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Detection and Differentiation of CVI988 (Rispens Vaccine) from Other Serotype 1

Isabel M. Gimeno, John R. Dunn, Aneg L. Cortes, Abd El-Galil El-Gohary, Robert F. Silva
Avian diseases 2014 v.58 no.2 pp. 232-243
chickens, quantitative polymerase chain reaction, feathers, neoplasms, DNA primers, serotypes, plasmids, vaccination, base pair mismatch, Gallid alphaherpesvirus 2, genes, early diagnosis, Marek disease, single nucleotide polymorphism
The serotype 1 Marek’s disease virus (MDV) is the causative agent for Marek’s disease (MD), a lymphoproliferative disease of chickens of great concern to the poultry industry. CVI988 (Rispens vaccine), an attenuated serotype 1 MDV, is currently the most efficacious commercially available vaccine for preventing MD. However, it is difficult to detect and differentiate CVI988 when other serotype 1 MDVs are present. To facilitate the detection of CVI988, we developed two sets of primers for a mismatch amplification mutation assay (MAMA) PCR that targeted the single nulceotide polymorphism associated with the H19 epitope of the phosphorylated protein 38 gene. The PCR was very specific. One primer set (oncogenic primers) amplified DNA from 15 different serotype 1 MDVs except CVI988. The other primer set (CVI988 primers) amplified DNA from CVI988 but not from any of the other 15 serotype 1 MDVs. A real-time PCR assay was developed using MAMA primers, and specificity and sensitivity was evaluated in vitro and in vivo. Mixtures of plasmids (CVI988 plasmid and oncogenic plasmid) at various concentrations were used to evaluate the sensitivity/specificity of MAMA primers in vitro. Both primer sets were able to amplify as little as one copy of their respective plasmid. Oncogenic primers were highly specific and only amplified CVI988 plasmid when the concentration of oncogenic plasmid was very low (1 3 101) and CVI988 plasmid was very high (1 3 106). Specificity of CVI988 primers was not as high because they could amplify oncogenic plasmids when the concentration of CVI988 plasmid was 1 3 103 and the concentration of oncogenic 1 3 102. Validation of MAMA primers in in vivo samples demonstrated that oncogenic primers can be used for both early diagnosis of MD in feather pulp (FP) samples collected at 3 week of age and confirmation of MD diagnosis in tumors. CVI988 primers could be used to monitor CVI988 vaccination in samples with a low load of oncogenic MDV DNA (latently infected samples or negative) but not in samples with a high load of oncogenic MDV DNA (tumors). Our results suggest that monitoring CVI988 vaccination in FP samples collected at 1 week of age ensures the specificity of the CVI988 primers.