A multicentre surveillance study for WELV infection was implemented across six provincial-level administrative divisions (Heilongjiang, Liaoning, Inner Mongolia Autonomous Region, Henan, Shandong, and Yunnan), representing four major eco-climate zones in China with confirmed WELV-positive tick distributions (Table S1). Patient recruitment occurred at six sentinel hospitals: Inner Mongolia General Forestry Hospital, Heilongjiang Red Cross Sengong General Hospital, First Affiliated Hospital of Jinzhou Medical University, 154th Hospital in Henan province, Yantai Qishan Hospital in Shandong province, and Yunnan Institute of Endemic Disease Control and Prevention. Eligibility criteria required participants to have either (1) documented outdoor field exposure or (2) reported tick bite incidents within 30 days preceding symptom onset. Whole blood and serum samples were collected for dual assessment: WELV-RNA detection via quantitative reverse transcription PCR (qRT-PCR) and WELV-specific IgM/IgG antibody quantification through enzyme-linked immunosorbent assay (ELISA). Clinical records were systematically reviewed to extract epidemiological data, clinical data, laboratory findings, and therapeutic interventions. The study protocol received ethical approval from the Academy of Military Medical Science Institutional Review Board, with written informed consent obtained from all participants prior to enrolment.
Molecular detection of WELV-RNA
Molecular screening for WELV-RNA was performed according to our established protocols [12]. Specifically, total RNA was extracted from three sample types: (1) pooled tick specimens, (2) homogenized tissue composites (liver, spleen, lung, kidney) from wild small animals, (3) serum samples from domestic animals and human patients, using the QIAamp Viral RNA Mini Kit (Qiagen, Germany). Primary viral detection employed qRT-PCR targeting conserved regions of the L and S genomic segments [12]. The screen for WELV was performed by qRT-PCR targeting the L and S segments using cut-off cycle threshold (CT) values for positive and negative of ≤35 and >35, respectively. The qRT-PCR-positive specimens underwent further genetic characterization through amplification of a 407-bp L gene fragment, a 454-bp S gene fragment, or a 444-bp M gene fragment using the PCR System 9700 (Applied Biosystems) (Table S3) [15]. Amplicons were purified and subjected to bidirectional Sanger sequencing. All nucleic acid extraction and amplification incorporated parallel processing of positive controls (WELV strain) and negative controls (nuclease-free water) to ensure accuracy and reliability.
Whole genome sequencing of WELV
Next-generation sequencing of WELV positive samples was performed according to a previously published protocol [12]. Briefly, sequencing library was constructed using MGIEasy RNA Library Prep Kit (Beijing macroµ-test Bio-Tech, China). Paired end (2 × 150 bp) sequencing of the RNA library was conducted on MGI2000 platform (Beijing macroµ-test Bio-Tech). The sequencing reads were de novo assembled using MEGAHIT (version 1.2.9).
Serological test of WELV antibody
WELV-specific antibody was detected by ELISA as previously described [12]. For IgG detection, 96-well plates were coated with 10 ng/well WELV-nucleocapsid protein (NP), followed by incubation with serum samples diluted 1:200 in Western Antibody Dilution Buffer (Cwbio, China). Species-specific detection systems were implemented: horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1:8,000; Jackson ImmunoResearch, USA) for human samples, complemented by rabbit anti-bovine IgG (Bioss, China) and rabbit anti-canine IgG (Bioss) for domestic animal specimens. After TMB substrate development, optical density (OD) at 450 nm was measured using a microplate reader. IgM detection followed analogous protocols with modifications: serum dilution at 1:80 and HRP-conjugated goat anti-human IgM (1:6,000; Jackson) for human samples. All assays included in-plate reference sera and negative controls. Samples were analysed in duplicate, with positivity defined as mean OD450 exceeding twice the negative control mean.
Pathogen screening
To investigate potential co-infections in human patients, serum and whole blood samples were systematically screened for other 10 tick-borne pathogens using PCR or real-time RT–PCR assays. Bacterial targets included Anaplasma phagocytophilum [16], A. capra [16], Ehrlichia chaffeensis [17], spotted fever group rickettsia (SFGR) [18], Borrelia burgdorferi sensu lato [19]. Viral screening encompassed YEZV [9], tick-borne encephalitis virus (TBEV) [20], ALSV [21], SGLV [9], and severe fever with thrombocytopenia syndrome virus (SFTSV) [15].
