Pathogenesis of visna. IV. Spinal fluid studies.

Visna is a persistent retrovirus infection of sheep which produces a chronic progressive paralytic disease after an incubation period lasting from months to years. The cerebrospinal fluid (CSF) was repeatedly sampled in a group of Icelandic sheep which were infected intracerebrally and followed up to 42 months. Minimal levels of infectious virus were isolated from the cerebrospinal fluid (CSF) up to 4 months after infection after which CSF neutralizing antibodies appeared in many sheep. These antibodies varied in titer and in some animals exceeded serum antibody levels which were moderate to high. CSF antibody is apparently produced within the CNS by local proliferation of B cell clones, and is accompanied by the appearance of considerable numbers of plasma cells in the neural parenchyma. Some sheep raised serum antibody to a second serotype of visna virus and in a number of these animals heterotypic antibody was also found in the CSF. An increase in CSF leukocytes often occurs within 1 to 3 months following infection and may then persist or wane. A persistent high level of CSF cells is an indicator of progressive CNS disease and such animals are more likely to yield virus, have higher CSF antibody levels, more severe CNS lesions, and an enhanced risk of clinical illness (progressive paralysis). CSF cells are predominantly macrophages and lymphocytes, with a consistent minority of plasma cells.

Pathogenesis of central nervous system lesions in visna: cell-mediated immunity and lymphocyte subsets in blood, brain and cerebrospinal fluid.

There are several indications that central nervous system (CNS) lesions in visna are immune-mediated and that cell-mediated immunity (CMI) may be of importance in the initiation of the lesions. To study the role of CMI in the pathogenesis of CNS lesions, five sheep were infected by intracerebral inoculation with visna virus and observed for 1 year. The following parameters were monitored at regular intervals: (1) neutralizing and ELISA antibodies; (2) visna virus-specific stimulation of lymphocytes from peripheral blood; (3) lymphocyte subpopulations in peripheral blood, cerebrospinal fluid (CSF) and brain at sacrifice. The CNS lesions were graded and compared with other parameters. The time course and titers of antibodies did not correlate with the severity of CNS lesions whereas the CMI did, indicating that CMI may play an important role in lesion development. The correlation of the number of CD8-positive cells in the CSF with the severity of lesions and the reversed ratio of CD4/CD8-positive cells in the diffusely infiltrated neuroparenchyma indicates that the CD8-positive T lymphocyte may be an important effector cell in the induction of CNS lesions.

Cerebrospinal fluid immunoglobulins in sheep with visna, a slow virus infection of the central nervous system.

Icelandic sheep were injected intracerebrally with visna virus, which produces a persistent infection of the CNS accompanied by encephalomyelitis and focal demyelinating lesions. Studies were conducted on two groups of sheep, with short-term infections (25 sheep sampled 1-3 months after infection) and long-term infections (14 sheep sampled 5-6 years after infection). Quantitative determination of CSF immunoglobulin levels 5 years after infection indicated that IgM concentration was usually elevated, IgG2 was occasionally elevated and IgG1 was rarely elevated. CSF oligoclonal bands were seen in about half the sheep examined 5 years after infection. There was a correlation between high titers of CSF antiviral antibody and both elevated CSF IgM concentration and CSF oligoclonal bands. Serum/CSF IgG1 ratios indicated that the blood-brain barrier was apparently intact in long-term visna infection, consistent with intrathecal synthesis of IgM and of antiviral antibody. The alterations in CSF immunoglobulins in visna resemble those found in other persistent CNS virus infections and in multiple sclerosis.

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells.

Two different recombinant visna virus (VV) gag-baculoviruses were constructed for the expression of precursor VV Gag in insect cells. Both recombinant Gag viruses expressed proteins migrating on SDS PAGE at the predicted rate for VV Gag precursor, Pr50gag. However, differences were seen in the morphology of the virus-like particles produced. Monoclonal antibody directed against the VV Gag capsid protein (p25) and sera from sheep infected with ovine lentiviruses reacted to both 50-kDa proteins. A recombinant VV env-baculovirus was constructed, substituting sequences encoding the signal peptide of VV Env with the murine IFN-gamma analogue. Sera from ovine lentivirus infected sheep reacted in immunoblots with two proteins of approximately 100 and 200 kDa found in the plasma membrane of insect cells infected with env-recombinant virus. Sheep immunized with either the recombinant Gag or the Env proteins developed high antibody titers to VV in ELISA. The serum of sheep and ascitic fluid of mice immunized with the recombinant Gag reacted with native Pr50gag and the processed Gag proteins in immunoblots, whereas serum of the recombinant Env immunized sheep reacted with VV gp135 and a putative oligomer of gp135. The immunized sheep responded specifically to visna virus by lymphocyte proliferation in vitro.

