Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany1
Groupe des Bunyaviridés, Unité des Arbovirus et Virus des Fièvres Hèmorragiques, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris, France2
Author for correspondence: Michael Frese. Fax +49 761 203 6626. e-mail frese{at}ukl.uni-freiburg.de
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Abstract |
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Introduction |
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The laboratory rat, Rattus norvegicus, has frequently been used as an animal model to study the pathogenesis of RVFV infection. Peters & Anderson (1981 ) and Peters & Slone (1982
) observed dramatic differences in disease manifestation in experimentally infected rats of different inbred strains. They demonstrated that rats, depending on their genetic background, either survive an RVFV infection without any symptoms or die as a result of fulminant hepatitis or encephalitis. For example, WistarFurth rats (WF/mai) die with extensive hepatic necrosis no later than 3 to 5 days after infection with RVFV, whereas Lewis rats (LEW/mai) are highly resistant to fatal hepatic disease, although a significant percentage (about 16%) develop encephalitis later on in the infection (Peters & Slone, 1982
; Anderson et al., 1987
). Genetic analysis revealed that the resistance to RVFV-induced fatal hepatitis observed in LEW/mai rats is inherited by a single Mendelian dominant gene (Peters & Anderson, 1981
), although the resistance gene has not yet been identified. However, interferon (IFN) seems to play a crucial role in the establishment of a resistant phenotype in rats (Rosebrock & Peters, 1982
; Rosebrock et al., 1983
; Anderson & Peters, 1988
). Peritoneal macrophages obtained from RVFV-resistant LEW/NHsdBR rats allow RVFV to replicate efficiently, but stimulation of the cell cultures with IFN type I (
/
) prior to infection inhibited RVFV growth (Rosebrock & Peters, 1982
; Rosebrock et al., 1983
). In contrast, IFN is not able to induce resistance in macrophages of RVFV-susceptible WF/HsdBR rats (Rosebrock & Peters, 1982
; Rosebrock et al., 1983
). Furthermore, injection of anti-IFN type I antibodies into LEW/mai rats led to a dramatically increased sensitivity to RVFV infections (Anderson, 1988
).
Here, we report that inbred rats from a European breeding colony (mol rats) do not respond to RVFV challenges in the same way as their American relatives (mai rats) do. WF/mol rats survived RVFV infections and LEW/mol rats died of acute hepatitis. Cross-breeding of WF/mol and LEW/mol rats revealed that resistance is segregated as a single dominant gene. It is presently not understood exactly how this gene contributes to the RVFV-resistant phenotype of WF/mol rats. However, cell culture experiments using primary hepatocytes suggest that the multiplication of RVFV is impaired in the liver of WF/mol rats. Furthermore, we have found no evidence to support the hypothesis that the resistance gene is regulated by IFN type I.
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Methods |
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Cells.
Primary hepatocyte cultures were established from adult WF/mol and LEW/mol rats as described previously (Schramm et al., 1993 ; Schmider et al., 1996
). Primary cultures of cortical glial cells were established from 1-day-old WF/mol and LEW/mol rats essentially as described (McCarthy & de Vellis, 1978
). Both hepatocytes and glial cells were cultured in Dulbeccos modified Eagles medium containing 200 U/ml penicillin, 200 µg/ml streptomycin and 10% foetal calf serum. Rat glioblastoma C6 cells (CCL-107) and human fibroblast MRC-5 cells (CCL-171) were obtained from the ATCC. African green monkey kidney (Vero) cells have been described previously (Frese et al., 1996
).
Viruses.
The ZH548 strain of RVFV (Meegan, 1979 ) was grown in MRC-5 cells and stock virus contained 5x106 p.f.u./ml as determined in Vero cells. The attenuated MP12 strain of RVFV (Caplen et al., 1985
) was grown in Vero cells and contained 4·4x107 TCID50/ml as determined in Vero cells.
Experimental virus infections.
Adult rats (at least 12 weeks old) of either sex were infected intraperitoneally with 103, 104 or 105 p.f.u. RVFV strain ZH548. After inoculation of the virus, the animals were monitored at least daily for clinical symptoms. Confluent primary hepatocyte cell monolayers or glial cell monolayers were infected with the MP12 strain of RVFV at a multiplicity of 0·1 TCID50 per cell, incubated for 1 h at 37 °C and washed to remove free virus. Fresh medium was added and cells were further incubated at 37 °C. Samples of the supernatant were taken at the times indicated and virus titres were then determined on Vero cells. Titres were calculated as reciprocals of the TCID50.
