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.

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.Caprine arthritis-encephalitis virus (CAEV) of goats and maedi-visna virus (MVV) of sheep belong to the genus Lentivirus of the family Retroviridae (18,22,39). Strong evidence indicates that cross-species transmission from sheep to goats and vice versa occurs under field conditions (16, 24, 33). Therefore, these viruses are no longer considered to be species specific and are referred to as small-ruminant lentiviruses (SRLV).The majority of infected animals mount a strong immune response to these viruses but remain persistently infected. Only one-third of infected goats develop overt clinical disease, and in sheep the percentage of animals with clinical symptoms differs greatly between breeds, strongly suggesting that, in both species, genetic factors play a key role in determining the clinical outcome of infection (11,28,29).In several countries, eradication programs have been initiated to control SRLV-induced diseases with the aim of eliminating these viruses (23). The Swiss CAEV eradication program, initiated in 1984, has reduced the seroprevalence from 60 to 80% to less than 1% and eliminated clinical cases in the goat population (14, 15). However, in the last phase of the eradication program, detection and elimination of the remaining virus carriers appear to be very difficult. The serological tools currently used (37, 38) are of limited use when applied to screen a population with a low seroprevalence. In the absence of reliable tools to directly detect SRLV, consistent serological diagnoses depend on the costly use of a combination of tests run in parallel (4). Major problems in SRLV serology are slow seroconversions and low titers of antibody to Gag in some animals. In this respect, the strong immunogenicity of the envelope glycoprotein (Env) and especially the rapid seroconversion induced by this antigen in infected animals make it an ideal candidate for diagnostic applications (2, 19). Goats and sheep infected with SRLV develop high titers of antibodies to several conformational and linear ...

BackgroundIn past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus.ResultsUsing GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter.ConclusionCombining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

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