Páll A. Pálsson

Cultivation of visna virus in tissue culture

The virus of Visna, a slow, demyelinating leucoencephalitis of sheep, has been cultivated in tissue culture. The cells employed are derived from the chorioid plexus of sheep. The virus causes characteristic cytopathic changes in the culture, so that the method may be used to detect virus activity and measure the activity of virus containing material. Virus which had undergone 3, 11, and 12 passages in TC was injected intracerebrally into sheep and found to produce typical Visna lesions. Neutralizing antibody has been detected in sera from a certain proportion of sheep affected with Visna. The rate of virus multiplication in tissue culture after inocula of varying size has been studied. Small inocula tend to give rise to a mild infection which persists in the culture for long periods of time without destroying more than a certain proportion of the cells. The possible relationship between this relatively stable balance between virus and cells and the extraordinarily slow progress of Visna in the CNS of sheep is discussed.

Antigenic Drift in Visna: Virus Variation During Long-term Infection of Icelandic Sheep

A group of 20 Icelandic sheep were infected intracerebrally with visna virus strain 1514, and 209 virus isolates were obtained from the blood, cerebrospinal fluid, and central nervous system (CNS) over a period of 7 years, during which eight animals developed clinical signs of visna necessitating sacrifice. (i) Using type-specific antisera, it was found that 12 (16%) of 76 isolates tested escaped neutralization. These 12 variant viruses were distributed randomly among animals and over time, and did not replace the infecting strain even though all sheep developed homotypic antibody within 3 months of infection. The one exception was sheep no. 1557 (an animal without clinical visna), where the last six isolates were variants. (ii) A total of 35 blood and CNS isolates from seven of these sheep (including five with clinical visna) were tested against serial samples of their own sera. Autologous antisera neutralized all isolates tested with the exception of isolates from sheep 1557. None of the isolates obtained at sacrifice from the five sheep with clinical visna escaped neutralization with autologous antisera. These data suggest that although variant viruses are encountered at considerable frequency during long-term infection of Icelandic sheep, the variants usually do not replace the infecting strain. Antigenic drift does not appear to be essential for virus persistence or for the development of clinically evident CNS lesions.

Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimentsl visna induced by virus strains of increased neurovirulence

SummaryIcelandic sheep were infected by intracerebral inoculation with visna virus strains of increased neurovirulence. The character and severity of pathological lesions were studied in brains from four sheep that developed clinical signs 5 to 12 weeks after infection. Viral antigens were identified by immunostaining using mouse monoclonal antibodies against two core proteins and the Avidin-Biotin method of detection. The pathological lesions were in general more severe than observed following infection with the parent strain K1514. Primary demyelination, a late manifestation of infection with K1514, was detected. Thus, in addition to causing more severe pathological lesions, these neurovirulent strains apparently have an increased potential to induce primary demyelination. Viral antigens were detected in lymphocytes, plasma cells, macrophages, endothelial cells, pericytes, fibroblasts and choroidal epithelial cells. Neurons and glial cells were antigen negative. The spectrum of infected cells in the brain was similar to that observed in infections with human immunodeficiency virus. These results do not support the view that the demyelination is caused by immunological damage to infected oligodendrocytes. A perturbation of the function of oligodendrocytes through a non-productive infection could be the underlying pathogenetic mechanism and/or a non-specific demyelination due to the intense inflammatory reaction.

