key: cord-0988738-e9i1atxb authors: Atkins, G. J.; Balluz, I. M.; Glasgow, G. M.; Mabruk, M. J. E. M. F.; Natale, V. A. I.; Smyth, J. M. B.; Sheahan, B. J. title: Analysis of the molecular basis of neuropathogenesis of RNA viruses in experimental animals: relevance for human disease? date: 2008-05-12 journal: Neuropathol Appl Neurobiol DOI: 10.1111/j.1365-2990.1994.tb01167.x sha: d454318f2ab633ce3a82f96a1ada52441b200040 doc_id: 988738 cord_uid: e9i1atxb RNA viruses with segmented genomes were the first model used for molecular analysis of viral neuropathogenesis, since they could be analysed genetically by reassortment. Four viruses with non–segmented genomes have been used as models of neurovirulence and demyelinating disease: JHM coronavirus, Theiler's virus, Sindbis virus and Semliki Forest virus (SFV). Virus gene expression in the central nervous system of infected animals has been measured by in situ hybridization and immunocytochemistry. Cell tropism has been analysed by neural cell culture. Infectious clones have been constructed for Theiler's virus, Sindbis virus and SFV, and these allow analysis of the sequences involved in the determination of neuropathogenesis, through the construction of chimeric viruses and site–specific mutagenesis. Measles and rubella viruses have been studied in animal systems because of their importance for human disease. The importance of two recently discovered mechanisms of neuropathogenesis, antibody–induced modulation of virus multiplication, and persistence of virus in the absence of multiplication, remains to be assessed. In the last 3 0 years or so, a large amount of effort devoted to elucidating the molecular mechanisms of animal virus multiplication has been rewarded with considerable success. This work has been based on the use of cell culture systems, although our knowledge of some viruses, such as papilloma viruses and hepatitis B virus, is based on sequence analysis only. In contrast, molecular mechanisms of multiplication in the animal host, and the interactions of the virus with the host to produce disease (i.e. pathogenesis) have been largely neglected. Often, viruses which exhibit similar mechanisms of multiplication in cell culture differ in the type of disease induced in the animal host. An example is the picornavirus family: although foot-and-mouth disease, hepatitis A, polio and rhino (common cold) viruses are similar in their multiplication strategies in cell cultures, the diseases produced in their animal hosts are quite different. Until recently, studies of viral pathogenesis were largely descriptive but, due to the introduction of new techniques, it is now possible to approach the problem of the genetic control of pathogenesis at the molecular level. For RNA viruses, these techniques have been the use of nucleic acid and antibody probes, complementary DNA (cDNA) sequencing and the construction of infectious clones. As with earlier studies of virus multiplication, the first advances are being made with model systems. Such systems have been utilized in the analysis of the molecular control of neurovirulence, immune-mediated demyelination and teratogenicity. It is our intention here to review current work being carried out on the pathogenesis of RNA virus infection of the central nervous system (CNS); we intend to concentrate on the advances being made in determination of the genetic and molecular basis of pathogenicity for the CNS by RNA viruses. Detailed consideration of the immune response to such viruses, although a fascinating subject, is beyond the scope of the present review. The preferred hosts for molecular analysis of viral pathogenesis are laboratory mice and rats. This is because these animals are easy to breed and maintain, and well-characterized inbred strains are available. This is particularly true for laboratory mice, and the use of inbred strains minimizes variation in the response to infection. The 6rst RNA viruses to be utilized for molecular analysis of neuropathogenesis were those with segmented genomes, such as reovirus, arenaviruses and bunyaviruses. This is because it was possible to assign the genetic control of pathogenic characteristics to specific genome segments by performing the appropriate crosses in cell culture and analysing reassortment. The construction of infectious clones, combined with sequence analysis, has facilitated molecular analysis of the pathogenicity of positive-stranded RNA viruses with non-segmented genomes. An infectious clone is constructed by first sequencing cDNA clones of the virus, produced by reverse transcription. The sequence of the entire virus genome is required: cDNA fragments are then joined in the correct order, to give a DNA copy of the virus genome. Using a suitable expression plasmid, a bacteriophage promotor (usually derived from SP6 or T7) is placed adjacent to the virus genome. The cDNA corresponding to the virus genome may then be transcribed in vitro using the appropriate RNA polymerase, to give infectious RNA. This RNA is used to infect cultured