Gudmundur Georgsson

PrP gene polymorphism and natural scrapie in Icelandic sheep.

The association between scrapie and polymorphism of the prion protein (PrP) gene was studied in the Icelandic sheep breed. Polymorphism of the three codons, 136, 154 and 171, that are important for scrapie susceptibility was determined. A BspHI restriction analysis was used to study the alleles of codons 136 and 154, while density gradient gel electrophoresis (DGGE) was used to analyse codon 171 and detect new polymorphisms. The PrP allelic variant, VRQ (amino acids at codons 136, 154 and 171), was found to be highly statistically associated with scrapie, whereas the allelic variant, AHQ, was never found in scrapie-affected animals, a finding that is statistically significant. Iceland has a few scrapie-free regions, which are a part of a quarantine network. Homozygotes for the VRQ variant were found there at a low frequency, indicating that genetic susceptibility is not enough for scrapie to develop and further evidence for the infectious nature of the disease. A comparison of PrP genotypes between sheep outside and within the scrapie-free zones revealed an increase in the AHQ allelic variant in the latter. No polymorphism was found at codon 171 in a total of 932 sheep studied, all individuals having the glutamine allele. Two novel, rare PrP alleles were found using DGGE at codons 138 and 151, i.e. S138N and R151C. Their relevance to scrapie is still unclear, but the former was found in scrapie-affected sheep as well as healthy sheep, whereas the latter was only found in healthy sheep.

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.

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.

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

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.

Duplicated Sequence Motif in the Long Terminal Repeat of Maedi-Visna Virus Extends Cell Tropism and Is Associated with Neurovirulence

ABSTRACT Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.

Search for healthy carriers of scrapie: an assessment of subclinical infection of sheep in an Icelandic scrapie flock by three diagnostic methods and correlation with PrP genotypes

Summary. Subclinical infection in scrapie of sheep, characterized by a long incubation period, may be of importance for the spread of the disease. We screened brain samples from all 65 sheep in a scrapie-affected flock for subclinical infection and correlated with results of PrP genotyping, which is of relevance for the epidemiology and the question, whether by breeding for resistant genotypes one would be breeding for healthy carriers. The sensitivity of three methods was compared, i.e. histopathological examination for vacuoles (HP), immunohistochemical staining (IHC) and Western blotting (WB) for PrPSc. Five sheep showed definite clinical signs and histological scrapie lesions, and signs of infection were detected in 25 of 60 asymptomatic sheep, by HP and/or IHC and WB. The IHC was slightly more sensitive than HP and WB. Sheep with subclinical infection were, with one exception, either homo- or heterozygotes for 136-V, as were four of the five sheep with clinical scrapie. The incidence of the VRQ allelic variant in the flock was unusually high compared to the Icelandic sheep population probably contributing to the high prevalence of both clinical and subclinical infection in the flock. Neither sheep with definite scrapie nor detectable subclinical infection, were of the resistant AHQ genotype, indicating that Icelandic AHQ sheep are not healthy carriers of scrapie infection.