Detection and serotyping of bluetongue viruses in Khyber Pakhtunkhwa Province, Pakistan.

This study investigated the presence of bluetongue virus (BTV) RNA and its serotypes in clinically healthy ruminants in Pakistan. A total of 673 blood samples were randomly collected from apparently healthy sheep, goats, buffaloes and cattle across various localities of the Khyber Pakhtunkhwa, Pakistan, between 2019 and 2021. BTV RNA detection was performed using pan-BTV reverse-transcriptase polymerase chain reaction (RT-PCR). Positive samples were further analyzed using serotype-specific real-time RT-PCR assays and nCounter NanoString technology. Overall, 118 samples scored positive for BTV RNA, indicating a prevalence of 17.5% among apparently healthy animals. Multivariate mixed effects logistic regression analysis revealed a significant difference (P < 0.05) in the prevalence of BTV RNAemia between cattle and buffaloes, age groups and season. However, no significant difference (P > 0.05) in the prevalence of BTV RNAemia was observed among sheep, goats and buffaloes. Of the 99 BTV RNA-positive samples, 57 (57.6%) were successfully serotyped, revealing eight different serotypes of BTV (i.e. BTV-1, 2, 4, 5, 12, 16, 21, 26). BTV-16 was the most prevalent, followed by BTV-4, 1, 5, 12, 2, 21 and 26. Notably, BTV-1, 5, 12, 21 and 26 were identified for the first time in Pakistan. Two samples scored positive for more than one serotype simultaneously; one for BTV-2 and BTV-12 and the other for BTV-4 and BTV-16. Further research is recommended to characterise the genotypes of circulating BTV strains in Pakistan.