Background: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load. This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously. Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens. The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 μL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility. The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV. This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT‐ddPCR assay for SADS-CoV detection.

Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). Firstly, the mAb against N protein of AKAV was produced and characterized by western blot (WB) and indirect immunofluorescence (IFA) assays. Then, the linear epitope of AKAV N protein against the mAb was identified and the mAb was applied to establish a double antibody sandwich ELISA (DAS-ELISA). One AKAV N-reactive monoclonal mAb was generated and designated as 2D3. WB and IFA assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168–182 of AKAV N protein. The double antibody sandwich ELISA (DAS-ELISA) established based mAb 2D3 was able to detect both the purified AKAV N protein (with detection limit of 6.25 ng/ml) and AKAV-infected cell culture supernatant (with detection limit of 250 TCID₅₀/ml). Taken together, we successfully prepared a mAb 2D3 against AKAV N protein and identified its corresponding linear epitope, and then established a DAS-ELISA for the detection of AKAV antigen. A produced mAb against AKAV N protein was used to define a linear epitope of AKAV and establish a DAS-ELISA for AKAV antigen detection.

Akabane virus (AKAV) is divided into five genogroups (I to V), and strains of different genogroups exhibit marked differences in pathogenicity. We isolated a genogroup II AKAV strain, TJ2016, in China in 2016, but its virulence remains unknown. The pathogenic potential of other genogroup II strains isolated in China also remains uncharacterized. The objectives of this study were to determine the pathogenicity of TJ2016. Kunming or Balb/c mice at 7 days or 8 weeks of age were inoculated with TJ2016 by intracerebral (IC), intraperitoneal (IP), subcutaneous (SC), or intramuscular (IM) routes. Clinical signs, pathological alterations, and AKAV distributions in the inoculated mice were monitored and analyzed. Virus inoculations by the IC route resulted in 75% ~ 100% mortality of the inoculated mice regardless of the mouse strains or ages. Virus inoculations by the IP route killed 75% to 100% of the suckling mice but killed no adult mice. All the mice inoculated via SC and IM routes survived until the end of the trial. AKAV was detected only in the brains of the mice that died or were euthanized before the end of the experiment. The AKAV antigens were only identifiable within neuronal cells. Brain lesions such as proliferation and infiltration of microglial cells, perivascular cuffing (PVC) of lymphocytes and macrophages, neuronal degeneration/necrosis, vascular dilatation and congestion, etc., were observed only in the mice that died or were euthanized before the end of the experiment. We characterized the virulence of TJ2016 by inoculating suckling and adult mice via different routes and established experimental mouse models, which holds significant implications for vaccine/drug development and further research on viral pathogenesis.