Interferon treatment.
Primary hepatocyte cell monolayers were either stimulated overnight with 100, 1000 or 5000 IU/ml of Cytimmune rat IFN type I (Lee Biomolecular Research) or left untreated.
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Results |
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Multiplication of RVFV in unstimulated hepatocyte and glial cell cultures
A rapid invasion of the liver is characteristic of RVFV infections in rats (McGavran & Easterday, 1963 ; Peters & Slone, 1982
). The multiplication of RVFV in hepatocytes is responsible for liver necrosis and contributes mainly to the high-titre plasma viraemia (Anderson et al., 1987
; Anderson & Smith, 1987
). Host defence mechanisms that protect the liver against RVFV infection or slow down virus replication in hepatocytes would have a huge impact on the development of the disease. To compare the multiplication of RVFV in hepatocytes from LEW/mol and WF/mol rats, primary hepatocyte cultures were established from both rat strains and infected with RVFV strain MP12 at 0·1 TCID50 per cell. Hepatocytes obtained from susceptible LEW/mol rats produced large amounts of RVFV (Fig. 1A
). About 36 h p.i., virus titres in the cell culture supernatant reached a peak of 2·4x106 TCID50/ml (mean titre of four experiments). At the same time-point, reduced virus titres (1·7x104 TCID50/ml) were detected in supernatants of hepatocytes derived from WF/mol rats. A virus-induced cytopathic effect (CPE) was observed in hepatocytes from both rat strains, but CPE was delayed and less pronounced in hepatocytes from WF/mol rats (data not shown).
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Taken together, the results indicate that WF/mol rats possess an innate resistance mechanism to RVFV that inhibits virus multiplication in hepatocytes but not in glial cells.
Multiplication of RVFV in IFN-treated hepatocyte cultures
Previous reports suggest differences between RVFV-resistant LEW/mai and susceptible WF/mai rats concerning the IFN-induced antiviral response (Rosebrock & Peters, 1982 ; Rosebrock et al., 1983
; Anderson & Peters, 1988
). Therefore, we investigated the effect of IFN type I on the multiplication of RVFV in cultured hepatocytes from LEW/mol and WF/mol rats. Primary hepatocytes were isolated from both rat strains and were either stimulated overnight with 100, 1000 or 5000 IU/ml of rat IFN type I or were left untreated. Subsequently, the cells were challenged with RVFV strain MP12 at an m.o.i. 0·1 and virus titre in the cell culture supernatant was determined 24 h p.i. Hepatocytes from both rat strains responded to the IFN treatment by inhibiting RVFV multiplication in a dose-dependent manner (Fig. 2
). Compared to untreated cells, hepatocytes from LEW/mol and WF/mol rats that were stimulated with 5000 IU/ml of IFN produced only 2% and 1% progeny virus, respectively. These results indicate that hepatocytes from RVFV-resistant WF/mol rats do not exhibit a stronger IFN-induced antiviral response than hepatocytes from susceptible LEW/mol rats. We suggest, therefore, that the resistance gene is not regulated by IFN type I.