cells, either by chemical transfection or electroporation, and infectious virus produced. Using an infectious clone, it is possible to apply recombinant DNA techniques to an RNA virus. Thus it is possible to use site-specific mutagenesis and the construction of recombinant (chimeric) viruses, containing sequences derived from different strains, in the analysis of pathogenicity. Infectious clones to enable the molecular analysis of neuropathogenesis have now been constructed for Theiler's virus (Theiler's murine encephalomyelitis virus, a picornavirus) and the togaviruses Sindbis virus and Semliki Forest virus (SFV). Although no infectious clone has yet been reported, murine coronavirus has long been used as a model for analysis of neuropathogenesis, and this system is ripe for exploitation at the molecular level. Model systems are usually based on animal viruses which efficiently infect laboratory mice and rats: most human viruses do not have this ability. Systems which require the use of primates, such as poliovirus and HIV. are beyond the scope of this review. However, some rodent experiments have been carried out with measles virus, and rubella virus has been studied in neural cell culture. The classical early experiments on the molecular basis of pathogenicity were carried out with reovirus, a doublestranded RNA virus, and with lymphocytic choriomeningitis (LCM) virus, an arenavirus, and La Crosse virus, a bunyavirus. Arenaviruses and bunyaviruses have negative or ambisense (positive and negative) single-stranded RNA genomes. Reoviruses have a genome consisting of 10 segments. The proteins coded by each of these segments, and their function in the multiplication of the virus is known. Also, the segments may be separated on gels and recognized by their size. Hybrid viruses can be constructed by infecting cells in culture with two different reovirus strains. Reassortment then occurs, with the formation of virions containing differing numbers of segments from each of the parental viruses. Such hybrid viruses can be isolated, propagated, and their pathogenic characteristics measured. By monitoring the segments which each hybrid isolate contains, it is possible to assign pathogenic characteristics to specific genome segments. Reovirus is naturally a respiratory and enteric pathogen: however, for molecular analysis of pathogenicity, infection of the neonatal mouse CNS is used as a model system. Two reovirus serotypes are used as models: type 1 and type 3. After intracerebral inoculation into neonatal mice, type 3 produces a lethal meningoencephalitis. which typically includes extensive damage to neurons. In contrast, type 1 does not damage neurons, but infects the ependymal cells lining the ventricles and produces ependymitis. Infected animals also frequently develop hydrocephalus. Analysis of reassortment has demonstrated that the cell tropism of reovirus (i.e. ability to infect neurons or ependymal cells) is determined by the virus S1 gene, which is a distinct genome segment. This gene codes for the sigma outer capsid protein, and is also the protein which recognizes the reovirus cell receptor [87, 881 The S 1 gene also determines the mechanism of spread of the virus following inoculation into the hindlimb of neonatal mice. Both type 1 and type 3 reovirus spread to the spinal cord following hindlimb inoculation. However, for type 3 this spread is through axonal transport, whereas for type 1 it is haematogenous [82] . Although the S1 gene is the main pathogenicity determinant for reovirus, experiments on temperaturesensitive (ts) mutants of this virus illustrate an important point. Ts mutants will multiply at a permissive temperature, but multiplication is restricted at a higher, nonpermissive temperature. The basis of such mutations is usually a missense substitution of an amino acid in the affected protein, resulting from a single base change. Such mutations usually attenuate the pathogenicity of the virus. Thus for virulent virus, such mutation may allow survival of the affected animals due to reduced multiplication rate of the virus. Such mutations may occur in several virus genes 1651. However, one effect of ts mutations may be to unmask pathogenic characteristics which are obscured by death in the case of the more virulent wild-type strain. LCM virus is an arenavirus whose genome consists of two segments. designated small and large. It has long been used as a model of persistent infection in mice. Infection of neonatal mice leads to life-long persistent infection. which is due to immune tolerance of the virus. For adult mice. infection leads either to death or viral clearance. Persistence occurs in most tissues of the mouse: in the CNS, persistent infection of the pituitary results in stunting of growth due to inhibition of growth hormone secretion. The infection of the pituitary is not cytolytic and can only be detected through the use of nucleic acid and antibody probes. This has led to a new concept in viral pathogenicity: inhibition of 'household' functions, such