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Am. J. Trop. Med. Hyg., 69(6), 2003, pp. 648-651
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT: DETECTION OF JAPANESE ENCEPHALITIS VIRUS IN MOUSE PERIPHERAL BLOOD MONONUCLEAR CELLS USING AN IN SITU REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION

CHING-KAI CHUANG, SHYAN-SONG CHIOU, LI-CHING LIANG, AND WEI-JUNE CHEN
Department of Public Health and Parasitology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan; Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan

 

ABSTRACT

Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin-labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2–3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides.

Japanese encephalitis (JE) virus is one of approximately 70 flaviviruses that belong to the family Flaviviridae.1 The JE virus is transmitted between vertebrates by mosquitoes in nature,2 causing approximately 10% of the susceptible population to be infected in Southeast Asian countries each year.3 Most infections of JE virus are subclinical.4 However, some cases may lead to a fatal outcome and survivors may have neurologic or psychological sequalae.5–7 A variety of JE virus strains have been identified from many different countries; the T1P1 strain used in this study was isolated from Armigeres subalbatus in an unconventional ecosystem in Taiwan.8

Diagnosis of JE by clinical symptoms is hardly feasible, especially in the acute stage of infections. Nowadays, confirmation of JE virus infection depends mostly on serologic assays such as IgM antibody detection by enzyme-linked immunosorbent assays.9,10 According to the known pathogenesis of JE, the virus enters peripheral blood mononuclear cells (PBMCs) including monocytes/macrophages as the primary site for replication in human and relevant vertebrate hosts. Subsequently, the virus migrates to the central nervous system (CNS) in which brain inflammation or encephalitis may occur.10 However, isolation of JE virus from peripheral blood is generally not possible because of the short period and low titer of viremia in the acute stage.10 Molecular diagnosis using a reverse trancriptase-polymerase chain reaction (RT-PCR) to amplify gene fragments of viral RNAs has recently been used to diagnose flavivirus infections.11–13 Unfortunately, the copy number of viral RNA in the blood of JE patients is usually low, making difficult RNA extraction from this source.

Flaviviral antigens have been detected in PBMCs of convalescent blood samples, indicating the possible persistence of the viruses in leukocytes.14–18 This represents the potential for the RT-PCR to detect viral RNAs in PBMCs.12 To avoid the obstacle of RNA extraction, we thus designed an approach from which E (envelope) gene fragments of JE virus inside PBMCs were amplified in situ.

In this study, baby hamster kidney (BHK)-21 cells were used to optimize conditions for conducting the in situ RT-PCR. Cells that were infected with the T1P1 strain of JE virus (multiplicity of infection = 5) for 24 hours were transferred from flasks into microtubes, fixed with 4% paraformaldehyde for 15 minutes, and then permeabilized with 0.1% Triton X-100 for five minutes. Most cells treated in this manner remained intact. The primer pair (PE1/ PE2) designed by Chung and others19 was used to amplify a specific fragment (291 basepairs) of JEV envelope protein. The sequence of the sense primer (PE1) was 5'-AGTTAACATCAGGCCAC-CTGA-3' and that of the complementary primer (PE2) was 5'-GTTCCATCTCGACCAGCAC-3'. The 5'-end of primer PE1 was labeled with digoxigenin (DIG). The subsequent RT-PCR was performed in a thermal cycler (PCR Express; Hybaid, Middlesex, United Kingdom) using the Reverse-iTTM One-Step RT-PCR Kit (ABgene, Epsom, Surrey, United Kingdom). For reverse transcription, the sample mixture was incubated at 42°C for one hour followed by an additional incubation at 94°C for three minutes. The reaction was subsequently run for 40 cycles at 94°C for 30 seconds, 50°C for one minute, and 72°C for one minute, followed by an additional elongation at 72°C for 10 minutes. The cells were then washed with phosphate-buffered saline (PBS), pH 7.4, for 10 minutes, blocked with 5% skim milk for 15 minutes, and washed with PBS for 10 minutes.

To visualize the target cDNA sequence, 50 µL of diluted (1:500 in PBS) goat anti-DIG antibody conjugated with alkaline phosphatase (Roche, Mannheim, Germany) was added to the microtubes and incubated for one hour. Subsequently, the cells were washed twice with 0.02 M Tris-base, 0.5 M NaCl, pH 9.5 for 10 minutes followed by another wash with buffer (0.1 M Tris-HCl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5). Substrate (nitroblue tetrazolium chloride/5-bromo-3-chloro-3-indolyl-phosphate [NBT/BCIP]) was then added to the microtubes and incubated for 10 minutes. The cells were washed with PBS, removed from the tubes, and distributed onto 12-well slides. A dark brown product representing the existence of virus can be recognized in at least 80% of cells that were infected with JE virus but not in uninfected cells (Figure 1Go). Other fixatives such as cold acetone or 1% formalin plus a digestion with 5 µg/mL of proteinase K (the alternative permeability enhancer) frequently caused cell aggregation and/or destruction, leading to a failure in reading the results.