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Discussion |
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Other observations, however, seem to indicate that the IFN-induced antiviral response is involved in the resistance of certain rat strains to RVFV infections. These observations are (i) the injection of IFN type I-specific antibodies into LEW/mai rats leads to a dramatically increased sensitivity to RVFV infections (Anderson, 1988 ); (ii) macrophages from RVFV-resistant LEW/NHsdBR rats but not from RVFV-susceptible WF/HsdBR rats exhibit resistance to RVFV after stimulation with IFN type I (Rosebrock & Peters, 1982
; Rosebrock et al., 1983
), suggesting that an IFN-induced antiviral mechanism is responsible for the RVFV-resistant phenotype; (iii) the multiplication of RVFV is inhibited by MxA, a human IFN-induced GTPase (Frese et al., 1996
); and (iv) preliminary data indicates that the rat Mx2 protein also possesses antiviral activity against bunyaviruses, including RVFV (M. Sandrock, M. Frese, G. Kochs and O. Haller, unpublished results). These findings clearly demonstrate that the IFN-induced antiviral defence is indispensable for resistance to RVFV infections. A similar conclusion applies for the mouse model since genetically targeted knockout mice, lacking the
-subunit of the IFN-
/
receptor, are highly susceptible to attenuated strains of RVFV that do not normally kill mice (Bouloy et al., 1999
). Although the genetic resistance against RVFV described here seems not to rely on an IFN response, IFN is certainly important for the outcome of the disease in both RVFV-susceptible rats and RVFV-resistant rats. Spontaneously transformed embryonic thymus cells derived from both LEW/mai and WF/mai rats, for example, are able to inhibit the formation of RVFV-plaques after exposure to IFN type I (Anderson & Peters, 1988
). Furthermore, the treatment of susceptible WF/mai rats with IFN improves the chances of surviving an RVFV infection (Anderson, 1988
). Since RVFV was inhibited in hepatocytes from both resistant WF/mol and susceptible LEW/mol rats following IFN treatment, we support the view that at least a part of the IFN-induced antiviral defence against RVFV also operates in susceptible rats. Furthermore, the present data provide no evidence for an IFN-regulated expression of the RVFV resistance gene in WF/mol rats.
RVFV is able to cross the bloodbrain barrier and infect neurons and glial cells. In humans, RVF is sometimes associated with retinitis and meningoencephalitis (Laughlin et al., 1979 ; Siam et al., 1980
). In rats infected via peripheral routes, RVFV may enter the central nervous system and cause encephalitis, even in genetically resistant animals that do not develop hepatitis (Peters & Slone, 1982
; Anderson et al., 1987
). Moreover, intracerebral inoculation kills resistant as well as susceptible animals as a result of an acute encephalitis. Here, we show that rat C6 glioblastoma cells and primary glial cells from both WF/mol and LEW/mol rats were fully permissive to RVFV. These data may explain why the central nervous system of resistant rats is susceptible to RVFV infection.
Finally, a word of caution to researchers using rats as animal models seems appropriate. In an attempt to characterize a gene locus that confers resistance to RVFV, we discovered that two inbred strains did not exhibit the resistant phenotype described previously in the literature. It had been reported that LEW/mai but not WF/mai rats are resistant to RVFV-induced hepatitis (Peters & Slone, 1982 ; Anderson et al., 1987
). The present data show, however, that LEW and WF rats obtained from the Danish breeder M&B behaved differently, i.e. LEW/mol rats were highly susceptible to RVFV infections whereas WF/mol rats were resistant to RVFV infections (Table 2
). The ZH501 strain of RVFV (Meegan, 1979
) was used in previous challenge experiments with LEW/mai and WF/mai rats, whereas we used strain ZH548 to infect LEW/mol and WF/mol rats. Therefore, it might be argued that the observed differences in host resistance may be attributed to specific pathogenic properties of the two virus strains rather than to genetic differences of the rat strains used. This is very unlikely as no substantial differences were found between RVFV strain ZH501 and strain ZH548 concerning (i) virulence for susceptible WF/mai rats; (ii) ability to form plaques in hepatocytes from resistant LEW/mai rats or susceptible WF/mai rats; and (iii) sensitivity to IFN in cell culture (Anderson & Peters, 1988
). Thus, rats that seemingly represent the same inbred rat strain may show quite opposite phenotypes upon RVFV infection, depending on the breeding stocks from which they are derived. The observation that LEW/NIco and WF/Ico rats from the French breeder IFFA CREDO (LArbresle, France) are both resistant to RVFV infections (M. Bouloy, unpublished results) further complicates the picture and indicates a high degree of genetic variability among the inbred rat strains LEW and WF. The results are in line with a previous genetic characterization of 156 rat substrains using 39 markers (Bender et al., 1994
). Interestingly, Bender and colleagues reported that 12 out of 13 LEW substrains have identical markers but LEW/mol rats differ from the other LEW rats by several gene loci. Furthermore, extensive substrain differences were found among WF rat markers (Bender et al., 1994
). Therefore, a revision of the nomenclature and a better genetic characterization of rat breeding stocks is needed in order to improve the standardization of animal experiments in the future.
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Acknowledgments |
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References |
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Received 26 April 2000;
accepted 2 August 2000.
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