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Detection of viral genes and their products in chronic neurological diseases

The suspicion that viruses are involved in multiple sclerosis (MS) now largely depends on epidemiological evidence. Numerous attempts to transmit MS have been unsuccessful, as have searches for viruslike particles or antigens. The April 1983 workshop “Detection of Virus Genes and Their Products in Chronic Neurological Disease,” held at Woods Hole, Massachusetts, under the auspices of the National Institutes of Health and National Multiple Sclerosis Society, brought together fifty-six scientists from eight countries to reexamine the issue of viral infection in MS for two reasons: the new information on persistent viral infections and the subtle mechanisms that link infection to disease, and the newly available technical opportunities to penetrate these mysteries. Using these instruments to full advantage depends on knowing what to look for, and there was considerable discussion of contemporary views of viral persistence in vivo. These are summarized in the Figure. Within the framework of the virus life cycle, there are four classes of mechanisms that, first, allow a virus to elude immune surveillance and, second, allow the host to survive for long periods. Both of these requirements must be satisfied in persistent infections. The most basic mechanism (type I) is analogous to lysogeny, with global repression of the viral genome such that the infected cell escapes detection and destruction because little if any antigen is present to focus the attack by immune and inflammatory cells. The slow infections visna and subacute sclerosing panencephalitis (SSPE) exemplify type I persistence. In types I1 and I11 surface antigenic targets are also greatly reduced, but by mechanisms that include antibody-mediated modulation of envelope components, and alterations in the synthesis or stability of the matrix polypeptide required for virus assembly. These mechanisms entrap the virus in the cell and place it beyond the reach of host defenses. Types I1 and 111 persistence also are exemplified by SSPE. In type IV persistence the final phase of virus maturation involving host cell proteolytic cleavage of a protein essential for infectivity, such as the fusion protein of paromyxoviruses, iails to take place, and virus continues to be harbored in cells. Persistent infection of lymphocytes by this mechanism raises the possibility of episodic disease if these cells should enter the nervous system and gain access to the requisite proteases. In type I persistence the only discernible evidence of infection is the viral genome and varying numbers of transcripts. In this situation hybridization methods are the appropriate means of investigation (Table 1). If infected cells are numerous, analysis of nucleic acid extracted from tissues by solid-phase methods will detect virus genes and integrated genomes. By using cloned probes from defined regions of the genome, additional information can be obtained about whether all or part of the genome is present and expressed. At the practical level the use of probes from small regions of the genome, or probes from reiterated regions of the genome, amplifies the signal and greatly enhances the sensitivity of the probe. In many chronic infections there are only a few infected cells in the tissues. Methods that rely on extracted nucleic acids will fail to detect virus genes because of dilution. The method of choice in this situation is in situ hybridization of individual cells. Results obtained with MS and non-MS neural tissue illustrate the relative sensitivity of these methods: measles virus-like nucleotide sequences have been detected by in situ hybridization in a few cells in tissue sections, but not in nucleic acids extracted from the tissues. One limitation of in situ hybridization is the small area of tissue subjected to analysis. This problem of sampling can be overcome by “hybridization histochemistry,” a technique in which the tissue is placed against x-ray film after hybridization to a radioactive probe. Darkening of the film serves as a guide to regions of the tissue that can be fruitfully examined microscopically, after coating the tissue with radioauto-

