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

Abstract

Various PCR-based assays for rabbit viruses have gradually replaced traditional virologic assays, such as virus isolation, because they offer high-throughput analysis, better test sensitivity and specificity, and allow vaccine and wild-type virus strains to be fully typed and differentiated. In addition, PCR is irreplaceable in the detection of uncultivable or fastidious rabbit pathogens or those occurring in low quantity in a tested sample. We provide herein an overview of the current state of the art in the molecular detection of lagomorph viral pathogens along with details of their targeted gene or nucleic acid sequence and recommendations for their application. Apart from the nucleic acids-based methods used for identification and comprehensive typing of rabbit viruses, novel methods such as microarray, next-generation sequencing, and mass spectrometry (MALDI-TOF MS) could also be employed given that they offer greater throughput in sample screening for viral pathogens. Molecular methods should be provided with an appropriate set of controls, including an internal amplification control, to confirm the validity of the results obtained.

Genotyping of rabbit hemorrhagic disease virus detected in diseased rabbits in Egyptian Provinces by VP60 sequencing

“…PCR also facilitates virus gene sequencing so that all vaccine and wild-type virus strains can be fully typed and differentiated. PCR can also be used for the detection of pathogens that are difficult to be isolated using traditional methods and those that are present in low titer in test samples [ 24 ]. The virus VP60 gene has been selected as a target [ 25 ] for reverse-transcription PCR (RT-PCR) assay which is currently used as a general screening tool for rabbit and hare caliciviruses.…”

Real-Time PCR Confirms Infection with Lagovirus europaeus

“…The real-time PCR method is a technique that successfully replaces classic PCR in virological research. Due to its high efficiency and the possibility of quantifying a small number of viral particles, it is widely used in research on L. europaeus, including its diagnostics [25]. The list of primers used in published analyses using the real-time PCR method is presented in Table 2.…”

A Review on the Methods Used for the Detection and Diagnosis of Rabbit Hemorrhagic Disease Virus (RHDV)

“…Several in-house protocols have been developed for the use of real-time RT-PCR, from genotype-specific to multiplex detection and quantification of lagoviruses, each adapted to the needs and epidemiological background of the region, e.g., [7,27,31,134,154,155]. A comprehensive review on such methods has been recently reported by Kwit and Rzeżutka (2019) [138]. Commercial kits are also available from, for example, Ingenetix and Genesig.…”

“…In addition, it was successfully used for RHDV detection from formalin-fixed, paraffin-embedded archived tissues further allowing retrospective studies [ 135 ]. However, nRT-PCR is more time-consuming and requires more a priori information on the target sequence than conventional RT-PCR [ 138 ].…”

“…It is also an alternative for limited-budget laboratories as it does not require any specialized equipment, but an isothermal water bath, and positive results can be easily detected by electrophoresis appearing as a ladder-like pattern in the gel or as a fluorescence emitting signal under UV light due to SYBR Green I staining [ 144 ]. While RT-LAMP seemed a promising method for the detection of RHDV, it has not been tested in strains other than Chinese [ 138 ] and requires a good knowledge on the sequence strains in order to design reliable primer sets.…”