A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

Seasonality and risk factors for myxomatosis in pet rabbits in Great Britain.

Myxomatosis is a highly contagious, frequently fatal viral disease affecting both wild and domesticated European rabbits across many areas of the world. Here we used electronic health records (EHRs) collected from pet rabbits attending a sentinel voluntary network of 191 veterinary practices across Great Britain (GB) between March 2014 and June 2019 to identify new features of this disease's epidemiology. From a total of 89,408 rabbit consultations, text mining verified by domain experts identified 207 (0.23 %) cases where myxomatosis was the only differential diagnosis recorded by the attending practitioner. Cases occurred in all months but February and were distributed across the country. Consistent with studies in wild rabbits, the majority of cases occurred between August and November. However, there was also evidence for considerable variation between years. A nested case control study identified important risk factors for myxomatosis within this pet animal population including season, sex, age, vaccination status and distance to likely wild rabbit habitats. Female entire rabbits were twice as likely to be a case (odds ratio (OR) 1.98, 95 % confidence interval (CI) 1.26-3.13, p = 0.003), suggesting a novel role for behaviour in driving transmission from wild to domesticated rabbits. Vaccination had the largest protective effect with vaccinated rabbits being 8.3 times less likely to be a case than unvaccinated rabbits (OR = 0.12, 95 % CI 0.06-0.21, p = <0.001).

Detection of viral DNA of myxoma virus using a validated PCR method with an internal amplification control.

Recognition of myxomatosis is usually based on clinical symptoms, but amyxomatous cases of the disease require the use of laboratory methods. Nowadays PCR assays are routinely employed for detection of MYXV DNA, but none of them have had their diagnostic usefulness conclusively confirmed through validation. The aim of the study was the development and validation of a PCR with an internal amplification control (IAC) for intravital and postmortem detection of viral DNA of myxoma virus. To avoid false negative results a chimeric internal amplification control (IAC) was prepared and incorporated into the PCR and amplified by the same primer set as the target DNA (M071L). The optimal concentration of particular ingredients in the PCR mixture (including IAC concentration and volume of DNA sample) was determined. To minimize the risk of amplicon carry-over contamination, uracil N-glycosylase was added to the reaction. Before proper validation the robustness of the IAC-PCR was verified. Validation of the method encompassed the following parameters: the analytical and diagnostic specificity (ASp, DSp) and sensitivity (ASe, DSe) of the assay, repeatability, and intra-laboratory reproducibility. The assay LOD was established at 2 TCIU of the virus particles/0.2 ml tissue homogenate with a 100% capacity to detect different MYXV strains (ASp). The method was characterized by good DSp of 0.955 (0.839-0.999 CI) and DSe of 0.976 (0.914-1.00 CI). In addition, it was repeatable and reproducible and confirmed its suitability for the detection of MYXV in clinical material. The IAC-PCR developed meets OIE validation requirements for virological methods and can be used in diagnostic or epidemiological studies of rabbit myxomatosis.

Development and validation of a real time PCR for the detection of myxoma virus based on the diploid gene M000.5L/R.

The myxoma virus (MYXV) causes severe infections in European rabbits that may reach mortality rates up to 100% depending on the viral strain. The typical symptoms and lesions induced by the virus are usually enough to permit the correct clinical diagnosis. However, in peracute forms the infection may be accompanied by unspecific symptoms. Sudden death may also occur without evident clinical signs of myxomatosis. Likewise, a clinical diagnosis of atypical forms of myxomatosis (amyxomatous) is often complicated and delayed due to the scarceness of skin lesions. As the disease control often depends on an early and unequivocal diagnosis of MYXV, laboratorial methods play a relevant role in the confirmation of MYXV infection. This study describes the development and validation of a novel, high accurate real time polymerase chain reaction assay (rtPCR) for the detection of MYXV. Primers were designed to amplify a 125-bp within the gene M000.5L/R, which is duplicated in the termini of the genome and is unique among Leporipoxvirus. The assay was negative for SFV and other poxviruses and was able to detect 2.6 copies of MYXV DNA proving the effectiveness, specificity and sensitivity of this diagnosis tool. The rtPCR has been applied successfully in INIAV laboratory for routine diagnosis of myxomatosis since 2005.

