Genetically Diverse Severe Fever with Thrombocytopenia Syndrome Virus Circulates in Shelter and Companion Dogs in South Korea

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne zoonotic pathogen that continues to cause a substantial public health burden in South Korea, yet the molecular epidemiology of SFTSV in dogs, particularly shelter populations, remains poorly characterized. To address this gap, blood samples from 715 dogs, including companion and shelter animals, were collected nationwide in 2024 and screened for SFTSV using RT-PCR targeting the S, M, and L genomic segments, followed by sequencing, phylogenetic analysis, and virus isolation. SFTSV was detected in 16 dogs (2.2%), with significantly higher prevalence in autumn, in the southern region, in shelter dogs, and in younger animals. A localized cluster of six infected dogs was identified in a southern shelter during autumn, and phylogenetic analysis revealed the circulation of three genotypes (B2, D, and F). Live virus was successfully isolated from one shelter dog, and this isolate belonged to genotype F, representing the first isolation of this genotype from dogs in South Korea. These findings demonstrate that dogs are exposed to genetically diverse SFTSV strains circulating in tick populations and support the use of canine surveillance, particularly in shelters, as part of a One Health approach to monitor and mitigate the risk of SFTSV transmission.

1. Introduction

Severe fever with thrombocytopenia syndrome virus (SFTSV), recently renamed Huaiyangshan banyangvirus, is a tick-borne zoonotic virus belonging to the genus Banyangvirus within the family Phenuiviridae (order Bunyavirales) [1]. The viral genome consists of three single-stranded, negative-sense RNA segments (L, M, and S), and SFTSV strains are classified into multiple genotypes (A–F), with genotype B predominating in South Korea while other genotypes, including D and F, co-circulate at lower frequencies [2]. Recent large-scale phylogenetic and phylodynamic analyses using comprehensive sequence datasets from East Asia have further refined the classification and evolutionary history of SFTSV. In particular, Sang et al. demonstrated that SFTSV has circulated in China, South Korea, and Japan for several centuries and proposed a more robust lineage classification based on extensive L, M, and S segment analyses, highlighting substantial genetic diversity and frequent reassortment events across regions [3]. In recent years, South Korea has experienced a persistently high and increasing number of human SFTS cases [4,5], underscoring the ongoing risk of exposure to infected ticks and the widespread circulation of the virus in the environment. Because SFTSV is maintained in complex enzootic cycles involving ticks, wildlife, domestic animals, and humans, effective control and risk assessment require a One Health approach integrating human, animal, and vector surveillance.

In South Korea, SFTSV is transmitted primarily by ixodid ticks, with Haemaphysalis longicornis recognized as the principal vector species nationwide, while H. flava, Ixodes nipponensis, and Amblyomma testudinarium have also been implicated [6]. Both transstadial and transovarial transmission of SFTSV have been demonstrated in H. longicornis, supporting the role of ticks as long-term reservoirs in the natural transmission cycle [7]. Although many SFTSV-infected dogs are asymptomatic, clinical illness ranging from mild hematological abnormalities to severe or fatal disease has been reported, indicating that dogs can act as susceptible hosts as well as sentinels of environmental viral circulation [8,9].

In South Korea, nationwide monitoring programs have demonstrated that SFTSV circulates extensively in ticks [6] and is detected in a wide range of animal hosts, including livestock and companion animals. Dogs and cats are of particular concern because of their close contact with humans and their frequent exposure to outdoor environments where ticks are abundant. Several studies have reported SFTSV infection and seropositivity in dogs [8,9,10,11,12] and cats [11,13], and severe or fatal clinical cases have been documented, highlighting their potential role as both victims of infection and indicators of environmental viral activity.

The public health relevance of canine SFTSV infection is further amplified by the rapidly increasing population of abandoned and rescued dogs in South Korea. Dogs housed in shelters frequently originate from outdoor or free-roaming environments and are often exposed to tick-infested habitats before and during their admission. In addition to tick-borne pathogens, shelter dogs [14,15] and cats [16] in South Korea are known to harbor a wide range of zoonotic gastrointestinal parasites and protozoa which reflect intense environmental and fecal exposure in these settings. Moreover, molecular surveillance studies have demonstrated that shelter dogs in South Korea are commonly infected with multiple tick-borne bacteria, such as Anaplasma phagocytophilum, Rickettsia spp., and Hepatozoon canis, further highlighting their role as sentinels of vector-borne and zoonotic disease circulation [17,18]. Importantly, many of these dogs are subsequently adopted into households, creating a direct interface between animals with complex pathogen exposure histories and humans. This raises substantial One Health concerns regarding zoonotic transmission and underscores the need for systematic molecular surveillance of tick-borne viruses, including SFTSV, in both shelter and companion dogs.

