Establishment of pyrosequencing technology to detect White Spot Syndrome Virus (WSSV) in cultured aquatic animals.

This study aimed to establish pyrosequencing methods to detect white spot syndrome virus (WSSV). One pair of polymerase chain reaction (PCR) primers, and one pyrosequencing primer, were designed for WSSV. The pyrosequencing reaction system and conditions were optimized and a pyrosequencing method for detecting WSSV was successfully established. This method was able to specifically detect WSSV in eight viruses, with high sensitivity. The minimum detectable limit for nucleic acid was 23 copies/µL. The method was verified by detecting WSSV in 1881 batches of samples collected from domestic and imported shrimps. The detection results were more sensitive than conventional PCR. This research has therefore provided a new detection method for monitoring, and controlling aquatic animal virus diseases.

Effect of common carp (Cyprinus carpio) TLR9 overexpression on the expression of downstream interferon-associated immune factor mRNAs in epithelioma papulosum cyprini cells.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs) and play an important role in the antiviral response. To determine the effect of common carp TLR9 (CcTLR9) overexpression on the expression of down-stream interferon associated immune factors in epithelioma papulosum cyprini (EPC) cells, may provide useful information for the further investigation on the anti-virus effect mediated by TLR9 in fish. In this study, we constructed an overexpression vector, pEGFP-N1-CcTLR9, by cloning the CcTLR9 gene and inserting it into an expression vector pEGFP-N1. Both plasmids DNA of pEGFP-N1 and pEGFP-N1-CcTLR9 were transfected into EPC cells, and the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin mRNA at 0, 6, 12, 24, 48 and 72h post-transfection were determined by real-time quantitative PCR (Q-PCR). Overexpression of the CcTLR9 gene in EPC cells upregulates the expression of IRF3, IRF7, ISG15, Mx1, PKR, and Viperin mRNA, and this was more significant for Viperin, ISG15, and IRF7, and least significant for PKR. Thus, fish TLR9 activates IRF7 signaling to induce I-IFN secretion and the subsequent upregulation of IFN-stimulated genes.