Innate resistance to flavivirus infection in mice controlled by Flv is nitric oxide-independent

Ondine J. Silvia1, Geoffrey R. Shellam1 and Nadezda Urosevic1

Department of Microbiology, University of Western Australia, Nedlands, WA 6907, Australia1

Author for correspondence: Nadezda Urosevic. Fax +61 8 9346 2912. e-mail nadia{at}cyllene.uwa.edu.au


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Innate resistance to flaviviruses in mice is active in the brain where it restricts virus replication. This resistance is controlled by a single genetic locus, Flv, located on mouse chromosome 5 near the locus encoding the neuronal form of nitric oxide synthase (Nos1). Since nitric oxide (NO) has been implicated in antiviral activity, its involvement in natural resistance to flaviviruses has been hypothesized. Here we present data on NO production before and during flavivirus infection in both brain tissue and peritoneal macrophages from two flavivirus-resistant (Flvr) and one congenic susceptible (Flvs) mouse strains. This study provides evidence that NO is not involved in the expression of flavivirus resistance controlled by Flv since: (a) there is no difference in brain tissue NO levels between susceptible and resistant mice, and (b) lipopolysaccharide-induced NO does not abrogate the difference in flavivirus replication in peritoneal macrophages from susceptible and resistant mice.


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Flaviviruses are important human pathogens causing a variety of diseases ranging from mild febrile illnesses to severe encephalitis and haemorrhagic fever. In laboratory mice these viruses are neurotropic and this provides a suitable animal model to study the pathophysiology of virus-induced encephalitis (Theiler, 1930 ). Furthermore, several laboratory mouse strains have been reported to possess natural resistance to flaviviruses, providing excellent models to study the mechanism of innate host resistance to flaviviruses (Sabin, 1952 ; Groschel & Koprowski, 1965 ; Brinton, 1983 ; Sangster et al., 1993 ; Urosevic et al., 1997 ; Shellam et al., 1998 ). A single genetic locus, flavivirus resistance (Flv), has been shown to confer flavivirus resistance to mice (Sabin, 1952 ; Green, 1989 ). We have mapped this locus to mouse chromosome 5 (Sangster et al., 1994 ; Urosevic et al., 1995 ), to a distal region encompassing a number of genetic loci including Nos1 (Lee et al., 1995 ). The Nos1 locus is known to encode a neuronal form of nitric oxide synthase (NOS) (Lee et al., 1995 ), an enzyme involved in the production of the potentially antiviral compound nitric oxide (NO).

Three different types of nitric oxide synthase have been identified: neuronal (NOS1), macrophage (NOS2) and endothelial (NOS3) (Nathan, 1992 ). Of these, the genes encoding NOS1 and NOS3 map to distal and proximal regions of mouse chromosome 5, respectively (Gregg et al., 1995 ; Lee et al., 1995 ), and are constitutively expressed in the mouse brain and in endothelial cells, respectively (Bredt et al., 1991 ; Lowenstein et al., 1992 ). While both NOS1 and NOS3 enzymes are Ca2+ and calmodulin regulated, the third enzyme, NOS2, which is located on chromosome 11 and expressed in macrophages, is inducible and Ca2+/calmodulin independent (Bredt et al., 1991 ; Xie et al., 1992 ; Gerling et al., 1994 ). All three enzymes, although differently regulated, catalyse the conversion of arginine to citrulline with NO as a major product. NO is an active molecule and a potent effector implicated in a variety of biological functions ranging from peripheral vasodilatation and platelet aggregation to neuromodulation and neurotransmission in the CNS (Nathan, 1992 ). Furthermore, there is recent evidence suggesting that NO exerts antiviral activity both in vitro and in vivo, possibly contributing to innate immune responses to various viruses including flaviviruses (Karupiah et al., 1993 ; Lin et al., 1997 ; Reiss & Komatsu, 1998 ). NO exhibits a number of properties which could be advantageous to its innate antiviral activity. Among these are its ability to penetrate cells and spread easily between neighbouring cells, the independence of its action from the acquired immune responses and the inability of viruses to develop resistance to such a small compound (Nathan, 1992 ; Karupiah et al., 1993 ).

