Sample collection and preparation were conducted in accordance with the WOAH Terrestrial Manual 2024 (Chapter 1.1: Specimen Collection, Submission, and Preparation). Biological specimens of ixodid ticks were collected from livestock such as cattle, sheep, and goats across farms and private holdings in three regions of Kazakhstan: Zhambyl (382 samples, 11.7%), Turkestan (2,690 samples, 81.9%), and Kyzylorda (209 samples, 6.4%) in 2024. In the Turkestan region, tick sampling was additionally stratified by administrative district, with collections performed in Shardara, Arys, Maktaaral, and Baitibek districts to enable localized analysis of virus circulation patterns. Sampling was conducted as single-timepoint examinations, involving 10 to 30 animals per herd. Particular attention was given to body regions where ticks are known to commonly aggregate, including the neck, axillae, groin, perianal area, udder, and tail base.

Host preference analysis was performed by examining 10% of animals within selected herds. The average tick burden per host species was calculated using the formula:

where BB = host species, KK = number of ticks on the host species, and SS = total ticks collected from all host species.

Tick collection was conducted exclusively from cattle in private household farms located in endemic southern regions of Kazakhstan. A total of 3,281 adult ticks were collected, including 382 samples (11.7%) from Zhambyl, 2,690 samples (81.9%) from Turkestan, and 209 samples (6.4%) from Kyzylorda regions. Following morphological identification, the collected specimens were classified into nine tick species belonging to the family Ixodidae.

Ticks were manually removed using surgical forceps to avoid damaging mouthparts and preserved in 70% ethanol. Morphological identification was confirmed microscopically using taxonomic keys (32). All procedures were performed in accordance with biosafety protocols 3.1.1027-01: Guidelines for the Collection and Laboratory Analysis of Arthropod Vectors (33).

A total of 3,281 adult ticks were analyzed, including Hyalomma scupense (27.5%), Hyalomma anatolicum (19.6%), Dermacentor marginatus (15.2%), Boophilus calcaratus (6.6%), Dermacentor pictus (5.6%), Haemaphysalis otophila (0.7%), Haemaphysalis punctata (0.5%), Rhipicephalus bursa (0.4%), Dermacentor niveus (0.2%), Argas persicus (23.7%), and Alveonasus lahorensis (0.03%). Ticks were homogenized in 2.0 mL DNase/RNase-free tubes (Eppendorf) with zirconia-silica beads (6 mm) and DMEM medium (Capricorn Scientific) using a LabSafer TS-48/64 homogenizer. Homogenates were freeze-thawed three times, clarified by centrifugation (600 × g, 10 min), and supernatants pooled by species for downstream analysis.

Total nucleic acids were extracted using the PureLink Microbiome DNA/RNA Purification Kit (Invitrogen, USA) following the manufacturer’s protocol.

Detection of arboviruses in tick samples was performed using different PCR-based approaches depending on the viral genome type. Ticks were pooled into groups of 3–5 individuals according to species, sex, and sampling location prior to nucleic acid extraction and subsequent molecular analysis. For the RNA viruses—CCHFV, SBV, and BTV—we employed either one-step RT-PCR or nested RT-PCR using virus-specific primers to enhance analytical sensitivity and detect low viral loads. Real-time RT-PCR (AmpliSens® kits, Russia) was additionally used for confirmation of CCHFV-positive samples. For the DNA virus LSDV, conventional PCR targeting the P32 gene was performed using primers described by El-Nahas et al. (34). For phylogenetic analysis, partial fragments of the S segment (~536 bp) and L segment (~476 bp) of the CCHFV genome were amplified. All procedures were performed in a BSL-3 facility compliant with WHO biosafety guidelines.

BTV and SBV were detected by one-step reverse transcription quantitative PCR (RT-qPCR), whereas LSDV, a DNA virus was tested by a quantitative PCR assay. We used commercial kits specific for each virus: Virotype® BTV RT-PCR Kit and Virotype® SBV RT-PCR Kit (Qiagen, Germany); and VetMAX® LSDV/Capripox PCR Kit (Thermo Fisher Scientific, USA). Reactions were performed according to the manufacturer’s instructions.

