Infectious Salmon Anaemia Virus (ISAV) Ringtest: Validation of the ISAV Diagnositic Process using Virus-spiked Fish Tissues and ISAV TaqMan® Real-time RT-PCR

Abstract

The incidence of white spot syndrome virus (WSSV) in the Indian farmed frozen shrimp products viz., whole, headless (HL) and peeled and undeveined (PUD) by polymerase chain reaction (PCR) was studied. The viability of WSSV in farmed frozen shrimps was investigated by conducting bio-inoculation studies. The products were screened for WSSV by both the single step (for the primer sets 1s5 & 1a16 and IK1 & IK2) and nested PCR (for the primers IK1 & IK2-IK3 & IK4). Single step PCR detected WSSV in 18% of samples, while the nested PCR detected WSSV in 73% of samples. The frozen material, which gave positive results for WSSV by PCR, was further confirmed for the viability of WSSV by the bio-inoculation studies. Mortality (100%) was observed within the 45 h of intra-muscular post injection (P.I) into the healthy wild live WSSV-free shrimps (Penaeus monodon). These results show that the WSSV survived the freezing process and the cold storage and could cause infection in native shrimp farms if such products are reprocessed in the importing countries.

Rapid differentiation of infectious salmon anemia virus avirulent (HPR0) from virulent (HPRΔ) variants using multiplex RT-qPCR

“…However, the much higher percentage of agreement among all 3 laboratories (88.4%) of "high-infected" positive samples suggests that both our new multiplex and the traditional method 39 would have a similar level of success in screening suspect fish to monitor ISAV outbreaks, which matches a previous study that found inter-laboratory agreement greater at higher ISAV viral titers. 23 We selected the particular fluorophores for the multiplex assay probes with portability between instruments in mind; KRB and UMCEDRL have QuantStudio 5 instruments, in contrast to the CFX 96/384 used at NCWMAC. Yakima yellow, which we used for the HPR0-HE22 when paired with BHQ3, is a cost-effective equivalent of VIC that will work on many different qPCR instruments and has a narrower spectral profile than the commonly used HEX fluorophore.…”

“…However, the much higher percentage of agreement among all 3 laboratories (88.4%) of “high-infected” positive samples suggests that both our new multiplex and the traditional method 39 would have a similar level of success in screening suspect fish to monitor ISAV outbreaks, which matches a previous study that found inter-laboratory agreement greater at higher ISAV viral titers. 23…”