Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells

Abstract

A reverse transcnptase-polymerase chain reaction (RT-PCR) assay was developed and applied to the detection and differentiation of viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in organ samples and cultured cells, regardless of the serotype. This method was developed by selecting primer sets corresponding to highly conserved regions of the glycoprotein G-gene sequences of the 2 vlruses. The very fast RNA extraction, reverse transcnption and PCR permitted us to read the agarose gels within 7 to 9 h after samples, cultured cells and whole fish arrived, which is of great importance when there is reason to believe that VHSV or IHNV may be present This is also the first report of a large-scale field trial companng the RT-PCR assay in trout from 30 German fish farms (a total of 330 rainbow trout) with the usual virus isolation and identification method in order to evaluate the efficiency of the RT-PCR assay for general use in fish health management programs. RT-PCR followed by semi-nested PCR uslng RNA dlrectly extracted from fish tissue turned out to be the most sensitive method. It recognized 9 fish farms a s VHS-positive and 7 as IHN-positive. This is 3 VHS-and 4 IHN-farms more than detected by the traditional virus isolation method. By directly examining the tissue by means of a PCR test ~t was possible to detect viral RNA in acutely and subacutely to chronically diseased fish as well as in asyrnptomatlc VHS/IHNcarner fish Therefore, this effectlve and powerful assay for detecting VHSV and IHNV by means of PCR has great advantages compared with the presently used procedures KEY WORDS: RT-PCR . VHSV/IHNV . Differential diagnosis in organ samples INTRODUCTIONAquatic rhabdoviruses such as viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are significant pathogens for salmonids. VHSV and IHNV often causes diseases with high mortality among salmonids, especially in rainbow trout Oncorhynchus mykiss.Since its first isolation (Jensen 1965) VHSV has been widely detected in Europe. VHSV was first isolated in 1988 in chinook salmon Oncorhynchus tshawytscha and in coho salmon 0. kisutch on the West coast of North America (Brunson et al. 1989, Hopper 1989. Virus isolations from Pacific cod Gadus macrocephalus and Alaskan Pacific herring Clupea harengus pallasi were reported in 1992 and 1993, respectively (Meyers et al. 1992(Meyers et al. , 1993. It was concluded that VHSV had been enzootic in the North Pacific region for some time (Batts et al. 1993). Even in the British Isles, thought to be VHSV-free, VHSV was isolated from a turbot Scophthalmus maximus farm in Scotland (Ross et al. 1994). Experimental infection showed that this turbot isolate was of low virulence for rainbow trout 0 Inter-Research 1998 Resale of full article not permitted

Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

“…After purification with the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA), amplified PCR products were cloned into the pCR 2.1 vector (Invitrogen, Groningen, the Netherlands) before transformation into Escherichia coli TOP10 (Invitrogen, Groningen, the Netherlands) using standard protocols. Nucleotide sequences of cloned G genes were obtained with ID4 primer (5'-CTC TGG ACA AGC TCT CCA AGG-3') [8], and M-13 forward and reverse primers. Three independent clones from each isolate were used for sequence analysis, and the resulting sequences were assembled with Genetyx Win Ver.…”

Is There Any Species Specificity in Infections with Aquatic Animal Herpesviruses?–The Koi Herpesvirus (KHV): An Alloherpesvirus Model

“…The rainbow trout were divided into four groups including fish kept at 15°C (n=10) and 20°C (n=10) water temperature for the infection trial and two additional tanks containing fish (n=5, respectively) kept as negative controls at the same temperatures. The fish were confirmed free from notifiable pathogens like viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) by RT-PCR [29] and additionally with the conventional PCR and nested PCR [30] according to Engelsma et al…”

Identification of spring viraemia of carp virus (SVCV) by combined RT-PCR and nested PCR

“…Reverse transcription PCR (RT-PCR) based on the G gene has been used to differentiate between the rhabdoviruses of viral haemorrhagic septicaemia and infectious haematopoietic necrosis (viruses classified within the genus Novirhabdovirus; Bruchhof et al 1995, Miller et al 1998, Strommen & Stone 1998, Guillou et al 1999. Rowley et al (2001) used primers derived from the G gene of SVCV to obtain nucleotide data from cyprinid rhabdoviruses in which virus neutralisation showed a narrow antigenic similarity to PFRV.…”