Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium1
Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany2
Division of Histopathology, University Hospitals, B-3000 Leuven, Belgium3
Author for correspondence: Johan Neyts. Fax +32 16 33 73 40. e-mail johan.neyts{at}rega.kuleuven.ac.be
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Abstract |
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Introduction |
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We report here a convenient model for studying flavivirus encephalitis, and the therapy thereof, using the Montana Myotis leukoencephalitis virus (MMLV) (BSL-2 according to the ACAV; SALS). MMLV was first isolated in 1958 from a mouse bitten under laboratory conditions by a naturally infected little brown bat (Myotis lucifugus) captured in western Montana. The virus was subsequently isolated from saliva, brain and various other tissues from other bats of the same species. Serological studies suggested that the virus belonged to the flaviviruses (Bell & Thomas, 1964 ). However, the virus was subsequently forgotten by the scientific community.
The genus Flavivirus contains both viruses that are transmitted by mosquitoes or ticks (arboviruses) and viruses with no known vector (NKV) (Chambers et al., 1990 ). In the accompanying manuscript we have described the complete genomic sequence of MMLV. Phylogenetic analysis has confirmed the classification of MMLV in the cluster of the NKV flaviviruses, as was suggested previously by Kuno et al. (1998)
based on the sequence of a fragment of the NS5 gene of approximately 1 kb. We have also demonstrated that: (i) MMLV has the same genomic organization as other flaviviruses, and (ii) in the genes that encode the serine protease/helicase and RNA-dependent RNA polymerase (which are important targets for antiviral therapy), the virus contains the same conserved motifs for enzymatic activity as those flaviviruses that are pathogenic to humans. The animal model presented here may be attractive for the study of antiviral strategies against flavivirus infections, in particular flavivirus encephalitis.
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Methods |
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Animals.
Eight- to twelve-week-old SCID mice and immunocompetent NMRI (Naval Medical Research Institute) mice weighing 1620 g were used for all experiments. All animals were bred at the Rega Institute under specific pathogen-free conditions. Experiments on animals have been approved by and were in accordance with the guidelines of the Ethical Committee on Vertebrate Animal Experiments of the Katholieke Universiteit Leuven.
Compounds.
Mycophenolic acid (MPA) was purchased from Sigma and ribavirin [1-(-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide] was from ICN. EICAR (5-ethynyl-1-
-D-ribofuranosylimidazole-4-carboxamide) was synthesized by Dr A. Matsuda (Hokkaido University, Sapporo, Japan). Tiazofurin and selenazofurin were provided by Dr R. Cooney and Dr D. G. Johns (National Cancer Institute, NIH, Bethesda, MD). Polyinosinicpolycytidylic acid [poly(I)·poly(C)] was purchased from SigmaAldrich.
Plaque reduction assay.
Serial dilutions of the test compounds were added to confluent Vero cell cultures grown in 96-well microtitre plates. Cells were infected with approximately 50 p.f.u. of either YFV, dengue fever virus (DENV), or MMLV. Cultures were further incubated at 37 °C for 7 days, after which they were fixed with 70% ethanol and stained with 2% Giemsa solution. Plaques were then counted under an inverted microscope. The antiviral activity of the compounds was expressed as the 50% effective concentration (EC50), i.e. the concentration required to reduce plaque formation by 50%.
Titration for infectious virus content.
MMLV-infected mice were sacrified at various days after infection by ether anaesthesia. Brains were dissected aseptically, and 10% (w/v) tissue homogenates were prepared in MEM supplemented with 2% FCS. Confluent Vero cell cultures grown in 96-well plates were infected with 10-fold serial dilutions of the tissue homogenates and incubated at 37 °C for 1 h, after which the inoculum was removed. Cultures were then washed twice with warm medium and further incubated at 37 °C for 79 days, after which the cultures were evaluated for virus-induced CPE. The titre was expressed as the 50% cell culture infective dose (CCID50), i.e. the infectious dose required to infect 50% of the cells.
RTPCR.