Despite accumulating evidence of SFTSV infection in dogs, important gaps remain in our understanding of the molecular epidemiology and genetic diversity of SFTSV circulating in the canine population of South Korea. In particular, little is known about the range of viral genotypes infecting dogs and the extent to which dogs may harbor genetically distinct SFTSV strains reflecting the diversity present in local tick populations.

Therefore, the present study was conducted to investigate the prevalence, epidemiological characteristics, and genetic diversity of SFTSV in dogs in South Korea, with a particular focus on abandoned shelter dogs and companion animals. Through molecular detection, virus isolation, and phylogenetic analysis, we aimed to clarify the role of dogs as incidental hosts and sentinels for SFTSV circulation and to provide data relevant to One Health-based surveillance and risk assessment of this emerging tick-borne zoonosis.

2. Results

2.1. Identification of SFTSV in Dog Blood Samples

Specific reverse transcription–polymerase chain reaction (RT-PCR) targeting the S, M, and L genomic segments of SFTSV detected viral RNA in 16 of 715 dog blood samples (2.2%; 95% confidence interval [CI]: 1.2–3.3) (

Table 1. Prevalence of SFTSV detected by PCR in dogs in South Korea.

Group	Category	No. Tested	No. Positive (%)	95% CI 1	${\mathit{\chi }}^2$ (df 2) (p-Value 3)
Seoson	Spring	220	2 (0.9)	0–2.2	8.482 (3) 0.0370
	Summer	205	4 (2.0)	0.1–3.8
	Autumn	185	9 (4.9)	1.8–8.0
	Winter	105	1 (1.0)	0–2.8
Region	Northern	275	2 (0.7)	0–1.7	8.357 (2) 0.0153
	Central	250	5 (2.0)	0.3–3.7
	Southern	190	9 (4.7)	1.7–7.8
Gender	Female	320	8 (2.5)	0.8–4.2	0.182 (1) 0.6698
	Male	395	8 (2.0)	0.6–3.4
Source	Shelter dogs	310	13 (4.2)	2.0–6.4	9.556 (1) 0.0020
	Pet dogs	405	3 (0.7)	0–1.6
Age	Young (<3)	275	11 (4.0)	1.7–6.3	7.433 (2) 0.0243
	Adult (3–7)	310	5 (1.6)	0.2–3.0
	Older (>7)	130	0
Total		715	16 (2.2)	1.2–3.3

¹ CI: confidence interval. ² df: degree of freedom. ³ p-values < 0.05 were considered statistically significant.

). The prevalence of SFTSV was significantly higher in autumn (9/185; 4.9%; 95% CI: 1.8–8.0), followed by summer (4/205; 2.0%; 95% CI: 0.1–3.8), spring (2/220; 0.9%; 95% CI: 0–2.2), and winter (1/105; 1.0%; 95% CI: 0–2.8) (p = 0.0370). Regionally, SFTSV was detected most frequently in the southern region (9/190; 4.7%; 95% CI: 1.7–7.8), followed by the central region (5/250; 2.0%; 95% CI: 0.3–3.7) and the northern region (2/275; 0.7%; 95% CI: 0–1.7), with a significantly higher prevalence in the southern than in the northern region (p = 0.0153). According to sex, SFTSV prevalence was higher in female dogs (8/320; 2.5%; 95% CI: 0.8–4.2) than in male dogs (8/395; 2.0%; 95% CI: 0.6–3.4), although the difference was not statistically significant (p = 0.6698). By source, SFTSV RNA was detected significantly more frequently in shelter dogs (13/310; 4.2%; 95% CI: 2.0–6.4) than in pet dogs (3/405; 0.7%; 95% CI: 0–1.6) (p = 0.0020). By age, SFTSV RNA was detected most frequently in young dogs (<3 years; 11/275; 4.0%; 95% CI: 1.7–6.6), followed by adult dogs (3–7 years; 5/310; 1.6%; 95% CI: 0.2–3.0), whereas no positive cases were identified in dogs older than 7 years (p = 0.0243).

