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. 2016 Jul:233:23-36.
doi: 10.1016/j.jviromet.2016.03.002. Epub 2016 Mar 10.

Real-time PCR for differential quantification of CVI988 vaccine virus and virulent strains of Marek's disease virus

Susan J Baigent  1 , Venugopal K Nair  2 , Hervé Le Galludec  3
Affiliations

Real-time PCR for differential quantification of CVI988 vaccine virus and virulent strains of Marek's disease virus

Susan J Baigent et al. J Virol Methods. 2016 Jul.

Abstract

CVI988/Rispens vaccine, the 'gold standard' vaccine against Marek's disease in poultry, is not easily distinguishable from virulent strains of Marek's disease herpesvirus (MDV). Accurate differential measurement of CVI988 and virulent MDV is commercially important to confirm successful vaccination, to diagnose Marek's disease, and to investigate causes of vaccine failure. A real-time quantitative PCR assay to distinguish CVI988 and virulent MDV based on a consistent single nucleotide polymorphism in the pp38 gene, was developed, optimised and validated using common primers to amplify both viruses, but differential detection of PCR products using two short probes specific for either CVI988 or virulent MDV. Both probes showed perfect specificity for three commercial preparations of CVI988 and 12 virulent MDV strains. Validation against BAC-sequence-specific and US2-sequence-specific q-PCR, on spleen samples from experimental chickens co-infected with BAC-cloned pCVI988 and wild-type virulent MDV, demonstrated that CVI988 and virulent MDV could be quantified very accurately. The assay was then used to follow kinetics of replication of commercial CVI988 and virulent MDV in feather tips and blood of vaccinated and challenged experimental chickens. The assay is a great improvement in enabling accurate differential quantification of CVI988 and virulent MDV over a biologically relevant range of virus levels.

Keywords: CVI988/Rispens; Marek’s disease virus; Real-time PCR; Single nucleotide polymorphism; Vaccination; pp38.

