| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Division of Immunology and Genetics, The John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia
Correspondence
Mohammed Alsharifi
mohammed.alsharifi{at}imvs.sa.gov.au
or
mohammed.alsharifi{at}anu.edu.au
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Present address: Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Royal Adelaide Hospital, Adelaide, SA 5000, Australia 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Pathak & Webb, 1974; Soilu-Hanninen et al., 1994). SFV can be divided into virulent (vSFV) and avirulent (aSFV) (Atkins et al., 1999) strains; both are lethal in weanling mice, however, they differ in their pathogenesis in adult mice. Infection of adult mice with aSFV is mostly asymptomatic, characterized by low virus titres in the brain and demyelination that peaks at 14–21 days post-infection (p.i.) (Atkins et al., 1999; Fazakerley et al., 1993). In comparison, vSFV infection is characterized by detectable virus titres in extra-neural tissues, high titres in the brain and rapid and lethal encephalitis (Atkins et al., 1999; Fazakerley et al., 1993).
Previously, we compared the susceptibility of mice deficient in granule exocytosis, Fas or both cytotoxic pathways of natural killer (NK) and cytotoxic T (Tc) cells to infection with vSFV to that of wild-type (wt) C57BL/6 (B6) mice (Alsharifi et al., 2006). We showed that B6 mice, deficient in perforin, granzyme (gzm) A, gzmB or gzmAxB, were marginally more resistant to low dose vSFV infections relative to wt mice. This resistance increases significantly in mice lacking functional Fas (Fas–/–), Fas.L (gld), perforin plus gzmA and B (perfxgzmAxB–/–), or Fas plus gzmA and B (FasxgzmAxB–/–). Mice deficient in perforin plus Fas.L (perf–/–xgld) are even more resistant to vSFV infection. This is reflected in only approximately 20 % mortality compared with approximately 70 % mortality in wt mice (Alsharifi et al., 2006). These results were interpreted in terms of Fas- and granule exocytosis-mediated immunopathology outweighing the beneficial effects of the cytolytic effector pathways in the control of SFV infection. SFV replication in tissues of all knockouts, with the exception of perf–/–xgld, reach similar levels to that detected for the wt mice (unpublished data). Therefore, based on the results presented here, perf–/–xgld mice represent a third category of SFV susceptibility and our interpretation needs to be modified to accommodate the unexpected low virus replication in tissues of perf–/–xgld mice relative to that in immunocompetent mice or mice deficient in perf or Fas.L alone.
Clinical symptoms after SFV infection, such as ruffled fur, hunched posture and hind-limb paralysis are similar in B6, perfxgzmAxB–/–, gld, Fas–/– and FasxgzmAxB–/– mice. In contrast, in SFV-infected perf–/–xgld mice no clinical signs of morbidity were observed. In fact, perf–/–xgld mice were healthy throughout the course of infection and their life span was within the expected duration of these animals, which is approximately 10 weeks (Licon Luna et al., 2002). The impaired cytolytic activity in mice defective in both the Fas- and exocytosis-mediated pathways of cytolysis and hence minimal immune-mediated tissue damage in the CNS appeared to be the most likely mechanism to account for their increased resistance to SFV infection. Although a differential efficiency of virus replication in the different mouse strains could not be excluded.
To investigate the underlying factors associated with this increased resistance of perf–/–xgld mice to vSFV infection, groups of mice (B6 and perf–/–xgld) were infected, intravenously (i.v.), with 1 p.f.u. vSFV. At 2 day intervals, mice were sacrificed, tissues (serum, spleen, muscle and brain) were aseptically harvested, snap-frozen in liquid nitrogen or dry ice and stored at –70 °C. Tissue suspensions (10 %, weight/volume) were homogenized in ice-cold Hanks' balanced salt solution (pH 7.6) containing 0.2 % BSA, clarified by centrifugation (18 000 g for 5 min at 4 °C) and supernatants were stored in aliquots at –70 °C. Virus titres were determined by plaque formation on semi-confluent monolayers of Vero cells as described previously (Licon Luna et al., 2002). The limit of virus detection by plaque assay in tissues and serum samples of infected mice was 102 p.f.u. g–1 or ml–1, respectively. As a control, brain, spleen and muscle tissues from uninfected animals were homogenized in the presence of a known amount of virus. Surprisingly, the kinetics and level of vSFV replication in extraneural and brain tissues of perf–/–xgld mice were markedly lower or undetectable than those in B6 wt mice (Fig. 1). Whilst SFV was present in serum of B6 mice on days 2 and 4 p.i., with virus titres ranging from 102 to 105 p.f.u. ml–1, no viraemia was detectable in perf–/–xgld mice. SFV was also present in some spleens of wt mice harvested at 2, 4 and 6 days p.i., but not in spleens from perf–/–xgld mice. Given that tissue titres were often not significantly higher than the corresponding virus titres in serum, the contribution of virus-containing blood to titres in the tissue samples cannot be excluded.