Viral RNA was amplified using the BioMaster One-Step RT-PCR–Extra kit (2×) (Biolabmix, Russia) following the manufacturer’s instructions. The first-round RT-PCR was carried out in a 20 μL reaction volume containing 5 μL of extracted RNA and the following primer sets targeting the S, M, and L segments of the CCHFV genome (Table 1). Thermal cycling conditions were as follows: reverse transcription at 50 °C for 30 min; initial denaturation at 95 °C for 5 min; followed by 35 cycles of 95 °C for 40 s, 58 °C for 40 s, and 72 °C for 90 s; with a final extension at 72 °C for 5 min. The second-round PCR was performed in a 20 μL reaction containing 5 μL of the first-round product and the following in Table 1 primers. Thermal cycling conditions were: initial denaturation at 95 °C for 5 min; followed by 35 cycles of 95 °C for 1 min, 49 °C for 40 s, and 72 °C for 90 s; with a final elongation step at 72 °C for 5 min.

PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Visualization was performed using the GelDoc imaging system (Bio-Rad, USA) and analyzed with Image Lab™ Software (Bio-Rad, USA).

The sequences were assembled using SeqMan software (DNASTAR, USA; version 6.1) and aligned using the MAFFT algorithm. The reference sequences published in the manual by Lukashev et al. (47) were used for the analysis. Evolutionary models were estimated in the MEGA 11 program, the best-fitting model for nucleotide sequences was Tamura et al. (35). The Tamura–Nei model was selected as the best-fit nucleotide substitution model based on Bayesian Information Criterion (BIC). Phylogenetic trees were constructed using the maximum likelihood method, incorporating the nearest neighbor interchange (NNI) heuristic algorithm and an automatically generated initial tree based on the default NJ/MP approach. Branch reliability was measured using bootstrap analysis with 1,000 repetitions. The percentage identity between sequences was calculated in the MegAlign program, which is part of the Lasergene 6.0 package (DNASTAR, USA). Phylogenetic trees based on the partial S and L genome segments of the studied CCHFV isolates are presented in Supplementary Figures 1, 2. The primer sequences used for amplification of the target regions of viral genomes, as well as the PCR conditions, are presented in Table 1.

To assess the prevalence of arboviral infections in ixodid ticks, a comprehensive molecular screening was conducted using real-time reverse transcription PCR (RT-qPCR) for the detection of viral RNA/DNA, including BTV, SBV, LSDV, and CCHFV. All samples tested negative for BTV, SBV, and LSDV. Only CCHFV RNA was detected in the tick samples, and therefore, only this virus was further analyzed by nested RT-PCR and sequencing.

A total of 3,281 adult ticks (2,627 females and 654 males) were collected from cattle herds across three regions of Kazakhstan: Zhambyl (382 samples, 11.7%), Turkestan (2,690 samples, 81.9%), and Kyzylorda (209 samples, 6.4%). Ticks were sampled from livestock using standardized protocols, focusing on body regions with high tick aggregation (e.g., neck, axillae, groin, udder, and tail base). All specimens were transported to the National Reference Center for Veterinary Medicine for further analysis. The sampling location and quantity are shown in Figure 1.

Morphological identification confirmed the presence of nine species from the Ixodidae family—Dermacentor marginatus (D. marginatus), Dermacentor pictus (D. pictus), Dermacentor niveus (D. niveus), Hyalomma anatolicum (H. anatolicum), Haemaphysalis punctata (Hae. punctata), Hyalomma scupense (H. scupense), Rhipicephalus bursa (R. bursa), Boophilus calcaratus (B. calcaratus), and Haemaphysalis otophila (Hae. otophila). Additionally, two species from the Argasidae family were identified: Alveonasus lahorensis (A. lahorensis) and Argas persicus (A. persicus).

Epidemiological surveys and sample collection were conducted during scientific expeditions to Zhambyl, Turkestan, and Kyzylorda regions. The collected ticks were screened for arboviral pathogens using RT-qPCR assays targeting conserved genomic regions of BTV, SBV, LSDV, and CCHFV.

The species distribution of ticks in southern Kazakhstan revealed the following prevalence - H. scupense (27.5%), H. anatolicum (19.6%), D. marginatus (15.2%), B. calcaratus (6.6%), D. pictus (5.6%), Hae. Otophila (0.7%), H. punctata (0.5%), R. bursa (0.4%), and D. niveus (0.2%). Among Argasidae, Argas persicus was the most abundant (23.7%), while A. lahorensis was rare (0.03%).