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Figures

Fig. 1
Fig. 1
Location of primers and probes in MDV-1 pp38 gene sequences. The pp38 gene sequence shows two single nucleotide differences (G/A SNPs) between CVI988 and Md5 (a representative virulent strain). The bold SNP X (#320) represents a consistent difference between CVI988 and virulent strains. The bold italicised SNP Y (#326) is not consistent in all virulent strains. Sequences of the primers (underlined text) were: forward primer 5′-GAGCTAACCGGAGAGGGAGA-3′, and reverse primer 5′-CGCATACCGACTTTCGTCAA-3′, and the amplicon size was 99 bp. Two 15-mer probes (light grey shading), designed to the minus strand, covered the region containing SNP #320: pp38-CVI (5′-CCCACCGTGACAGCC-3′, Tm = 57.7) and pp38-Vir(1) (5′-CCCACTGTGACAGCC-3′, Tm = 54.1). A third (17-mer) probe, pp38-Vir(3) included both SNPs #320 and #326 (5′-CTCCCACTGTGACAGCC-3′, Tm = 57.1). A fourth probe (pp38-Generic, 5′-GCTACCGCCTGAGCC-3′, designed to the plus strand, Tm = 57.4) which targets a region common to pp38 of all MDV-1 strains, is shaded dark grey.
Fig. 2
Fig. 2
Specificity of probes for CVI988 and virulent MDV-1. Change in fluorescence (delta Rn) when pp38-CVI, pp38-Vir(3) and pp38-Generic probes were used in 40-cycle q-PCR using 100 ng DNA from either CVI988-infected CEF or RB-1B-infected CEF as a template. The thick horizontal line with an arrowhead at each end is the threshold (default value 0.2). (a) pp38-CVI probe detects CVI988 DNA but not RB-1B DNA; NTC is no-template control. (b) pp38-Vir(3) probe detects RB-1B DNA, while the signal from the CVI988 PCR product is very low. By raising the threshold line to 0.6, CVI988 is scored negative in this reaction. (c) pp38-Generic probe detects both CVI988 DNA and RB-1B DNA. (d) The specificities of pp38-CVI, pp38Vir-A(3) and pp38-Generic probes were tested against a range of Mardivirus strains in a 40-cycle q-PCR. The 40-Ct value (the cycle threshold value, Ct, subtracted from 40) is proportional to the amount of target DNA present. A 40-Ct value of 0 indicates that fluorescence did not cross the threshold, i.e. the probe did not bind to the PCR product. ‘att MDV1′ = attenuated MDV-1, vMDV1 = virulent MDV-1, vvMDV1 = very virulent MDV-1, vv+MDV1 = very virulent plus MDV-1.
Fig. 3
Fig. 3
Assay sensitivity, efficiency, linearity and reproducibility. The probes were tested in q-PCR assays using serial dilutions of DNA extracted from CVI988-infected CEF or RB-1B-infected CEF. Mean 40-Ct values from triplicate assays, with 95% confidence limits for the regression and the R2 value, are shown for (a) pp38-CVI probe with serial dilutions of CVI988CEF DNA, and (b) pp38-Vir(3) probe with serial dilutions of RB-1B CEF DNA. The underlying points for 40-Ct values from triplicate assays are shown using three different symbols to distinguish the three assays (open squares, black diamonds and grey triangles).
Fig. 4
Fig. 4
Quantitative accuracy of detection in virus mixtures using pp38 SNP q-PCR assays. DNA was prepared from CEF cells infected with pCVI988, and from CEF cells infected with RB-1B, and each was diluted to approximately 5 × 105 virus genomes per 4 μl. (a) DNA of pCVI988-infected CEF was diluted in DNA of RB-1B-infected CEF (open triangles) or in DNA from non-infected CEF (black squares). (b) DNA of RB-1B-infected CEF was diluted in DNA of pCVI988 infected CEF (open triangles) or in DNA from non-infected CEF (black squares). Samples were run in q-PCR using either pp38-CVI probe or pp38-Vir(3) probe. The figures show mean 40-Ct values with standard error bars for duplicate dilution series and duplicate reactions (only upper error bars are shown, for clarity).
Fig. 5
Fig. 5
Validation of pp38 SNP q-PCR assays against BAC and US2 q-PCR assays. Each data point shows values for an individual chicken (log10 genomes per 104 cells). (a) Comparison of BAC q-PCR and pp38-CVI q-PCR to measure pCVI988 in vaccinated/non-challenged and vaccinated/challenged birds (n = 74). (b) Comparison of US2 q-PCR and pp38-Vir(3) q-PCR to measure RB-1B in challenged/non-vaccinated and challenged/vaccinated birds (n = 79). (c) Comparison of pp38-Generic q-PCR with additive results from pp38-CVI and pp38-Vir(3) q-PCRs to measure total MDV-1 in all vaccinated and/or challenged birds (n = 102). (d) Comparison of additive results from pp38-CVI and pp38-Vir(3) q-PCRs, with additive results from BAC and US2 q-PCRs to measure total MDV-1 in all vaccinated and/or challenged birds (n = 102).
Fig. 6
Fig. 6
Time-course of replication of commercial CVI988 vaccine and RB-1B challenge virus in individual chickens. One-day-old Rhode Island Red chickens were divided between three experimental groups. Group A (n = 8) received CVI988 vaccine only, Group B (n = 14) received RB-1B challenge virus only, and Group C (n = 10) was both vaccinated and challenged. Feather and blood samples were collected from each chicken at regular intervals. DNA was subjected to pp38 SNP/ovo duplex q-PCR, using either pp38-CVI or pp38-Vir(3) as the pp38 detection probe, and the level of each virus quantified as genomes per 104 cells. Mean virus levels (with 95% confidence intervals) for each group and each sample type at each time-point are shown for CVI988 vaccine virus (a) and RB-1B challenge virus (b). Values of 0.6 (baseline value) are considered negative. Only upper confidence intervals are shown for clarity.
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