Phylogenetic reconstruction of WELV
Amino acid and nucleotide sequences of WELV obtained in this study were aligned with reference strains from the Nairoviridae family downloaded from GenBank (Table S4 and S5) using ClustalW (v2.1) in MEGA (v11). Phylogenetic trees were constructed using the maximum-likelihood method with the best-fitting model determined by the ModelFinder program implemented in IQ-TREE (v1.6.12), and bootstrap values were calculated based on 1,000 replicates. The resulting phylogenetic trees were visualized and refined using the Interactive Tree of Life (iTOL) (http://itol.embl.de/personal_page.cgi). The sequences generated in this study have been deposited in GenBank under accession numbers PV470891-PV470909 (full-length genome sequence), PQ757393-PQ757547, PQ849658-PQ849659, PV470879-PV470882 (partial L gene), PQ757339-PQ757392 (partial S gene), and PV4708867-PV470878 (partial M gene). The viral metagenomic data are available through the NCBI sequence reads archive (SRA) under accession number PRJNA1251011.
Statistical analysis
The WELV-RNA prevalence (95% confidence intervals) in ticks was calculated by maxim likelihood estimation (MLE) using the PooledInfRate (www.cdc.gov/mosquitoes/php/toolkit/mosquito-surveillance-software.html; accessed on 29 May 2024). Geospatial mapping of sampling locations was performed using Google Maps coordinates. Statistical comparisons of categorical variables employed Pearson's χ2 or Fisher's exact tests, as appropriate, with two-tailed significance thresholds set at P < 0.05. All analyses were conducted in R (V 4.0.5), ensuring reproducibility through script-based workflows.
Results
Detection of WELV in questing and feeding ticks
A total of 33,091 questing and feeding ticks, representing 5 genera and 11 species, were tested in 5,333 pools based on their species and collection locations. WELV-RNA was determined in 0.86% (275/5,333) (95% CI: 0.77-0.97%) of the tick pools representing 9 species, including 3.45% of Haemaphysalis tibetensi, 3.43% of Haemaphysalis qinghaiensis, 3.07% of Ha. concinna, 2.08% of Dermacentor nuttalli, 1.17% of Hyalomma asiaticum, 0.63% of Ixodes persulcatus, 0.58% of Haemaphysalis longicornis, 0.56% of Dermacentor silvarum, and 0.25% of Rhipicephalus microplus (Table 1 and Table S6). Positive detections were recorded in all six sampled eco-climate regions, with significant geographical heterogeneity: Ha. qinghaiensis that dominated in Qinghai-Tibet had high prevalence of 15.65%; followed by Ha. concinna (3.07%) in northeastern China, Ha. longicornis in central China (1.59%), and Ha. tibetensi (3.45%) in southwestern China (Figure 1 and Table S6). WELV-RNA positive rate exhibited marked seasonality (χ2 test, P < 0.001), peaking in summer (0.99%), followed by spring (0.82%), autumn (0.09%), with no positive in winter (Figure 1 and Table 2).
Table 1.
Positive rate of Wetland virus in patients, ticks, wild small and domestic animals by seven eco-climate regions.
