ABSTRACT
Bovine coronavirus (BCoV) is a major cause of infectious disease in cattle, causing huge economic losses to the beef and dairy industries worldwide. BCoV can infect humans and multiple other species of animals. A rapid, reliable, and simple test is needed to detect BCoV infection in suspected farms. In this study, we developed a novel multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combination assay, targeting a highly conserved region of the viral nucleocapsid (N) gene for BCoV detection. The MIRA-LFD assay was highly specific and sensitive, comparable to a published reverse transcription quantitative PCR (RT-qPCR) assay for BCoV detection. Compared with the published RT-qPCR assay, the κ value of the MIRA-LFD assay in the detection of 192 cattle clinical samples was 0.982. The MIRA-LFD assay did not require sophisticated instruments and the results could be observed with eyes. Our results showed that the MIRA-LFD assay was a useful diagnostic tool for rapid on-site detection of BCoV.
ABSTRACT
While discussing the ideal candidates of viral restriction factor, the interferon (IFN) and interferon-stimulated genes (ISGs) could be considered potential targets. However, numerous viruses have evolved multiple strategies to modulate the host innate immune signaling for optimal infection, including the porcine epidemic diarrhea virus (PEDV), a coronavirus spreading widely around the world with high morbidity and mortality in piglets. The immunosuppression mediated by PEDV infection creates an impediment for studying the host-virus interactions and screening the antiviral ISGs. Here, the PEDV variant strain 85-7C40 was screened using the continuous passaging, which showed significantly attenuated viral replication compared with its parent on MARC-145 cells. The comparative transcriptome analysis (accession nos. SRR13154018 to SRR13154026) indicated that 85-7C40 infection led to enhanced immune response on MARC-145 cells, particularly to the IFN antiviral signaling, which mediated the stronger activation of numerous ISGs. Numerous ISGs activated by 85-7C40 showed antiviral effects against the wild-type strain infection, particularly the IFI44 (an ISG upregulated specifically by the 85-7C40 infection) and OASL (upregulated higher in 85-7C40 than 85-7-infected cells), exhibited powerful antiviral activity. IFI44 promoted the production of RIG-I, while the OASL interacted directly with RIG-I, and then they both activated the phosphorylation of STAT1, indicating that they restricted PEDV replication by positively regulating the type I IFN response. Our results provided insight into the ISGs with antiviral activity against PEDV infection and also expanded our understanding of the innate immune response to PEDV infection, which may promote the development of novel therapeutics. IMPORTANCE Host innate immune responses, particularly interferon (IFN) antiviral signaling, can activate diverse downstream ISGs to exert antiviral effects. However, porcine epidemic diarrhea virus (PEDV) infection has evolved multiple strategies to escape from this immune clearance. The immunosuppression mediated by PEDV infection creates an impediment for studying the host-virus interactions. We screened a PEDV variant strain, 85-7C40, which induced enhanced immune responses on MARC-145 cells and thus mediated the stronger activation of numerous ISGs. The laboratory-generated variant might induce inconsistent immune responses with a natural wild-type strain during infection, while numerous ISGs activated by 85-7C40 showed antiviral effects against the wild-type strain infection, particularly the IFI44 and OASL, restricted PEDV replication by positively regulating the type I IFN response. These findings were suggestive of the immune-enhanced variant being capable of using as an ideal viral model for screening the efficient antiviral proteins and elucidating the underlying mechanisms between PEDV and host innate immune responses.