as protein and nucleic acid synthesis, leads to cell death: however, inhibition of 'luxury' functions, such as growth hormone synthesis, allows cell survival but affects the viability of the animal [ 3 2 ] . Although this may be a general disease mechanism, this concept should be interpreted with caution, as LCM virus causes an unusual infection, and there is as yet no evidence that such mechanisms operate in human disease. The establishment of persistence and associated suppression of the antiviral cytotoxic T-lymphocyte response has been mapped to the small RNA segment. as has the suppression of growth hormone [6 71. Indeed, persistence has been associated with a single amino acid change in the envelope glycoprotein of the virus [70] . Although LCM virus causes persistent infection in mice, it can be neurovirulent in guinea-pigs. Neurovirulence in guinea-pigs maps to the large RNA segment: however, neurovirulence for guinea-pigs is directly related to the rate of virus multiplication [67] and not to differences in cell tropism. La Crosse virus is a bunyavirus whose genome consists of three segments, labelled large, medium and small. It is a mosquito-transmitted arbovirus. and can therefore infect both vertebrate and invertebrate hosts. Reassortment experiments involving parental viruses of differing pathogenicity have shown that both neurovirulence and ability to infect mosquitoes are determined by the medium segment. These markers vary independently and experiments with monoclonal antibodies have shown that they are probably determined by sites within the G 1 envelope glycoprotein. However, as with other systems, mutations in other segments may modulate virulence by influencing multiplication rate [22] . JHM coronavirus is a strain of mouse hepatitis virus type 4. It is an enveloped virus whose genome consists of a single molecule of single-stranded RNA of positive polarity. In susceptible mouse strains it causes a virulent systemic infection which involves the CNS as well as the liver. However, attenuated mutants such as temperature-sensitive (ts) mutants can produce a n altered CNS disease [25, 331. The wild-type virus produces a lethal encephalitis in most infected mice due to infection of neurons. However, mutants such as ts8 produce chronic and recurrent demyelination, which involves both virus persistence and infection of oligodendrocytes [33] . In rats, infection of susceptible strains such as Lewis rats leads to chronic demyelination and virus persistence. This is associated with sensitization of T-lymphocytes to myelin basic protein 1861. In primary glial cell cultures derived from rats, type 1 astrocytes and microglial cells are the initial target cells, whereas oligodendrocytes are rarely infected [4 71. Although no infectious clone for JHM coronavirus is yet available, two approaches have indicated that virion structural proteins are important determinants of pathogenicity. In the first approach, monoclonal antibodies were raised against the E2 envelope protein. Some of these neutralized the infectivity of the virus, and when passively administered to mice, the animals developed chronic demyelination rather than lethal encephalitis [12] . Escape mutants which were resistant to neutralization could also be selected using these antibodies. Such mutants had an altered E2 protein and caused chronic demyelination in mice rather than lethal encephalitis The second approach involved utilizing the low level of recombination which occurs between virus genomes in coronavirus-infected cells. JHM coronavirus produces panencephalitis in mice but mild hepatitis, whereas the A59 strain of mouse hepatitis virus produces focal encephalitis and severe hepatitis. A panel of recombinant viruses derived from the JHM and A59 strains, and containing different proportions of RNA from the two parental strains was constructed. It was found that the main determinants of pathogenesis were localized to the 3' portion of the genome (about 25%), which encodes the viral structural proteins [ 3 5 ] . [ I l l . Theiler's murine encephalomyelitis (abbreviated here to Theiler's virus) is a non-enveloped picornavirus. Its genome consists of a single molecule of positive-sense single-stranded RNA. Virulent strains of Theiler's virus such as GDVII induce a lethal encephalitis when given intracerebrally to adult mice. However, avirulent strains such as DA or BeAn induce a biphasic disease. In the first few days following infection, the virus infects neurons and induces an acute encephalomyelitis. The amount of damage is small, and most mice survive: however, in the second phase of the disease virus is found in glial cells of the white matter, and virus persists in these cells. This persistence is associated with immune-mediated demyelination (Figure 1 ), so Theiler's virus has been used as a model for human multiple sclerosis (MS) [89] . The cell tropism of Theiler's virus for the murine CNS has been investigated by two methods. In brain cell cultures, Theiler's virus lytically infects neurons and oligodendrocytes and persistently infects astrocytes and macrophages [14, 23, 531. Using in situ hybridization. infection of these cells can also be shown in the animal [5] . There appears to be no difference in cell tropism between virulent and avirulent strains. In terms of the genetic determinants of pathogenicity of Theiler's virus, investigations have centred on the molecular control of virulence, persistence and demyelination. The complete nucleotide sequences of the DA, BeAn and GDVII strains of Theiler's virus have been determined [54. 