Viral RNA was extracted from 140 µl of either the cell culture supernatant, or the serum, lymphocytes or macrophages of infected animals using the QIAamp Viral RNA kit (Qiagen). For the isolation of RNA from cell cultures, cell debris was first spun down with two consecutive centrifugation steps at 1500 g in a Minifuge-T. For the isolation of RNA from tissue samples, the RNeasy Mini kit (Qiagen) was used. Prior to reverse transcription (RT), 31·5 µl purified viral RNA was mixed with 10 µl 5x RT buffer (Amersham Pharmacia Biotech), 0·5 mM each of dATP, dTTP, dGTP and dCTP, and 1·2 µM of reverse primer (5' GGGAGGCTGAGTAAGCATACACAAGC 3', nt 38343859) and denaturated at 92 °C for 1 min followed by chilling on ice. RT reaction mixtures contained this denatured RNA plus 95 U human placenta RNase inhibitor (HPRI; Amersham Pharmacia Biotech), 2·4 U RAV-2 reverse transcriptase (Amersham Pharmacia Biotech) and RNase-free water added to give a final volume of 50 µl. The reaction mixture was incubated at 45 °C for 1·5 h. The PCR reaction mixture was prepared as follows: 5 µl cDNA, 5 µl 10x PCR buffer, 2 µl dNTP mix (100 µM of each dNTP), 1·2 µM forward primer (5' CGAGGTCAAAAACCCAAGGAGG 3'), 1·2 µM reverse primer (5' CCACAGCATGGGCTCAGATGTT 3') and 3·5 U Super Taq (HT Biotechnology). The following PCR programme was run: 10 min at 95 °C, 30 cycles of 30 s at 94 °C, 30 s at 60 °C, 1 min at 72 °C and, for the final extension, 10 min at 72 °C. The primers were specific MMLV primers that anneal in the NS1NS2A genes (forward primer at nt 32813302 and reverse primer at nt 36393660).
Semi-quantitative RTPCR.
A calculated amount of 5 ng of total RNA isolated from tissue fragments was used in a 50 µl semi-quantitive RTPCR assay using the OneStep RTPCR kit (Qiagen). GAPDH (glyceraldehyde phosphate dehydrogenase) served as an internal control and a primer ratio MMLV/GAPDH of 2/1 was used. Amplification primers were GAPDH forward primer: 5' GGTGAAGGTCGGTGTGAACG 3'; GAPDH reverse primer: 5' ATGTTCTGGACAACCCCACG 3'; MMLV forward primer: 5' GCACGAAGCTGCTGAGAGTGCC 3'; MMLV reverse primer: 5' TGAAAGCTGTGTAGGCGCTCCA 3'. After amplification (annealing at 60 °C, 30 cycles) and separation of the amplicons (GAPDH 609 bp, MMLV 978 bp) by agarose gel electrophoresis, the gel was scanned and the two bands were quantified using the ImageMaster software (Hoeffer Pharmacia Biotech).
Quantification of MMLV RNA by 5' nuclease real-time RTPCR.
RNA preparation was performed using the QIAamp Viral RNA kit (Qiagen), according to the manufacturer's instructions. For elution of RNA, the columns were incubated with 50 µl of RNase-free water at 80 °C. Primers MMLVS2 (5' TCCGTAGGAAGCGTTGGTGT 3') and MMLVAs1 (5' ATTCCATTACTTCTGCCACTTCGA 3') were used to amplify a 170 bp segment in the 5' non-coding/core region of MMLV. For detection of PCR products, probe MMP (5' AAGAGCGACGTGGTTTCCAGCC 3') was used. It was labelled with FAM (6-carboxy-fluorescein) at the 5' end and TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at the 3' end. The probe was phosphorylated at its 3' end to prevent elongation during PCR. RTPCR was carried out using the Superscript One-Step RTPCR System with Platinum Taq (Life Technology). A 20 µl reaction volume contained 10 µl of 2x reaction buffer, 2·5 mM additional magnesium sulfate, 300 nM each of primers MMLVS2 and MMLVAs1, 200 nM of probe MMP, 0·8 µg BSA (SigmaAldrich) and 0·4 µl of Superscript Reverse Transcriptase/Platinum Taq enzyme mix. Prepared RNA (2 µl) was added to each reaction. Thermal cycling in a LightCycler (Roche Molecular Biochemicals) involved reverse transcription at 45 °C for 20 min, denaturation at 95 °C for 5 min, followed by 45 cycles of 95 °C for 5 s and 57 °C for 35 s. (Wittwer et al., 1997 ).