| Eco-climate regions | Total | P-value | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Northeastern China | Inner Mongolia-Xinjiang | Northern China | Central China | Southwestern China | Qinghai-Tibet | Southern China | |||
| WELV-RNA | |||||||||
| Wild animal | – | 0/335 (0) | 6/822 (0.73) | 0/229 (0) | 2/50 (4) | 0/44 (0) | 0/13 (0) | 8/1493 (0.54) | 0.05a |
| Domestic animal | 17/900 (1.89) | – | 1/682 (0.15) | 11/996 (1.10) | – | – | – | 29/2578 (1.12) | 0.005b |
| Goat/sheep | 17/800 (2.13) | – | 1/558 (0.18) | 10/949 (1.05) | – | – | – | 28/2307 (1.21) | 0.005b |
| Cattle | 0/100 (0) | – | 0/100 (0) | 1/47 (2.13) | – | – | – | 1/247 (0.40) | 0.19a |
| Dog | – | – | 0/24 (0) | – | – | – | – | 0/24 (0) | – |
| Tickc | 84/324/4576 (2.12) | 23/300/1909 (1.25) | 125/3826/24198 (0.53) | 22/511/1839 (1.20) | 2/215/375 (0.53) | 19/157/194 (9.83) | – | 275/5333/33091 (0.86) | < 0.001d |
| Patient | 45/1131 (3.98) | – | 4/547 (0.73) | 17/2065 (0.82) | 5/178 (2.81) | – | – | 71/3921 (1.81) | < 0.001d |
| WELV IgG | |||||||||
| Domestic animal | 29/900(3.22) | – | 6/682 (0.88) | 28/996 (2.81) | – | – | – | 63/2578 (2.44) | 0.007b |
| Goat/sheep | 29/800 (3.63) | – | 6/558 (1.08) | 28/949 (2.95) | – | – | – | 63/2307 (2.73) | 0.015b |
| Cattle | 0/100 (0) | – | 0/100 (0) | 0/47 (0) | – | – | – | 0/247 (0) | – |
| Dog | – | – | 0/24 (0) | – | – | – | – | 0/24 (0) | – |
| Patient | 95/1131 (8.40) | – | 19/547 (3.47) | 48/2065 (2.32) | 2/178 (1.12) | – | – | 164/3921 (4.18) | < 0.001b |
Note: Data are No. of positive samples/ No. of total tested samples (%).
Fisher's Exact test.
Chi-squared test.
Data are No. of positive pools/No. of tested tick pools/No of tested ticks. The WELV-RNA prevalence in ticks was calculated by maximum likelihood estimation (MLE) using the program PooledInfRate.
Chi-squared test (Yate's correction).
Table 2.
Positive rate of Wetland virus in patients, ticks, wild small and domestic animals by seasons.
| Seasons | Total | P-value | ||||
|---|---|---|---|---|---|---|
| Spring | Summer | Autumn | Winter | |||
| WELV-RNA | ||||||
| Wild animal | 4/294 (1.36) | 3/588 (0.51) | 1/399 (0.25) | 0/212 (0) | 8/1493 (0.54) | 0.18a |
| Domestic animal | 3/751 (0.40) | 22/1318 (1.67) | 4/509 (0.79) | – | 29/2578 (1.12) | 0.02b |
| Goat/Sheep | 3/551 (0.54) | 22/1318 (1.67) | 3/438 (0.68) | – | 28/2307 (1.21) | 0.07b |
| Cattle | 0/200 (0) | – | 1/47 (2.13) | – | 1/247 (0.40) | 0.19a |
| Dog | – | – | 0/24 (0) | – | 0/24 (0) | – |
| Tickc | 128/1367/16677 (0.82) | 146/3678/14955 (0.99) | 1/203/1053 (0.09) | 0/85/406 (0) | 275/5333/33091 (0.86) | < 0.001b |
| Patient | 29/1357 (2.14) | 30/1868 (1.61) | 11/662 (1.66) | 1/34 (2.94) | 71/3921 (1.81) | 0.49a |
| WELV IgG | ||||||
| Domestic animal | 12/751 (1.60) | 37/1318 (2.81) | 14/509 (2.75) | – | 63/2578 (2.44) | 0.20b |
| Goat/sheep | 12/551 (0.54) | 37/1318 (2.81) | 14/438 (3.20) | – | 63/2307 (2.73) | 0.60b |
| Cattle | 0/200 (0) | – | 0/47 (0) | – | 0/247 (0) | – |
| Dog | – | – | 0/24 (0) | – | 0/24 (0) | – |
| Patient | 70/1357 (5.16) | 79/1868 (4.23) | 12/662(1.81) | 3/34 (8.82) | 164/3921 (4.18) | < 0.001a |
Note: Data are No. of positive samples/ No. of total tested samples (%).
Fisher's Exact test.
Chi-squared test.
Data are No. of positive pools/No. of tested tick pools/No of tested ticks. The WELV-RNA prevalence in ticks was calculated by maximum likelihood estimation (MLE) using the program PooledInfRate.
Comparative analysis of five tick species (De. nuttalli, De. silvarum, Ha. concinna, Rh. microplus, Ha. longicornis) among 18,524 questing and 12,585 engorged ticks (10,027 collected from goats; 1190 from dogs; 700 from cattle; 324 from yak; 307 from hedgehogs; 22 from chicken; and 15 from rabbits), revealed distinct ecological patterns. Feeding ticks exhibited higher WELV prevalence than questing ticks across four species (De. nuttalli, De. silvarum, Ha. concinna, Ha. longicornis), with statistical significance in De. nuttalli (χ2 test, P = 0.01) (Table S7). Notably, ticks collected from WELV IgG-positive goats demonstrated trend towards higher positive rate than those collected from WELV IgG-negative goats (2.82% vs. 0.66%), however with no statistical significance (Fisher's Exact test, P = 0.14).