56, 59, 601. Using this sequence information, infectious clones have been constructed for these three strains of virus [18, 29, 45, 68, 801. These infectious clones have been used to construct recombinant (chimeric) virus and hence to map viral genes controlling neurovirulence, persistence and demyelination, by fragment exchange. The results indicate that all three phenotypes are determined by the capsid proteins of the virus [6, 10, 13, 17, 18, 46, 791. The observation that the 5' non-translated region is important in the determination of neurovirulence first appeared to be due to an inadvertent base deletion which occurred during the construction of chimeric virus [64] . A threenucleotide insertion in this region which inhibits translation also reduces neurovirulence [8] and other results also suggest that the 5' non-translated region influences neurovirulence and persistence through its effect on translation [77] . That growth rate of the virus may alter CNS disease has been shown by analysis of a neutralization escape mutant of the DA strain. This has a single amino acid change which maps to amino acid 101 of the VP1 protein. This mutant replicates poorly in the CNS and causes only mild demyelination [9 1-93]. Sindbis virus is an RNA-containing, enveloped togavirus. which is mosquito-transmitted in its natural state. Its genome consists of a single molecule of positive-stranded RNA. It has been used as a model for many years for the study of the molecular biology of virus multiplication in cell culture [71] , mainly in the USA (a different togavirus. Semliki Forest virus, has been used in Europe. see below). Laboratory strains of Sindbis virus are avirulent for adult mice. although a neurovirulent strain has been isolated 1411. Most laboratory strains are lethal for neonatal mice 1751. In pathogenicity experiments in mice, the virus is usually given by intracerebral inoculation. A neuroadapted strain, virulent for adult mice, has been isolated by one group of workers [24] . Other studies have utilized neonatal mice [ 551. Using in situ hybridization and immunocytochemistry. it has been shown that neurons are the main target cells in the CNS 1281, and immune-mediated demyelination is not induced. However, the basis of a paralytic disease induced in SJL mice [50] has not so far been characterized. Some temperature-sensitive mutants of Sindbis virus are avirulent for neonatal mice, although weight gain is inhibited [9] . The importance of the E2 protein in the determination of pathogenesis was first shown using rapidly penetrating mutants of Sindbis virus, which are also attenuated. Such mutants have an altered E2 glycoprotein, as shown by changes in monoclonal antibody binding [58, 691 , and a pathogenesis domain has been defined on the E2 protein [58, 63, 741 . The E2 protein is one of the two Sindbis virus envelope glycoproteins, and probably recognizes the host cell receptor(sj. The receptor shows a prevalence in the CNS of mice which is age-dependent [83] , and one receptor at least has been identified as high affinity laminin receptor [85] . Whether the receptors described in these two studies are the same is not yet clear. However, both the E l and E2 proteins are involved in Sindbis virus infectivity, since monoclonal antibodies to both proteins can neutralize virus infectivity; neutralizing and non-neutralizing antibodies to both proteins can passively protect mice against lethal challenge, showing that both are important determinants of pathogenicity [48] . In initial experiments, it was shown that both infectious virus and viral RNA were cleared from the CNS within 3 weeks after infection. This was not due to the action of cytotoxic T-lymphocytes, but to antibodymediated restriction of virus gene expression in neurons [ 3 7 ] . The technique originally used to detect the virus genome was in situ hybridization. However, it was later shown using the polymerase chain reaction, a more sensitive method, that low levels of Sindbis virus RNA do persist in the CNS after clearance of infectious virus [36]. This low-level persistence does not appear to be associated with any detectable clinical signs, and the significance of this finding is not clear at present, A laboratory strain of Sindbis virus has been sequenced and an infectious clone produced [66] . A single amino acid change in the E2 protein controls virulence. This change affects the binding of the virus to mouse brain cells [81] . Changes in both the E l and E2 proteins