Division of Immunology and Cell Biology, John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, ACT 2601, Australia
Correspondence
Mario Lobigs
Mario.Lobigs{at}anu.edu.au
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ABSTRACT |
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MAIN TEXT |
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The earliest host responses to viral infections are non-specific and involve among other cytokines the induction of interferons (IFNs) (Biron & Sen, 2001; Goodbourn et al., 2000
). Type I IFNs, IFN-
/
, are produced by leukocytes and fibroblasts, respectively, in response to infection and activate the transcription of IFN-inducible genes which leads to the induction of antiviral pathways within hours. IFN-
is made exclusively by natural killer and T cells and has important immunoregulatory functions (Boehm et al., 1997
). Most of the antiviral activity of IFN-
is mediated by nitric oxide (NO) synthesized by monocytes, following induction of the enzyme NO synthase by the cytokine (Guidotti & Chisari, 2000
; MacMicking et al., 1997
; Reiss & Komatsu, 1998
). The contribution of IFN-
/
in recovery from infection with encephalitic flaviviruses has been shown in vivo by the therapeutic and prophylactic effects of administration of IFN-inducers (Haahr, 1971
; Taylor et al., 1980
; Vargin et al., 1977
) or IFN (Brooks & Phillpotts, 1999
; Pinto et al., 1988
). On the other hand, it remains unresolved whether IFN-
, for instance via induction of NO, plays an exacerbating or protective role in encephalitic flavivirus infection. Whilst an immunopathological contribution of NO was noted in experimental infections with MVE and tick-borne encephalitis virus (Andrews et al., 1999
; Kreil & Eibl, 1996
), others have found increased mortality of JEV-infected mice when NO synthase was inhibited (Lin et al., 1997
; Saxena et al., 2000
, 2001
). Here we have employed mice deficient in type I or type II IFN responses as a consequence of gene knockout in a model of flavivirus encephalitis to investigate the role of IFNs and NO in pathogenesis and recovery from infection with MVE.
Infection of 6-week-old B6 mice with 0·1 to 105 p.f.u. of MVE (prototype strain MVE-1-51) intravenously (i.v.) gives mortality in up to 50 % of animals, despite low or undetectable virus growth in extraneural tissues (Licon Luna et al., 2002). A lack of the IFN-
receptor dramatically increased the susceptibility of mice to MVE (Fig. 1
A). Infection with 102 p.f.u. i.v. of IFN-
receptor knockout mice (IFN-
-R-/-; B6 congeneic),which are completely unresponsive to type I IFNs (Müller et al., 1994
), was lethal in 100 % of animals. The average time to death (ATD) of these mice (5·6 days) was significantly shorter (P=0·004; MannWhitney test) relative to control B6 mice (13·2 days) infected with the same dose of MVE. In contrast, mice deficient in IFN-
production (IFN-
-/-; B6 congeneic) (Dalton et al., 1993
) showed only a slight increase in mortality relative to the control group (P=0·045, Fisher's exact test; Fig. 1A
) after infection i.v. with 102 p.f.u. of MVE. Mortality in a group of 28 infected IFN-
-/- mice was 60 % in comparison to 30 % mortality in a group of B6 mice infected in parallel. The ATD (11·6 and 13·2 days, respectively) was not significantly different between the two groups (P=0·14).
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To analyse the role of IFNs in the control of virus growth in extraneural tissues and in turn, the rate of neuroinvasion, 6-week-old B6, IFN--/- and IFN-
-R-/- mice were infected i.v. with 102 p.f.u. of MVE and serum and tissues collected at the times p.i. shown in Fig. 2
. Spleen and brain tissue was processed as described (Licon Luna et al., 2002
); blood was taken from the tail vein of animals under anaesthesia or by heart puncture of animals that were sacrificed, allowed to clot at room temperature for 10 min, kept on ice for 20 min, centrifuged at 2000 r.p.m. for 10 min in an Eppendorf microfuge, and serum collected for storage at -70 °C. Virus titres were determined by plaque formation on Vero cells (Licon Luna et al., 2002
). Infection of B6 wild-type (wt) mice gave no detectable viraemia at 2, 4, 6, 8 and 9 days p.i.; only one mouse showed a low virus titre in serum (3·5x102 p.f.u. ml-1) at 24 h p.i. No virus was detected in all but one (2x103 p.f.u. g-1 on day 4 p.i.) of the spleen samples harvested between days 2 to 9 p.i. In the absence of IFN-
, virus growth in extraneural tissues was also poor. However, the observation that all spleen samples (n=5) taken at 4 days p.i. from IFN-
-/- mice had detectable virus (103 to 104 p.f.u. g-1) provides evidence for an increased viral load in the knockout relative to wt mice. It should be noted that an inhibitory effect of spleen homogenates on growth of JEV serotype flaviviruses at low dilutions (
0·5 % homogenate) results in an underestimation of the true viral load by plaque assay (Lee & Lobigs, 2002
). This is relevant for MVE titres in the spleen in the range of 103 to 104 p.f.u. g-1, which may be underestimated by up to 10-fold. No such inhibitory effect is apparent for virus detection in serum.
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In 6-week-old mice deficient in IFN-/
responses, injection of 102 p.f.u. of MVE i.v. resulted in a fulminant infection. At 2 days p.i. all animals were viraemic (mean titre=8x103 p.f.u. ml-1) and had virus in spleen (mean titre=8x104 p.f.u. g-1) but not brain. The viral load in the spleen increased massively in the following 2 days of infection (mean titre on day 4 p.i.=7·5x108 p.f.u. g-1) and exceeded viral titres in serum 1000-fold, suggesting that MVE grows efficiently in the spleen of IFN-
-R-/- mice. Virus could be recovered from the brain at 4 days p.i. with viral titres ranging from 5x103 to 2x108 p.f.u. g-1. Given that in each mouse the viral load in the brain was equal or greater than that in the serum, it is unlikely that virus detection in brain samples was due to viraemic blood contamination.
To evaluate the contribution of IFN- and NO to inflammation and pathology in the brains of 6-week-old mice infected with MVE (102 p.f.u., i.v.), brain tissue from mice sacrificed at 8 and 9 days p.i. was histologically examined (Fig. 3
and Table 1
). Virus titres in the brain were determined in parallel. Detection of virus by plaque assay in brain samples was mostly indicative of the presence of inflammatory infiltrates and necrotic cells in the brain parenchyma and leptomeninges on days 8 and 9 p.i. in B6 wt, IFN-
-/- and i-NOS-/- mice. However, the severity of inflammatory and histopathological manifestations, which were more prominent at 9 days p.i., showed no correlation with the magnitude of viral titres and was not markedly different between the mouse strains. Flavivirus-immune CD8+ T cells, which are found in the brains of infected mice as early as 5 days p.i. (Liu et al., 1989
), secrete IFN-
upon in vitro stimulation with viral antigens (Liu & Chambers, 2001
; Regner et al., 2001
), and IFN-
is produced in the CNS of infected mice (Liu & Chambers, 2001
), suggesting that the stimulation of the cytokine is not down-regulated in flaviviral infections.
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Received 13 June 2002;
accepted 29 October 2002.