Two tick species (Ha. qinghaiensis and Ha. longicornis) collected at the same location were examined across all three life stages (adult, nymph and larval). Life-stage comparison in Ha. qinghaiensis showed comparable prevalence, whereas Ha. longicornis displayed stage-dependent infection difference: highest for adult (0.90%), followed by nymph (0.21%) and larval (0.05%) (χ2 test with Yate's correction, P = 0.03) (Table S8).
Detection of WELV in wild small animals and domestic animals
Molecular screening of 1,493 wild small mammals (32 species, 8 families) identified WELV-RNA in 0.54% (8/1,493) of them, exclusively in Muridae family: Apodemus chevrieri (4.08%, 2/49) and Rattus norvegicus (1.00%, 6/602) (Table 3 and Table S9). No obvious histomorphological abnormalities were observed from the WELV-positive rodents during their dissection. Spatial clustering emerged in southwestern China (4.00%) and northern China (0.73%) (Fisher's exact test, P = 0.05), with seasonal positive detections limited to spring through autumn, while not in winter (Figure 1 and Tables 1 and 2).
Table 3.
Detection of Wetland virus RNA and IgG antibodies in human patients and animals.
| Hosts | No. of tested | No. (%) RNA positive |
No. (%) IgG positive |
|---|---|---|---|
| Patients | 3921 | 71 (1.81) | 164 (4.18) |
| Domestic animals | 2578 | 29 (1.12) | 63 (2.44) |
| Goat/sheep | 2307 | 28 (1.21) | 63 (2.73) |
| Cattle | 247 | 1 (0.40) | 0 (0) |
| Dog | 24 | 0 (0) | 0 (0) |
| Wild animals | 1493 | 8 (0.54) | – |
Domestic animal surveillance revealed WELV-RNA in 1.12% (29/2,578) of apparently healthy livestock, including 1.21% (28/2,307) of goats/sheep, 0.40% (1/247) of cattle, and none of the 24 dogs (Table 3). Positive detection was observed in all three sampled eco-climate regions, with a higher prevalence in northeast China (1.89%, 17/900) compared to central China (1.10%, 11/996) and northern China (0.15%, 1/682) (χ2 test, P = 0.005) (Table 1). A significant difference was observed in WELV-RNA prevalence among domestic animal types (Fisher exact test, P < 0.001), with goats/sheep having a higher WELV-RNA positive rate compared to all other animals (1.21% vs. 0.37%; χ2 test with Yate's correction, P = 0.35) (Figure 1 and Table 1). A seasonal pattern of WELV infection was also determined (χ2 test, P = 0.02), with the highest prevalence determined in summer (1.67%), followed by autumn (0.79%) and spring (0.40%) (Figure 1 and Table 2).
Serological profiling detected WELV-IgG exclusively in goats/sheep (2.73%, 63/2,307), which showed significant regional trends (northeastern China: 3.63%, 29/800 > central China: 2.95%, 28/949 > northern China: 1.08%, 6/558; χ2 test, P = 0.015), while no seasonal difference (χ2 test, P > 0.05) (Figure 1 and Tables 1–3). No antibodies were detected in cattle or dogs.
WELV infection in human patients
Between April 2015 and October 2024, a total of 3,921 febrile patients (median age 59 years, range 3–89 years; 53.00% female) were recruited from four eco-climate regions (Figure 1, Table S10). WELV-RNA was determined in 1.81% (71/3,921) of the patients, across all four sampled regions, with the highest prevalence observed in northeastern China (3.98%, 45/1,131), followed by southwestern China (2.81%, 5/178), central China (0.82%, 17/2,065), and northern China (0.73%, 4/547) (Figure 1 and Table 1). Seasonal distribution was observed, with 83.10% (59/71) of human WELV infections occurring between May and September and 60.56% (43/71) reported a history of tick bites. WELV specific IgM and IgG antibodies were present in 60.56% (43/71) and 59.15% (42/71) of these patients, respectively.