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  		    FIGURE 1. Baby hamster kidney-21 cells infected with the T1P1 strain of Japanese encephalitis virus for 24 hours were fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.1% Triton X-100 for five minutes, followed by an in situ reverse transcriptase-polymerase chain reaction in microtubes. A, Positive samples (arrows) among virus-infected cells. B, Negative samples from mock-infected cells. (Magnification x 100.)

 
For virus detection in infected mice, PBMCs were isolated from 1–2 mL of whole blood of female ICR mice (4–5 weeks old) intravenously inoculated with 100 µL of PBS (control group) or 1 x 106 plaque-forming units/mouse of JE virus in Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) solution.17 Isolated cells were fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.1% Triton X-100 for five minutes as reported earlier for BHK-21 cells.

The present study is the first to reproducibly detect viral RNA in PBMCs of JE virus-infected mice with an in situ RT-PCR. The uniqueness of the technique was conducting the RT-PCR in tubes instead of on slides.20–24 It has largely reduced the possibility of losing cells from slides. The results in Table 1Go show that positive PBMCs (dark brown on the edge of a cell or in a whole cell) appeared in two of four pools of mouse blood (3–5 mice/pool) collected at day 1 post-inoculation (Figure 2AGo). Although the type of infected cells was not specifically identified, they were probably composed of monocytes/macrophages and lymphocytes.16 In the four pools of blood collected at day 3 post-inoculation, all were positive (Figure 2AGo), although the number of positive cells was reduced. In contrast, no positive cells was detected in blood collected at day 5 post-inoculation (Figure 2BGo). This implies that the viremic stage of JE virus infection in mice is limited to 2–3 days. Moreover, the virus titer could be relatively low since a low titer of neutralizing antibodies (the reciprocal geometrical mean ± SD titer was estimated to be 15.1 ± 1.46) has been obtained from five mice intravenously inoculated with JE virus.


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TABLE 1
Viral RNA detection in peripheral blood mononuclear cells (PBMCs) and brain tissues of mice intravenously inoculated with the T1P1 strain of Japanese encephalitis virus*
 


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  		    FIGURE 2. A, Completely (arrow) and partially (arrowhead) positive peripheral blood mononuclear cells in a mouse blood pool (3–5 mice/pool) collected at day 1 or day 3 post-inoculation with Japanese encephalitis virus. B, Cells from blood collected at day 5 post-inoculation or mock-infected mice were not infected. (Magnification x 1,000.)

 
The presence of JE virus in brain tissues was also examined. The results showed that viral RNA was absent in mouse brain until day 3 post-inoculation (Table 1Go). It is presumed that virus migration from peripheral blood into the CNS occurs between day 3 and day 5 post-inoculation. Because clinical symptoms, primarily paralysis, appeared only in certain inoculated mice and were usually not observed until day 7 post-inoculation, structural degradation and functional abnormality of the CNS may not occur unless the virus has entered and replicated in the brain.

The degradation of RNA may occur in infected specimens, thus reducing the amount of RNA that can be extracted from cells infected by the virus.25 Therefore, an in situ PCR or RT-PCR is advantageous because there is no need for extraction of nucleic acids. Moreover, this type of technique has been demonstrated to be capable of detecting a low number of RNA copies, even a single copy, from intact virus-infected lymphocytes without much loss of sensitivity and specificity.22 A previous report using this technique has shown that the percentage of PBMCs infected with human immunodeficiency virus-1 ranged from 0.1% to 13.5%.21 In addition to viral RNAs, the in situ RT-PCR can also be useful in detecting intracellular cytokines or inducible nitric oxide synthase mRNA.20,23 The in situ RT-PCR may also be used as a tool to study the cellular immune response without need of efficient mRNA extraction,26 especially for samples containing small amounts of mRNA. More significantly, conducting the RT-PCR in tubes has been shown to be convenient, accurate, and efficient in contrast to the conventional in situ RT-PCR, which was conducted mostly on slides.20,21,23 Theoretically, this in situ technique could be applied to the diagnosis of other flaviviral infections such as dengue that normally infect mononuclear cells at the early or persistent phase of infection.17,27

Received February 17, 2003. Accepted for publication July 25, 2003.

Acknowledgments: We thank Mei-Hwei Fan-Chiang and Jing-Gi Hwang for their technical assistance.

Financial support: This work was supported by a grant from Chang Gung Memorial Hospital (CMRP 1212) and in part by the Department of Health, Republic of China (DOH88-TD-1018).

Authors’ addresses: Ching-Kai Chuang, Li-Ching Liang, and Wei-June Chen, Department of Public Health and Parasitology, College of Medicine, Chang Gung University, 259 Wen-Hwa First Road, Kwei-San, Tao-Yuan 33332, Taiwan. Shyan-Song Chiou, Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei 10018, Taiwan.

Reprint requests: Wei-June Chen, Department of Public Health and Parasitology, College of Medicine, Chang Gung University, 259 Wen-Hwa First Road, Kwei-San, Tao-Yuan 33332, Taiwan, Fax: 886-3-211-8700, E-mail: wjchen{at}mail.cgu.edu.tw.

 

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