reduce virulence, and a gradient of virulence exists involving changes in both proteins [42, 61, 621 . One amino acid change in the E2 protein, previously defined using monoclonal antibodies, has a marked effect on virulence. However, changes in this site which decrease virulence do not decrease binding of the virus to mouse brain cells [62, 741. The 3' and 5' non-coding regions of the Sindbis virus genome may also play a role in pathogenesis [78] . These regions are involved in the binding of the viral polymerase for the synthesis of negative and positive RNA strands, and the 5' region is also involved in the initiation of translation. Host proteins may be important in these processes, and cellular proteins have been shown Like Sindbis virus, Semliki Forest virus (SFVj IS an RNA-containing togavirus. Its genome consists of a single molecule of positive-stranded RNA and it is mosquito-transmitted in its natural state. It has been used for many years in European laboratories as a model for the study of the molecular biology of virus multiplication [29] . Some naturally occurring strains of SFV are virulent for adult mice when given peripherally. and avirulent strains or mutants often induce demyelination in the CNS which is immune-mediated ( Figure 1 ). Avirulent strains or mutants also may infect the developing fetus and induce fetal death or teratogenesis [ 31. a. Viral antigen in putative oligodendrocytes and cell processes in an area of spongiform degeneration in the spinal cord. 6 days after intraperitoneal infection with the M9 mutant of SFV. Immunogold silver staining using SPV specific IgG. b, Viral RNA in putative oligodendrocytes (arrows) in an area of spongiform degeneration in the white matter of the cerebellum, 6 days after intraperitoneal infection with the M9 mutant of SFV. In situ hybridization using a ["S]-labelled RNA probe complementary to SFV RNA. c. Viral antigen in neurons and in neuronal processes in the hyppocampus, 4 days after intranasal infection with the virulent SFV4 strain (derived from a n infectious clone). Immunoperoxidase staining using SFV-specific IgC. d. Viral RNA in neurons between bundles of myelinated fibres in the thalamus, 4 days after intranasal infection with the virulent SFV4 strain (derived from an infectious clone). I n situ hybridization using a biotinylated cDNA probe specific for SFV (red) and alkaline phosphatase immunocytochemistry using anti-myelin basic protein (MBT) antibody (blue). All illustrations x 364. T-cell mediated autoimmunity to myelin basic protein strains, damage to neurons exceeds a lethal threshold. Figure 3 ) show that virulent and avirulent A 7 strain persists in the CNS [16] . In SJL mice, which strains have similar cell tropism for the CNS; they both have a T-suppressor defect, lesions of demyelination infect oligodendrocytes and neurons. but not astrocytes. persist in the CNS of a proportion of infected mice after Virulent strains are, however, more cytopathic for neuinfection with an avirulent mutant, but infectious virus rons than avirulent strains or mutants [ 7 ] . This has also is cleared [76] . been shown by experiments involving neural cell cul-By light and electron microscopy it has been shown ture. In mixed glial cell cultures, rapid destruction of that both avirulent strains and mutants infect both oligodendrocytes occurs. Virulent strains such as L10 oligodendrocytes and neurons in the CNS. The origin (ori) of replication of the plasmid is shown. To produce infectious RNA. the plasmid is linearized with the restriction enzyme Spel. and RNA transcribed using bacteriophage SP6 polymerase. The location of the SP6 promoter is shown. The SFV non-structural genes nsP1, nsP2, nsP3 and nsP4, and the structural genes E l , E2. E3 (the three envelope protein genes) and C (the core protein gene) are shown in order of transcription. 6K is a small protein which is not incorporated into virions. cytopathic effect than an avirulent strain such as A7 or an avirulent mutant such as M9 [4, 201. Defective interfering (DI) particles have also been shown to modulate SFV virulence [3]. These are deletion mutants which can only replicate in the presence of a wild-type helper virus, but which interfere with its multiplication. Different DI clones vary in their ability to protect mice from lethal infection (M. Thomson and N. Dimmock, personal communication). SFV is one of the few good models of viral teratogenesis. Avirulent strains such as A7 induce rapid fetal death when given peripherally to pregnant mice [l]. The mutant ts22, derived from A7, is teratogenic [27] and induces skeletal, skin and CNS defects [43] . For the CNS. ts22 induces open neural tube defects in a minority of infected fetuses: the majority of fetuses show abnormal development of the neuroepithelium. These neural tube defects are indirectly induced by infection of mesenchy-ma1 cells adjacent to the neural tube, and not by infection of the neuroepithelium itself [44] . The genome of a laboratory strain of SFV has been completely sequenced