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.     

Viral and cellular specificities of caprine arthritis encephalitis virus Vif protein

The caprine arthritis encephalitis virus (CAEV) Vif protein is necessary for a productive infection of susceptible goat cells. The vif gene is conserved among all primate and most nonprimate lentiviruses. However, previous reports demonstrated that, in their respective host cells, primate Vif deleted lentiviruses could not be complemented by nonprimate Vif proteins, suggesting that species-specific restrictions between Vif and the virus-producing cells may modulate the Vif function on viral infectivity. Here we bring further support to this hypothesis since we show that CAEV Vif, when expressed in goat cells, is able to increase the infectivity of Vif deleted human immunodeficiency type-1 virus and of murine leukemia virus. Moreover, we demonstrate in vitro interactions between different Vif proteins and NC domains of heterologous Gag precursors, supporting the notion that species specificity of lentiviral infection is not due to molecular interactions between Vif and viral components.     

Sequence comparison on gag gene of caprine arthritis encephalitis virus from Korea

Caprine arthritis encephalitis (CAE) is a lentiviral infection in goats characterized by arthritis. Although CAE has been identified in most goat-rearing countries around the world, Korea had been considered to be free from this disease. Here, we determine the partial sequence on the gag gene of caprine arthritis encephalitis virus (CAEV) suspect from Korea using nested PCR. Genetic analysis of our isolate showed 93.5 and 90.9% sequence homology with the standard strain CAEV Cork and lentivirus strain, respectively. Our results show that the virus is closely related to other CAEV isolates, and therefore, suggest that the causative agent was CAEV. This is the first outbreak of CAE to be ever reported in a local farm in South Korea.     

Specific G2 arrest of caprine cells infected with a caprine arthritis encephalitis virus expressing vpr and vpx genes from simian immunodeficiency virus

Primate lentivirus (HIV and SIV) vpr accessory genes encode 12- to 14-kDa proteins which induce cell cycle arrest at the G2 phase of infected cells, preventing them from going through mitosis. Members of the HIV-2/SIVmac/SIVsmm group also encode a second closely related accessory protein called Vpx. Vpx and HIV Vpr are critical for virus replication in nondividing cells due to their participation in nuclear import of the preintegration complex. Caprine arthritis encephalitis virus (CAEV) and maedi visna virus are the natural lentiviruses of domestic goat and sheep, respectively, and their genomes do not carry vpr and vpx genes. In this study, we generated chimeric CAEV-based genomes carrying vpr and vpx genes from SIVmac239 and tested their ability to induce G2 cell cycle arrest in infected caprine cells. CAEV-pBSCAvpxvpr is the chimeric genome that was shown to be infectious and replication competent. Our data demonstrated that CAEV-pBSCAvpxvpr-infected goat synovial membrane cell monolayer developed more cytopathic effects and a high proportion of cells remained in the G2 phase of cell cycle. This G2 arrest was observed both at the early and at the late stages of infection, while minimal effect was observed with the parental CAEV-pBSCA. These results, described for the first time in mammalian cells other than those of primates, indicate that Vpr-induced G2 cell cycle arrest is not restricted to only primate cells. Thus, conservation of Vpx/Vpr protein functions in caprine cells suggests a possible role for these proteins in the virus life cycle and its ability to adapt to new hosts. The data presented here thus raise a pertinent question about the biological significance of the conservation of Vpr and Vpx functions in caprine cells despite the high phylogenic distance between primates and small ruminants.     

Immunohistochemical detection of the p27 capsid protein of caprine arthritis-encephalitis virus (CAEV) in bone-marrow cells of seropositive goats

Bone-marrow samples were collected from 48 CAEV-seropositive, symptomless goats (30 kids, 18 adults). The samples were formalin-fixed and processed for histological examination. In addition, all samples were examined immunohistochemically with a monoclonal antibody (1A7) against the p27 capsid protein of maedi-visna virus, an antibody which cross-reacts with the Ca-p27 of CAEV. Samples from 16 goats (10/30 kids, 6/18 adults) showed positive immunolabelling of bone-marrow stromal cells (fibrocytes, endothelial cells and adipocytes) and of scattered macrophages, whereas haematopoietic cells were negative. The detection of viral Ca-p27 protein in bone-marrow fibrocytes was consistent with previous in-vitro studies which indicated that such cells are semi-permissive for CAEV infection. It is speculated that bone-marrow stromal cells represent a viral reservoir in symptomless animals.     