Fifty-five patients with WELV-RNA monoinfection (median age: 54 years old; range 10-82; 63.64% female) exhibited a median treatment delay of 5 days (range: 2-31) from symptom onset. Among these patients, 36.36% (20/55) required hospitalization. The most common clinical manifestations at the time of hospital admission included fever (100%, 20/20), malaise (55%, 11/20), gastrointestinal manifestations (50%, 10/20), myalgia (35%, 7/20), headache (30%, 6/20), dizziness (15%, 3/20), arthritis (10%, 2/20), and back pain (5%, 1/20) (Table 4). Petechiae and localized lymphadenopathy were displayed among 5% (1/20) of the patients; and neurological symptoms such as dysphoria, lethargy, and coma were reported in seven patients. Laboratory profiling revealed haematologic dysregulation at admission (lymphopenia in 65% of patients; thrombocytopenia in 75%; leukopenia in 85%); alongside elevated level of high-sensitivity C-reactive protein (hs-CRP, 80%), d-dimer (45%), lactate dehydrogenase (LDH, 20%), fibrinogen (15%), alanine aminotransferase (ALT, 55%), aspartate aminotransferase (AST, 35%). Despite the viral aetiology, 95% (19/20) received empiric antibiotics, while 65% (13/20) underwent ribavirin therapy (Table 4).
Table 4.
Clinical characteristics and treatments of patients with Wetland virus infection.
| Characteristic | Monoinfected patients (N = 55) | Coinfected patients (N = 16)a |
|---|---|---|
| Age, years | 54 (10-82) | 62 (9-78) |
| <60 | 38 (69.09) | 7 (43.75) |
| ≥60 | 17 (30.91) | 9 (56.25) |
| Sex, female | 35 (63.64) | 11 (68.75) |
| Occupation | ||
| Farmer | 28 (50.91) | 10 (62.50) |
| Worker | 5 (9.09) | 2 (12.50) |
| Unknown | 22 (40) | 4 (25.00) |
| Clinical signsb | ||
| Fever | 20 (100.00) | 14 (100.00) |
| Malaise | 11 (55.00) | 12 (85.71) |
| Myalgia | 7 (35.00) | 9 (64.29) |
| Headache | 6 (30.00) | 3 (21.43) |
| Dizziness | 3 (15.00) | 1 (7.14) |
| Arthritis | 2 (10.00) | 0 (0) |
| Back pain | 1 (5.00) | 0 (0) |
| Gastrointestinal manifestations | 10 (50.00) | 9 (64.29) |
| Petechiae | 1 (5.00) | 2 (14.29) |
| Lymphadenopathy | 1 (5.00) | 7 (50.00)c |
| Neurological symptoms | 7 (35.00) | 4 (28.57) |
| Laboratory abnormalities on admissionb | ||
| Leukopaenia | 17 (85.00) | 9 (64.29) |
| Thrombocytopenia | 15 (75.00) | 12 (85.71) |
| Lymphopenia | 13 (65.00) | 11 (78.57) |
| Hs-CRP >3 mg/liter | 16 (80.00) | 12 (85.71) |
| Fibrinogen >4.0 g/liter | 3 (15.00) | 3 (21.43) |
| D-dimer >0.55 mg/liter | 9 (45.00) | 7 (50.00) |
| LDH >245 U/liter | 4 (20.00) | 6 (42.86) |
| AST levels, > 40 U/liter | 7 (35.00) | 9 (64.29) |
| ALT levels, > 40 U/liter | 11 (55.00) | 10 (71.43) |
| Treatmentsb | ||
| Antibiotics | 19 (95.00) | 13 (92.86) |
| Ribavirin | 13 (65.00) | 6 (42.86) |
| Mortality rate | 3 (5.45) | 2 (12.50) |
Note: Data are median (range) or n (%).
Coinfection involved SFTSV (13 patients), TBEV (two patient) and spotted fever group Rickettsia (one patient). LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Hs-CRP, high-sensitivity C-reactive protein.
Shown are collected clinical characteristics, laboratory abnormalities on admission and treatments of hospitalized patients with WELV-RNA positive and complete medical records.
P = 0.004. Fisher's exact test.
