Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus.

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

Isolation, complete genome sequencing, and phylogenetic analysis of the first Chuzan virus in China.

A Chuzan virus (CHUV), defined as GX871 here, was isolated from blood from a sentinel cattle firstly in China, and its full-length genome was sequenced in this study. The GX871 genome included 10 segments and 18914 bp, one base fewer than the CHUV prototype strain K-47 due to a one-base deletion in the 5' non-coding region of segment 8. A frameshift mutation was detected in a short coding region (1010-1026 nt) corresponding to the VP1 protein; this frameshift resulted in a five-amino acid mutation from 336CVLSY340 to 336YGAKL340. In addition, there were a one-base deletion at 1713 nt and a one-base insertion at 1682 nt in the 3' non-coding region of segment 5. Based on phylogenetic analysis of the deduced VP2 amino acid sequences, Palyam serogroup viruses were classified into three groups. The Chinese CHUV isolate GX871 was categorized into the same group as CHUV prototype strain K-47. The phylogenetic tree was divided into three clusters according to the geographical distribution of the partial nucleotide sequences of VP7, and this arrangement might define the geographical gene pool of CHUV.