A novel tick-borne orthonairovirus called the Yezo virus (YEZV), primarily transmitted by the Ixodes persulcatus tick, has been recently discovered and poses significant threats to human health. The YEZV is considered endemic in Japan and China. Clinical symptoms associated with this virus include thrombocytopenia, fatigue, headache, leukopenia, fever, depression, and neurological complications ranging from mild febrile illness to severe outcomes like meningitis and encephalitis. At present, there is no treatment or vaccine readily accessible for this pathogenic virus. Therefore, this research employed an immunoinformatics approach to pinpoint potential vaccine targets within the YEZV through an extensive examination of its structural proteins. Three structural proteins were chosen using specific criteria to pinpoint T-cell and B-cell epitopes, which were subsequently validated through interferon-gamma induction. Six overlapping epitopes for cytotoxic T-lymphocytes (CTL), helper T-lymphocytes (HTL), and linear B-lymphocytes (LBL) were selected to construct a multi-epitope vaccine, achieving a 92.29% coverage of the global population. These epitopes were then fused with the 50S ribosomal protein L7/L12 adjuvant to improve protection against international strains. The three-dimensional structure of the designed vaccine construct underwent an extensive evaluation through structural analysis. Following molecular docking studies, the YEZV vaccine construct emerged as a candidate for further investigation, showing the lowest binding energy (−78.7 kcal/mol) along with favorable physiochemical and immunological properties. Immune simulation and molecular dynamics studies demonstrated its stability and potential to induce a strong immune response within the host cells. This comprehensive analysis indicates that the designed vaccine construct could offer protection against the YEZV. It is crucial to conduct additional in vitro and in vivo experiments to verify its safety and effectiveness.

Haemaphysalis is the second largest genus of hard ticks (Acari: Ixodidae), both in terms of diversity and global distribution. Despite their wide occurrence, genetic information on this genus remains scarce. This study investigated the genetic diversity, phylogenetic relationships, and associated pathogens of Haemaphysalis ticks infesting small ruminants across northern Pakistan. A total of 613 ticks were collected from 95 infested goats and sheep. Four species were identified based on morphological and molecular analyses, including Haemaphysalis (Ha.) cornupunctata, Ha. danieli, Ha. montgomeryi, and a distinct Haemaphysalis sp. closely related to Ha. sulcata. Species identification was confirmed using mitochondrial 16 S rDNA and cox1 markers. Molecular confirmation of unidentified Haemaphysalis sp. was detected across multiple districts of northern Khyber Pakhtunkhwa, indicating close relation to Ha. sulcata. Pathogen screening revealed Rickettsia hoogstraalii (gltA and ompB) in Haemaphysalis sp. and Ha. danieli, Theileria sinensis-like (18 S rDNA) in Ha. montgomeryi and Ha. danieli, and Anaplasma bovis (16 S rDNA) in Ha. cornupunctata and Ha. montgomeryi. This study provides the first molecular evidence of T. sinensis-like in Ha. montgomeryi and Ha. danieli, R. hoogstraalii in Ha. danieli and Haemaphysalis sp., and A. bovis in Ha. cornupunctata and Ha. montgomeryi from Pakistan. Phylogenetic analyses supported species-level relationships and revealed substantial genetic divergence within Haemaphysalis. These findings highlight the importance of locality-based molecular surveillance for resolving cryptic diversity and improving understanding of tick–pathogen associations in mountainous regions of Pakistan.