2.2. Results of Virus Isolation

Of the 16 SFTSV-positive samples, only one sample, which was collected from a shelter dog, was positive in the virus isolation assay. Replication of SFTSV in Vero E6 cells was confirmed by RT-PCR targeting the S, M, and L genomic segments using cell culture supernatants, and the amplified products were successfully sequenced, confirming the identity of the isolated virus. In addition, Vero E6 cells inoculated with this isolate exhibited specific immunoreactivity to SFTSV by indirect immunofluorescence assay (IFA). Strong cytoplasmic staining was observed when the cells were probed with both anti-SFTSV rabbit polyclonal and anti-SFTSV mouse monoclonal antibodies, whereas no specific signal was detected in the negative control cells. These findings confirmed that the isolated virus was virologically and antigenically consistent with SFTSV (Figure 1).

2.3. Molecular Characterization and Phylogenetic Analysis

Phylogenetic analysis based on partial nucleotide sequences of the S (Figure 2), M (Figure 3), and L (Figure 4) segments showed that the SFTSV strains identified in this study clustered with previously reported reference strains and were classified into three genotypes: B2, D, and F. Notably, the virus isolated from a shelter dog belonged to genotype F. Among the 16 SFTSV-positive samples, genotype B2 was the most prevalent (62.5%, 10/16), followed by genotype D (25.0%, 4/16) and genotype F (12.5%, 2/16).

For the S segment, the B2, D, and F genotype strains showed 98.5–100%, 99.7–100%, and 100% nucleotide identity within each genotype, respectively. Comparison with reference SFTSV strains in GenBank revealed nucleotide identities of 98.9–99.4% for genotype B2, 99.1–99.7% for genotype D, and 99.7–100% for genotype F.

For the M segment, the B2, D, and F genotype strains showed 99.5–100%, 99.8–100%, and 100% nucleotide identity within each genotype, respectively, and shared 98.1–98.5% (B2), 98.2–100% (D), and 98.8–100% (F) nucleotide identity with reference sequences in GenBank.

For the L segment, the B2, D, and F genotype strains showed 99.1–100%, 99.3–100%, and 100% nucleotide identity within each genotype, respectively, and exhibited nucleotide identities of 98.7–100% (B2), 99.3–100% (D), and 98.8–100% (F) compared with GenBank reference strains.

To further examine the relationship between viral genotypes and epidemiological variables, genotype distributions were compared according to season, geographic region, source, and age group (Table S1). Although genotype B2 was detected across multiple seasons and regions, genotype F was identified exclusively in shelter dogs sampled during autumn in the southern region. Genotype D was detected sporadically without a clear seasonal or regional clustering pattern. Statistical analysis using Fisher’s exact test did not reveal significant associations between genotype and any epidemiological variable (p > 0.05 for all comparisons), likely due to the limited number of PCR-positive cases (n = 16).

The nucleotide sequences generated in this study were deposited in GenBank under accession numbers PX897703–PX897718 for the S segment, PX897719–PX897734 for the M segment, and PX897735–PX897750 for the L segment.

No amplification was observed in negative controls in any RT-PCR run. Independent recombinant clones derived from the same sample showed identical nucleotide sequences for each genomic segment analyzed. Visual inspection of sequence chromatograms revealed no ambiguous base calls or excessive substitution patterns suggestive of hypermutation. Comparative alignment across the S, M, and L segments did not indicate evidence of recombination.

3. Discussion

Previous surveillance studies in South Korea have demonstrated that SFTSV circulates widely among arthropods [6,19,20], companion animals [8,9,10,11,12,13], livestock [21,22], and humans [2,5]. In the present study, SFTSV RNA was detected in 2.2% of dog blood samples, and the detection rate varied significantly according to season, geographic region, source (shelter vs. pet), and age, while no significant difference was observed by sex. These epidemiological patterns are highly consistent with the ecology of ticks, the primary vectors of SFTSV, and with previously reported spatial and temporal trends of SFTS in South Korea. This study was not designed to estimate the true national prevalence of SFTSV in all dogs in South Korea; rather, it aimed to characterize viral circulation, epidemiological patterns, and genetic diversity of SFTSV in dogs sampled across multiple regions and exposure settings.