rus Genes and Their Products in Chronic Neurological Disease," held at Woods Hole, Massachusetts, under the auspices of the National Institutes of Health and National Multiple Sclerosis Society, brought together fifty-six scientists from eight countries to reexamine the issue of viral infection in MS for two reasons: the new information on persistent viral infections and the subtle mechanisms that link infection to disease, and the newly available technical opportunities to penetrate these mysteries.
Using these instruments to full advantage depends on knowing what to look for, and there was considerable discussion of contemporary views of viral persistence in vivo. These are summarized in the Figure. Within the framework of the virus life cycle, there are four classes of mechanisms that, first, allow a virus to elude immune surveillance and, second, allow the host to survive for long periods. Both of these requirements must be satisfied in persistent infections. The most basic mechanism (type I) is analogous to lysogeny, with global repression of the viral genome such that the infected cell escapes detection and destruction because little if any antigen is present to focus the attack by immune and inflammatory cells. The slow infections visna and subacute sclerosing panencephalitis (SSPE) exemplify type I persistence. In types I1 and I11 surface antigenic targets are also greatly reduced, but by mechanisms that include antibody-mediated modulation of envelope components, and alterations in the synthesis or stability of the matrix polypeptide required for virus assembly. These mechanisms entrap the virus in the cell and place it beyond the reach of host defenses. Types I1 and 111 persistence also are exemplified by SSPE. In type IV persistence the final phase of virus maturation involving host cell proteolytic cleavage of a protein essential for infectivity, such as the fusion protein of paromyxoviruses, iails to take place, and virus continues to be harbored in cells. Persistent infection of lymphocytes by this mechanism raises the possibility of episodic disease if these cells should enter the nervous system and gain access to the requisite proteases.
In type I persistence the only discernible evidence of infection is the viral genome and varying numbers of transcripts. In this situation hybridization methods are the appropriate means of investigation (Table 1). If infected cells are numerous, analysis of nucleic acid extracted from tissues by solid-phase methods will detect virus genes and integrated genomes. By using cloned probes from defined regions of the genome, additional information can be obtained about whether all or part of the genome is present and expressed. At the practical level the use of probes from small regions of the genome, or probes from reiterated regions of the genome, amplifies the signal and greatly enhances the sensitivity of the probe.
In many chronic infections there are only a few infected cells in the tissues. Methods that rely on extracted nucleic acids will fail to detect virus genes because of dilution. The method of choice in this situation is in situ hybridization of individual cells. Results obtained with MS and non-MS neural tissue illustrate the relative sensitivity of these methods: measles virus-like nucleotide sequences have been detected by in situ hybridization in a few cells in tissue sections, but not in nucleic acids extracted from the tissues.
One limitation of in situ hybridization is the small area of tissue subjected to analysis. This problem of sampling can be overcome by "hybridization histochemistry," a technique in which the tissue is placed against x-ray film after hybridization to a radioactive probe. Darkening of the film serves as a guide to regions of the tissue that can be fruitfully examined microscopically, after coating the tissue with radioauto-
. How are persistent infections related to disease? Table 2 indicates that in some cases the relationship is direct, eg., demyelination as a consequence of lytic infection of the oligodendrocyte by coronaviruses or papovaviruses. With recombinant DNA techniques, in the case of herpes simplex virus it is now possible to define precisely the region of a virus genome directly responsible for neurovirulence. In other instances the link between infection and disease is not easily discerned: for example, if the virus causes dysfunction of cells that elaborate hormones with distant target organs, or if the virus sets in motion a pathological process whose effects extend beyond the boundaries of the initial infection. In the demyelinating phase of Theiler's virus infection of mice, the viral genome persists in glial cells, and the correlation between foci of inflammation, demyelination, and cells with viral antigen suggests that disease is related to a virus-induced immune attack on white matter. In other animal mod- els of demyelination, such as rats infected with coronavirus, the virus apparently sensitizes the animal to myelin basic protein, and infection leads to an autoimmune leukoencephalomyelitis.

Replication of Genomes
The most difficult situations to analyze are those in which the virus initiates a pathological change but need not be present at the time of manifest disease, or in which episodes of disease follow introduction of virus from an extraneural reservoir. Such indirect pathogenetic mechanisms make it virtually impossible to satisfy the molecular equivalents of Koch's postulates: constant association of a virus genome with disease, and in a location that plausibly accounts for the pathological findings. The discoveries of herpes simplex and measles virus genes in the brains of MS patients and control subjects can be interpreted as adventitious infection, or, alternately, as residual evidence of the source of an autoimmune process directed to white matter.
The cause of MS remains an enigma, but the conference ended with optimism, founded in the formidable powers of the new technology, and the successes of the past ten years in the analysis of the relationship among virus genes, host genes, and cancer.