Heather Dyson

Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis

The course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination.

Characterization of the cellular and cytokine response in the central nervous system following Semliki Forest virus infection

Cytokines are important mediators in the pathogenesis of central nervous system (CNS) inflammatory diseases including multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE), viral encephalitis and virus induced demyelinating diseases. We have used immunohistochemical techniques to characterize the mononuclear cell infiltrate and cytokine profiles in the CNS following infection of mice with the demyelinating A7(74) strain of Semliki Forest virus (SFV), an important viral model of MS. Mononuclear cell infiltrates in the CNS, first observed at 3 days and maximal during clearance of infectious virus, were comprised predominantly of CD8+ lymphocytes. F4/80+ macrophage/microglia and CD45/B220+ B lymphocytes were most numerous during the subsequent phase of demyelination. CD4+ T-lymphocytes were observed at low levels throughout infection. By immunostaining MHC class I, IL-1beta , IL-3 and TGF beta1 were constitutively expressed in normal mice and were upregulated following infection. MHC class II, IL-1alpha, IL-2, IL-2R, TNF-alpha and IL-6 were strongly upregulated in the CNS of SFV-infected mice and mice with chronic relapsing EAE. The spatial and temporal distribution of these cytokines during the course of disease was analysed. Whereas IL-1alpha, IL-1beta, IL-10, and TGF beta1 were observed on day 3 following infection GMCSF, IL-2 and TNF alpha were first apparent at day 7 when the cellular infiltration in the CNS was most intense. In contrast IFN gamma and IL-6 were first observed on day 10 prior to the demyelination phase of disease. Cytokines in the lesions of demyelination suggest a role in the pathogeneisis of myelin damage. Based on cytokine profiles no clear bias of either a Th1 or Th2 response was observed in the CNS during infection.

Susceptibility to a neurotropic virus and its changing distribution in the developing brain is a function of CNS maturity

Many major physiological changes occur within the rodent central nervous system (CNS) during the first few postnatal weeks. These include axonogenesis, synaptogenesis and myelination. Concomitant with CNS development over this period, there is a decrease in susceptibility to many neurotropic virus infections in that infection of suckling animals results in lethal encephalitis whereas infection of weanling animals is not lethal. The events underlying this dramatic change in susceptibility have been unclear. Here we demonstrate that age-related virulence of the neurotrophic alphavirus, Semliki Forest virus is dependent upon ability of the infection to spread in the CNS. This is not determined by maturity of interferon, or specific immune responses or the blood brain barrier, but by maturity of neuronal systems. Detailed study of the course of infection in the cortex, hippocampus and cerebellum during their postnatal development indicates that as these and other neuronal systems mature they become resistant to spread of the virus and the pattern of infection changes from widespread to focal.

Kinetic and phenotypic changes in murine lymphocytes infected with murine gammaherpesvirus-68 in vitro

Primary infection with murine gammaherpesvirus-68 (MHV-68), as with other members of the gammaherpesvirus subfamily, is characterized by a lymphoproliferative phase. MHV-68 causes acute splenomegaly and an infectious mononucleosis-like syndrome in which there is expansion of the CD8+ T cell subset. In long-term infections, MHV-68 is associated with lymphoma development. In order to elucidate the mechanisms underlying the proliferative processes, the events following infection of murine splenocytes or purified murine B lymphocytes in vitro have been examined. MHV-68 infection prolonged the viability of murine splenocytes and stimulated cellular proliferation. Unlike Epstein-Barr virus and herpesvirus saimiri, MHV-68 did not cause growth transformation. Growth transformation did not occur even when cells with a predisposition to transformation were infected or when culture conditions were selected to enhance the viability of the cells. Following MHV-68 infection, the latency-associated viral tRNAs were transcribed. However, transcription of the other known latency-associated gene, M2, was not observed. In addition, there was no evidence of productive virus replication either by staining with antibodies specific for late virus antigens or by in situ hybridization for early and late mRNAs. In contrast to Epstein-Barr virus- and herpesvirus saimiri-infected lymphocytes, where episomal genomes are seen, Gardella gel analysis indicated that the primary lymphocytes infected by MHV-68 in vitro contained only linear virus DNA. This DNA was nuclease sensitive, indicating that, while MHV-68 was efficiently uncoated, its circularization in vitro was extremely inefficient. These results are discussed in terms of the host-virus interaction.

2'-Deoxy-5-ethyl-β-4'-thiouridine inhibits replication of murine gammaherpesvirus and delays the onset of virus latency

The antiviral thionucleoside analogue 2′-deoxy-5-ethyl-β-4′-thiouridine (4′-S-EtdU) was shown to be a more potent inhibitor of gammaherpesvirus infection than acyclovir. This compound inhibits replication of murine herpesvirus (MHV)-68 in the lungs of mice when given 3 days post-infection. However, as with other nucleoside analogues, it was unable to prevent the establishment of latency, despite delaying the onset of latent infection in the spleen. In contrast, virus persistence in the lung was inhibited following drug treatment, although persistence was re-established in mice when treatment was suspended after 12 days. These data suggest that 4′-S-EtdU is a highly effective inhibitor of murine gammaherpesvirus replication and as such provides a powerful tool to study the pathogenesis of this virus in vivo.

Virus induced demyelination

The effect of intravitreally injected cytokines (TNFa, TNF/3, IL-1, IL-6, IFN--/) on glia in mouse optic nerves was determined, by using intracellular dye-injections to characterise their whole-cell morphology. Following intravitreal injection of TNFa or TNFI3, oligodendrocytes initially developed swellings along the length of their myelin segments, and internodal myelin segments became increasingly attenuated, until, in extreme examples, individual oligodendrocytes lost their full complement of myelin segments; oligodendrocyte changes were not observed with IL-1, IL-6 or IFN-7. Widespread astrocyte reactivity, characterised by astrogliosis and an upregulation of GFAP, was observed following intravitreal injection of TNFa, TNF/3, IL-1 or IL-6; IFN-7 alone had no effect. Similar glial changes have been seen during demyelination following inoculation with Semliki Forest virus. This study supports a role for TNF in T-cell mediated demyelination and for TNF, IL-1 and IL-6 in mediating astrogliosis and reactivity.