Seasonal analysis showed that SFTSV detection in dogs was highest in autumn, followed by summer, with lower prevalence in spring and winter. This pattern closely mirrors the seasonal dynamics of tick populations in South Korea, where larval ticks increase markedly from late summer to early autumn and human SFTS incidence typically peaks in October [6]. Dogs that spend time outdoors during this period are therefore more likely to be exposed to infected ticks, supporting the observed seasonal increase in SFTSV detection in autumn.

Regionally, SFTSV was most frequently detected in the southern region, followed by the central region, with the lowest prevalence in the northern region. This pattern is in strong agreement with Korean tick surveillance data, which consistently demonstrate higher tick densities and higher SFTSV minimum infection rates in southern and southwestern regions characterized by warmer climates, grasslands, and mountainous terrain [6]. These ecological conditions support dense tick populations and diverse wildlife hosts, creating favorable environments for sustained SFTSV transmission. Dogs residing in or originating from these areas are therefore more likely to be exposed to infected ticks.

In South Korea, dogs categorized as shelter dogs are typically captured following reports of free-roaming or stray behavior and therefore represent animals with recent outdoor exposure prior to shelter admission. A particularly striking finding was the significantly higher prevalence of SFTSV in shelter dogs compared with pet dogs. This pattern is well supported by studies of other tick-borne pathogens in Korean dogs [17], which consistently show higher infection rates in shelter or stray dogs than in household pets. Shelter dogs are more likely to have experienced prolonged outdoor exposure, roaming, or contact with tick-infested environments prior to or during admission to shelters. In contrast, pet dogs are typically maintained in controlled environments and often receive regular tick prevention, which greatly reduces their risk of exposure. These results indicate that environmental exposure, rather than inherent host susceptibility, is the dominant determinant of SFTSV infection risk in dogs.

An important epidemiological feature of this study was the occurrence of a localized infection cluster in a shelter located in the southern region during autumn, where six of the 16 SFTSV-positive dogs were detected within the same period. All six dogs were housed in the same kennel, indicating that they were exposed to a shared environment. Notably, genotyping revealed that two of these dogs were infected with genotype F, whereas the remaining four were infected with genotype B, suggesting that the cluster likely resulted from exposure to multiple infected ticks carrying different viral lineages rather than a single point-source infection. All six dogs within the shelter cluster were sampled on the same day in September 2024, precluding temporal ordering of infections. Within-genotype phylogenetic comparison showed that the four genotype B2 sequences from the cluster were more closely related to each other than to other genotype B2 sequences detected outside the cluster, supporting localized exposure rather than independent introductions. One of the genotype F-infected dogs exhibited a particularly high viral load, from which live SFTSV was successfully isolated. Although virus isolation from the remaining dogs was unsuccessful, the temporal and spatial clustering strongly indicates intense exposure to infected ticks within the kennel environment. In addition, the close proximity of the dogs raises the possibility that limited short-range transmission among animals infected with the same genotype may have occurred, particularly within the genotype B-infected group, through contact with infectious secretions or contaminated surfaces. Although tick bites are considered the primary route of SFTSV transmission, non-tick-borne transmission has been reported in veterinary and household settings, including cases of human infection following close contact with infected companion animals in the absence of known tick exposure [23]. Furthermore, experimental studies have demonstrated efficient intraspecies transmission of SFTSV under co-housing conditions, supporting the plausibility of contact-associated transmission once the virus is introduced into a confined environment [24]. This cluster therefore illustrates how shelter environments may serve as focal points for both multiple independent tick-borne introductions and potential contact-associated transmission. Such conditions may facilitate local amplification of SFTSV and increase the risk of exposure for animal handlers and the surrounding community, underscoring the importance of rapid detection and isolation of infected dogs in high-density shelter settings.

