Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.

Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.

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.

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.

In vivo and in vitro infection with two different molecular clones of visna virus.

The behavior of two genetically different molecular clones of visna virus KV1772-kv72/67 and LV1-1KS1 was compared in vivo and in vitro. On intracerebral inoculation, clone KV1772-kv72/67 induced a similar response in five sheep as has already been reported with neurovirulent derivates of visna virus. Virus was frequently isolated from blood, cerebrospinal fluid (CSF), and lymphoid organs and induced characteristic central nervous system (CNS) lesions. A strong humoral immune response was detected by ELISA, immunoblotting, and neutralization. Six sheep infected with clone LV1-1KS1 showed a completely different picture. No virus could be isolated from blood or CSF during 6 months of infection. At sacrifice all organs were virus-negative except the CNS of one sheep. None of the six sheep developed significant neutralizing antibodies and only low titer antibodies were detected by ELISA and immunoblotting. Minimal CNS lesions were present in one sheep. The molecular clones were also tested in sheep choroid plexus cells (SCP) and macrophages. In macrophages LV1-1KS1 replicated to a significantly lower titer but induced much more cell fusion than KV1772-kv72/67. The clones replicated equally well in SCP cells. Thus, these molecular clones of visna virus, which differ only by 1% in nucleotide sequence, showed a profound difference in replication and pathogenicity both in vitro and in vivo. These results can be used to map viral genetic determinants important for host-lentivirus interactions.

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.

[Visna and AIDS--various comparative aspects]. / Visna og AIDS--nogle komparative aspekter.

Visna, a lingering meningo-encephalitis in sheep, was one of the diseases on which B. Sigurdsson based his theory of a special group of disorders called slow infections. The cause of the disease, a retrovirus, was isolated in 1957. Visna is now classified in a subgroup of retroviridae, lentivirinae, together with virus types in animals and the human immune deficiency virus HIV. Notwithstanding that Visna has been eradicated among Icelandic sheep for 25 years research into this virus continues since it bears many similarities to HIV and also MS.

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.

Maedi-visna in sheep: host-virus interactions and utilization as a model.

Controlled animal experiments with the ovine maedi-visna virus, the prototype lentivirus, have been carried out for almost 40 years. This non-oncogenic virus leads to a life-long, persistent infection with slow development of lesions in the lungs and in the central nervous system. The virus is present in many cells in a DNA provirus state and its replication and expression is highly restricted in vivo. The basic biological features of maedi-visna virus are quite similar to those of HIV and this ovine lentiviral disease may be useful as a model for infection with human lentiviruses.

Detection of visna virus antigens and RNA in glial cells in foci of demyelination.

Visna is a slow virus infection of sheep in which the characteristic pathological change is demyelination in foci of inflammation. The latter is thought to be the result of an immunopathological process directed against cellular and antigenic targets that have been difficult to define because of restricted viral gene expression. A new simultaneous detection assay is used to demonstrate viral RNA in cells identified unambiguously as oligodendrocytes and astrocytes. These cells were found in inflammatory foci. With a new strain of virus that causes a rapid form of visna in Icelandic sheep, viral antigens were demonstrated in cells in the inflammatory lesions. These findings are consistent with the postulated immunopathological mechanism of demyelination: cells that maintain intact myelin sheaths in the central nervous system are destroyed by the inflammatory response to viral antigens expressed in these cells.

Primary demyelination in visna. An ultrastructural study of Icelandic sheep with clinical signs following experimental infection.

Two 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.

An ultrastructural study of the cerebrospinal fluid in visna.

An electron microscopic examination was done on 8 samples of cerebrospinal fluid (CSF) from Icelandic sheep infected by the intracerebral route with visna virus. The specimens were collected 1 month, 2 months, and 4 years after infection. A differentail cell count done on low-power electron micrographs showed that the cellular exudate was composed of mononuclear cells mainly macrophages and lymphocytes with a few plasma cells. Macrophages were with one exception more numerous than lymphocytes and an increased proportion of macrophages showed evidence of phagocytosis with time after infection. Reactive lymphocytes were in general more numerous than small lymphocytes. Various stages in the maturation of plasma cells were observed. The cellular composition in the CSF is compatible with the view that visna is an immunopathological process. Myelin figures and fragments of myelinated axons were observed in two specimens indicating an active myelin-breakdown. The possibility that escape of myelin into the CSF may lead to sensitization to myelin antigens and perpetuation of this chronic neurologic affection is discussed. Visna virions could not be demonstrated.

The ultrastructure of early visna lesions.

The ultrastructure of visna, a slowly progressive menigo-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.