Neuropathologic aspects of lentiviral infections.

Studies of lentiviral infections of various animals and man have shown that all may invade the CNS and induce pathological lesions. This is well established in infections with VV, CAEV, SIV, HIV-1, and FIV. Although VV and CAEV do not cause an overt immunodeficiency, they share several features pertinent for the establishment of neuropathologic lesions with those that induce immunodeficiency. This holds especially true for the initial steps and early CNS lesions. 1) Infection of the CNS is from the blood stream. Although a definite proof of how the different viruses cross the blood-brain barrier remains to be brought forward there are indications that it may occur through migration of infected monocytes and/or lymphocytes into the brain. Furthermore free virus may enter the CNS, either directly or through infection of endothelial cells. 2) The lesion pattern at least in initial stages is similar; that is, it consists of meningitis, perivascular infiltrations especially of the deep white matter, and inflammation of the choroid plexus. In visna a local amplification of the inflammatory response is frequently observed in choroid plexus often with formation of active lymphoid follicles. Multinucleated giant cells are prominent in HIV-1 and SIV infections, but rare in VV, and practically nonexistent in infections with FIV and CAEV, possibly a reflection of differences in virus replication. Myelin breakdown is a feature of various lentiviral infections but its mechanisms and morphological expression may vary. Sharply demarcated plaques of primary demyelination seem to be unique for VV infection and vacuolar myelopathy for infection with HIV-1. 3) The main target cells in the brain are cells of the monocyte/macrophage/microglial lineage. In visna infected monocytes are found but evidence for infection of the enigmatic resident microglial cells is still lacking. Infection, especially productive, of neuroectodermal cells is rare, but may, however be important for viral persistence. Infection of endothelial cells occurs in the various lentiviral infections and may play a part in viral entry into the CNS and contribute to tissue damage. 4) The discrepancy between the frequency of productively infected cells and cell types infected and extent and character of pathological lesions, indicates that a mechanism other than the direct effect of the virus contributes to the evolution of CNS lesions. In HIV-1 infection evidence, mainly obtained by in vitro studies, indicates that lesions are mediated by cytokines and other toxic factors secreted by inflammatory or glial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Pathogenesis of central nervous system lesions in visna cell-mediated immunity and lymphocyte subsets in blood, brain, and cerebrospinal fluid.

The time course and titers of antibodies did not correlate with the severity of CNS lesions whereas the CMI did, indicating that CMI may play an important role in lesion development. The correlation of the number of CD8 positive cells in the CSF with the severity of lesions and the reversed ratio of CD4/CD8 positive cells in the diffusely infiltrated neuroparenchyma indicates that the CD8 positive T cells may be an important effector cell in the induction of CNS lesions.

Treatment of visna virus infection in lambs with the acyclic nucleoside phosphonate analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA).

Nucleoside and nucleotide analogues, which are inhibitors of human immunodeficiency virus reverse transcriptase, are highly active inhibitors of visna virus replication in cell cultures. One such analogue, the acyclic nucleoside phosphonate PMEA, has also been found to have a prophylactic effect on visna virus infection in lambs. In the present study, lambs were injected subcutaneously with 10 mg/kg PMEA three times a week starting 4 weeks after inoculation with visna virus, when brain infection had been established. After 3 weeks of treatment there was a reduction in the amount of virus isolated from blood cells of PMEA-treated lambs compared to controls and during the remaining 7 months of drug treatment there was significantly less virus isolated from the blood cells of treated lambs than of controls. Antibody response against visna virus was also slower in the treated than in the untreated control group. On the other hand, there was no difference in the amount of virus isolated from various organs of the two groups and the severity of CNS lesions in sheep treated with PMEA for 8 months was comparable to that found in untreated controls, even though PMEA reached concentrations in the cerebrospinal fluid which were well in excess of the EC50 value of the drug for visna virus.