During PCR, the probe MMP was digested by the 5' exonuclease activity of Taq DNA polymerase when specifically annealed to the generated MMLV PCR product (Holland et al., 1991 ). Probe digestion liberated the FAM from the TAMRA dye, causing an increase in FAM-specific fluorescence during PCR (Livak et al., 1995
). FAM fluorescence was measured on detection channel F1 (530 nm) and divided by fluorescence measured on channel F2 (640 nm) for normalization. As a quantification standard for real-time PCR, a strong positive MMLV stock was diluted in virus-negative human plasma prior to RNA preparation. One PCR unit (PCRU) of MMLV RNA was defined as the lowest possible dilution that could be amplified in five out of five replicate reactions. The quantification procedure using the LightCycler has been previously described (De Silva et al., 1998
).
Animal experiments.
NMRI mice or SCID mice were inoculated with 104 p.f.u. of MMLV via either the intraperitoneal (i.p.; 200 µl), intracerebral (i.c.; 50 µl) or intranasal (i.n.; 20 µl) route. The animals were examined daily for signs of morbidity and mortality. The statistical significance of differences in the mean day of death was assessed by means of the Students t test and differences in the number of survivals by means of the 2 test with Yates correction. To study the progression of MMLV infection in NMRI mice and SCID mice, the animals were infected intraperitoneally with MMLV and two to four mice were sacrified every 3 days. Blood samples were taken and total RNA was extracted from the serum and subsequently used for RTPCR. Brain, salivary glands, lung, liver, kidney, pancreas, seminal vesicles and spleen were removed and subjected to immunostaining.
In some experiments, half of the infected animals were implanted on day 0 or on day 14 following infection with a mini-osmotic pump (Alzet Model 2002, Alza Corporation, California, USA) filled with ribavirin at a concentration of 83·3 µg/µl, resulting in a continuous subcutaneous calculated release of 50 mg/kg/day of ribavirin. Mice were monitored for 4 weeks after virus challenge.
Electron microscopy.
Vero cells that had been infected with MMLV and that exhibited an extensive CPE were fixed in a buffered glutaraldehyde solution for 30 min, washed with isotonic phosphate buffer, post-fixed with 1% OsO4 solution for 1 h, collected, dehydrated and embedded in Dow epoxy resin for electron microscopy. Sections (1 µm) stained with toluidine blue were examined with a light microscope. Sample sections (50 nm) of areas of interest were collected on grids, stained with uranyl acetate and lead citrate, and examined with a Philips CM10 electron microscope.
Histopathology.
Moribund animals with severe signs of paralysis were euthanized with ether anaesthesia and were transcardially perfused with 20 ml of a buffered 4% formaldehyde solution for histology. Fixed tissue samples were embedded in paraffin and further processed using standard methods.
Preparation of digoxigenin-labelled cRNA.
MMLV cDNA encompassing 655 nucleotides of the NS5 region of the MMLV genome (forward primer 5' TCATCCAGAGAAGAATTG 3'; reverse primer 5' TTCAAAAAGCCAAAGCTTTCAAATTC 3') was cloned into the transcription vector pGEM-T (Promega). To generate runoff transcripts, the plasmid was linearized with NotI (Promega) for 1·5 h at 37 °C. The linearized plasmid DNA was precipitated with 3 M sodium acetate (pH 4·6) and 2·5 vols ethanol. After centrifugation, the pellet was washed in 70% ethanol and dissolved in RNase-free water at a concentration of approximately 100 ng/µl.
Transcription reactions (20 µl) consisted of 2 µl water, 10 µl linearized cDNA (1 µg), 4 µl 5x transcription buffer (Promega), 2 µl 10x Dig Labelling Mix (Roche Diagnostics) and 2 µl T7 RNA polymerase (Promega). The reaction was incubated for 2 h at 37 °C. The cRNA derived by T7 polymerase in vitro transcription was isolated on a Mini Quick Spin RNA column (Boehringer Mannheim) and the eluate was frozen at -80 °C until use.
In situ hybridization.
Paraffin-embedded tissue was sectioned, hydrated, fixed, denaturated and acetylated according to standard procedures (Breitschopf et al., 1992 ). The sections were permeabilized by digestion with proteinase K (100 µg/ml) (Boehringer Mannheim) for 20 min. Prehybridization was at 60 °C for 30 min. Hybridization solution was prepared by adding 200 ng of digoxigenin (DIG)-labelled cRNA per ml of hybridization buffer (EasyHyb, Roche Diagnostics). The hybridization solution (150 µl/section) was added, the section was heated for 4 min at 95 °C to denature the probe and hybridization was allowed to occur at 60 °C for 46 h in a humidified chamber. The sections were incubated in 2x SSC (0·15 M NaCl, 0·015 M sodium citrate) for 12 h and washed with 1x SSC. On completion of the hybridization step, the sections were incubated in buffer solution containing 0·5% (w/v) blocking reagent (Boehringer Mannheim) at room temperature for 15 min, followed by incubation with alkaline phosphatase-conjugated anti-DIG polyclonal antiserum (1:500 in blocking buffer) for 1 h and incubated overnight with the NBT/BICP reagent (Roche Molecular Biochemicals).