Sixteen patients presented with co-infection involving WELV-RNA and other tick-borne pathogens, predominantly SFTSV (13 cases), TBEV (two cases), spotted fever group Rickettsia (one case). Comparative analysis revealed significantly higher prevalence of lymphadenopathy (50.00% vs. 5.00%, Fisher's Exact test, P = 0.004) and hospitalization rate (87.50% vs.36.36%, χ2 test, P < 0.001) compared to those with WELV monoinfection (Table 4). Mortality was recorded in 7.04% (5/71) of WELV-RNA positive patients, comprising three cases of monoinfection and two cases of co-infections with SFTSV. The survived patients did not exhibit any significant sequelae during prospective follow-up observations.
Phylogenetic analysis of WELV
We performed meta-transcriptomic sequencing on WELV-positive samples, yielding six complete WELV genomes (L, M, and S segments; GenBank: PV470891-PV470908) from Ha. concinna ticks and a full-length S segment from the sheep (GenBank: PV470909). Phylogenetic reconstruction using Tofla virus (TFLV) as the outgroup revealed that WELV strains cluster within the Hazara genogroup of the Orthonairovirus genus, forming a distinct monophyletic lineage separate from other Hazara orthonairoviruses (Figure 2(A)–(C)).
Figure 2.
Phylogenetic tree and mutation of Wetland virus. The phylogenetic trees were estimated on the basis of complete AA sequences of L protein (A), GPC (B), and N (C) from WELV strains, as well as other recognized species in the Nairoviridae family, with the use of 1000 bootstrap replications. The best-fitting model was determined by the ModelFinder program implemented in IQ-TREE based on the Bayesian information criterion (BIC), the LG + G + F model for the L sequences, the LG + G + I model for the M sequences, the LG + G model for the S sequences. The scale bars denote the number of protein substitutions per site. WELV sequences that were obtained in this study and previous report are marked by red and blue dots, respectively. (D) Phylogenetic tree based on six L protein of tick-derived WELV strains in this study together with sequences of 10 human and tick-derived WELV strains from GenBank. (E) Structure and mutation of WELV RdRP CDS. The CDS contains OTU and RdRP domain. The mutations are highlighted in yellow, blue, purple, red, green, and pink, dependent on the amino acid. CDS = coding sequence. OTU = ovarian tumour protease. RdRP = RNA-dependent RNA-polymerase. E = Glu. G = Gly. K = Lys. R = Arg. T = Thr. A = Ala.
The L protein-based phylogeny resolved two major WELV clades: Clade I includes the 2019 human-derived strain (GenBank: XH033407) and our Ha. concinna-derived strains from Inner Mongolia (2019–2023); Clade II comprises one Ha. concinna-derived sequence from this study, two Ha. concinna-derived sequences from Heilongjiang/Inner Mongolia (GenBank: XJP49237, XJP49240), and two De. nuttalli-derived sequences from Inner Mongolia (GenBank: XJP49238-39) (Figure 2(D)). The human derived sequence exhibited 99.36-100% and 99.22-99.34% amino acid similarity of L protein to tick-derived sequences in Clade I and II, respectively.
Our genomic characterization identified four conserved functional domains across all WELV isolates: an ovarian tumour domain–like cysteine protease (vOTU, aa35-161), a topoisomerase-like domain (KLXXY), a C2H2-type zinc-finger domain (aa 601-624), and a leucine zipper motif. However, critical amino acid substitutions differentiated the clades in both the vOTU and RNA-dependent RNA polymerase (RdRp) domains. Within the N-terminal vOTU region, Clade I strains predominantly exhibited Lys at aa70 and aa85, contrasting with Glu at aa70 and invariant Arg at aa85 in Clade II. The RdRp domain (aa2042-2774) showed further divergence, with Clade I maintaining Lys at aa2130 and Thr at aa2341 versus Glu and Ala at these positions in clade II (Figure 2(E)).
To resolve broader evolutionary relationships, we performed comparative phylogenetic analyses using partial gene sequences from diverse hosts (Figures S1-S3). By examining 407-bp L gene fragments (n = 161), 444-bp M gene regions (n = 12), and 454-bp S gene segments (n = 54) obtained from ticks (n = 194), rodents (n = 5), goats (n = 2), and patients (n = 26), we observed remarkable sequence stability. Tick-derived WELV strains in this study maintained 99.73-100% (L gene), 98.65-100% (M), and 99.55-100% (S) nucleotide identity with the Ha. concinna-associated WELV strains (GenBank: OR860403, OR860413 and OR860397) from our previous study [12]. Human-derived sequences showed comparable conservation, exhibiting 98.92-100% (L), 99.76-99.77% (M), and 99.54-99.85% (S) identity to prior human-associated references (GenBank: OR860402, OR860408 and OR860396) [12].
