The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 μg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 2¹⁷, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Crimean-Congo hemorrhagic fever (CCHF), which has a fatality rate of 20–30%, is widely prevalent in several regions in Asia, Europe, and Africa and has spread to a wider range of areas in recent years. At present, there is a lack of safe and effective vaccines for the prevention of CCHF. In this study, we prepared three vaccine candidates, rvAc-Gn, rvAc-Np, and rvAc-Gn-Np, that encoded the CCHF virus (CCHFV) glycoprotein Gn and the nucleocapsid protein (Np) on the surface of baculovirus using an insect baculovirus vector expression system (BVES) and evaluated their immunogenicity in BALB/c mice. The experimental results showed that both CCHFV Gn and Np were expressed by the respective recombinant baculoviruses and anchored to the viral envelope. BALB/c mice were immunized, and all three recombinant baculoviruses showed significant humoral immunity. At the cellular level, the level of immunity in the rvAc-Gn group was significantly higher than that in the rvAc-Np and rvAc-Gn-Np groups, and the rvAc-Gn-Np coexpression group exhibited the lowest level of cellular immunity. In conclusion, the strategy of coexpressing Gn and Np in the baculovirus surface display system did not result in improvements in immunogenicity, whereas the recombinant baculovirus displaying Gn alone could induce significant humoral and cellular immunity in mice, indicating that rvAc-Gn has potential as a CCHF vaccine candidate. This study thus provides new ideas for the development of a CCHF baculovirus vaccine.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), which can cause severe clinical disease and even death in humans. In recent years, the disease has spread to a wider area, posing a major public health threat to China as well as the Middle East, Europe and Africa, and there is no safe and effective vaccine to prevent the disease. Recently, it has been shown that using the Zera fusion to target proteins can enhance immunogenicity and improve the potential for developing viral vaccines. Based on this finding, in this study, two vaccine candidates, Zera-Gn and Zera-Np, were prepared using an insect baculovirus system expressing CCHFV glycoprotein (Gn) and nucleocapsid protein (Np) fused with Zera tags, and evaluated for immunogenicity in BALB/c mice. The obtainedresults showed that both Zera-Gn and Zera-Np recombinant nanoparticles were successfully expressed, and Zera-Gn had good induction of humoral and cellular immunity in mice, and its immunogenicity was significantly higher than that of Zera-Np. The results indicated that Zera-Gn self-assembled nanoparticles prepared by fusing Zera tags with CCHFV spike-in protein Gn have the potential to be a candidate vaccine for CCHF, and this study provides a reference for the development of Zera self-assembled nanoparticle vaccine for CCHF.

Background/Objectives: Mycobacterium tuberculosis is the causative agent of tuberculosis and the leading cause of death from a single infection with the microorganism. Tuberculosis remains globally one of the major diseases leading to high mortality rates, with serious implications for public health and economic development. Therefore, tuberculosis prevention and control is crucial for global health and socio-economic stability. The development of effective preventive vaccines remains an urgent task in the fight against tuberculosis. Methods: The Mycobacterium tuberculosis antigen PPE18 was fused to Zera, and Bacmid was extracted and transfected into Sf9, which was purified and characterized for the formation of nanoparticle protein bodies. BALB/c mice and calves were immunized, and the immunogenicity of the nanoparticle vaccine was assessed by serum antibodies and splenic lymphocytes. Results: Zera-71CA-mCherry can be expressed in Sf9 cells, forming 0.5–1.2 μm protein bodies. Excising the mCherry sequence, Zera-71CA/Zera-PPE18 candidate nanoparticle-immunized mice were able to elicit serum antibody levels and the proliferation of splenic lymphocytes, and immunized calves were determined to have high levels of serum antibody levels, and IFN-γ and TNF-α levels. Conclusions: The results indicated that Zera-71CA/Zera-PPE18 recombinant nanoparticles had good immunogenicity as a subunit vaccine in both BALB/c mice and calves and are potential candidates for further development as effective subunit vaccines.

Dairy cows are economically significant ruminants in China, and the dairy industry is closely linked to food safety and the agricultural economy. However, various factors such as pathogenic microorganisms often lead to frequent diseases in dairy cows. Furthermore, as potential hosts for diverse viruses, dairy cows can harbor zoonotic pathogens, which pose a threat to public health. The Shaanxi–Gansu–Ningxia region boasts abundant natural resources and extensive pastures. It is a major animal husbandry base in Northwest China, and dairy farming plays a significant role in the local economy. However, research on dairy cow virus diversity in this region remains limited; epidemic prevention and control capabilities are constrained, and the risk of disease outbreaks is elevated. In this study, 790 dairy cow samples were collected from 13 large-scale farms and free-range households in the Shaanxi–Gansu–Ningxia region from 2021 to 2023. Sample types consisted of nasal and anal swabs. Six viral metagenomic libraries were constructed and analyzed using high-throughput sequencing and bioinformatics methods, leading to the identification of 51 viral families. These comprised 16 positive-sense single-stranded RNA virus families, one Retroviridae family, four double-stranded RNA virus families, 21 double-stranded DNA virus families, and nine single-stranded DNA virus families. Among these, RNA viruses were represented by families such as Astroviridae, Coronaviridae, Caliciviridae, Picornaviridae, and Picobirnaviridae; DNA viruses were primarily detected in Circoviridae, Papillomaviridae, Genomoviridae, and Smacoviridae. Alpha diversity analysis revealed no significant differences in viral diversity and abundance among the three regions (p > 0.05); however, significant differences were observed in the read counts and proportions of RNA and DNA viruses across the provinces. Phylogenetic analysis further indicated that viruses carried by dairy cows exhibit considerable genetic diversity and pose potential cross-species transmission risks. This study established a reference database for the dairy cow virome in the Shaanxi–Gansu–Ningxia region, elucidated the phylogenetic relationships of key viruses, and provided a scientific basis for future monitoring and prevention of dairy cow viruses.