Channel catfish calicivirus (ChCV) has emerged as a novel viral pathogen threatening the sustainability of channel catfish (Ictalurus punctatus) aquaculture. The absence of rapid, on-site detection methods has hindered effective disease prevention and control. In this study, a visual RPA-CRISPR/Cas12a detection platform targeting the conserved non-structural protein (NS) gene of ChCV was established, and the key reaction parameters were systematically optimized. The optimal combination was identified as the F3/R3 primer pair with crRNA3-2. The optimal reaction conditions included RPA amplification at 37°C for 30 min, followed by CRISPR/Cas12a detection at 37°C for 30 min using 250 nM ssDNA reporter, 50 nM crRNA3-2 and 200 nM Cas12a. Specificity analysis showed that the established system specifically recognizes ChCV without cross-reactivity with other common aquatic pathogens affecting channel catfish, including channel catfish virus (CCV, also known as Ictalurid herpesvirus 1, IcHV1), channel catfish reovirus (CCRV), Edwardsiella tarda, Aeromonas hydrophila and Aeromonas veronii. Sensitivity evaluation demonstrated that the fluorescence visualization mode of the system achieved a limit of detection (LOD) of 2 copies/μL, while the lateral flow dipstick (LFD) mode exhibited an LOD of 2 × 102 copies/μL. Both detection modes were significantly more sensitive than conventional PCR. When applied to 20 clinical samples, the RPA-CRISPR/Cas12a system yielded a positive detection rate of 60%, substantially higher than that of conventional PCR (25%). In conclusion, the RPA-CRISPR/Cas12a system established in this study exhibits high specificity, ultra-sensitivity and operational simplicity, making it a promising tool for rapid and field-deplorable detection of ChCV.
Chu Ma, Yong Zhou, Xin Ren et al.· Journal of Fish Diseases· 0 citations
Spring viremia of carp virus (SVCV) poses a major threat to carp aquaculture due to the lack of effective antiviral strategies. This study investigates the antiviral potential of baicalin in epithelioma papulosum cyprini (EPC) cells and zebrafish. Baicalin exhibits antiviral activity during both pretreatment and post-infection treatment and shows direct virucidal activity during virus–compound co-incubation. Moreover, baicalin alleviated oxidative stress, preserved mitochondrial function, and reduced apoptosis in infected cells. Proteomic analyses revealed that baicalin suppressed SVCV replication by modulating the AMPK/mTOR signaling pathway, as evidenced by decreased AMPK phosphorylation, increased mTOR phosphorylation, and subsequent inhibition of autophagy. These findings demonstrate that baicalin significantly suppresses SVCV replication and is associated with modulation of the AMPK/mTOR-mediated autophagy pathway. These findings identify baicalin as a promising antiviral candidate against SVCV and provide a foundation for further evaluation in economically important aquaculture species.
Mingyang Xue, Mengwei Zhang, Yang Hu et al.· Frontiers in Immunology· 0 citations
Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a major pathogen causing substantial economic losses in global tilapia aquaculture. The type VII secretion system (T7SS), present in Actinobacteria and Firmicutes, secretes effector proteins implicated in bacterial virulence, yet its functional mechanisms remain poorly defined. Here, we constructed an essC deletion mutant (∆essC) in S. agalactiae strain HN016 to investigate the role of T7SS in virulence. The ∆essC exhibited impaired growth and declined intracellular magnesium ion concentration in THB or magnesium-limited chemically defined medium (1 mM Mg2+-CDM); these defects were complemented by addition of wild-type culture supernatant. Transcriptomic and quantitative reverse transcription polymerase chain reaction (qRT–PCR) analyses revealed altered sugar metabolism and significant downregulation of capsule biosynthesis genes (cpsA, cpsB, and cpsD) in ∆essC. Consistent with this, the mutant produced markedly less capsular polysaccharide and displayed impaired capsule integrity. Furthermore, cellular assays confirmed that compared with HN016, the ∆essC mutant exhibited significantly reduced adhesion capacity, immune evasion ability, and cytotoxicity. More importantly, the ∆essC mutant showed attenuated virulence in vivo, with reduced bacterial loads in host tissues and diminished ability to cross the blood–brain barrier (BBB). Our findings provide the first evidence that the T7SS influences magnesium homeostasis and is essential for maintaining capsule integrity, both of which contribute critically to pathogenicity. This study identifies T7SS as a potential target for novel therapeutic strategies against streptococcal disease in aquaculture.
Fengyan Li, Chen Xu, Weiyi Ma et al.· Veterinary Research· 0 citations
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