and an infectious clone produced [40] (Figure 4 ). The infectious virus produced from this clone is virulent when given intranasally to adult mice. Two mutants in the E2 glycoprotein have been shown to be attenuated: for one mutant, infection of developing fetuses occurs, and for the other immune-mediated damage to the CNS occurs [21] . Measles and rubella viruses have been studied in animals because of their importance to human disease. Measles virus is a negative-stranded paramyxovirus. Rubella virus is a togavirus like Sindbis virus and SFV: however, in its natural state it infects only humans and no vector is involved in its transmission cycle. When inoculated intracerebrally with measles virus, neonatal rats die from encephalopathy. However, adult Lewis rats develop a subacute encephalomyelitis which is associated with virus persistence [ 391. Restricted expression of the viral envelope protein genes occurs both in the CNS of infected rats [73] and in cultured rat glial cells [72] , and may be associated with the regulation of persistence. Antibody-induced restriction of measles virus gene expression also occurs in infected rats [38] and in cell culture [90] . Rubella virus does not multiply well in experimental animals. However, it does infect cultured mouse embryos [26] and cultured rat neural cells. In neural cell culture, oligodendrocytes only are infected and the virus does not multiply in neurons or astrocytes [2, 521. One of the main reasons for the use of animal model systems is to gain insight into mechanisms of human disease. Animals are used to perform experiments which would be difficult, unethical or impossible using humans. However, caution must be exercised in interpreting results from animal models in terms of human disease, since a comparison only can be made and no definitive conclusions reached. In terms of RNA virus models, comparisons have been made to two human diseases which affect the CNS: multiple sclerosis (MS) and viral encephalitis. Teratogenesis has also been studied for SFV. MS is an autoimmune, demyelinating disease of the human CNS. Its aetiology is unknown, although viruses and genetic factors have been implicated. Evidence implicating viruses in MS has been equivocal [84] , but if a virus is involved, two mechanisms could operate: the disease could be triggered by a virus, which then disappears, or a persistent virus infection could be involved. The animal models described here indicate how such mechanisms could operate in MS. Two of the models, Theiler's virus and JHM coronavirus, induce autoimmune demyelination which is linked to virus persistence. For SFV, persistence of infectious virus may not be involved but the virus may act as a trigger. Two mechanisms of RNA virus pathogenesis have been described recently which may be of relevance to MS and other CNS diseases. These are antibody-induced modulation and persistence of the virus genome in neurons in the absence of virus multiplication. Further work with model systems is necessary to gain further insight into the relevance of these mechanisms for virus persistence and pathogenesis of human neurological disease. The use of infectious clones is advancing our understanding of the molecular determination of neurovirulence. The main model systems used so far have been Theiler's virus, Sindbis virus and SFV. The use of these systems may enhance our understanding of pathogenic mechanisms in human encephalitis, and may lead to improved virus vaccines. The use of adult animals in infection experiments, and avoidance of intracerebral inoculation, allows the study of the interaction of the virus with a fully developed immune system and the use of natural routes of infection, but this may not be possible for some systems. So far, the work has been confined to sequence analysis. Two types of mutation have been analysed. Mutations which impair the function of a viral protein attenuate the virus, but may lead to the unmasking of pathogenic mechanisms which are obscured by death for virulent viruses. Mutations which do not impair virus multiplication but determine cell tropism and hence disease syndromes have also been described, and are of fundamental importance in understanding the molecular control of pathogenicity. Such mutations have so far only been described in genes coding for viral structural proteins. The study of structure-function relationships between viral and cellular nucleic acids and proteins, and their rBle in pathogenesis, is just beginning. Effect of alphavirus infection on mouse embryos Multiplication of rubella and measles viruses in primary rat neural cell cultures: relevance to a postulated triggering mechanism for multiple sclerosis Molecular basis of Sindbis virus neurovirulence in mice Terdtogenicity of the Semliki Forest virus mutant ts22 for the foetal mouse -induction of skeletal and skin defects Effect of infection with the ts22 mutant of Semliki Forest virus on development of the central nervous system in the fetal mouse Molecular cloning of the complete genome of Theiler's virus, strain DA, and production of infectious transcripts Genetic mapping of the ability of Theilers virus to persist and demyelinate. 