|
To confirm these observations of suppressed SFV replication in perf–/–xgld mice, we also tested tissue titres following infection with the non-lethal aSFV strain. Groups of B6 wt and perf–/–xgld mice were infected i.v. with 103 p.f.u. of aSFV, and brain, spleen, muscle and serum were harvested on days 2, 4, 6 and 8 p.i. In B6 wt mice, aSFV replicated to significantly higher titres in the spleen on days 2 and 4 p.i. (Fig. 2) compared with that of vSFV (Fig. 1
). In addition, aSFV was detected in the CNS of wt mice, but was cleared between 6 and 8 days p.i. without reaching titres greater than 105 p.f.u. g–1 and without causing death (Fig. 2). The low virus titres in the brain are a distinct feature of aSFV infection, where replication in neurons is restricted (Fazakerley et al., 1993). A comparison of growth of aSFV in B6 wt and perf–/–xgld mice clearly demonstrates reduced viral replication in all tissues of the mutant mice tested, except that viraemia was comparable to that of wt mice on day 2 p.i. These results suggest that the absence of clinical symptoms and increased survival rate of perf–/–xgld mice during vSFV infection is the result of impaired viral replication rather than reduced immune-mediated pathology due to defective cytolytic effector function.
|
) and NK cells have been implicated in immunopathology (Alsharifi et al., 2006). However, this does not explain the markedly reduced SFV replication and spread. Perf–/–xgld mice have previously been shown to suffer from autoimmune diseases (Spielman et al., 1998). Thus, components of the innate immune response may play a role in the observed phenomenon. Perf–/–xgld mice have been reported to suffer from hyper-gamma-globulinemia (Spielman et al., 1998), but no SFV neutralizing activity was detected in the serum of naïve perf–/–xgld mice (unpublished data), which rules out a contribution of natural antibodies to the lower virus titres in tissues of perf–/–xgld mice.
High numbers of activated macrophages have been found in the pancreas and ovaries of perf–/–xgld mice. This is thought to be responsible for female infertility of these mice (Spielman et al., 1998). Thus, activated macrophages and their associated cytokine [tumour necrosis factor alpha (TNF-
)], which is an important cofactor for NK cell gamma interferon (IFN-
) production in vivo (Bancroft et al., 1989), may contribute to the resistance of these mice to SFV infection. IFN- plays a crucial role in the resistance to SFV infection, since mice of the 129 strain became highly susceptible as a result of IFN- receptor deficiency (Alsharifi et al., 2006). This is supported by circumstantial evidence from SJL mice, where low SFV titres in the brain are associated with high levels of TNF- and IFN- compared with B6 mice where high SFV titres in the brains are associated with low levels of TNF- and IFN- (Mokhtarian et al., 1996). IFN- is required for macrophage activation, but must be accompanied by TNF- to achieve full macrophage activation (reviewed by Mosser, 2003). In addition, a synergistic effect of both IFN- and TNF- has been reported to mediate an antiviral effect in vitro (Wong & Goeddel, 1986).
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Atkins, G. J., Sheahan, B. J. & Liljestrom, P. (1999). The molecular pathogenesis of Semliki Forest virus: a model virus made useful? J Gen Virol 80, 2287–2297.
Bancroft, G. J., Sheehan, K. C., Schreiber, R. D. & Unanue, E. R. (1989). Tumor necrosis factor is involved in the T cell-independent pathway of macrophage activation in scid mice. J Immunol 143, 127–130.[Abstract]
Fazakerley, J. K. (2002). Pathogenesis of Semliki Forest virus encephalitis. J Neurovirol 8 (Suppl 2), 66–74.[CrossRef][Medline]
Fazakerley, J. K., Pathak, S., Scallan, M., Amor, S. & Dyson, H. (1993). Replication of the A7(74) strain of Semliki Forest virus is restricted in neurons. Virology 195, 627–637.[CrossRef][Medline]
Licon Luna, R. M., Lee, E., Müllbacher, A., Blanden, R. V., Langman, R. & Lobigs, M. (2002). Lack of both Fas ligand and perforin protects from flavivirus-mediated encephalitis in mice. J Virol 76, 3202–3211.
Mokhtarian, F., Wesselingh, S. L., Choi, S., Maeda, A., Griffin, D. E., Sobel, R. A. & Grob, D. (1996). Production and role of cytokines in the CNS of mice with acute viral encephalomyelitis. J Neuroimmunol 66, 11–22.[CrossRef][Medline]
Mosser, D. M. (2003). The many faces of macrophage activation. J Leukoc Biol 73, 209–212.
Müllbacher, A. & Blanden, R. V. (1978). Murine cytotoxic T-cell response to alphavirus is associated mainly with H-2Dk. Immunogenetics 7, 551–561.[CrossRef]
Pathak, S. & Webb, H. E. (1974). Possible mechanisms for the transport of Semliki forest virus into and within mouse brain. An electron-microscopic study. J Neurol Sci 23, 175–184.[CrossRef][Medline]
Soilu-Hanninen, M., Eralinna, J. P., Hukkanen, V., Roytta, M., Salmi, A. A. & Salonen, R. (1994). Semliki Forest virus infects mouse brain endothelial cells and causes blood-brain barrier damage. J Virol 68, 6291–6298.
Spielman, J., Lee, R. K. & Podack, E. R. (1998). Perforin/Fas-ligand double deficiency is associated with macrophage expansion and severe pancreatitis. J Immunol 161, 7063–7070.
Wong, G. H. & Goeddel, D. V. (1986). Tumour necrosis factors and β inhibit virus replication and synergize with interferons. Nature 323, 819–822.[CrossRef][Medline]
Received 21 November 2007;
accepted 7 April 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) and perf–/–xgld (
) mice. Groups of mice were infected, i.v. with 1 p.f.u. per mouse of vSFV and tissues were collected at the time points indicated. Virus content was determined by plaque assay on Vero cells. Dotted line indicates detection limit and each symbol represents an individual mouse.