Although all dogs included in this study were clinically normal at the time of blood sampling, follow-up information was available for the 16 SFTSV-positive dogs after molecular confirmation. Among them, 10 dogs remained asymptomatic throughout the observation period. In contrast, the six SFTSV-positive dogs identified within the shelter cluster subsequently exhibited mild and transient clinical signs, including low-grade fever, lethargy, and occasional vomiting. No severe or fatal outcomes were observed. These findings indicate that SFTSV infection in dogs is often subclinical, but mild clinical illness may occur, particularly under conditions of intense exposure such as those encountered in shelter environments.

Young dogs (<3 years) showed the highest prevalence of SFTSV, followed by adult dogs, whereas no infections were detected in older dogs. This age-related pattern parallels observations from studies of other canine tick-borne pathogens in South Korea, including H. canis and Anaplasma spp., which are also most frequently detected in younger animals [17]. Younger dogs tend to be more active, roam more widely, and have less consistent tick prevention, leading to greater exposure to tick habitats. In contrast, older dogs may have reduced outdoor activity and possibly partial immunity from previous exposures, resulting in lower detection rates.

Although a slightly higher prevalence was observed in female dogs, the difference was not statistically significant, indicating that sex itself is not a major determinant of SFTSV infection in dogs. This supports the conclusion that exposure-related factors—such as environment, roaming behavior, and management practices—are more important than intrinsic biological differences between sexes.

Previous molecular epidemiological studies have demonstrated that SFTSV circulating in South Korea is genetically diverse and can be classified into multiple genotypes corresponding to the A–F classification system. In humans, genotype B—particularly the B2 sublineage—has consistently predominated [2], whereas genotypes D and F have been detected less frequently [5]. This genotype structure is consistent with recent comprehensive evolutionary analyses of SFTSV in East Asia, which revealed long-term circulation, extensive genetic diversity, and frequent reassortment among viral lineages across China, South Korea, and Japan [3], providing a broader evolutionary framework for interpreting the multiple genotypes detected in dogs in the present study. Animal-derived SFTSV strains in South Korea have largely mirrored the genotype distribution observed in humans and ticks. Genotype B has been reported in mites [19], ticks [6], dogs [8,9,10,11,12], and cats [11,13], while more recent investigations have identified additional genotypes, including genotype D in dogs [10] and cats [13] and genotype F in dogs [12] and cats [13]. These findings indicate that multiple SFTSV genotypes co-circulate among animal hosts in South Korea, reflecting ongoing viral diversification and reassortment within the enzootic transmission cycle. Although phylogenetic reconstruction in this study was performed using the maximum likelihood method implemented in MEGA, the concordant clustering patterns observed across multiple genomic segments, together with genetic distance analyses and model selection-based justification of the nucleotide substitution model, support the robustness of genotype assignments.

In dogs, several studies have documented SFTSV infection based on RT-PCR, serology, and virus isolation. Earlier investigations in South Korea consistently identified genotype B as the dominant lineage in dogs [8,9,10,11,12], and the limited number of virus isolates obtained to date clustered within the Japanese clade, which corresponds to genotype B under the current classification scheme. Subsequent molecular surveys detected additional genotypes, including genotypes D [10] and F [12], in PCR-positive dogs; however, these genotypes were not recovered as infectious virus isolates, leaving the biological relevance of non-B genotypes in canine hosts uncertain.

In contrast to previous studies, the present work provides several novel contributions to the understanding of SFTSV infection in dogs in South Korea. First, this study represents the largest nationwide molecular survey of SFTSV in dogs to date, encompassing 715 samples collected across multiple regions and seasons. Second, while earlier investigations detected genotypes D and F only at the molecular level, we successfully isolated live SFTSV belonging to genotype F from a shelter dog, providing the first direct evidence of productive infection of this genotype in canine hosts. Third, the identification of a localized infection cluster involving multiple genotypes within a single shelter highlights the complex and dynamic nature of tick-borne exposure in high-risk environments. Together, these findings extend previous surveillance-based observations and demonstrate that dogs, particularly shelter dogs, can harbor and amplify genetically diverse SFTSV lineages circulating in the environment. Despite the successful isolation of infectious SFTSV, several limitations of the present study should be acknowledged. Although virus isolation provides direct biological evidence of productive infection in canine hosts, we did not perform subsequent in vitro characterization such as viral growth kinetics, replication efficiency, or host range assessment. As a result, the pathogenic potential and replication dynamics of the isolated genotype F strain could not be evaluated in detail. Importantly, the primary objective of this study was molecular surveillance and epidemiological characterization of SFTSV circulating in dogs, rather than experimental assessment of viral fitness or virulence. Future studies incorporating comparative growth analyses, cell tropism, and in vivo infection models will be necessary to fully elucidate the biological properties and pathogenic significance of SFTSV genotypes detected in canine hosts.

