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

Ikeno S, Suzuki MO, Muhsen M, Ishige M, Kobayashi-Ishihara M, Ohno S, Takeda M, Nakayama T, Morikawa Y, Terahara K, Okada S, Takeyama H, Tsunetsugu-Yokota Y - Front Microbiol (2013)

Bottom Line: 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.Here, we developed a sensitive one-step quantitative reverse transcription (qRT)-PCR that simultaneously measures nucleocapsid (N) and human RNase P mRNA levels.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, National Institute of Infectious Diseases Tokyo, Japan ; Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology/Waseda University Graduate School of Collaborative Education Curriculum Tokyo, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: For the detection of MV infection in clinical specimens, Hummel et al. (2006) established a sensitive qRT-PCR system that used primer and probe sets targeting the MV-N gene. In our humanized mouse model, it is necessary to analyze endogenous mRNA expression in human PBMCs to determine the level of human cell-associated MV infection in mouse blood. We initially assumed that hCD45 expression would be suitable to discriminate human hematopoietic cells from co-existing mouse hematopoietic cells in vivo. On that basis, we designed human-specific primer and TaqMan probe sets for hCD45 and compared their usefulness with a primer/probe set for a widely used housekeeping gene, RNase P. RNA was extracted from humanized (hu-mouse) or non-humanized (non-hu-mouse) murine splenocytes, and the level of mRNA was measured by one-step qRT-PCR. Both hCD45 and RNase P primer/probe sets detected mRNA expression of target genes from human PBMCs present in hu-mouse spleen, at similar sensitivities, but neither set detected expression in non-hu-mouse (Figure 1A). Thus, both primer/probe sets are human-specific. Next, we enriched CD14+ monocytes and T cells from PBMCs by positive and negative magnetic-bead selection, respectively, and then determined the copy numbers of hCD45 and RNase P in these cell fractions from each of five donors. In Figure 1B, the expression levels of hCD45 (left panel) and RNase P (right panel) in monocytes and T cells are depicted relative to the level in each donor’s PBMCs. Because RNase P expression was less affected by cell type than CD45 expression (*P < 0.05), in subsequent experiments we exclusively used RNase P primer/probe sets as an endogenous control for mRNA expression.


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

Ikeno S, Suzuki MO, Muhsen M, Ishige M, Kobayashi-Ishihara M, Ohno S, Takeda M, Nakayama T, Morikawa Y, Terahara K, Okada S, Takeyama H, Tsunetsugu-Yokota Y - Front Microbiol (2013)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795360&req=5

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).
Mentions: For the detection of MV infection in clinical specimens, Hummel et al. (2006) established a sensitive qRT-PCR system that used primer and probe sets targeting the MV-N gene. In our humanized mouse model, it is necessary to analyze endogenous mRNA expression in human PBMCs to determine the level of human cell-associated MV infection in mouse blood. We initially assumed that hCD45 expression would be suitable to discriminate human hematopoietic cells from co-existing mouse hematopoietic cells in vivo. On that basis, we designed human-specific primer and TaqMan probe sets for hCD45 and compared their usefulness with a primer/probe set for a widely used housekeeping gene, RNase P. RNA was extracted from humanized (hu-mouse) or non-humanized (non-hu-mouse) murine splenocytes, and the level of mRNA was measured by one-step qRT-PCR. Both hCD45 and RNase P primer/probe sets detected mRNA expression of target genes from human PBMCs present in hu-mouse spleen, at similar sensitivities, but neither set detected expression in non-hu-mouse (Figure 1A). Thus, both primer/probe sets are human-specific. Next, we enriched CD14+ monocytes and T cells from PBMCs by positive and negative magnetic-bead selection, respectively, and then determined the copy numbers of hCD45 and RNase P in these cell fractions from each of five donors. In Figure 1B, the expression levels of hCD45 (left panel) and RNase P (right panel) in monocytes and T cells are depicted relative to the level in each donor’s PBMCs. Because RNase P expression was less affected by cell type than CD45 expression (*P < 0.05), in subsequent experiments we exclusively used RNase P primer/probe sets as an endogenous control for mRNA expression.

Bottom Line: 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.Here, we developed a sensitive one-step quantitative reverse transcription (qRT)-PCR that simultaneously measures nucleocapsid (N) and human RNase P mRNA levels.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, National Institute of Infectious Diseases Tokyo, Japan ; Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology/Waseda University Graduate School of Collaborative Education Curriculum Tokyo, Japan.

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.

No MeSH data available.


Related in: MedlinePlus