Affinity (tropism) of caprine arthritis encephalitis virus for brain cells.

One of the constraints in unraveling the mysteries blurring the advancement of research in the quest to totally put HIV problems under control is getting the appropriate animal model that would truly simulate human cases. This problem is more apparent in studies involving the central nervous system. Consequently, a viable animal model to generate information for the production of drugs and vaccines for the prevention and or control of lentiviral induced dementia in affected host animals is pertinent and vital. In this study, explant cultures prepared from the brain of new-born goat-kid were infected with CaprineArthritis Encephalitis (CAE) virus- a retrovirus affecting goats. The specific brain cell types infected by the (CAE) virus were determined using reverse-transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM techniques). TEM showed that in 85 - 90% cases, microglia were the cells specifically infected by the virus. Amplification of the genomic sequence of the envelope and the gag genes by RT-PCR confirmed the presence of CAEV proviral DNA in the brain cells of affected animals. No productive infection of the astrocytes was observed. The results of this study showed a lot of similarities in the tropism of CAE virus infection of goat brain cells to that of HIV infection in humans thus suggesting the potential usefulness of the caprine model for the study of HIV neuropathology. The goat model system as a non-primate model therefore could be more adaptable as a simple animal model than primate models with their complexity of anthropological, environmental and safety problems.

Demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats.

Recombination of different strains and subtypes is a hallmark of lentivirus infections, particularly for human immunodeficiency virus, and contributes significantly to viral diversity and evolution both within individual hosts and within populations. Recombinant viruses are generated in individuals coinfected or superinfected with more than one lentiviral strain or subtype. This, however, has never been described in vivo for the prototype lentivirus maedi-visna virus of sheep and its closely related caprine counterpart, the caprine arthritis-encephalitis virus. Cross-species infections occur in animals living under natural conditions, which suggests that dual infections with small-ruminant lentiviruses (SRLVs) are possible. In this paper we describe the first documented case of coinfection and viral recombination in two naturally infected goats. DNA fragments encompassing a variable region of the envelope glycoprotein were obtained from these two animals by end-limiting dilution PCR of peripheral blood mononuclear cells or infected cocultures. Genetic analyses, including nucleotide sequencing and heteroduplex mobility assays, showed that these goats harbored two distinct populations of SRLVs. Phylogenetic analysis permitted us to assign these sequences to the maedi-visna virus group (SRLV group A) or the caprine arthritis-encephalitis virus group (SRLV group B). SimPlot analysis showed clear evidence of A/B recombination within the env gene segment of a virus detected in one of the two goats. This case provides conclusive evidence that coinfection by different strains of SRLVs of groups A and B can indeed occur and that these viruses actually recombine in vivo.

Cultured early goat embryos and cells are susceptible to infection with caprine encephalitis virus.

Zona-pellucida-free embryos at 8-16 cell stage were co-cultured for 6 days in an insert over a mixed cell monolayer infected with CAEV-pBSCA. Embryos were washed and transferred to an insert on CAEV indicator goat synovial membrane cells for 6 h, then they were washed and cultivated in B2 Ménézo for 24 h, finally, embryo cells were dissociated and cultivated on a feeder monolayer for 8 days. After 5 weeks, multinucleated giant cells typical of CAEV infection were observed in indicator GSM cell monolayers. In the acellular medium, the early embryonic cells produced at least 10(3.25) TCID50/ml over 24 h. The monolayer of cultivated embryonic cells developed cytopathic lesions within 8 days, and CAEV RNA, CAEV proviral DNA and protein p28 of the capsid were detected. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with CAEV and that infection with this virus is productive.

Gene transfer system derived from the caprine arthritis-encephalitis lentivirus.

Lentiviruses are attractive candidates for therapeutic vectors, because of their ability to infect non-dividing target cells. Vectors based on HIV-1 efficiently transfer gene expression to a variety of dividing or quiescent cells, but are subject to reservations on safety grounds. Caprine arthritis encephalitis virus (CAEV) is a lentivirus inducing only minor pathology in its natural host and in related species after cross-species transmission. To test the CAEV potential as vector for gene transfer, a cassette expressing the green fluorescent protein (GFP) under control of a CMV promoter was inserted into the CAEV genome, producing the pK2EGFPH vector. When pseudotyped with vesicular stomatitis virus (VSV)-G envelope protein, this vector allowed efficient transfer of GFP expression in human cells (up to 86% of GFP-expressing cells into the TE671 cell line). Three vectors carrying different parts of the viral gag, pol and env genes were then developed, together with a CAEV packaging system. These vectors allowed delimitation of the minimal CAEV sequences necessary for an improvement of vector production compared to the previously described CAEV-based vectors [Mselli-Lakhal et al., 1998. Defect in RNA transport and packaging are responsible for low transduction efficiency of CAEV-based vectors. Arc. Virol. 143, 681-695]. While our previous vectors were produced in a helper/vector system, the present vectors are produced in a helper/free system. However, these vector titers remain lower than those obtained with other lentiviral vectors carrying equivalent packaging sequences. We discuss on possible reasons of such differences and possible improvements.

Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis-encephalitis virus (CAEV).