Viral skin diseases of the rabbit.

This article describes the viral skin diseases affecting the domestic rabbit, the most important being myxomatosis. Transmission and pathogenesis, clinical signs, diagnosis, treatment, and control are described and the article will be of interest to veterinary practitioners who treat rabbits. Shope fibroma virus, Shope papilloma virus, and rabbitpox are also discussed.

Epidemiology of viral haemorrhagic disease and myxomatosis in a free-living population of wild rabbits.

From January 1993 to June 1996, the epidemiology of myxomatosis and viral haemorrhagic disease (VHD) was studied in a free-living population of wild rabbits (Oryctolagus cuniculus) in Spain by means of serological surveys and radiotracking. Myxomatosis was endemic and associated with the breeding period. Its serological pattern was characterised by a 100 per cent prevalence of antibodies in adult rabbits and a rapid increase in antibodies in young rabbits in their first year. No mortality from myxomatosis was detected in adults, and mortality in young rabbits could not be estimated because of interference by predators and scavengers and the deaths of many radiotagged rabbits inside their burrows. VHD was also an endemic disease associated with the breeding period. Adults had a higher prevalence of antibodies against VHD than young rabbits, reaching values of 80 to 90 per cent. During the study, there was an increase in rabbit numbers as a result of a decrease in mortality from predation which was associated with an increase in mortality due to VHD and in the prevalence of antibodies to VHD. Mortality from VHD was lower in rabbits with VHD antibodies than in seronegative rabbits, but some mortality from the disease was also detected in seropositive rabbits. The annual mean mortality rate due to VHD in adult rabbits was estimated to be 21.8 per cent.

Development of an ELISA for detection of myxoma virus-specific rabbit antibodies: test evaluation for diagnostic applications on vaccinated and wild rabbit sera.

An enzyme-linked immunosorbent assay (ELISA) was developed and compared with 2 reference diagnostic tests (indirect immunofluorescence [IF] and complement fixation) to detect myxoma virus-specific antibodies in sera from 50 rabbits experimentally vaccinated with an attenuated strain of myxoma virus or with a Shope fibroma virus. The ELISA was highly specific (100% specificity) and sensitive (100%, 21 days after homologous vaccination). In a comparison of the ELISA with the IF test in 128 wild rabbits from France, discrepant results were obtained in only 11 (8.6%) animals, which were positive with the ELISA and negative with the IF test. The higher sensitivity and the good specificity of the ELISA was confirmed in a serologic survey of 118 rabbits from 2 Kerguelen (Indian Ocean) islands, where the prevalence of myxomatosis varied considerably. The ELISA is an alternative serologic test for diagnosis, vaccine evaluation, and seroepidemiologic surveys of myxomatosis.

[Reaction of the complement fixation test on microtitration plates: application to the serology of myxomatosis. Comparative study of the results with the reaction of indirect immunofluorescence]. / Réaction de fixation du complément en plaques de microtitration: application à la sérologie de la myxomatose. Etude comparative des résultats avec la réaction d'immunofluorescence indirecte.

The authors describe a complement fixation technique on microtitration plates, using an antigen prepared from myxomas induced in rabbits. Compared with indirect immunofluorescence this technique was less cumbersome, more economical, easier to read and (as a conventional procedure) applicable in all laboratories. Results obtained with 165 serum samples tested by both methods showed good correlation and a specificity at least equal to that of indirect immunofluorescence. Taking into account its lower sensitivity, the positive threshold value for complement fixation under the described experimental conditions was a dilution of 1:4 (H50).