Rapid diagnosis of canine respiratory coronavirus, canine influenza virus, canine distemper virus and canine parainfluenza virus with a Taqman probe-based multiplex real-time PCR.

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/µL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/µL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.

Enhancing tomato disease resistance through endogenous antifungal proteins and introduced nematode-targeting dsRNA of biocontrol agent Bacillus velezensis HS-3.

BACKGROUND: As a type of biological control agent (BCA), Bacillus velezensis possesses the efficacy of inhibiting pathogenic microorganisms, promoting plant growth, and overcoming continuous cropping obstacles (CCOs). However, there is limited reporting on the optimization of the cultivation conditions for such biocontrol agents and their role as double-stranded RNA (dsRNA) delivery vectors. RESULTS: In this study, a Bacillus velezensis strain HS-3 was isolated from the root zone of tomato plants with in vitro anti-Botrytis cinerea activity. The investigation into active compounds revealed that HS-3 predominantly employs proteins with molecular weights greater than 3 kDa for its antifungal activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified various proteases and chitosanase, further suggesting that HS-3 most likely employs these enzymes to degrade fungal cell walls for its antifungal effect. To optimize the production of extracellular proteins, fermentation parameters for HS-3 were systematically optimized, leading to an optimized medium (OP-M). HS-3 cultured in OP-M demonstrated enhanced capacity to assist tomato plants in withstanding CCOs. However, the presence of excessive nematodes in diseased soil resulted in the disease severity index (DSI) remaining high. An RNA interference mechanism was further introduced to HS-3, targeting the nematode tyrosine phosphatase (TP) gene. Ultimately, HS-3 expressing dsRNA of TP in OP-M effectively assisted tomatoes in mitigating CCOs, reducing DSI to 2.2% and 17.8% of the control after 45 and 90 days of growth, respectively. CONCLUSION: The advantages of Bacillus velezensis in crop disease management and the mitigation of CCOs become even more pronounced when utilizing both optimized levels of endogenous enzymes and introduced nematode-targeting dsRNA. © 2024 Society of Chemical Industry.

Knockout and Replacement Gene Surgery to Treat Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa.

Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP.