Tick-borne viruses (TBVs) pose significant public health and economic threats. Pakistan has endemic Crimean-Congo hemorrhagic fever virus (CCHFV), but evidence suggests broader TBV circulation. This study assessed the seroprevalence of thirteen TBVs (seven are members of the genus Orthonairovirus and six are members of the genus Bandavirus) in humans, livestock, and rats in Punjab, Pakistan. Serum samples (n = 794: 321 livestock, 253 human, and 220 rat) were collected from the Narowal, Lahore, and Faisalabad districts. Antibodies to viral NPs were detected using the luciferase immunoprecipitation system (LIPS). The overall seroprevalence was 19.14% (152/794); it was highest in livestock (27.10%), then humans (20.55%), and then rats (5.91%). The highest seroprevalence rates were 3.12% for CCHFV in livestock, 3.56% for Yezo virus (YEZV) in humans, and 0.91% for Tamdy virus (TAMV) and Tacheng tick virus 1 (TcTV-1) in rats. Neutralizing antibodies were detected against CCHFV (1 cattle, 4 humans), Bhanja virus (BHAV) (3 livestock, 1 rat), TAMV (1 cattle), Guertu virus (GTV) (1 cattle), and Dabie bandavirus (2 cattle). Sixteen samples showed antibodies to both orthonairoviruses and bandaviruses, indicating co-exposure. Further analysis showed that seropositivity was not randomly distributed. Livestock kept in commercial farming systems and people working mainly outdoors had distinctly higher exposure to TBVs than subsistence livestock and indoor workers. The results supported the circulation of TBVs among hosts within the close socio-economic/ecological integration area of Pakistan. These findings confirm the circulation of CCHFV, SFTSV, GTV, and TAMV; provide the first serologic evidence of BHAV in Pakistan; and underscore the need for further investigation into the potential circulation of additional TBVs. All results demonstrated that multiple TBVs have been circulating among humans, livestock, and rodents in Pakistan.

Viruses within the Bandavirus genus (Phenuiviridae family), phylogenetically divided into SFTSV and Bhanja groups, pose public health risks through cross-species transmission. Despite their significance, a systematic comparison of their infectivity, pathogenicity, and antigenicity is lacking. This study evaluates these key properties for five representative species: Severe fever with thrombocytopenia syndrome virus (SFTSV), Guertu virus (GTV), and Heartland virus (HRTV) from the SFTSV group; and Bhanja virus (BHAV) and Lone Star virus (LSV) from the Bhanja group. The five bandaviruses demonstrated efficient replication kinetics across a panel of human and mammalian cell lines, suggesting a broad cellular tropism. Wild-type C57BL/6 mice developed transient viremia (2-10 dpi) without overt disease, followed by neutralizing antibody production. Cross-neutralization was robust within each phylogenetic group but restricted between groups, supporting two separate serogroups. In contrast, IFNAR^-/- C57BL/6 mice exhibited significant hematological abnormalities and uniformly lethal infection, with the 50% lethal dose-based virulence ranking as SFTSV > LSV > GTV >BHAV >HRTV. Pathological analysis revealed that SFTSV caused severe multi-organ damage, with predominant spleen tropism; GTV and HRTV induced similar but milder spleen-tropic lesions; and LSV and BHAV caused multi-organ injuries primarily affecting the liver and spleen. Our findings demonstrate that the variation in virulence among Bandavirus species is closely linked to their antigenic divergence and evolutionary lineages. The established infection models provide valuable platforms for investigating virus-host interactions and pathogenesis. These insights facilitate the development of vaccines and antiviral therapies, and improve public health preparedness against emerging tick-borne pathogens.

Mosquitoes and biting midges harbor diverse viruses, but the factors shaping their viromes remain incompletely understood. We analyzed 35 pooled libraries from seven host groups across 15 locations in Yunnan, China, sampled in June 2025, using total-RNA sequencing, viral annotation, phylogenetics, and community analyses. We retained 1295 viral contigs grouped into 221 annotation-defined viral taxonomic units (VTUs) spanning 24 families; 107 VTUs were supported only by protein homology. Banna virus-related sequences, an Aedes-associated flavivirus, and four putative components of a divergent Jingmenvirus-related candidate were identified. Host group was associated with family-level virome composition after adjustment for location and sequencing depth (R² = 0.213, p = 0.0052), and the association persisted across alternative feature definitions. Culex tritaeniorhynchus showed the greatest cumulative VTU richness, although its ranking changed when library number was equalized. Most VTUs detected in this host occurred at one sampled site, without a significant monotonic distance-decay pattern. This study expands knowledge of mosquito-associated and biting midge-associated viral diversity in Yunnan, identifies representative lineages for further characterization, and documents host-associated differences in the detected viromes.