On the role of monocytes/macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy.

The pathogenesis of the deposition of a variant cystatin C as amyloid in hereditary cystatin C amyloid angiopathy (HCCAA) is not known. To address this question the synthesis and secretion of cystatin C in cultured monocytes from 9 carriers of the mutated cystatin C gene (5 symptomatic and 4 asymptomatic) was examined. The quantity of cystatin C in cells and supernatants was determined by the ELISA method, Western blots were done and selected samples immunostained for cystatin C. Monocytes from individuals carrying the gene defect synthesized cystatin C that was apparently not truncated, a form found in the cerebral amyloid deposits in HCCAA, but showed a distinctly lower rate of cystatin C synthesis than monocytes from healthy controls. The main difference was that the quantity of cystatin C was significantly lower in the supernatants in monocyte cultures from carriers of the gene defect than from healthy controls, possibly due to a partial block in its secretion. This abnormal processing of the cystatin C could explain the low cerebrospinal fluid levels of cystatin C in HCCAA and might be a part of the pathogenetic pathway of amyloid deposition. Furthermore it could, through a lower extracellular concentration of this inhibitor of cysteine proteinases, contribute to destruction of the amyloidotic blood vessels, leading to the most serious clinical manifestation in HCCAA, intracerebral hemorrhage.

Evaluation of local toxicity after repeated intranasal vaccination of guinea-pigs.

In intranasal vaccination it is important that the adjuvant does not have any toxic effect on the sensitive nasal mucosa. In this study a histological and clinical evaluation of the effects of two different adjuvants in a vaccine containing detoxified diphtheria (DT) and tetanus toxoid (TT) in guinea pigs was done. The guinea pigs were divided in four groups and treated daily for 14 days with different formulations. Group I with saline, Groups 2 and 3 with the vaccines in a non-ionic surfactant formulation containing glycerides and Group 4 with tetraethyleneglycol formulation containing glycofurol. The guinea pigs in Groups 1, 2 and 4 were sacrificed on day 15 and Group 3, 1 week later and the tissues processed for histological examination. The animals remained healthy during the treatment and minor clinical signs, such as nose-blowing, decreased with time. The histological appearance, including the development of lymphoid tissue, was comparable in all groups. A specific toxic effect on the nasal mucosa by the different vaccine and adjuvant formulations was not observed.

Human and ovine lentiviral infections compared.

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the "slowness" of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.

Quantitative assessment of the astrocytic response in natural scrapie of sheep.

Scrapie of sheep and goats belongs to the spongiform encephalopathies, a term derived from the characteristic vacuolar degeneration in the central nervous system. Astrocytosis has been described, but a systematic quantitative study has not been made. Such a study is important in resolving the still controversial issues as to whether the astrocytic response is caused directly by the infectious agent or represents a secondary reaction to tissue damage. In this study the numbers of astrocytes in 12 sheep with scrapie and 12 healthy sheep of a similar age were assessed and compared. Sections from eight planes of the brain were immunostained with anti-glial fibrillary acidic protein (GFAP) for astrocytes and their numbers counted in 40 areas of 1 mm2. The main findings were as follows. (1) A significant increase of astrocytes was detected in sheep with scrapie. (2) Astrocytosis was not usually related to the severity of the characteristic vacuolar lesions, indicating that it was at least partly due to a direct effect of the infectious agent but was not a secondary response to the neuronal damage. (3) The astrocytic response varied considerably between individual affected sheep; this may have been due to differences in agent strains, host response, or both.

Precipitating antibodies in experimental visna and natural progressive pneumonia of sheep.

Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.

Radiology in the Nordic Countries.

NEMT, Nordic Evaluation of Medical Technology, conducted a study in 1988-89 on the use and diffusion of diagnostic radiology technologies in Denmark, Finland, Iceland, Norway and Sweden, i.e. the Nordic Countries. The study analysed the responses to a questionnaire sent to all Nordic radiology departments. Our findings show a variation from about 500 to nearly 900 radiology examinations per 1000 inhabitants among the Nordic Countries. Some of the differences are explained by unique structural factors of the health care system in each country, even if they all provide comparable public health services. Other differences are explained by variations in medical practice, accessibility to new imaging modalities, and replacement policies. This paper summarizes the results of the study.