Immunohistochemistry.
Primary antiserum was produced in New Zealand rabbits after immunization with a suspension of UV-inactivated MMLV and complete Freund's adjuvant. Tissue sections were rehydrated and blocked with 2% skimmed milk. The sections were then incubated with 200 µl of primary rabbit antiserum (dilution 1:100) for 1 h at 37 °C. The sections were rinsed in PBS and incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody diluted 1:500 in PBS for 30 min at 37 °C. After rinsing, endogenous peroxidase activity was quenched by incubation overnight in 70 ml buffer (0·1 M acetate and 0·15 ml of a 3% H2O2 solution) containing 20 mg AEC (3-amino-9-ethyl-carbazole) (SigmaAldrich) in 5 ml N,N-dimethylformamide.
To monitor MMLV infection in the brain, sections were stained with haematoxylin and eosin (H&E) according to standard procedures.
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Results |
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Discussion |
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We have presented a model for the study of antiviral strategies against flavivirus encephalitis employing the Montana Myotis leukoencephalitis virus. MMLV is highly pathogenic to mice and has been classified as a biosafety level II pathogen by SALS (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s71.htm). As we report in the accompanying paper (Charlier et al., 2002 ), MMLV has the same overall genome organization as flaviviruses of clinical importance, and in those genes that are considered to be interesting antiviral targets (i.e. the NS3 gene, which encodes an NTPase/helicase and serine protease, and the NS5 gene encoding an RNA-dependent RNA polymerase), it has the same conserved motifs as flaviviruses that are infectious to humans.
MMLV can be readily propagated in cell culture and the virus has a susceptibility to a selection of antiviral drugs, including ribavirin and its 5-ethynyl analogue, EICAR (Leyssen et al., 2000 ), that is comparable with that of YFV and DENV, which further points to the relevance of MMLV for the study of antiviral strategies against flaviviruses.
MMLV is neuroinvasive in SCID mice, but not in immunocompetent mice, in which it is only neurovirulent. In SCID mice infected via the peripheral route with MMLV, the virus is detected in the brain and serum; no viral antigens or viral RNA are detectable in solid organs (by means of immunohistochemistry or in situ hybridization) nor in macrophages or other blood-borne cells (by means of RTPCR) (data not shown). Virus titres in the serum of MMLV-infected SCID mice start to rise well before the virus is detected in the brain. It may be assumed that this viraemia is the result of low-level virus replication in peripheral organs, a level that is, however, too low to be detected by in situ hybridization or immunohistochemistry. Virus replication in these organs was not detectable by means of RTPCR or titration for infectious virus content, since, despite extensive perfusion of the animals, traces of blood resulted in false positive signals for all organs. M. Halevy and collegues concluded that WNV replicates in as yet unidentified target tissues following peripheral inoculation of SCID mice with the virus (Halevy et al., 1994 ). Modoc virus (MODV), a flavivirus isolated from the white-footed deer mouse (Johnson, 1967
), is, like MMLV, neuroinvasive in SCID mice, but, unlike MMLV, replicates in peripheral organs such as the salivary glands and in the spleen (Leyssen et al., 2001
). In a recent study, a neuroadapted strain of YFV, 17D, derived from a multiple mouse brain-passaged virus, proved neuroinvasive in SCID mice. Unlike MMLV, this virus exhibited an efficient growth in peripheral tissues of SCID mice (Chambers & Nickells, 2001
). In addition to isolation from the brain, JEV was also isolated from the liver and spleen of newborn mice infected via transplacental transmission of intraperitoneally infected pregnant mice (Mathur et al., 1981
). It remains largely unclear which determinants influence the organ tropism of flaviviruses in peripheral organs. As is the case for the murine MMLV and WNV models, in patients with WNV encephalitis, virus was not detected in major organs such as lung, liver, spleen and kidney (Nash et al., 2001
; Sampson et al., 2000
; Shieh et al., 2000
).