To determine if NO is involved in flavivirus resistance controlled by Flv, we have monitored the brain tissue levels of NO before and after flavivirus infection in flavivirus-susceptible C3H/HeJARC (Flvs) mice and in two congenic, flavivirus-resistant mouse strains, C3H.PRI-Flvr and C3H.M.domesticus-Flvr-like. Inbred C3H/HeJARC mice are a lipopolysaccharide (LPS)-responsive subline of the LPS-unresponsive C3H/HeJ mouse strain, which has been maintained as a separate colony in Western Australia for more than 28 years (Silvia & Urosevic, 1999 ). These mice were used as an acceptor strain for the flavivirus resistance Flvr-like allele of wild Mus domesticus during the creation of the flavivirus-resistant mouse strain C3H.M.domesticus-Flvr-like (Urosevic et al., 1999 ). The other resistant mouse strain, C3H.PRI-Flvr, was developed by backcross breeding of resistant PRI mice to mice of C3H/He background more than 30 years ago (Groschel & Koprowski, 1965 ). These two resistant mouse strains are congenic to C3H/HeJARC mice, and they carry chromosomal segments encompassing Flv which are of different genetic origins (Urosevic et al., 1999 ). In addition to Flv, these polymorphic chromosomal segments also encompass Nos1, while Nos2 and Nos3 are located within the non-polymorphic chromosomal segments which are of the same genetic origin (The Mouse Genome Database, The Jackson Laboratory, Maine, USA. Website: www.informatics.jax.org; visited March 2000; Flaherty, 1981 ). While there is evidence suggesting the existence of different Flv alleles in these three congenic mouse strains (Sangster et al., 1993 , 1998 ; Urosevic et al., 1999 ), it is not known whether similar allelism at the Nos1 locus may exist, conferring various levels of basal NO synthesis in the brains of these mice. Furthermore, it is not known whether Flv itself exerts any influence upon NO synthesis controlled by the inducible Nos2 gene.

In order to answer these questions we initially monitored the levels of NO synthesis in the brains of these three congenic mouse strains before and after flavivirus infection. Mice of the C3H/HeJARC, C3H.PRI-Flvr and C3H.M.domesticus-Flvr-like strains were injected intracerebrally (i.c.) with either a mouse osmolality phosphate-buffered saline (MOBS) or 105·2 infectious units (IU) of Murray Valley encephalitis (MVE) virus strain OR2. At various days post-infection (p.i.), groups of three to five mice of each strain were sacrificed and used to monitor both virus titres and NO levels in brain tissue homogenates (1:10, w/v). Virus titres were determined by 50% tissue culture infective dose (TCID50) assay and were shown to be significantly lower in the brains of flavivirus-resistant than in the brains of flavivirus-susceptible mouse strains (Fig. 1A). In contrast, the brain tissue NO levels, as determined by indirect measurement of the stable nonvolatile breakdown product nitrite () using the Griess reagent (Promega) did not show any significant difference between susceptible and resistant mouse strains before and after virus infection (Fig. 1B). This indicates that different levels of flavivirus replication observed in the brains of susceptible and resistant mice, which are controlled by Flv, do not correlate with the extent of either constitutive or inducible NO synthesis in the brains of these mice.



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Fig. 1. Virus replication and NO levels in the brains of flavivirus-susceptible and -resistant mice during infection with MVE virus strain OR2. Mice were inoculated i.c. with either saline or 105·2 IU of MVE virus. Prior to virus infection (day 0) and at various times p.i. three to five mice of each strain were sacrificed, and their brains removed and homogenized in MOBS (1:1, w/v). Virus titres in brain homogenates were determined by TCID50 assay in Vero cells (A), while levels were determined by the Griess assay (B). The difference in virus titres between susceptible and two resistant mouse strains was determined to be significant by Student’s t-test (0·005<P<0·01 at days 1 and 3 p.i. and P<<0·001 at day 5 p.i.), while the same test did not reveal any significant difference in levels in the brains of these mice at any time-point before or after virus infection (P>>0·1).

 
These findings are in agreement with the studies of other groups in which only late effects of the virus or viral proteins on the expression of Nos2 and Nos1 have been observed in rodent brain infected with either a flavivirus (Andrews et al., 1999 ) or other RNA viruses (Grzybicki et al., 1997 ; Bagetta et al., 1997 ). This suggests that NO is more important in chronic rather than acute virus infections and that it may contribute more to inflammatory rather than antiviral processes in the virus-infected rodent brain.

