Quantitative PCR technique for detecting lymphocytic choriomeningitis virus in vivo

Abstract

Quantitative PCR (QPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models. We have developed a QPCR assay to accurately quantify lymphocytic choriomeningitis virus (LCMV) in infected mice. We first validated this assay using plasmid DNA and LCMV viral stocks. We then demonstrated that the LCMV QPCR assay can detect LCMV in serum and tissues of chronically infected mice (LCMV-clone 13), with greater sensitivity than conventional plaque assay. Subsequently, we demonstrated that the QPCR assay can detect LCMV in tissues of CD40L(-/-) mice during a low grade chronic infection with LCMV Armstrong. Finally, we improved the assay further such that it was approximate 1000-fold more sensitive than plaque assay for detection of the presence of LCMV in tissue.

Figure 1. Quantitative LCMV RT-PCR (QPCR)

(a-d) NP primers. (e-g) GP primers. (a) QPCR LCMV NP standard curve using serial dilutions of an NP encoding plasmid. Dotted line indicates threshold used to establish a positive signal. Each curve is the amplification product from an amount of plasmid template 10-fold greater than the next sample to the right. (b) Melt curves of amplified NP PCR products from the standard curve samples, demonstrating the presence of a single amplication product with the expected melting temperature. (c) Quantitation of the NP standard curve. (CT = cycle threshold) (d) QPCR of LCMV_arm viral stock (LCMV RNA copies/ml), in comparison with conventional plaque assay (PFU/ml). (e) QPCR LCMV GP standard curve using serial dilutions of an GP encoding plasmid. Each curve is the amplification product from an amount of plasmid template 10-fold greater than the next sample to the right. (f) Melt curves of amplified GP PCR products from the standard curve samples, demonstrating the presence of a single amplication product with the expected melting temperature. Data is representative of more than three independent experiments. (g) Quantitation of the GP standard curve. (CT = cycle threshold).

Figure 2. Serum viral loads in LCMV_cl13 infected mice

Comparison between QPCR (LCMV RNA copies/ml, “RNA”) and plaque assay (PFU/ml, “PFU”). Assays were run in parallel on samples divided into two aliquots. (a) Day 30 post-infection. (b) Day 60 post-infection. (c) Day 90 post-infection. Means are indicated by solid bars. Dashed lines indicate detection threshold for each assay. Data is representative of more than three independent experiments.

Figure 3. Serum and tissue viral loads in LCMV_cl13 infected mice

Serum (a), kidney (b), and liver (c) samples were taken from a group of seven mice at day 30 after infection with LCMV_cl13. QPCR (LCMV RNA copies/ml, “RNA”) and plaque assay (PFU/ml, “PFU”) were directly compared. Assays were run in parallel on samples divided into two aliquots. Means are indicated by solid bars. Dashed lines indicate detection threshold for each assay. Data is representative of more than three independent experiments.

Figure 4. LCMV_arm viral loads in CD40L^-/- mice at day 8 postinfection

Wildtype (WT) and CD40L^-/- mice were infected i.p. with LCMVarm, and viral loads were measured at day 8 postinfection. QPCR (LCMV RNA copies/ml, “RNA”) and plaque assay (PFU/ml, “PFU”) were directly compared. Assays were run in parallel on samples divided into two aliquots. Means are indicated by solid bars. Dashed lines indicate detection threshold for each assay. (a) Serum. (b) spleen. (c) liver. Data is representative of two independent experiments.

Figure 5. LCMV_arm viral loads in CD40L^-/- mice at day 30 postinfection

(a) Serum. (b) Spleen. (c) Liver. (d) Kidney. Assays were run as described in Figure 4. Data is representative of two independent experiments. Dashed lines indicate detection threshold for each assay, or the threshold is below the baseline shown.

Figure 6. LCMV_arm viral loads in CD40L^-/- mice at day 90 postinfection

(a) Serum. (b) Spleen. (c) Liver. (d) Kidney. Assays were run as described in Figure 4. Dashed lines indicate detection threshold for each assay, or the threshold is below the baseline shown.

Figure 7. Immune responses in CD40L^-/- mice

LCMV-specific CD8 T cells (a,e,i), CD4 T cells (b,f,j), plasma cells (c,g,k), and germinal centers (d,h) were quantified in the wildtype (“WT”) and CD40L^-/- mice from Figures 4,5, and 6 at days 8 (a-d), 30 (e-h), and 90 (i-k) after infection with LCMV_arm. N = 4/group. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

Figure 8. Comparison of primer pairs NP1 (original), NP2, and GP

Serum, liver, and kidney samples were tested from two representative LCMV_cl13 infected mice at day 30 postinfection. (ns = nonspecific products, as determined by melt curve)

Figure 9. LCMV_arm viral loads, using GP primers

Wildtype mice infected with LCMV_arm were tested at day 8 and day 30 post-infection for LCMV using GP QPCR primers. Infected mice (“+”) were compared against uninfected mice (“—”) in all cases. (a) Serum. (b) Lymph node (inguinal). (c) Spleen. (d) Kidney. Dashed line indicates detection threshold.