Pathology of visna

In the period 1935--51 sporadic cases of a chronic disease, affecting the central nervous system of sheep, were observed over wide areas in the southwestern and southern districts of Iceland. Usually only a few sheep were affected on each farm, but sometimes a considerable percentage of the flock showed symptoms of this disease, named Visna, which means wasting. M~edi i, a slow by progressing, highly lethal pneumonia of sheep was extremely prevalent in the same areas during the same period. The losses from Msedi overshadowed completely the losses from gisna, and therefore the farmers generally did not pay very much attention to the losses from Visna, particularly as the signs of Visna very often developed in sheep which had already shown signs of lVi~edi for some time, and were therefore considered poor risks anyway. ?r was introduced into Iceland in 1933, but did not become widespread until 5 or 6 years later. I t is believed that Visna became prevalent at the same time, and there is no doubt that the peak incidence of Visna coincided with heavy incidence of M~edi in the flocks. During the years 1949 to 1951, all sheep in the south-west and south part of Iceland were destroyed in order to eradicate M ~edi from the country, and these areas were restocked with sheep from areas where Mmdi and Visna were not known to occur. Since then Visna has not been found in Iceland. Whether the cpizootic of Visna occurred quite independently of ~ e d i or whether lVi~edi was in some way instrumental in provoking the overt Visna eases, remains, however, an open question. Another disease, affecting the central nervous system of sheep, was known to have been enzootic in certain areas of Northern Iceland for a long time 2. This disease named Rida, which means ataxia or tremor, appeared to be a contagious disease with a very protracted course. The lesions of Rida were first described by H~LLGnIMSSON in 1938 a. Later Smv~])ssoN 4 described this disease as a chronic encephalitis of sheep, with an incubation of 8 to 18 months. The onset of the disease was marked by uncertainty

The effect of post-infection immunization on the severity of experimental visna

Abstract Visna is a slow retrovirus infection of sheep which produces both inflammatory and demyelinating lesions of the central nervous system. A prior study indicated that immunosuppression markedly reduced the early inflammatory lesions. To further explore immunological determinants in the pathogenesis of visna, infected sheep were immunized 3 and 5 weeks after intracerebral virus infections and killed at 8 to 9 weeks. When infected autologous cells in Freunds complete adjuvant (FCA) were used as immunogen, there was no evidence of an accelerated immune response and no effect on lesion severity. When purified concentrated virus in FCA was used as immunogen, there was an accelerated immune response and a modest increase in lesion severity. Because visna virus replication is very slow in vivo, it was hypothesized that the extent of the antigen target might be a limiting parameter in disease progression. Comparison of 2 virus inocula, 10 8 and 10 5 TCD50, indicated that the larger dose was associated with more severe lesions. This is consistent with the view that the size of antigen target limits lesion severity, and could explain why immunization has only a modest influence on the extent of CNS pathology.

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.

Pathogenesis of Visna

Visna, the prototype of slow infections (Sigurdsson, 1954), is a menin-goencephalitis of sheep, caused by a retrovirus (Petursson et al., 1979) which belongs to the group of lentiviruses and is related to the recently isolated human virus that causes AIDS (Sonigo et al., 1985).

Primary demyelination in visna

SummaryTwo Icelandic sheep with clinical signs of visna appearing 6–7 years after intracerebral infection with visna virus were killed, fixed by perfusion and the central nervous system lesions examined by light and electron microscopy. Both sheep showed similar pathological changes. In the brain there was a severe periventricular inflammatory process with small foci of liquefaction necrosis and scattered small granulomas. In some areas of inflammation there was evidence of primary demyelination but it was not prominent. In the spinal cord there were focal plaques of primary demyelination. At the ultrastructural level the spinal cord lesions showed unambiguous primary demyelination with many naked axons; various stages of remyelination with peripheral type of myelin were also common.These observations indicate that the CNS lesions of visna, as seen in Icelandic sheep, fall into two categories: (a) an inflammatory process which often begins within weeks of infection and which occurs in the majority of infected animals in the absence of clinical paresis; and (b) focal demyelinating lesions of the spinal cord which are seen in sheep with clinical paresis but are uncommon in animals prior to onset of clinical signs. Both types of lesions may coexist.

Biological and Genetic Differences Between Lung- and Brain-Derived Isolates of Maedi-Visna Virus

During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.

The ultrastructure of early visna lesions

SummaryThe ultrastructure of visna, a slowly progressive meningo-encephalomyelitis of sheep, was studied in animals sacrificed one month after intracerebral inoculation of visna virus.The major pathological changes, representative of those seen during the first year after infection, consist of inflammation and minor focal destructive lesions of grey and white matter. The inflammatory infiltrates, both subependymal and perivascular as well as of the choroid plexus, were composed mainly of lymphocytes and macrophages with varying numbers of plasma cells. The demyelination seen was of the secondary or Wallerian type. There was no evidence of primary demyelination. Visna virions were not seen in any of the CNS material studied.The ultrastructural findings are compatible with the view that lesions in visna may be induced by a cell-mediated immune response. However, changes characteristic of an autoimmune reaction to myelin antigens were not observed.