The tight regulation of NO synthesis in the brain is not surprising considering the importance of its role in neurotransmission and neuromodulation within the CNS. In contrast, NO synthesis in macrophages is induced by various stimuli, including bacterial LPS, interferon-{gamma} and different combinations of these agents with tumour necrosis factor (TNF)-{alpha} and -{beta}, interferon-{alpha} and -{beta}, and IL-1 (Nathan, 1992 ). It has been suggested that NO is an effector molecule mediating cytotoxic actions of macrophages against tumour cells and micro-organisms (Xie et al., 1992 ). Since its production in macrophages is controlled by the high output pathway (MacMicking et al., 1997 ), NO may represent one of the major mechanisms by which virus replication in macrophages is controlled.

Peritoneal macrophages from flavivirus-resistant mice have been shown to be a major in vitro model of the Flv-controlled virus resistance in addition to mouse embryo fibroblasts (Goodman & Koprowski, 1962 ; Brinton, 1983 ; Silvia et al., 1997 ). In the current study we have monitored the in vitro effect of virus replication on NO production in mouse macrophage cultures derived from flavivirus-resistant and -susceptible mice to assess whether Nos2 is implicated in innate Flv-controlled resistance observed in vitro (Silvia et al., 1997 ). Peritoneal macrophages lavaged 3 days after intraperitoneal injection of mice with thioglycollate were treated with E. coli K235 LPS (Sigma) for 16 h before being infected with West Nile (WN) encephalitis virus strain Sarafend at an m.o.i. of 1. Three to five aliquots of replicate tissue culture supernatants were initially removed at 24 h post-treatment (p.t.) and then at every day afterwards for up to 8 days for determination of virus titres by TCID50 (Fig. 3) or levels with Griess reagent (Fig. 2), respectively. While LPS produced a stimulatory effect on NO production in macrophages derived from all three congenic C3H mouse strains at every day p.t. for up to 8 days starting at day 1, infection with WN virus alone did not significantly affect basal NO levels at any of these time-points (Fig. 2).



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Fig. 3. Growth of WN virus strain Sarafend in peritoneal macrophages from resistant and susceptible mice. Thioglycollate-elicited peritoneal macrophages were either incubated without LPS (A) or pre-treated with 1 µg/ml of LPS K235 for 16 h (B) before addition of virus at an m.o.i. of 1 for 1 h. The virus titres were determined by TCID50 assay in Vero cells. Mouse strains used in the assay were flavivirus-susceptible C3H/HeJARC ({blacksquare}) and two resistant strains, C3H.PRI-Flvr ({square}) and C3H.M.domesticus-Flvr-like ({diamondsuit}) strains. The broken line (- - -) represents the titres of the virus left to decay in culture medium only over the period of time.

 


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Fig. 2. Effect of LPS K235 and flavivirus WN strain Sarafend on NO production in peritoneal macrophages obtained from congenic flavivirus-susceptible and -resistant mouse strains. Peritoneal exudates were extracted from minimum of three mice of each strain 3 days post-treatment with 1 ml of 6% thioglycollate. After 16 h of pre-treatment with LPS the cells were washed in fresh medium without LPS and further incubated for 1 h in medium alone or with WN virus (m.o.i. 1). Twenty-four hours later the initial replicate samples were removed for measurement of levels by the Griess assay. Five replicate samples were then regularly taken at each day p.t. for up to 8 days. Only the measurements taken at 48 h p.t. are shown, since they are descriptive of the common trend observed at other time-points. The experiment was repeated twice and each point represents a mean value from 10 replicate samples.

 
The stimulatory effect of LPS on NO production observed in primary macrophage cultures derived from all flavivirus-resistant and -susceptible mouse strains is in agreement with the existence of Lps-responsive Tlr4 alleles in these mouse strains (Silvia & Urosevic, 1999 ). However, the peritoneal macrophages isolated from the resistant C3H.PRI-Flvr mice consistently showed a decreased response to LPS throughout the whole monitoring period when compared to macrophage cultures derived from resistant C3H.M.domesticus-Flvr-like and susceptible C3H/HeJARC mouse strains (Fig. 2). Although these three mouse strains are highly congenic, they carry different levels of polymorphism at Flv and a number of other chromosome 5 loci (Urosevic et al., 1999 ). While the resistant C3H.PRI-Flvr mice carry a polymorphic segment of 31 cM encompassing Flv, the other resistant mouse strain, C3H.M.domesticus-Flvr-like, carries a similar Flv allele on a smaller polymorphic segment (Urosevic et al., 1999 ). Since both resistance alleles are introduced onto a similar genetic background, we predict that the locus responsible for the control of LPS-induced NO responses is located on a chromosome 5 segment which is identical between C3H.M.domesticus-Flvr-like and C3H/HeJARC mice, and polymorphic in C3H.PRI-Flvr mice (Urosevic et al., 1999 ).