Cross-species comparisons uncovered minimal divergence thresholds of 97.31% (L) and 99.32% (S) among WELV strains from nine tick species (Figure S4, Table S11), demonstrating exceptional sequence preservation across arthropod vectors. Phylogenetic reconstruction incorporating 65 representative sequences (40 partial L, 8 M, 17 S from ticks; all available goat/human sequences) consistently positioned WELV within a monophyletic cluster of Hazara orthonairoviruses (Figures S1–S3).
Discussion
The current findings substantially broadens the recognized endemic range of WELV across seven biogeographic regions in China, delineated by distinct climatic and ecological parameters [22,23]. Beyond its initial detection in Ha. concinna and four other tick species (Ha. japonica, Ix. persulcatus, De. silvarum, Ha. longicornis), we identified five novel WELV tick vectors: Ha. qinghaiensis (mainly endemic to western China) [24]; Ha. tibetensis (mainly in Tibet and Gansu provinces) [25]; De. nuttalli (widely distributed from southern Siberia to northern China) [26]; Hy. asiaticum (prevalent across Asia); and Rh. microplus (subtropical and tropical cattle tick) [27]. Regional prevalence patterns revealed vector specialization, with Ha. qinghaiensis dominating Qinghai-Tibet (WELV-RNA positive rate of 15.65%), Ha. concinna in northeastern China (3.07%), and D. nuttalli in Inner Mongolia-Xinjiang (1.81%). This geographical partitioning suggests primary and secondary vector roles within local transmission cycles, particularly for Ha. qinghaiensis. Despite high natural prevalence, its vector competence requires experimental validation.
The ability of WELV to replicate in various cell types and be positively detected in multiple animal species underscores its capacity to productively infect different animal hosts [12]. Our study, for the first time revealed that a wide range of domestic (goats, sheep, cattle, dogs, chicken, and yaks) and wildlife (hedgehog) harbour WELV-positive feeding ticks in endemic regions. Serological evidence in goats/sheep (2.73% IgG positivity) and elevated WELV prevalence in ticks feeding on seropositive hosts suggest their role as amplification reservoirs. Spatial congruence in WELV detection among ticks, livestock, and humans across three eco-regions supports an enzootic “tick-wildlife-livestock-human” transmission model. Notably, only 60.56% of WELV infected patients recalled tick exposure, implying alternative routes or unrecognized vector interactions.
Furthermore, an extensive investigation of wild small mammal species across extended regions had identified additional reservoir hosts beyond the known hosts Myospalax psilurus in northeastern China. Specifically, WELV prevalence was higher in Apodemus genus (4.17%) from southwestern China and Rattus genus (1.13%) from northern China, both in the family Muridae. However, no correlation was found between WELV-RNA prevalences in wild small mammals and ticks in the same regions (Table 1), suggesting these mammals may maintaining adult tick populations but are not reproductive hosts for WELV transmission.
Human surveillance revealed 1.81% RNA and 4.18% IgG prevalence, peaking in northeastern China (3.98% RNA positivity), followed by southwest China (2.81%), central China (0.82%), and northern China (0.73%). Case distribution followed a bimodal seasonal pattern (April-May and September-October) (Figure S5), which might align with agricultural activities and tick abundance cycles. Phylogenetic clustering of human, tick, and livestock strains confirmed ecological overlap driving spillover risk.