It has been demonstrated for YFV, WNV, JEV, MVEV, TBEV and LIV that specific amino acid substitutions within the E protein are associated with loss of neuroinvasiveness in mice (Cecilia & Gould, 1991 ; Hasegawa et al., 1992
; Sumiyoshi et al., 1995
; McMinn et al., 1995a
, b
; Holzmann et al., 1990
; Jiang et al., 1993
; Chambers et al., 1998
; Chambers & Nickells, 2001
). Obviously the E protein plays a prominent role in neuropathogenesis of flaviviruses and the particular characteristics of the E protein of MMLV may thus determine the tropism of this bat virus.
MMLV-induced encephalitis in SCID mice is characterized by the presence of viral RNA in virtually all regions of the brain and infection is clearly confined to neurons. We did not detect other cells of the CNS that were infected with MMLV. In the brain of mice infected with JEV, major cytopathological changes were observed in neurons and developing neurons (Ogata et al., 1991 ; Hase, 1993
; Hase et al., 1993
; Wang et al., 1998
). Also, in rodent infection models with viruses such as MVEV, YFV and WNV, virus replication in the brain was mainly confined to neurons (Matthews et al., 2000
; Schlesinger et al., 1996
; Weiner et al., 1970
; Eldadah & Nathanson, 1967
). Presence of virus in all regions of the brain has also been observed in patients with Japanese encephalitis (Johnson et al., 1985
). In patients with West Nile (meningo)encephalitis, virus was detectable in neurons throughout the brain (Nash et al., 2001
; Nichter et al., 2000
; Sampson et al., 2000
; Shieh et al., 2000
) and, as seen in the MMLV model, also in the medulla oblongata. TBEV was detected in the thalamus, substantia nigra and cerebellum of the brain of patients with tick-borne encephalitis (Mazlo & Szanto, 1978
).
In the CNS of MMLV-infected SCID mice, no or little cell infiltration was observed. Since SCID mice do not carry (functional) B and T cells, infiltration of the brain by lymphocytes is not possible. Yet, SCID mice do harbour functional macrophages as well as natural killer (NK) cells; however, no or little infiltration of these cells in the brain of infected animals was observed. In WNV-infected mice, two populations of inflammatory cells were detected in the central nervous system: NK cells and cytotoxic T cells (Liu et al., 1989 ). In normal mice, JEV infection caused prominent inflammatory changes with leukocytic infiltration and perivascular cuffing (Hase et al., 1990
). The fact that no or little inflammation was observed in the brain of MMLV-infected SCID mice points to the fact that direct viral damage is the main (or sole) reason for brain dysfunction and virus-induced mortality. This may make the MMLV model particularly attractive for the study of antiviral strategies against flaviviruses. Indeed, a protective effect in the MMLV model will probably be solely attributable to a direct inhibitory effect of the compound on virus replication in the brain. If such a molecule can prove sufficiently protective in the MMLV model, it may be interesting to study its effect in animal (including monkey) models with more pathogenic flaviviruses, in which an inflammatory response is also involved in the pathology of the disease.
To validate the MMLV model for future use in antiviral drug studies, we sought to prove that protection against MMLV disease progression correlates with an inhibitory effect on virus replication. Treatment with ribavirin had a weak inhibitory effect on the replication of MMLV in vitro (as for YFV and DENV) and did not protect mice from MMLV-induced morbidity and mortality, nor did it cause a reduction in viral RNA in the brain. This is in line with the finding that ribavirin does not result in any protective effect in rhesus monkeys infected with dengue virus type 1 (Malinoski et al., 1990 ). Ribavirin has also never been shown to be protective against flaviviruses in man. Ribavirin, therefore, did not prove useful to validate the MMLV model for antiviral drug studies. For this reason, we made use of the interferon-
/
inducer poly(I)·poly(C). This molecule has previously been shown to elicit a protective effect against JEV and MODV infection in mice and/or monkeys (Leyssen et al., 2001
; Harrington et al., 1977
; Singh & Postic, 1970
; Worthington et al., 1973
). The interferon inducer delayed MMLV-induced mortality and reduced virus titres, which further demonstrates that the MMLV model may be useful in both prophylactic and therapeutic studies.
In conclusion, the MMLV mouse model has several clinical, histopathological and virological features reminiscent of flavivirus infections, in particular flavivirus encephalitis, in humans. Therefore, the MMLV model may be valuable for the study of antiviral strategies against infections with flaviviruses, in particular those causing encephalitis.
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Acknowledgments |
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References |
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Received 14 December 2001;
accepted 10 March 2002.