When the LPS pre-treated macrophages derived from these three congenic LPS-responsive mouse strains were infected with the flavivirus WN virus strain Sarafend for 1 h, further changes in NO production were observed (Fig. 2). While in the absence of LPS treatment WN virus alone did not significantly affect NO production in macrophages of all three mouse strains, it caused significant changes in NO production of LPS pre-treated cells (Fig. 2). Infection with WN virus caused either a significant increase (Student’s t-test, 0·002>P>0·001) in NO production in cells derived from C3H/PRI-Flvr mice or a decrease in NO production in the cells derived from C3H/HeJARC and C3H.M.domesticus-Flvr-like mice (0·002>P>0·001 and 0·10>P>0·05, respectively) (Fig. 2). A similar modifying effect of viral infection on NO production in mouse macrophages has been previously reported for another flavivirus, tick-borne encephalitis virus, and this effect was shown to be mediated by {alpha}/{beta}-interferons (Kreil & Eibl, 1995 ). While it is not likely that {alpha}/{beta}-interferon genes are differently regulated between C3H congenic mouse strains, as illustrated by the similar brain interferon levels (Hanson et al., 1969 ), it is more likely that the adverse effect of viral infection on LPS-stimulated NO production is mediated by some chromosome 5 locus other than Flv. Since both the LPS signalling pathway (Han et al., 1994 ) and a cascade of events leading to induction of iNOS in macrophages (Gao et al., 1998 ) are very complex, it is difficult to predict from the current study which step in this pathway is responsible for the difference observed. Consequently, our congenic C3H mouse strains may provide an excellent model to further dissect effects of LPS and virus on NO production in vitro.

In the current study we have also observed that the in vitro pre-treatment with LPS of macrophages from both flavivirus-resistant and -susceptible mice has restricted the replication of WN virus in these cells (Fig. 3). While there was still some residual virus replication observed in the cells derived from susceptible (C3H/HeJARC) mice, LPS stimulation completely obstructed WN virus replication in primary macrophages derived from flavivirus-resistant C3H/PRI-Flvr and C3H.M.domesticus-Flvr-like mouse strains (Fig. 3). This outcome may have resulted from either the decreased susceptibility of activated macrophages to virus infection, or may have been mediated by an antiviral compound such as NO. The inhibitory effect of NO on flavivirus RNA replication, protein synthesis and release from the monocyte/macrophage cell lines has been previously reported for Japanese encephalitis virus (Lin et al., 1997 ). However, regardless of what mechanism is responsible for this antiviral effect, it does not appear to abrogate Flv regulated effects on flavivirus replication.

In conclusion, data presented here indicate that NO is not implicated in Flv-controlled resistance in mice. This has been supported by the lack of NO responses to flavivirus infection in both mouse brain tissue and peritoneal macrophages in flavivirus-resistant and -susceptible mice before and during the acute phase of infection. While the virus titres in the brains of susceptible mice were significantly higher, resulting in much greater neuronal damage and more severe tissue inflammation than in the brains of resistant mice (data not shown), no significant effect of the virus on the brain tissue NO levels was observed in these mice. We have also presented some additional evidence suggesting the involvement of chromosome 5 genetic loci other than Flv in the control of LPS-induced NO responses in mouse peritoneal macrophages. This evidence suggests that natural resistance to flaviviruses and LPS-induced antiviral state are two distinct mechanisms, which involve independent signalling pathways in the cell.


   Acknowledgments
 
The authors thank Andrew McWilliams and Anthony Scalzo for critically reading this manuscript. This work was supported by the National Health and Medical Research Council of Australia.


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Received 17 May 2000; accepted 16 November 2000.