While WELV monoinfection typically mirrors the non-specific febrile presentation of other tick-borne pathogens, including SFTSV [28], YEZV [6,29], ALSV [21], and SGLV [9], our data reveal exacerbated clinical diseases during WELV coinfection with SFTSV, which represents the first report of SFTSV-WELV coinfection. SFTSV, known to induce a clinical spectrum from influenza-like illness to fatal haemorrhagic and neurological complications [28], demonstrated pathogenic synergy when coexisting with WELV. Coinfected patients exhibited amplified severity of overlapping pathologies, with lymphadenopathy incidence increasing tenfold, while LDH elevations – a biomarker of cellular damage – elevated proportionally to viral loads. These synergistic effects likely stem from compounded endothelial dysfunction and immune hyperactivation, mechanisms previously implicated in SFTSV-driven critical illness [33]. This differs from clinical effects of SFTSV interactions with SFGR (delayed recovery, elevated mortality) and Hantaan orthohantavirus (HTNV) (aggravated haemorrhagic manifestations) [30–32]. On the other hand, the overlapping symptom profile between WELV and SFTSV necessitate multiplex molecular testing in endemic regions to prevent misdiagnosis or underdiagnosis. Furthermore, the hospitalization rate in coinfected cohorts than monoinfections underscores the urgency for early empiric antiviral protocols.
Within the Hazara orthonairovirus genogroup, only four species have been characterized to date. WELV poses as the sole member with confirmed human infectivity, contrasting with TFLV and Meihua Mountain virus (MHMV), which remain restricted to wild boars and cattle [34,35]. Notably, WELV demonstrates exceptional zoonotic plasticity, been detected across three rodent genera and five livestock species (sheep, goats, pigs, horses, dogs), underscoring its broad host adaptability [12]. While all Hazara orthonairoviruses primarily utilize Haemaphysalis ticks as vectors, WELV exhibits exclusive transovarial transmission capability in Ha. concinna – a critical adaptation for environmental persistence. Its distinctive pathogenicity is further evidenced by lethal outcomes in diverse murine models and tropism for human cell lines (e.g. hepatocytes, neurons) as well as primate, canine, and rodent-derived cultures [12]. The current comprehensive phylogenetic analyses of WELV strains revealed remarkable genetic conservation patterns across diverse hosts and tick vectors. This conserved evolutionary pattern persisted despite the virus' capacity to infect multiple mammalian hosts, suggesting strong purifying selection pressures maintaining core genomic stability.
In reviewing the results of this study, two potential limitations should be kept in mind. First, wide sampling across regions compounded by sparse temporal data for poultry and livestock species had limited the application of multivariate statistical analysis. Second, the absence of epidemiological linkages among human cases – coupled with low incidence rates – precludes definitive conclusions regarding transmission routes. While current evidence points to sporadic, tick-mediated spillover, the small cohort size cannot exclude potential human-to-human transmission mechanisms observed in other nairoviruses. These gaps highlight the necessity for longitudinal, population-level surveillance to delineate WELV's full transmission potential.
The current findings underscore the imperative for healthcare networks in regions with documented WELV activity (human, vector, or animal) to incorporate WELV screening into differential diagnoses of acute febrile syndromes, particularly where SFTSV or other tick-borne pathogens are endemic. The established co-circulation patterns necessitate the implementation of multiplex diagnostic platforms capable of simultaneous detection of WELV, SFTSV, and associated pathogens – a critical advancement given the synergistic morbidity observed in coinfected patients. Furthermore, the geographical congruence between human seropositivity, livestock reservoirs, and vector prevalence maps argues for integrated One Health surveillance systems that monitor animal sentinels, tick populations, and human cases through coordinated frameworks.
Supplementary Material
Acknowledgments
The authors would like to thank all the subjects, their families, and collaborating clinicians for their participation.
Author contribution
Conceptualization: Shu-Zhen Han, Xiao-Ai Zhang and Wei Liu. Gathered data and collected the samples: Zhi-Jun Zhao, Hong-Xia Yuan, Xiao-Long Lv, Xiao-Hu Han, Zhi-Jun Hou, Ning Cui, Xin Yang, Han-Zheng Mu and Peng-Tao Bao. Laboratory tests: Guang-Qian Si, LZ, HZM, YDM and CP. Data analysis: Mei-Qi Zhang, Guang-Qian Si, Si-Qian Wu, Ming Chen, Lei Zhang, Hao Li, Xiao-Ai Zhang and Wei Liu. Writing – original draft: Hong-Xia Yuan, Xiao-Long Lv, Mei-Qi Zhang, Guang-Qian Si, Shu-Zhen Han, Xiao-Ai Zhang and Wei Liu. Writing – review and editing: Hong-Xia Yuan, Xiao-Long Lv, Mei-Qi Zhang, Guang-Qian Si, Shu-Zhen Han, Xiao-Ai Zhang and Wei Liu. All authors reviewed the manuscript and agreed with the final version. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplemental Material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2025.2502003.
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