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.

Potential vectors of bluetongue virus in high altitude areas of Yunnan Province, China.

BACKGROUND: Bluetongue disease of ruminants is a typical insect-borne disease caused by bluetongue virus (BTV) of the genus Orbivirus (family Reoviridae) and transmitted by some species of Culicoides (Diptera: Ceratopogonidae). Recently, the detection of BTV in yaks in high altitude meadows of the Shangri-La district of Yunnan Province, China, prompted an investigation of the Culicoides fauna as potential vectors of BTV. METHODS: A total of 806 Culicoides midges were collected by light trapping at three sites at altitudes ranging from 1800 to 3300 m. The species were identified based on morphology and the DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in Culicoides spp. A phylogenetic analysis was used to analyze the cox1 sequences of some specimens. RESULTS: Four species dominated these collections and cox1 barcoding revealed that at least two of these appear to belong to species new to science. Culicoides tainanus and a cryptic species morphologically similar to C. tainanus dominated low altitude valley collections while C. nielamensis was the most abundant species in the high-altitude meadow. A species related to C. obsoletus occurred at all altitudes but did not dominate any of the collections. BTV RT-qPCR analysis detected BTV RNA in two specimens of C. tainanus, in one specimen closely related to C. tainanus and in one specimen closely related to C. obsoletus by barcode sequencing. CONCLUSIONS: This study suggests that BTV in high altitude areas of Yunnan is being transmitted by three species of Culicoides, two of which appear to be new to science. This research may be useful in improving understanding of the effects of global warming on arboviral disease epidemiology and further study is important in research into disease control and prevention.

The serologic investigation and viral isolation of bluetongue virus in Shangri-La in Southwest China.

Bluetongue is an arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV). In China, BTV is relatively common in Yunnan Province with the exception of northern regions around Shangri-La, where the average altitude is approximately 3,450 metres. Recently, the seroprevalence of BTV has been measured in yaks in Shangri-La; therefore, this study investigated BTV infections in this area. The serological investigation in five villages in Shangri-La showed that there were sporadic BTV infections in yaks (20 of 507 positive) during 2014 to 2017, while the seroprevalence of BTV at three goat farms in a nearby river valley was 35%-65% in 2017. Subsequently, 20 sentinel goats were kept on two separate farms in the river valley and monitored for seroconversion between May and September of 2017. Five of the sentinel animals were tested positive for antibodies to BTV by C-ELISA during the study period, and 13 BTV isolates were isolated from ten sentinel animals. All isolates were identified as the same serotype, and the complete nucleotide sequence of one was determined. The genomic sequences showed that the isolated BTV strain belonged to serotype 21 and had approximately 99.8%-100% homology with three Indonesian BTV-21 strains (D151, RIVS-66 and RIVS-60) between their coding sequences (CDSs) except for Seg4 (99.5%). Besides, our data suggested that this BTV-21 strain might have also infected some local yaks and sheep.