These findings highlight the dominance of Hyalomma and Dermacentor species as potential vectors for arboviral transmission in the region. The high prevalence of H. scupense and H. anatolicum is particularly noteworthy, given their known role in transmitting CCHFV and other zoonotic pathogens.

To assess the prevalence and distribution of arboviral infections in Kazakhstan, tick pools were collected from three regions—Turkestan, Kyzylorda, and Jambyl—during 2024. The collected ticks were grouped by species and sex, and each pool was subjected to real-time RT-PCR (RT-qPCR) for the detection of BTV, SBV, and LSDV, and nested RT-PCR for the detection of CCHFV, including BTV, SBV, LSDV, and CCHV. Table 2 presents a detailed breakdown of the total number of ticks per species and region, along with the corresponding real-time RT-PCR and nested RT-PCR results. While real-time RT-PCR results for BTV, SBV, and LSDV were predominantly negative, CCHFV positivity varied across species and regions, with notable differences observed between female and male tick pools. These findings underscore the regional and taxonomic heterogeneity of arboviral infections in ticks and emphasize the need for ongoing surveillance in these endemic areas.

Tick pools (grouped by species and sex) for the presence of viruses from Turkestan, Kyzylorda, and Zhambyl regions were screened by RT-PCR for four arboviruses: CCHFV, BTV, SBV, and LSDV (Table 3). CCHFV was the only virus detected among all samples; no pools tested positive for BTV, SBV, or LSDV. Notably, all CCHFV-positive pools were from the Turkestan region and consisted of female ticks, with Dermacentor pictus showing the highest infection rate among the species tested.

The results presented in Table 3 indicate a marked heterogeneity in both tick abundance and arboviral infection rates across the three regions examined. In the Turkestan region—the area with the highest tick yield (2,685 individuals)—female pools exhibited notable rates of CCHFV positivity. CCHFV RNA was most frequently detected in D. pictus, with progressively lower positivity rates observed in H. anatolicum, H. scupense, and D. marginatus.

In both the Kyzylorda and Jambyl regions, overall tick numbers and the detection of arboviral infections were considerably lower than in Turkestan. This disparity suggests that Turkestan may represent a potential hotspot for CCHFV transmission, particularly within female tick populations of specific species. The absence of evidence for other arboviruses further underscores the predominant epidemiological relevance of CCHFV in these settings. Of the four targeted arboviruses, only CCHFV RNA was detected in the analyzed tick pools. All samples tested negative for BTV, SBV, and LSDV by PCR. Therefore, sequencing and phylogenetic analysis were conducted exclusively for CCHFV-positive samples.

Detailed analysis of CCHFV positivity by taxonomic classification and sex revealed notable differences among tick species. Within the Ixodidae family, the highest infection rate was observed in female D. pictus (18.58%), followed by D. marginatus (5.02%), H. anatolicum (3.45%), and H. scupense (4.55%). No CCHFV RNA was detected in male ticks or in any specimens from the Argasidae family. These findings are summarized in Table 2, which presents species-level tick abundance and CCHFV positivity rates based on pooled molecular screening.

The results presented in Table 2 highlight marked interspecific variation in CCHFV positivity among female Ixodidae ticks. Notably, D. pictus and D. marginatus exhibited higher rates of infection compared to other taxa, suggesting a potential role as primary vectors in the region. In contrast, ticks from the Argasidae family showed no detectable CCHFV RNA, indicating their limited involvement in the virus’s local transmission cycle. These data underscore the importance of species- and sex-specific surveillance in understanding the ecology of tick-borne viruses.

To further examine localized patterns of CCHFV circulation, PCR assays were conducted on ticks collected from four districts within the Turkestan region. The highest CCHFV positivity was observed in D. pictus from Shardara District (20.5%), followed by H. anatolicum from Arys District (15.0%), D. marginatus from Maktaaral District (14.3%), and H. scupense from Baitibek District (16.7%) (Table 4).

The results presented in Table 4 demonstrate considerable variation in CCHFV positivity among tick species across different localities within the Turkestan region. Notably, D. pictus showed the highest infection rate (20.5%), suggesting a prominent role in virus maintenance. D. marginatus, H. anatolicum, and H. scupense also exhibited moderate positivity rates (14.3–16.7%), highlighting species-specific and ecological contributions to virus circulation.