Tick-borne encephalitis virus interaction with the target cells

Summary The binding of tick-borne encephalitis virus to porcine kidney embryo cells was studied. Anti-idiotypic antibodies against TBE virus E protein precipitated gp 110 kDa which is predicted to be a cellular receptor for TBE virus.     

The polypyrimidine tract-binding protein (PTB) is required for efficient replication of hepatitis C virus (HCV) RNA

Our earlier work found that the polypyrimidine tract-binding protein (PTB) specifically interacts with the poly(U/C) tract of the hepatitis C virus (HCV) 3' untranslated region (3'UTR) [Luo, G., 1999. Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome. Virology 256, 105-118]. We report here that the phosphorylated form of PTB is associated with the membrane-bound HCV replication complex. To determine whether the PTB is required for HCV RNA replication, synthetic small interfering RNAs (siRNAs) were used to specifically knockdown PTB expression in the HCV replicon-harboring Huh7 cells. The level of PTB expression was efficiently reduced by PTB-specific siRNAs. Consequently, the levels of HCV proteins as well as HCV RNA were proportionally decreased by increasing concentrations of PTB siRNA. However, the translation mediated by the encephamyocarditis virus internal ribosome entry site was unaffected, suggesting that the inhibition of HCV RNA replication by PTB siRNA was not due to its effect on the EMCV IRES-mediated translation. Collectively, these findings demonstrate that PTB is required for efficient HCV RNA replication in the cell.     

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.     

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.     

Nonvirion (soluble) antigen of the tick-borne encephalitis virus

Immunoelectrophoretic analysis of the cells destroyed as a result of infection with tick-borne encephalitis virus showed a considerable portion of nonvirion ("soluble") antigen to remain associated with cell membranes and to be released after treatment of the cells with detergents. After treatment with nonionic detergents, the nonvirion antigen showed electrophoretic heterogeneity in immunoelectrophoresis, and after treatment with sodium dodecyl-sulphate behaved as a homogeneous population of protein molecules. Using radioactively labeled carbohydrate precursors, lipids, and RNA, the nonvirion antigen was demonstrated to be a complex structure: a membrane-containing ribonucleoprotein. The immunoprecipitation of proteins by means of antibodies ligated to CNBr-activated sepharose confirmed that protein NV 5 (p93) of tick-borne encephalitis virus was an antigenically active component of the nonvirion antigen. This protein was found to undergo proteolytic cleavage in immunochemical reaction. The possible role of the nonvirion antigen in reproduction of flaviviruses is discussed.     

Lack of trans-activation function for Maedi Visna virus and Caprine arthritis encephalitis virus Tat proteins

All lentiviruses contain an open reading frame located shortly upstream or inside of the env gene and encoding a small protein which has been designated Tat. This designation was mainly with respect to the positional analogy with the first exon of the trans-activator protein of the well studied human immunodeficiency virus type 1 (HIV-1). In this work we comparatively studied the trans- activation activity induced by Tat proteins of the small ruminant Maedi Visna virus (MVV) of sheep and Caprine arthritis encephalitis virus (CAEV) of goats on MVV and CAEV LTRs with that induced by the human lentivirus HIV-1 on its own LTR. The HIV-1 LTR alone weakly expresses the reporter GFP gene except when the HIV-1 Tat protein is coexpressed, the GFP expression is increased 60-fold. In similar conditions only minimal trans-activation increasing two- to three-fold the MVV and CAEV LTR activity was found with MVV Tat protein, and no trans-activation activity was detected in any used cell type or with any virus strain when CAEV Tat was tested. These results indicate that the small ruminant lentiviruses (SRLV) differ from the primate lentiviruses in their control of expression from the viral LTRs and put into question the biological role of the encoded protein named "Tat."     

Equine herpesvirus type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress

The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HDeltagK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HDeltagK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting M(r)s between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.