Caprine oviduct epithelial cells (COEC) are commonly used in in vitro goat embryo production protocols to stimulate early embryonic development. These feeder cells are usually collected from slaughterhouses from unknown serological status animals for caprine arthritis-encephalitis virus (CAEV) infection which is frequent in many regions of the world. Tissues derived from this source may be contaminated with CAEV and the use of such material in in vitro fertilisation systems may contribute to transmission of this pathogen to the cultured embryos and dissemination via embryo transfer (ET). The aim of this study was to determine the permissiveness of COEC to CAEV replication in vitro. Cells were isolated from goats from certified CAEV-free herds and then were inoculated with two CAEV strains: the molecularly-cloned isolate of CAEV (CAEV-pBSCA) and the French field isolate (CAEV-3112). Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using a hyperimmune serum. The CAEV proteins were correctly and properly processed by artificially-infected COEC and the titers of virus released into the supernatant reached 10(6) TCID(50)/ml 6 days post-inoculation. Although the macrophage lineage cells are the main centre of infection in the virus-positive animal, these findings suggest that epithelial cells may be important in the viral life cycle probably as a reservoir allowing the viral persistence, dissemination and pathogenesis. These results suggest also that the use in in vitro fertilisation systems of co-culture feeder cells that support efficient replication of CAEV to high titers could represent a serious risk for permanent transmission of virus to the cultured embryos and to the surrogate dam involved.

[Identification of caprine arthritis encephalitis virus using histopathological, immunohistochemica, and ultrastructural examination of tissues from seropositive goats in Mexico]. / Identificación del virus de la artritis encefalitis caprina mediante el estudio histopatológico, inmunohistoquímico y ultraestructural, en tejidos de cabras seropositivas en México.

This study demonstrated the presence of viral particles suggestive of arthritis-encephalitis virus through the clinic, serologic, pathologic, immunohistochemic and ultrastructural studies in dairy goats. In the postmortem studies the tissues used were: synovial membrane, lungs and mammary gland from goats with clinical signs characteristic of the disease and positive for serum antibodies to the virus.

The response of goat brain cells to infection by caprine arthritis-encephalitis virus.

Caprine arthritis-encephalitis virus is a lentivirus of goats causing encephalomyelopathy acutely in young goats and occasionally in late stages of chronically infected adult goats. To determine the specific brain cells infected by the virus and the response of other cell types, goat cerebrum explant cultures prepared from newborn goat kids were infected by virus application. Infection of brain cultures was also possible by coculture with virus-infected macrophages. Predominantly, microglia cells were specifically infected by the virus, resulting in death of more than 50% of the cells within 2 weeks of infection. Infected cells enlarged and formed multinucleated giant cells. Infection of oligodendrocytes was observed, but infrequently. Astrocytes were not infected. Proviral deoxyribonucleic acid (DNA) was detected in infected cultures by amplification of a 490 base-pair fragment of the viral gag gene by the polymerase chain reaction (PCR) technique. Virus budding from plasma membrane of infected microglia cells was demonstrated by transmission electron microscopy. The supernatant of infected brain cells induced cytopathic effect when applied to indicator cells. These observations suggest productive infection of brain cultures. Virus infection of neurons was not observed. Culture of neurons in virus-free supernatant of CAEV-infected microglia or mixed glia culture resulted in death of 50% of the neurons in 11 days, suggesting the production of neurotoxic substances by the virus-infected glial cells.

Caprine arthritis-encephalitis lentivirus SU is the ligand for infection of caprine synovial membrane cells.

Infection of goat synovial membrane (GSM) cells by caprine arthritis-encephalitis virus (CAEV) was inhibited by incubation of cells with CAEV gp 135 envelope glycoprotein (SU) expressed by recombinant vaccinia virus. Incubation of cells with protein expressed by a control recombinant vaccinia virus without the CAEV envelope gene did not inhibit CAEV infection. Removal of recombinant SU from blocking medium by adsorption with anti-SU IgG/protein G-Sepharose complexes resulted in loss of CAEV inhibition. Results support that CAEV infection of GSM cells is mediated by a specific interaction between SU and a cell surface receptor or receptor complex.

Pestivirus is a common contaminant in maedi-visna and caprine arthritis-encephalitis virus stocks.

Eight different laboratory stocks of maedi-visna or caprine arthritis-encephalitis virus were examined for the presence of pestiviruses by a fixed-cell immunoperoxidase assay with polyclonal and monoclonal antibodies. All of the viral stocks examined were found to contain noncytopathic pestivirus contaminants. The panel of monoclonal antibodies could not type the isolates as being more related to bovine virus diarrhea virus or border disease virus. However, the results did indicate that all isolates were not the same, except for two from the same laboratory where the source of pestivirus contamination may have been common.

Defective viral particles in caprine arthritis encephalitis virus infection.

Attempts to isolate full-length unintegrated circular forms of the caprine arthritis encephalitis virus (CAEV) genome yielded only a large number of molecules with deletions. The 3' borders of most of these deletions were near the U3 region of the long terminal repeat whereas the 5' edges were found at various upstream sites within pol or env. With one exception, gag sequences were always present. Analysis of molecular clones derived from integrated proviral CAEV genomes from the same infected cells showed a similar spectrum of deletions. The presence of transcriptionally active elements within the U3 domain of the defective genomes, as well as cis-acting elements within the leader sequences known to be required for efficient encapsidation of viral RNA, suggested that the defective viral DNA genomes could be transcribed into defective RNA molecules which could then be packaged into virions. Isopycnic density gradient centrifugation of supernatants of infected cell cultures indicated the presence of particles with densities less than that expected for intact virions (1.16 g/cc). Northern analysis revealed the presence of smaller viral-specific RNAs that lacked env sequences. These data, along with the structures of the molecular clones, suggest that CAEV stocks contain particles with defective genomes. The role of these particles in influencing the course of virus infection remains to be determined.