Diagnostic efficacy of molecular assays for the viral haemorrhagic septicaemia virus isolates from the Czech Republic

The diagnostic properties of the one-step real-time reverse-transcription polymerase chain reaction assay for viral haemorrhagic septicaemia virus detection were compared to methods currently in use in the Czech Republic, namely, virus isolation using the cell culture and conventional reverse-transcription polymerase chain reaction followed by the nested polymerase chain reaction. The assays were tested on a panel of 25 archived viral haemorrhagic septicaemia isolates and 8 archived infectious haematopoietic necrosis isolates obtained from monitoring and/or outbreaks of the diseases among farmed salmonids in the Czech Republic. The ability to detect the presence of the virus in the tissues of fish was tested on additional 32 field samples collected from the rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis). The real-time assay showed the highest analytic sensitivity by detecting the presence of viral nucleic acid in samples with 10-7 dilution, whereas the sensitivity of the conventional polymerase chain reaction peaked at 10-5. Diagnostic specificity of both molecular assays was confirmed by absence of cross-reactivity with the infectious haematopoietic necrosis virus isolates. This, along with consistent results in the detection of the virus in the fish tissues, confirms that the one-step real-time reverse-transcription polymerase chain reaction is currently an optimal stand-alone diagnostic method for the detection of the viral haemorrhagic septicaemia virus. Cell culture, real-time PCR, RT-PCR, rainbow trout, Rhabdoviridae Viral haemorrhagic septicaemia virus (VHSV) from the family Rhabdoviridae is the most important viral pathogen of salmonids worldwide. The enveloped single-stranded ribonucleic acid virus causes outbreaks of the viral haemorrhagic septicaemia (VHS) amongst over 80 freshand saltwater fish species and has an increasing economic impact due to clinical signs, high mortality from the disease and costly eradication measures (Skall et al. 2005). Intensive aquaculture production of rainbow trout (Oncorhynchus mykiss), which is the most economically relevant salmonid-related industry in the Czech Republic, is especially affected, with fish showing severe clinical signs such as haemorrhages in the muscle and organ tissue, exophthalmos, and ascites (Reichert et al. 2013). Neither reliable treatment nor vaccine against the VSHV is currently available. Control of the disease is based on prevention and surveillance programs (OIE Manual of Diagnostic Tests for Aquatic Animals 2016). Diagnostic methods for detection of the virus with high sensitivity and specificity are the key components of these programs. The currently used methods meet these criteria, but their disadvantages are high time requirements (isolation on the cell culture) or higher risk of sample contamination (conventional reverse-transcription polymerase chain reaction [RT-PCR], enzyme-linked immunosorbent assay [ELISA]) (Mackay 2004). ACTA VET. BRNO 2017, 86: 207-212; https://doi.org/10.2754/avb201786030207 Address for correspondence: Ľubomír Pojezdal National Reference Laboratory for Viral Diseases of Fish Veterinary Research Institute Hudcova 296/70, 621 00 Brno, Czech Republic Phone: +420 5 3333 1117 E-mail: pojezdal@vri.cz http://actavet.vfu. cz/ A legislative option to use one-step real-time RT-PCR as a stand-alone method of VHSV diagnosis exists since 2016 (Commission Implementing Decision (EU) 2015/1554), although this method has not yet been validated for use in reference laboratory in the Czech Republic. The aim of the present study was to test the real-time RT-PCR assay, as described by Jonstrup et al. (2013), on a panel of archived Czech VHSV isolates and field samples. Diagnostic sensitivity and specificity of the assay was compared to the diagnostic methods currently in use, namely isolation on the cell culture and conventional RT-PCR followed by nested PCR. Materials and Methods Tissue samples In total, 169, 33, and 19 specimens of rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and brook trout (Salvelinus fontinalis), respectively, were examined. The samples represented either fish farms with clinical outbreaks of the viral haemorrhagic septicaemia (VHS) or clinically healthy wild freshwater fish populations of the Southern Bohemian Region. The animals were collected from 2015 to 2017 and were pooled into 32 samples of up to 10 fish each, according to their species and place of origin. The spleen, anterior kidney and heart of the fish were dissected, homogenized in the Minimum Essential Medium Eagle (MEM) HEPES modification (Sigma-Aldrich, USA) at the 1:10 tissue to medium ratio and centrifuged at 3000 g for 14 min at 4 °C. The supernatant was used for further analysis.