Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses - PubMed (original) (raw)

Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses

P Desprès et al. J Virol. 1998 Jan.

Abstract

Apoptosis has been suggested as a mechanism by which dengue (DEN) virus infection may cause neuronal cell death (P. Desprès, M. Flamand, P.-E. Ceccaldi, and V. Deubel, J. Virol. 70:4090-4096, 1996). In this study, we investigated whether apoptotic cell death occurred in the central nervous system (CNS) of neonatal mice inoculated intracerebrally with DEN virus. We showed that serial passage of a wild-type human isolate of DEN virus in mouse brains selected highly neurovirulent variants which replicated more efficiently in the CNS. Infection of newborn mice with these neurovirulent variants produced fatal encephalitis within 10 days after inoculation. Virus-induced cell death and oligonucleosomal DNA fragmentation were observed in mouse brain tissue by day 9. Infected mouse brain tissue was assayed for apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and for virus replication by immunostaining of viral antigens and in situ hybridization. Apoptotic cell death and DEN virus replication were restricted to the neurons of the cortical and hippocampal regions. Thus, DEN virus-induced apoptosis in the CNS was a direct result of virus infection. In the murine neuronal cell line Neuro 2a, neuroadapted DEN virus variants showed infection patterns similar to those of the parental strain. However, DEN virus-induced apoptosis in these cells was more pronounced after infection with the neurovirulent variants than after infection with the parental strain.

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Figures

FIG. 1

FIG. 1

Replication of DEN virus in mouse brain. Litters of 2-day-old Swiss mice (Breeding Centre R. Janvier, Le Genest St-Isle, France) were inoculated i.c. with 20 μl of DEN virus in Leibovitz L-15 growth medium containing 2% heat-inactivated fetal bovine serum. (A) Mice were inoculated with 105 FFU of mouse-passaged DEN-1 virus (strain FGA/89) and observed daily for 21 days, and mortality was recorded (•). Eight days after inoculation, brains from three suckling mice were collected and weighed. Brain tissues were prepared as 10% (wt/vol) suspensions, and their infectivity was titrated by FIA (○). Titers are expressed as FFU per gram of brain tissue. (B and C) Newborn mice were inoculated with 5,000 FFU of FGA/89 (○), FGA/NA a5c (▪), or FGA/NA d1d (▴). For panel B, infected mice were observed daily for 21 days and mortality was recorded. For panel C, viral growth in the brains of infected mice was titrated. Each point represents the titration of pooled brain tissues extracted from three DEN virus-infected mice. (D) Oligonucleosomal DNA fragments in brain tissue suspensions were quantified by ELISA. Mouse brains were harvested in triplicate 9 days after infection. Brain tissue suspensions (20 mg) were incubated with lysis buffer, and the histone-associated DNA fragments released into the cytoplasmic fractions were quantified with a cell death detection ELISA kit according to the manufacturer’s protocol (Boehringer Mannheim Biochemicals). Optical density (O.D.) was measured at 405 nm.

FIG. 2

FIG. 2

Distribution of apoptosis in the brain regions of DEN virus-infected mice. Mouse brains were harvested 9 days after inoculation. For mouse brain sections, the brains were removed, covered in Tissue-Tek O.C.T. (Miles) embedding medium, and stored at −80°C. Parasagittal sections of the brain blocks (15-μm thickness) were cut on a cryostat (Jung Frigocut) and mounted onto Vectabond-precoated slides (Vector Laboratories). Sections were fixed in 3% paraformaldehyde in phosphate-buffered saline and stored at 4°C in 70% ethanol. Tissue sections mock infected (A) or infected with DEN virus (FGA/NA d1d) (B and C) were processed for TUNEL analysis. The TUNEL assay was performed with mouse brain sections as described in the instructions to an in situ cell death detection kit from Boehringer Mannheim Biochemicals. TUNEL-positive cells were observed by fluorescence. Cortical (A and B) and hippocampal (C) regions are shown. Magnification, ca. ×100.

FIG. 3

FIG. 3

Viral RNA detected by in situ hybridization in parasagittal sections of mouse brain harvested 9 days after inoculation. The DEN-1 cDNA, coding for proteins prM and E (residues 95 to 775 [6]) of FGA/NA d1d, was amplified by PCR and ligated into the mammalian expression vector pCI-neo (Promega) to generate the recombinant plasmid pCI/prM-E. Transcription with T3 RNA polymerase produced a DIG-labeled antisense transcript of 2,000 nucleotides complementary to the DEN virus genome. A negative control cRNA was generated by the same method with the β-galactosidase gene introduced into the plasmid pCI-neo (pCI/Lac-Z). To reduce the length of the DIG-labeled cRNA for in situ hybridization, the transcripts were treated by alkaline hydrolysis (26). In situ hybridization was performed as described previously (26). DIG-labeled RNA hybrids were detected by ELISA with an anti-DIG-alkaline phosphatase conjugate to catalyze a color reaction between X-phosphate solutions and nitroblue tetrazolium salt, according to the manufacturer’s instructions (Boehringer Mannheim Biochemicals). The tissue sections were incubated for 1 h in the dark and mounted on Vectabond-precoated slides (Vector Laboratories). Magnification, ca. ×64.

FIG. 4

FIG. 4

DEN virus-induced apoptosis in Neuro 2a cells. (A) Oligonucleosomal DNA laddering in DEN virus-infected Neuro 2a cells. Fragmented DNA was extracted from Neuro 2a cells 40 h after mock infection (M.I.) or infection with the indicated DEN virus (MOI of 400 FFU/cell) as previously described (7). To detect cleaved DNA by endonucleases, free 3′-OH DNA termini were labeled with DIG-labeled ddUTP in the presence of terminal deoxynucleotidyl transferase. DIG-labeled DNA samples were subjected to electrophoresis in a 1.8% agarose gel and transferred to Hybond-N nylon membrane, and the immunodetection protocol described in the DIG DNA labeling and detection kit (Boehringer Mannheim Biochemicals) was then used. Results from ethidium bromide staining (left) and immunodetection (right) of low-molecular-weight DNA are shown. (B) Detection of DEN virus-infected cells in the apoptotic state 30 h postinfection. Paraformaldehyde-fixed cells were permeabilized with Triton X-100, and viral antigens were visualized by an immunofluorescence assay with anti-DEN virus hyperimmune mouse ascites fluid. The proportion of DEN virus-infected cells with chromatin condensation was determined by propidium iodide staining as previously described (7).

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References

    1. Bhamarapravati N, Halstead S B, Sookavachana P, Boonyapaknavik V. Studies on dengue virus infection. 1. Immunofluorescence localization of virus in mouse tissue. Arch Pathol. 1964;77:538–543. - PubMed
    1. Chen W, Kawano H, Men R, Clark D, Lai C-J. Construction of intertypic chimeric dengue viruses exhibiting type 3 antigenicity and neurovirulence for mice. J Virol. 1995;69:5186–5190. - PMC - PubMed
    1. Chu M C, O’Rourke E J, Trent D W. Genetic relatedness among structural protein genes of dengue virus strains. J Gen Virol. 1989;70:1701–1712. - PubMed
    1. Clarke D K, Duarte E A, Moya A, Elena S F, Domingo E, Holland J. Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses. J Virol. 1993;67:222–228. - PMC - PubMed
    1. Craighead J E, Sather G E, Hammon W M, Dammin G J. Pathology of dengue virus infection in mice. Arch Pathol. 1966;81:232–239.

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