A total of 97 out of 760 ticks (12.7%) tested positive for CCHFV RNA based on pooled analysis. These findings underscore the importance of tick species composition, local environmental factors, and host–vector interactions in shaping arbovirus transmission dynamics. The data support targeted surveillance and vector control strategies in high-risk districts of southern Kazakhstan.

To confirm the identity of the detected viral RNA and assess the genetic diversity of circulating CCHFV strains, representative PCR-positive samples (n = 97) were subjected to Sanger sequencing. Phylogenetic trees were constructed in MEGA v12 using the maximum likelihood method with the Tamura-Nei substitution model, identified as the best-fit model based on Bayesian Information Criterion (BIC) scores. Bootstrap analysis with 1,000 replicates was performed to evaluate the robustness of the tree topology. Reference sequences were retrieved from GenBank and included strains from Central Asia (Uzbekistan, Tajikistan), East Asia (China), and the Middle East, enabling comparative assessment of the Kazakhstani isolates.

Pairwise nucleotide identity values are presented in Figure 2 for both the S and L segments. The sequences from the present study demonstrated 92.1–98.4% identity with regional strains from Uzbekistan, Tajikistan, and China, confirming their affiliation with the Asia-1 and Asia-2 genotypes. The high similarity with strains circulating in neighboring countries supports the hypothesis of transboundary virus exchange via livestock or tick migration.

To investigate the evolutionary relationships of the CCHFV strains detected in this study, phylogenetic trees were constructed based on partial nucleotide sequences of the S and L genome segments. Representative PCR-positive samples from the Turkestan region—CCHF_15407_KZ_Maktaaral_2024 (S segment) and CCHF_ Maktaaral_2024 (L segment)—were sequenced and analyzed alongside reference strains retrieved from GenBank.

The phylogenetic analysis of the S segment is shown in Supplementary Figure 1 demonstrated that the Kazakhstani isolate clustered within the Asia-1 genotype, showing close genetic relatedness to strains from Uzbekistan (AY277437, Hoftha, 1967) and India (JN572089, 2011). The high sequence identity (96.1–96.7%) and short evolutionary distance support the hypothesis of a shared regional lineage with persistent circulation in Central Asia.

In contrast, the tree based on the L segment (see Supplementary Figure 2) showed that the Kazakhstani isolate from CCHF_154sam S segment Kazakhstan_Arys_2024 grouped with strains from Asia-2 genotype, including reference sequences from Turkmenistan (KX013463), Afghanistan (MH128201), and Uzbekistan (AY347889). This clade suggests a distinct but geographically proximate lineage that likely reflects long-term endemicity and cross-border virus maintenance.

Together, these results indicate that multiple genetic variants of CCHFV are co-circulating in southern Kazakhstan. The phylogenetic placement of the isolates within Asia-1 and Asia-2 genotypes, and their clustering with strains from neighboring countries, provide compelling evidence of transboundary virus transmission, potentially facilitated by livestock trade and the movement of infected tick vectors.

The phylogenetic relationships were inferred using the Tamura-Nei substitution model and a heuristic search with nearest-neighbor interchange (NNI) in MEGA version 12. The isolate obtained in this study (CCHF_165_KZ_Shardara_2024) is highlighted in bold and clusters within the Asia-1/2 genotype clade. Bootstrap values (>70%) are indicated at major nodes. Reference sequences representing recognized genotypes (Europe 1–2, Asia 1–3, Africa 2–3) were included to illustrate the genetic positioning of the Kazakhstani isolate within the global diversity of CCHFV.

The tree was constructed using the Tamura-Nei substitution model and the maximum likelihood method in MEGA version 12, with bootstrap support values (>70%) indicated at key nodes. The isolates obtained in this study (CCHF_154sam_KZ_Arys_2024 and CCHF_75sam_KZ_Maktaaral_2024) are shown in bold and cluster within the Asia-2 genotype. Reference sequences from GenBank representing major global genotypes (Asia 1–2, Africa 1–3, Europe 1–3) were included to contextualize the phylogenetic placement of the Kazakhstani strains within the broader genetic diversity of CCHFV.