1 Virol Analysis of murine hepatitis UHM) strain tropism toward Lewis rat glial cells in vitro Monoclonal antibodies to the El-glycoproteins and E2-glycoproteins of Sindbis virus -definition of epitopes and efficiency of protection from fatal encephalitis Pathogenesis of in utero infection with abortigenic and non-abortigenic alphaviruses in mice Role of the immune response in Sindbis virus-induced paralysis of SJL/J mice Predisposition to EAE induction in resistant mice by prior infection with Semliki Forest virus Effect of rubella virus infection on the development of rat cerebellar cells in culture Cytotropism of Theiler's murine encephalomyelitis viruses in oligodendrocyte-enriched cultures Molecular cloning and sequence determination of DA strain of Theiler's murine encephalomyelitis viruses Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals Theiler's virus genome is closely related to that of encephalornyocarditis virus. the prototype cardiovirus Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2 Insights into Theiler's virus neurovirulence based on genomic comparison of the neurovirulent GDVII and less virulent BeAn strains Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses. 1 Virol Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro Attenuating mutations in glycoprotein-El and glycoproteiu-E2 of Sindbis virus produce a highly attenuated strain when combined in vitro Mutational analysis of a virulence locus in the E2 glycoprotein gene of Sindbis virus A single base deletion in the 5' noncoding region of Theiler's virus attenuates neurovirulence Neurotropic virus-host relationship alterations due to variations in viral genome as studied by electron microscopy Production of infectious RNA transcripts from Sindbis virus cDNA clones; mapping of lethal mutations, rescue of a temperaturesensitive marker, and in vitro mutagenesis to generate defined mutants Mapping arenavirus genes causing virulence Infectious cDNA clones of the DA strain of Theilers murine encephalomyelitis virus Sindbis virus mutations which coordinateiy affect glycoprotein processing, penetration. and virulence in mice Molecular basis of viral persistence -a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic lymphocyte-T response and establishment of persistence Replication of Togaviridae and Flaviridae Restricted expression of measles virus in primary rat astroglial cells Restriction of measles virus gene expression in acute and subacute encephalitis of Lewis rats Directed mutagenesis of a Sindbis virus pathogenesis site Pathogenesis of encephalitis induced in newborn mice by virulent and avirulent strains of Sindbis virus Multiplication of virulent and demyelinating Semliki Forest virus in the mouse central nervous system -consequences in BALB/c and SJL mice Influence of Theiler's murine encephalomyelitis virus 5' untranslated region on translation and neurovirulence Functions of the 5'-terminal and 3'-terminal sequences of the Sindbis virus genome in replication Determinants of persistence and demyelination of the DA strain of Theiler's virus are found only in the VP1-gene Molecular cloning of the complete genome of strain-GDVII of Theiler virus and production of infectious transcripts Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2-glycoprotein Distinct pathways of viral spread in the host determined by reovirus S 1 segment Identification of a putative alphavirus receptor on mouse neural cells Viral etiology of multiple sclerosis: where does the truth lie? High-&nity laminin receptor is a receptor for Sindbis virus in mammalian cells Adoptive transfer of EAE-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis Molecular basis of reovirus virulence: the role of the S1 gene. I'rm Absolute linkage of virulence with central nervous system cell tropism of reovirus to hemagglutinin Pathogenesis of Theiler's murine encephalomyelitis virus Antigenic modulation of measles subacute sclerosing panencephalitis virus in a persistently infected rat glioma cell line by monoclonal anti-haemagglutinin antibodies A neutralization-resistant Theilers virus variant produces an altered disease pattern in the mouse central nervous system Alteration of amino acid-101 within capsid protein Vp-1 changes the pathogenicity of Theilers murine encephalomyelitis virus Restricted virus replication in the spinal cords of nude mice infected with a Theiler's virus variant We thank Peter Liljestrom for contributing the basis of Figure 4 , and Christy King for help with photography.Studies on the molecular basis of RNA virus pathogenicity are supported in our laboratories by the Wellcome Trust, the Multiple Sclerosis Society of Ireland and the Health Research Board. For a detailed review of the pathogenesis of virus-induced demyelination, particularly immunological aspects. the reader is referred to Fazakerley JK and Buchmeier MJ Adv Virus Res 1993: 42: 249-324.