Metagenomic and serological evidence of emerging tick-borne viruses in livestock, humans, and rats in Pakistan.

Tick-borne viruses (TBVs) pose significant emerging threats to public and veterinary health worldwide. In Pakistan, the potential threats posed by TBVs extend far beyond Crimean-Congo hemorrhagic fever virus (CCHFV), which causes outbreaks and severe hemorrhaging with a high fatality rate among humans each year. However, the full extent of the tick-borne virome remains largely unexplored. This study presents the metagenomic profiling of viruses in livestock-associated ticks from Pakistan. Eighty-seven ticks belonging to the genera Ixodes, Rhipicephalus, Haemaphysalis, and Hyalomma species from livestock in Punjab. These ticks were subsequently grouped into 11 pools for RNA sequencing. Our analysis revealed extensive viral diversity, identifying sequences related to 31 viruses spanning at least 11 families. New strains of Jingmen tick virus (JMTV), brown dog tick phlebovirus 2 (BDTPV-2), and Liman tick virus (LMTV) were characterized, confirming their presence in the region. Serological surveys performed among 319 livestock, 253 humans, and 214 rats detected antibodies against these viruses, indicating host exposure. Notably, the presence of JMTV-neutralizing antibodies was confirmed in two livestock animals, one human, and one rat, providing evidence of productive infection. Our findings significantly expand the known diversity and distribution of TBVs in Pakistan, establish the preliminary baseline of the tick virome in the country, and provide serological evidence of cross-species exposure to emerging TBVs. This study highlights the underestimated risk of tick-borne viral zoonoses in Pakistan and underscores the urgent need for enhanced surveillance and risk assessment.