Although a high degree of nucleotide similarity was observed among some canine-derived SFTSV sequences, several lines of evidence argue against contamination during sample collection or laboratory processing. Samples were collected independently across multiple regions, institutions, and time points, and multiple genotypes (B2, D, and F) were detected, including within the same shelter on the same sampling day. The coexistence of distinct genotypes within a single localized cluster is inconsistent with a single-source contamination event. Furthermore, identical sequences were consistently obtained across multiple genomic segments and confirmed by cloning-based sequencing, supporting the authenticity of the detected viral sequences.

Similar levels of high intra-genotype nucleotide similarity have been reported in focal outbreaks and localized transmission settings of SFTSV in humans, animals, and ticks, where viral circulation occurs over short temporal and spatial scales [3,6,23]. These observations indicate that high sequence similarity is biologically plausible under conditions of recent introduction and limited local transmission and does not necessarily imply methodological artifacts.

In the present study, three SFTSV genotypes—B2, D, and F—were identified among PCR-positive dogs, indicating that genetically distinct viral lineages are actively circulating in the canine population of South Korea. Notably, infectious SFTSV was successfully isolated from a shelter dog, and this isolate was confirmed to belong to genotype F. This represents the first isolation of a genotype F SFTSV from a dog in South Korea and provides direct biological evidence that this lineage is capable of productive infection in canine hosts. Although genotype F has recently been detected in dogs at the molecular level, recovery of an infectious virus had not previously been achieved.

The isolation of genotype F from a shelter dog is epidemiologically plausible when considered in the context of tick ecology in South Korea. Nationwide tick surveillance has shown that SFTSV-infected ticks are widely distributed [6], with particularly high tick densities and infection rates in grasslands, forest edges, and cemetery areas—habitats that are commonly accessed by free-roaming or shelter dogs. Moreover, seasonal studies have demonstrated that tick larvae increase markedly from late summer to early autumn, coinciding with the peak period of SFTS incidence in humans [6]. Dogs housed in shelters or exposed to outdoor environments during this period are therefore likely to encounter multiple tick developmental stages carrying genetically diverse SFTSV strains.

This study has several limitations. Although genotype-level analyses were performed, the small number of PCR-positive dogs (n = 16) limited statistical power to detect significant associations between viral genotypes and epidemiological variables. Accordingly, genotype–epidemiological relationships should be interpreted cautiously, and larger-scale longitudinal studies will be required to confirm these patterns. Despite these limitations, descriptive integration of phylogenetic and epidemiological data suggests that multiple SFTSV genotypes co-circulate in canine populations and that shelter environments may facilitate exposure to diverse viral lineages within a short time frame. The detection of both genotype B2 and genotype F within a single shelter cluster sampled on the same day highlights the dynamic nature of local tick-borne transmission.

Taken together, these findings support the interpretation that shelter dogs are repeatedly exposed to infected ticks and may acquire SFTSV strains representing different genetic lineages, including relatively uncommon genotypes such as F. The successful isolation of a genotype F virus from a shelter dog extends previous PCR-based observations and underscores the value of canine surveillance as a sensitive indicator of SFTSV diversity circulating in the environment. From a One Health perspective, these results highlight the importance of implementing routine molecular surveillance of SFTSV in dogs, particularly in high-risk settings such as animal shelters. Specific measures may include systematic screening of shelter dogs upon admission, integration of canine surveillance data with ongoing tick and human SFTS monitoring programs, and targeted tick control strategies in shelter environments. In addition, education of shelter staff, veterinarians, and adopters regarding tick-borne risks and appropriate preventive measures may help reduce the risk of zoonotic transmission. Continuous monitoring of dogs with frequent outdoor exposure may therefore contribute to early detection of emerging or under-recognized SFTSV genotypes and support coordinated public health interventions aimed at mitigating SFTSV transmission at the human–animal–environment interface.

