Hyalomma ticks are known for their ability to transmit a wide range of pathogens, posing a significant threat to both human and animal health. The viral communities associated with Hyalomma asiaticum ticks in Kazakhstan remain largely unexplored. Using high-throughput sequencing on the Ion Torrent platform, nine RNA viruses belonging to seven families were identified. These viruses were associated with H. asiaticum ticks collected in 2024 in southern Kazakhstan. The detected viruses—including Bole tick virus 1 (Phenuiviridae), Bole tick virus 3 (Chuviridae), Bole tick virus 4 (Flaviviridae), Hubei toti-like virus 24 (Totiviridae), Kashgar totiv tick virus 1 (Totiviridae), Lonestar tick totivirus (Totiviridae), Qingyuan parti tick virus 1 (Partitiviridae), and Taishun tick virus (Rhabdoviridae)—had previously been reported only in China. This study provides the first documented evidence of the presence of RNA viruses previously unreported in Kazakhstan within H. asiaticum tick populations. A newly isolated Kazakhstan strain of Wad Medani virus (Sedoreoviridae), identified from H. asiaticum, shares genetic similarities with Russian strains, suggesting a common epidemiological landscape across Central Eurasia. The detection of novel RNA viruses in Kazakhstan highlights the need for ongoing surveillance, as their impact on human and animal health remains insufficiently understood.

Background: Brucellosis remains a significant zoonotic infection affecting approximately 500,000 people worldwide annually, with no licensed human vaccine available. Recombinant influenza virus vectors expressing Brucella spp. antigens represent a promising vaccine platform. However, transitioning from laboratory constructs to clinical candidates requires validation of scalable purification methods compliant with Good Manufacturing Practice (GMP) standards. This study aimed to develop and optimize a pilot-scale purification protocol for these vectors. Methods: Recombinant influenza A viruses (H5N1) expressing Brucella spp. antigens (Omp16, Omp19, L7/L12, Cu-Zn SOD) were propagated in MDCK cell culture. The optimized purification process included: (1) clarification; (2) ultrafiltration/diafiltration (100 kDa MWCO); (3) two-step chromatography (anion-exchange Q-Sepharose^® Fast Flow and multimodal Capto™ Core 700); and (4) sterile filtration. Process validation was performed across three independent pilot-scale batches (20 L each). Results: The purification process demonstrated high reproducibility for all constructs. Final preparations met established quality criteria: infectious titer ≥ 5.2 log₁₀ TCID₅₀/mL, hemagglutination activity 7.33 ± 0.58–8.33 ± 0.58 log₂, total protein content 157–305 μg/mL, residual host cell DNA < 10 ng/dose, and bacterial endotoxin levels ≤ 0.15 IU/mL. The overall recovery of infectious virus was 20–24%, an optimal value for multi-stage bioprocessing. Preservation of the target genetic insert was confirmed in all final preparations by PCR and sequencing. Conclusions: The developed integrated purification protocol yields vectors with high purification efficiency, preserving biological activity and meeting regulatory quality requirements for residual host cell DNA and endotoxins. The technological platform demonstrated versatility, robustness (inter-batch coefficient of variation for yield did not exceed 10–12%), and scalability, establishing a foundation for preclinical and clinical studies of candidate brucellosis vaccines.

Brucellosis is still an important public health problem as long as natural reservoirs of infection exist. Currently, live attenuated vaccines based on strains S19, RB51 and Rev1 are used for the prevention of brucellosis in animals, the main disadvantage of which is virulence for humans. However, animal immunization programs should be implemented to reduce the incidence of humans. The development of safe and effective new generation vaccines using “omix” technology is a promising direction of vaccinology. A number of immunogenic Brucella proteins that elicit both a humoral and cellular immune response has been identified. The aim of these research was to optimize the expression and purification conditions of the Brucella spp. recombinant proteins L7/L12 and SodC. As a result, expressing plasmids pET/Br-L7/L12 and pET/Br-SodC were obtained. The parameters of target genes expression in E. coli were established and the method for purification of recombinant proteins was optimized. Purification of the L7/L12 protein was performed under hybrid conditions on HisPur agarose using a binding buffer containing 6 M guanidine hydrochloride, a wash buffer with 20 mM imidazole and an elution buffer with 300 mM imidazole. Protein SodC was purified under denaturing conditions with the addition of 1 % Triton X-100 and 1 % sodium deoxycholate to the lysis buffer. Inclusions were solubilized with a buffer containing 8 M urea and 5 mM imidazole. The target protein was eluted from HisPur agarose with buffer containing 8 M urea and 100 mM imidazole. The use of modified purification protocols made it possible to obtain purified recombinant proteins with a yield of 13 mg/L for the L7/L12 protein and 10 mg/L for the protein SodC, respectively. The specificity of the proteins was confirmed by a Western blot. Immunization of mice with recombinant proteins led to the production of specific antibodies, the titer of which in ELISA was 1:20480 and 1:20480, respectively.

This article presents the results of studies on biological samples collected from 640 swabs taken from dogs and cats across various regions of the Republic of Kazakhstan. These samples were part of a monitoring study on the spread of coronavirus among domestic animals. Total RNAs were isolated using the magnetic sorption method with the ALPREP kit and subsequently analyzed with the ALSENSE-SARS-CoV-2 RT-qPCR kit. Real-time RT-PCR analysis revealed that 104 samples tested positive within 22–37 amplification cycles. These positive samples were then cultured in Vero cell lines to confirm the presence of the virus. The biological activity of the resulting virus-containing suspension was determined using the Reed-Muench method. During cultivation, one viral isolate with a biological activity of 5.83±0.08 lg TCID50/ml was obtained. A microphotograph of the virus was taken using an electron microscope to determine its size, shape, and structure, which confirmed its morphology corresponding to the Coronaviridae family. The data obtained further indicate that domestic animals can suffer from and carry coronavirus. It is becoming increasingly evident that the virus can infect and replicate in the organs of various farm and domestic animals.

The COVID-19 pandemic caused bу SARS-CoV-2 coronavirus has emphasised the need for rapid and accurate diagnosis to effectivelу control the spread of the disease. RT-PCR of RT is one of the most sensitive and specific methods for detection of virus RNA. However, the qualitу and efficiencу of RT-PCR are directlу related to the proper choice of primers and probes, and high accuracу and reliabilitу are related to the use of controls. The aim of this work is to develop primers and probes for the diagnosis of COVID-19 and to create a positive control sample for the test sуstem. This work applies modern approaches to the selection of targets for amplification, which include the analуsis of conserved regions of the viral genome. Bioinformatics tools were also used to select primers and probes for detection of SARS-CoV-2 virus. A sуnthetic construct containing target regions of the virus genome was used to create a control sample, which was successfullу cloned into a plasmid vector. As part of the studу, primers and a fluorescent probe specific to conserved regions of the SARS-CoV-2 virus genome were designed, taking into account genetic variations in the virus. The resulting positive control was successfullу tested in the assaу, confirming its efficacу and reproducibilitу. The developed primers and probes for SOVID-19 diagnosis can be used for epidemiological monitoring and control of SARS-CoV-2 spread with clinical and diagnostic purposes. The developed control sample can be used to standardise the diagnosis of COVID-19, improving the accuracу and reliabilitу of the assaу in clinical practice.