Development, Application and Epidemiological Analysis of a TaqMan-Based RT-qPCR Assay for Nairobi Sheep Disease Virus in China

Nairobi sheep disease virus (NSDV) is a typical tick-borne virus belonging to the genus Orthonairovirus in the family Nairoviridae, which causes Nairobi sheep disease (NSD) with mortality rates exceeding 70% in small ruminants such as sheep and goats. This pathogen has also been associated with human infections, highlighting its zoonotic potential. In this study, we developed a TaqMan-based reverse-transcription quantitative PCR (RT-qPCR) assay for NSDV detection in China. The assay indicated high analytical sensitivity; the limit of detection of 10 copies/µL was determined using recombinant plasmid DNA, representing the performance of the qPCR amplification step only, with a standard curve showing excellent linearity (R2 = 1.000). The assay exhibited good analytical specificity for NSDV, with no cross-amplification of other related pathogens. Intra-assay and inter-assay coefficients of variation for recombinant plasmid standards were consistently <1%, indicating high reproducibility. We then applied this RT-qPCR assay to quantify viral loads in multiple tissues from sheep experimentally infected with NSDV. Detectable NSDV nucleic acid was found in the heart, liver, spleen, lung, kidney, duodenum, jejunum, rectum, cecum, ileum, and lymph nodes at 3 and 7 days post-infection (dpi). Descriptive analysis showed higher NSDV RNA loads in the spleen and lung relative to other tissues at both 3 dpi and 7 dpi. The maximum RNA load of 104.428 copies/mg was detected in the spleen at 3 dpi, indicating that the spleen and lung represent the tissues with the highest NSDV RNA accumulation. Meanwhile, this assay was applied to conduct an epidemiological survey for NSDV nucleic acid in sheep and ticks across selected areas of Heilongjiang Province, China. The NSDV RNA detection proportions were 3.46% (33/955) in sheep and 2.88% (20/694) in ticks, respectively. Phylogenetic analysis revealed that the tick-derived NSDV sequence, designated NSDV/HLJ-1/tick/China/2023, forms a distinct lineage within the Chinese NSDV clade and shares relatively high nucleotide homology with the NSDV SZWH2 strain from China. In conclusion, this accurate and sensitive RT-qPCR assay provides a robust tool for laboratory-based NSDV detection and epidemiological surveillance.