Sensitive detection of measles virus infection in the blood and tissues of humanized mouse by one-step quantitative RT-PCR

Abstract

Live attenuated measles virus (MV) has long been recognized as a safe and effective vaccine, and it has served as the basis for development of various MV-based vaccines. However, because MV is a human-tropic virus, the evaluation of MV-based vaccines has been hampered by the lack of a small-animal model. The humanized mouse, a recently developed system in which an immunodeficient mouse is transplanted with human fetal tissues or hematopoietic stem cells, may represent a suitable model. Here, we developed a sensitive one-step quantitative reverse transcription (qRT)-PCR that simultaneously measures nucleocapsid (N) and human RNase P mRNA levels. The results can be used to monitor MV infection in a humanized mouse model. Using this method, we elucidated the replication kinetics of MV expressing enhanced green fluorescent protein both in vitro and in humanized mice in parallel with flow-cytometric analysis. Because our qRT-PCR system was sensitive enough to detect MV expression using RNA extracted from a small number of cells, it can be used to monitor MV infection in humanized mice by sequential blood sampling.

Keywords: EGFP expression; flow cytometry; humanized mouse; measles virus infection; quantitative RT-PCR.

FIGURE 1

Selection of an endogenous control for the analysis of MV-infected human PBMCs. (A) RNA was extracted from spleen cells of hNOJ and non-humanized NOJ, and one-step qRT-PCR was performed using primer and probe sets designed against the human-specific hCD45 and RNaseP mRNAs. To calculate copy numbers of these genes, the PCR products of human CD45 and RNase P were subcloned into plasmids and used as standard DNAs. (B) Human PBMCs from five donors were fractionated into CD14⁺ monocytes and T cells. RNA from these cell populations was extracted, and the expression levels of hCD45 and RNase P were analyzed by qRT-PCR. The graph depicts the expression levels in these fractionated cells relative to the levels in PBMCs (defined as 1). Statistical differences in hCD45 and RNase P expression among these cell populations were evaluated by non-parametric one-way ANOVA test (*P<0.05).

FIGURE 2

course of MV infection in vitro. Jurkat/hSLAM cells were infected with wild-type MV IC323-EGFP at MOI of 0.01, 0.05, and 0.25, washed, and harvested at the indicated time points. (A) Cells were stained with PE-conjugated anti-hSLAM mAb, fixed with 2% formalin/PBS, and GFP expression was analyzed. (B) RNA was extracted from cells, and expression levels of MV-N and RNase P were analyzed by one-step qRT-PCR. The copy numbers of MV-N and RNase P were determined, and the ratio of MV-N copies to RNase P copies is depicted on the vertical axis. (C) Correlation between the percentage of GFP⁺ Jurkat/SLAM cells and the time course of MV-N expression. Spearman’s rank correlation coefficient was used for statistical analysis.

FIGURE 3

Analysis of MV infection in vivo. Three hNOJ mice (127-1, -4, and -5) were infected intravenously with 2,000 pfu of the MV vaccine strain, AIK-C-EGFP. Mice were sacrificed at day 7 post-infection, and blood and bone marrow cells (BM) were obtained. (A) BM cells were stained with PB-anti-human CD45 mAb, fixed with 2% formalin/PBS, and GFP expression was analyzed. (B) PBMCs from blood and BM cells were lysed, and RNA was prepared. The expression MV-N and RNase P was analyzed as described in the legend for Figure 2B. (C) Correlation between the percentage of GFP⁺ cells among hCD45⁺ cells in BM and the level of MV-N expression in MV-infected hNOJ mice, at day 7 (n = 4) or day 10 (n = 4) p.i. Spearman’s rank correlation coefficient was used for statistical analysis.

FIGURE 4

Monitoring of MV replication in vivo. Two to three hNOJ mice per group were challenged with MV AIK-C-EGFP at 20,000 (squares), 2,000 (triangles), or 200 pfu (circles). The PBMCs of these mice were collected at day 3, 5, 7, 10, 14, and 21 p.i. (A) The level of MV-N expression in the blood of infected hNOJ. Vertical axis shows the level of MV-N relative to that of RNaseP, as described in the legend for Figure 2B. (B) For some mice (depicted using the same symbol as (A)), the number of human cells per 50 μl of blood used for RNA extraction and analyzed for MV-N expression was plotted on the X-axis. (C) An hNOJ mouse infected with MV at a low dose (gray closed circle, 200-2) exhibited an increased level of MV-N. At day 21 p.i., the mouse was sacrificed; cells from blood, spleen, BM, and mesenteric lymph node (MLN) were prepared. Cells were stained and analyzed as described in the legend for Figure 2A.