Detection and differentiation of rabbit hemorrhagic disease and European brown hare syndrome viruses by amplification of VP60 genomic sequences from fresh and fixed tissue specimens

Abstract

Two reverse transcription-PCR (RT-PCR) assays have been developed for the detection and differentiation of rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), two closely related caliciviruses. In order to select highly specific primers, comparative analysis was performed with a large number of RHDV and EBHSV genomic sequences. Regarding these data, primers were selected from similar regions of the VP60 genes to amplify a fragment of 316 nucleotides from the genome of RHDV and a fragment of 265 nucleotides from the genome of EBHSV. In sensitivity studies, as few as 10 copies of cloned viral genomic fragments were detected in each PCR assay, and no cross amplification was observed between the two viruses. The diagnostic value of the assays was confirmed with clinical material by testing fresh and formalin-fixed, paraffin-embedded liver and spleen specimens from a large number of geographically and temporally distant outbreaks. Thus, the two PCR assays provide highly specific and sensitive, novel means of direct detection of the two caliciviruses. In addition, by detecting the viruses in formalin-fixed, paraffinembedded tissues (PETs), the RT-PCR assays facilitate retrospective virological and epidemiological studies. For example, the identification of EBHSV in PET specimens collected in the 1970s indicates that this virus appeared in the hare populations several years before the first reports of European brown hare syndrome during the 1980s.

A pandemic strain of calicivirus threatens rabbit industries in the Americas

“…Likewise, serum and heparinized blood samples from all animals were negative by RT-PCR (Table 3 ). While these data indicate that liver tissue represents the best sample for RHD diagnosis by RT-PCR, other more sensitive methods using either a nested RT-PCR [ 46 ] or a realtime RT-PCR [ 47 ] may be used to detect RHDV in other tissues types, blood or even paraffin embedded tissue sections.…”

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain

“…Detection of EBHSV was carried out using nested RT‐PCR as described by Ros Bascuñana et al. (). Briefly, one step RT‐PCR was performed using the Qiagen kit following the manufacturer's instructions (PCR I) followed by a nested PCR (PCR II).…”

Molecular methods in detection and epidemiologic studies of rabbit and hare viruses: a review

“…40,92 Furthermore, nRT-PCR was able to reduce the effect of reaction inhibition and eliminate nonspecific amplification when rabbit and hare tissues were tested for RHDV and EBHSV. 92 Although nPCR increases the specificity of DNA amplification by reducing background amplification of nonspecific DNA, it requires more detailed information on the target sequence as well as lengthening the analysis compared to a single-round PCR. In addition, this technique is susceptible to carryover contamination with PCR products from a preceding amplification.…”