Host range of small-ruminant lentivirus cytopathic variants determined with a selectable caprine arthritis- encephalitis virus pseudotype system.

The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.

Plasmid DNA encoding caprine interferon gamma inhibits antibody response to caprine arthritis-encephalitis virus (CAEV) surface protein encoded by a co-administered plasmid expressing CAEV env and tat genes.

This study characterized immune responses of Saanen goats co-immunized with pUC18 based plasmids expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) and a second plasmid encoding caprine interferon gamma (IFNgamma). A previous study reported that immunization with pENV or pTAT-ENV induces IgG2 biased antibody responses to plasmid encoded CAEV surface envelope protein (SU). We show here that regional lymph nodes (LN) of pENV and pTAT-ENV immunized goats contain a dominant subset of SU activated IFNgamma+ Th1 lymphocytes. Co-immunization with pENV or pTAT-ENV and a caprine IFNgamma cDNA expression plasmid (pcIFNgamma) did not potentiate activation of SU responsive Th1 lymphocytes. However, the antibody response to SU encoded by pTAT-ENV was inhibited by co-immunization with pcIFNgamma. Results indicate that synergistic effects of CAEV Tat and IFNgamma suppress the primary adaptive B cell response to plasmid encoded SU.

Conservation of human immunodeficiency virus type 1 gp120 inner-domain sequences in lentivirus and type A and B retrovirus envelope surface glycoproteins.

We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47-54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand beta 25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for beta-strand 5 in the C2 region, which is antiparallel to beta 25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for beta 5 and part of the preceding strand beta 4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in beta 4. In addition, the beta 4/beta 5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel beta-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.

Immunization with plasmid DNA expressing the caprine arthritis-encephalitis virus envelope gene: quantitative and qualitative aspects of antibody response to viral surface glycoprotein.

Saanen goats were vaccinated intradermally with plasmid DNA expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) or vaccinia virus expressing CAEV env (rWR-63). Sera from all vaccinated goats immunoprecipitated CAEV surface (SU) and transmembrane (TM) glycoproteins with a dominant response to SU. Antibody response to CAEV SU induced by plasmid DNA was relatively biased toward IgG2, whereas vaccinia rWR-63 induced predominantly IgG1 antibodies to SU. Differential IgG isotype bias established by immunization with plasmid or vaccinia vectors was maintained following subcutaneous boost with purified CAEV SU in Freund's incomplete adjuvant (FIA). Goats injected with pUC18 control plasmid followed by immunization with SU-FIA also had IgG2 biased responses, whereas SU-FIA immunization of a goat primed with vaccinia rWR-SC11 without the CAEV env gene induced a predominant IgG1 response. We conclude that pUC based plasmids expressing the CAEV env gene promote stable type 1 biased immune responses to plasmid encoded SU. IgG2 biased response may be due to innate type 1 priming capacity of immunostimulatory CpG motifs in the pUC ampicillin resistance gene.

Sequence similarity between the envelope surface unit (SU) glycoproteins of primate and small ruminant lentiviruses.

Sequence similarity has been previously described in the transmembrane domain unit of envelope glycoproteins of primate and non-primate lentiviruses but similarity between the surface unit (SU) glycoprotein of these viruses is less clear or absent. Here we describe a consistent and significant sequence-similarity between the ovine/caprine lentivirus surface glycoprotein gp135 and the primate lentivirus gp120 in the region between variable loops V2 and V3. The biological relevance of this sequence similarity was indicated by clustering of conserved motifs in regions of structural importance in the human immunodeficiency virus type 1 gp120, conservation of cysteine residue pairs forming disulfide bonds and similar patterns of sequence variation in gp135 and gp120 between strains. The results indicate that SU glycoproteins from primate and small ruminant lentiviruses have structurally related domains.

Immune response to caprine arthritis-encephalitis virus surface protein induced by coimmunization with recombinant vaccinia viruses expressing the caprine arthritis-encephalitis virus envelope gene and caprine interleukin-12.

The objective of this study was to determine if interleukin (IL)-12 can focus an antigen specific type 1 immune response characterized by activation of Th1 lymphocytes and production of IgG2 antibodies in vivo. Saanen goats co-immunized with recombinant vaccinia viruses expressing caprine IL-12 (rRB-IL12) and the caprine arthritis-encephalitis virus (CAEV) envelope (env) gene (rWR-63) were evaluated for development of immune responses to the CAEV env encoded surface glycoprotein (SU). Immune responses were defined by: (i) SU antibody titers; (ii) the ratio of SU IgG1 and IgG2 antibodies; (iii) interferon gamma (IFNgamma) and IL-4 gene expression and proliferative response of SU stimulated lymph node mononuclear cells (LNMC). Apart from enhancement of IFNgamma and IL-4 gene expression in SU stimulated LNMC, rRB-IL12 did not affect the immune response to rWR-63 encoded SU. Thus, localized production of exogenous species specific IL-12 at the site of immunization did not focus initial priming of antigen reactive Th lymphocytes. These results are in contrast to previous studies using inbred mice and raise questions regarding the use of cytokine adjuvants to focus immune responses in outbred animals.

Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.

The rhoptry-associated protein 1 (RAP-1) expressed by all babesial parasites is encoded by tandemly arranged genes separated by discrete intergenic (IG) regions. We hypothesize that these IG regions regulate rap-1 gene expression. In Babesia bovis two identical rap-1 gene copies are separated by a 1.0-kb noncoding region which is also exactly conserved 5' to the rap-1 gene 1. In contrast, the complex B. bigemina rap-1 locus contains at least 5 polymorphic rap-1a genes separated by uncharacterized 3.38-kb regions. A genomic clone encoding the 3' sequence of rap-1 gene copy 1, the 1 kb IG region, and the 5' sequence of gene copy 2 was obtained by PCR amplification of DNA from the Mo7 biological clone of B. bovis and sequenced. This was follow by amplification and sequence analysis of the 3.38-kb region separating two B. bigemina rap-1a genes, revealing the presence of two different IG regions denominated IG-1 (0.7 kb) and IG-2 (1.3 kb), flanking a newly identified rap-1b orf. Sequence analysis and comparison among babesial rap-1 IG regions from B. bovis, B. bigemina, B. canis, and B. ovis revealed conservation of at least three putative regulatory boxes consistently positioned 5' of the start of the rap-1 orfs. To determine whether rap-1 IG regions contained a functional promoter, the entire 1-kb IG region from B. bovis was cloned into pCAT, a promoterless plasmid containing the cat gene. The IG region in the 5' --> 3' orientation strongly promoted transcription in vitro by homologous B. bovis RNA polymerases. The presence of conserved regions 5' to each rap-1 gene copy and among other babesial rap-1 IG regions and the in vitro promoter function in the 5' --> 3' orientation support a role for the IG region in rap-1 gene regulation.

Immunodominant T-cell antigens and epitopes of Babesia bovis and Babesia bigemina.

Despite convincing evidence that T cells are critical for both cellular and humoral immunity against haemoprotozoan parasites, the difficulty of performing meaningful experiments in cattle that would define the role of T cells in immunity to Babesia spp. has impeded research in this area. However, experiments performed ex vivo with immune T cells can reflect in-vivo events, and provide valuable insight into the nature of immunogenic proteins and the responding lymphocytes. The progress made towards identification of the immunogenic proteins and epitopes that stimulate anamnestic CD4+ type-1 (interferon-gamma-producing) T-cell responses in cattle immune to challenge with Babesia bovis or B. bigemina is the subject of the present review.

Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus.

The complexity of multigene families encoding rhoptry proteins and the generation of new variants in these families are constraints to development of vaccines incorporating rhoptry proteins. For example, the Babesia bigemina rhoptry associated protein (rap)-1 locus is composed of tandemly arranged genes including four polymorphic rap-1a genes and two classes of divergent genes, rap-1b and rap-1c. B. bigemina rap-1 polymorphism reflects recombination and gene conversion and results in multiple RAP-1 proteins with unique B- and T-cell epitopes. Is this complex locus structure and recombination a required feature of the rap-1 gene family among Babesia species? We addressed this question by analysis of the rap-1 locus in B. bovis. Sequence analysis of an 11 kb genomic clone representing the B. burn rap-1 locus revealed only two identical and continuous rap-1a gene copies, rap 1a-1 and rap-1a-2, located in a similar head to tail orientation. Using the conserved ig gene as a marker for the 3' boundary of the rap-1 locus, we conclude that divergent rap-1b and rap-1c genes, present in B. bigemina, are not similarly cis-linked to the B. bovis rap-1 locus. Analysis of the rap-1a genes 1 and 2 from each of multiple B. bovis strains from North and South America demonstrated RAP-1 size conservation with very limited amino acid sequence variation. The results suggest that the simple two gene arrangement in the B. bovis rap-1 gene family was generated by gene duplication and, in contrast to the B. bigemina rap-1 locus, both genes evolved together using homogenization mechanisms with point mutation as the single mechanism for gene variation. Three discontinuous non-rap-1 genes are closely cis-linked to the B. bovis rap-1 locus and the presence of multiple introns in these genes may limit rap-1 gene variation due to unequal crossing over. The different mechanisms likely involved in the evolution of the rap-1 family in B. bigemina versus B. bovis are reflected in the marked structural and antigenic polymorphism in the B. bigemina RAP-1 molecules as compared with the essentially monomorphic RAP-1 in B. bovis.

Helper T-cell epitopes encoded by the Babesia bigemina rap-1 gene family in the constant and variant domains are conserved among parasite strains.