4. Materials and Methods

4.1. Ethical Approval

Blood samples used in this study were residual diagnostic samples obtained from dogs by veterinary practitioners at local clinics and animal shelters during routine surveillance, treatment, and health monitoring, as well as during regular medical checkups after verbal consent was obtained from the owners. As this study utilized only leftover diagnostic samples collected for clinical and surveillance purposes and did not involve any experimental or invasive procedures on animals, approval from an Institutional Animal Care and Use Committee was not required.

4.2. Sample Size Determination and Sample Collection

Sample size was determined by power analysis based on an expected disease prevalence of 10%, an absolute precision of 5%, and a confidence level of 95%, assuming a simple random sampling design [25]. Based on this calculation, a minimum of 138 samples was required. A total of 715 blood samples were randomly collected from clinically normal dogs between January and December 2024 at local veterinary clinics and animal shelters located in multiple regions of South Korea. All samples were residual diagnostic specimens obtained during routine clinical care, surveillance, or health checkups, and each dog was sampled only once. Whole blood was collected into EDTA-containing tubes and transported to the laboratory under refrigerated conditions. Upon arrival, samples were processed immediately or stored at −20 °C until nucleic acid extraction. Information on geographic region, source (pet or shelter), sex, age, and season at the time of sampling was recorded for subsequent analyses. No longitudinal follow-up or repeated sampling of the same individuals across seasons was performed. To minimize the risk of cross-contamination, samples were handled individually using sterile, single-use consumables, and all laboratory procedures followed strict unidirectional workflows with physical separation of sample processing, nucleic acid extraction, PCR setup, and post-amplification analysis. No pooling of samples was performed at any stage, and each sample was processed and analyzed independently.

4.3. Molecular Detection of SFTSV

Total RNA was extracted from dog whole blood samples using the RNeasy Mini Kit (Qiagen, Melbourne, Australia) according to the manufacturer’s instructions. RT-PCR was performed using AccuPower RT-PCR Premix and AccuPower HotStart PCR Premix kits (Bioneer, Daejeon, Republic of Korea). For molecular detection of SFTSV, the viral L segment was amplified by RT-PCR using primers LF1 and LR2 [8], and the M segment was amplified by RT-PCR using primers MF3 and MR2 [20]. The S segment was detected using a nested PCR approach: the first-round RT-PCR was performed with primers NP2-F and NP2-R, followed by a second-round nested PCR using primers N2-F and N2-R, as previously described [26]. The expected amplicon sizes, primer sequences, target genes, and PCR cycling conditions are provided in Supplementary Table S2. The genomic positions of all primer sets were determined by alignment to reference SFTSV genomes (GenBank accession nos. KP202163–KP202165) and are summarized in Supplementary Table S2.

4.4. DNA Cloning

PCR amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen) and ligated into pGEM-T Easy vectors (Promega, Madison, WI, USA). The recombinant plasmids were transformed into Escherichia coli DH5α competent cells (Thermo Fisher Scientific, Wilmington, DE, USA) and incubated overnight at 37 °C. Plasmid DNA was extracted using a plasmid miniprep kit (Qiagen) according to the manufacturer’s protocol.

4.5. DNA Sequencing and Phylogenetic Analysis

Recombinant clones were selected and sequenced commercially by Macrogen (Seoul, Republic of Korea) using vector-specific primers. Cloning-based sequencing was performed instead of direct Sanger’s sequencing to minimize the risk of mixed chromatogram signals and to ensure sequence accuracy, particularly in samples with low viral load or potential co-circulating variants. Nucleotide sequences obtained in this study were aligned with reference sequences retrieved from GenBank using CLUSTAL Omega (version 1.2.1). Sequence editing and trimming were performed using BioEdit (version 7.2.5) to remove ambiguous regions. Positions containing gaps or poorly aligned sites were excluded prior to phylogenetic analysis. Phylogenetic trees were constructed using MEGA version 6.0 based on the maximum likelihood method with the Kimura two-parameter (K2P) model. Bootstrap analysis with 1000 replicates was conducted to assess the robustness of tree topology. Pairwise sequence comparisons were performed to evaluate nucleotide homology. The optimal nucleotide substitution model was evaluated using the built-in model selection function in MEGA version 6.0, which compares candidate models based on the Akaike Information Criterion and Bayesian Information Criterion. Among the tested models, the K2P model was selected as an appropriate model for the present datasets or showed no substantial difference in model fit compared with more complex models. Given the relatively short alignment lengths and limited sequence divergence within genotypes, the K2P model was considered suitable for maximum likelihood phylogenetic reconstruction. For each PCR-positive sample and each genomic segment, multiple independent recombinant clones were sequenced to assess sequence consistency. Based on this analysis, identical nucleotide sequences were obtained from clones derived from the same sample. Accordingly, a single representative consensus sequence per genomic segment and per dog was used for subsequent phylogenetic analyses.

