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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

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.
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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.

Mentions: Because wild-type MV mainly utilizes SLAM as the receptor for entry into lymphoid cells (Tatsuo et al., 2000), the kinetics of MV infection in Jurkat/hSLAM cells can be clearly visualized by flow cytometry. We infected Jurkat/hSLAM cells with a wild-type MV encoding EGFP (IC323-EGFP) at MOI of 0.01, 0.05, and 0.25. Cells were washed and harvested at 6, 12, 18, or 24 h after MV infection. A subset of the cells in each sample was analyzed by flow cytometry, and the remainder of the sample was used for RNA extraction. The mRNA levels of MV-N and RNase P were determined by qRT-PCR, and the level of MV-N mRNA relative to RNase P RNA was calculated. Representative results of three experiments are shown in Figure 2A (flow cytometry) and Figure 2B (qRT-PCR). Because of the rapid and strong cytopathic effect by MV at the highest MOI (0.25), we omitted the flow cytometry data corresponding to that condition. At MOI 0.01, a similar frequency of GFP+ cells was detectable at 12 and 18 h p.i., whereas at MOI 0.05, the GFP+ cell frequency was already high at 12 h p.i. Note that the level of hSLAM was not down-modulated by MV infection. Over the time course, relative MV-N expression level at all three MOIs increased in parallel over two orders of magnitude, indicating that these two methods yield comparable results (as shown in Figure 2C) and are useful for monitoring the replication kinetics of MV infection in vitro.


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC3795360&req=5

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.
Mentions: Because wild-type MV mainly utilizes SLAM as the receptor for entry into lymphoid cells (Tatsuo et al., 2000), the kinetics of MV infection in Jurkat/hSLAM cells can be clearly visualized by flow cytometry. We infected Jurkat/hSLAM cells with a wild-type MV encoding EGFP (IC323-EGFP) at MOI of 0.01, 0.05, and 0.25. Cells were washed and harvested at 6, 12, 18, or 24 h after MV infection. A subset of the cells in each sample was analyzed by flow cytometry, and the remainder of the sample was used for RNA extraction. The mRNA levels of MV-N and RNase P were determined by qRT-PCR, and the level of MV-N mRNA relative to RNase P RNA was calculated. Representative results of three experiments are shown in Figure 2A (flow cytometry) and Figure 2B (qRT-PCR). Because of the rapid and strong cytopathic effect by MV at the highest MOI (0.25), we omitted the flow cytometry data corresponding to that condition. At MOI 0.01, a similar frequency of GFP+ cells was detectable at 12 and 18 h p.i., whereas at MOI 0.05, the GFP+ cell frequency was already high at 12 h p.i. Note that the level of hSLAM was not down-modulated by MV infection. Over the time course, relative MV-N expression level at all three MOIs increased in parallel over two orders of magnitude, indicating that these two methods yield comparable results (as shown in Figure 2C) and are useful for monitoring the replication kinetics of MV infection in vitro.

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