4.6. Sequence Quality Control and Contamination Prevention

To ensure sequence quality and minimize the risk of contamination, all RT-PCR procedures were conducted using physically separated pre- and post-amplification work areas and aerosol-resistant pipette tips. Each RT-PCR run included a negative control (nuclease-free water) and a positive control [21]. To further reduce the possibility of PCR carry-over contamination, PCR amplicons selected for sequencing were cloned into plasmid vectors, and individual recombinant clones were sequenced. For each sample, multiple independent clones were selected for sequence analysis. Sequence chromatograms were manually inspected, and low-quality regions were trimmed prior to alignment. Comparative alignment across the S, M, and L genomic segments was used to assess sequence consistency.

4.7. Virus Isolation and Identification

To isolate SFTSV from dog blood samples, whole blood was mixed with an equal volume of Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with penicillin (5000 IU/mL) and streptomycin (5 mg/mL), clarified by centrifugation, and filtered through a 0.45 µm syringe filter. The resulting supernatants were inoculated onto monolayers of Vero E6 cells (ATCC No. CRL-1586; American Type Culture Collection, Manassas, VA, USA) in 6-well plates and incubated at 37 °C in a humidified incubator with 5% CO₂ for 2 h to allow virus adsorption. After adsorption, the inoculum was removed and replaced with DMEM supplemented with 2–10% fetal bovine serum as maintenance medium, and the cells were incubated for 7 days [27,28]. Cells were monitored daily for cytopathic effects. Cell culture supernatants were blind-passaged up to two times onto fresh Vero E6 monolayers. Aliquots of supernatants were collected at each passage and stored at −70 °C until further analysis. After viral adaptation and replication in Vero E6 cells, the presence of SFTSV was confirmed by RT-PCR targeting the S, M, and L genomic segments. The amplified products were subsequently sequenced to confirm viral identity and genotype. Viral antigen expression in infected cells was further verified by IFA using anti-SFTSV rabbit polyclonal and mouse monoclonal antibodies, as previously described [29]. All virus isolation experiments were performed in a biosafety level-3 laboratory in accordance with institutional biosafety regulations.

4.8. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism version 5.04 (GraphPad Software Inc., La Jolla, CA, USA). Associations between categorical variables were evaluated using Pearson’s chi-square test. A p-value ≤ 0.05 was considered statistically significant. Ninety-five percent confidence intervals (95% CIs) were calculated for all estimates.

5. Conclusions

This study demonstrates that SFTSV is actively circulating in dogs in South Korea, with a prevalence of 2.2% and significant associations with season, geographic region, source, and age, reflecting patterns of tick exposure and environmental risk. A localized cluster of infections in a southern shelter during autumn, involving multiple dogs housed in the same kennel, highlights the potential for intense exposure to infected ticks and local amplification of the virus in high-density settings. Importantly, we identified three SFTSV genotypes (B2, D, and F) in dogs and successfully isolated a genotype F virus from a shelter dog, representing the first isolation of this genotype from dogs in South Korea. The presence of both B and F genotypes within the same cluster suggests multiple tick-borne introductions with possible limited transmission among dogs infected with the same genotype. Together, these findings indicate that dogs—particularly shelter and free-roaming dogs—can serve as important incidental hosts and sentinels for monitoring SFTSV diversity in the environment. Integrated surveillance of dogs and ticks may therefore improve early detection of emerging viral lineages and support public health efforts to reduce the risk of zoonotic transmission.