Among important candidates for babesial vaccines are apical complex proteins, including rhoptry-associated protein 1 (RAP-1) from Babesia bovis and B. bigemina, which have been shown to induce partial immunity. Four variant B. bigemina rap-1 transcripts identified in a clone of the Mexico strain have highly conserved sequence in the central region but vary in sequence at the amino and carboxy termini (NT and CT) of the predicted proteins, resulting in different combinations of NT and CT domains in the individual gene products. Cattle were immunized with native protein consisting of the RAP-alpha1 variant, which contains NT-1 and CT-1 domains, and T-cell responses were characterized. We previously reported the identification of two T helper (Th) cell epitopes in B. bigemina RAP-1alpha1 protein (I. Hötzel, W. C. Brown, T. F. McElwain, S. D. Rodriguez, and G. H. Palmer, Mol. Biochem. Parasitol. 81:89-99, 1996). One epitope mapped to the constant domain of RAP-1 (amino acids [aa] 144 to 187), and one mapped to the CT-1 variable domain (aa 386 to 480). Th1-like clones responding to these epitopes proliferated differentially to different strains of B. bigemina, raising the possibilities that the T-cell epitopes may vary antigenically and that CT-1 may be differentially expressed with respect to the other RAP-1 CT domains in the different strains. In this report, we definitively map the T-cell epitope identified in the constant domain of RAP-1 to aa 159 to 187 (FVVSLLKKNVVRDPESNDVENFASQYFYM) and show that the predicted amino acid sequence is completely conserved among seven strains. The T-cell epitope in the CT-1 domain was mapped to aa 436 to 465 (VNSEKVDADDAGNAETQQLPDAENEVRADD), which is also completely conserved among eight strains of B. bigemina. We further show that the RAP-1alpha1-immunized cattle were protected against homologous B. bigemina challenge, thus suggesting an association between protective immunity and the helper T-cell response against the two epitopes. The immunogenic and highly conserved nature of these T-cell epitopes and their ability to stimulate functionally relevant Th cells that express gamma interferon support their inclusion in a vaccine.

Genetic variation in the dimorphic regions of RAP-1 genes and rap-1 loci of Babesia bigemina.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle. RAP-1 has two regions of sequence dimorphism at the carboxy and amino terminal ends, respectively. Neutralization-sensitive, surface-exposed B-cell epitopes are present in the amino terminal variant type 1 (NT-1), and CD4+ T-cell epitopes in the carboxy terminal variant type 1 (CT-1). Importantly, antibodies recognizing NT-1 epitopes do not cross react with NT-2 and CD4+ T-cells recognizing epitopes in CT-1 do not cross react with CT-2, suggesting that variation in dimorphic regions of RAP-1 is immunologically significant. We evaluated rap-1 locus structure and the extent of sequence variation in the dimorphic regions of rap-1 genes from geographically diverse strains of B. bigemina. All strains contained NT-1 and NT-2 the encoding sequences were highly conserved, with at least 99%, nucleotide identity among strains. However, the Puerto Rico strain encoded a hybrid NT-1/NT-2 sequence which appears to have originated by a gene conversion event. The 3' ends of rap-1 genes, which include the carboxy terminal variants, are conserved among strains. A new and conserved CT variant (CT-3), with a region of sequence identity to CT-2 and a sequence not related to either CT-1 or CT-2, was identified in all strains of B. bigemina. All but one strain encode both NTs and the three CT variants. The S1A strain, an attenuated strain from Argentina, does not encode CT-2. While NT-1 is associated only with CT-1, NT-2 can be associated with all three CT variants in RAP-1. Within the genome, rap-1 genes are arranged in tandem repeats but with different gene copy number and arrangements among strains. Collectively, the data suggest that gene conversion and unequal recombination events contribute to overall rap-1 sequence conservation among gene variants and strains but may also generate new rap-1 variants.

Dimorphic sequences of rap-1 genes encode B and CD4+ T helper lymphocyte epitopes in the Babesia bigemina rhoptry associated protein-1.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle and contains neutralization-sensitive B cell epitopes. RAP-1 variants containing blocks of sequence dimorphism in the amino and carboxy terminal ends are encoded by four nonallelic genes in B. bigemina. Epitopes recognized by RAP-1 specific monoclonal antibodies (MAbs) and bovine CD4+ T cell clones were mapped to determine whether these epitopes are localized in the amino and carboxy terminal dimorphic regions. Four B cell epitopes, including a neutralization-sensitive epitope, required both the amino terminal variant type 1 (NT-1) and non-dimorphic sequences for conformation. Intrachain disulfide bonds were required for at least one of these epitopes, since reduction and alkylation of cysteine residues abolished MAb binding. A fifth B cell epitope was mapped to the carboxy terminal variant type 1 (CT-1). As expected, the neutralizing MAb and two other MAbs requiring NT-1 for epitope binding recognized only the two RAP-1 variants with the NT-1 sequence, while the MAb binding an epitope in CT-1 did not bind RAP-1 variants with CT-2. In contrast, the fourth MAb requiring NT-1 for binding recognized all rap-1 gene products, indicating that dimorphic residues are not part of the epitope recognized by this MAb. Bovine CD4+ T cell clones characterized previously as responding in a strain dependent fashion recognized at least one epitope in CT-1, and did not cross-react with CT-2. A second group of bovine CD4+ T cell clones that responded to multiple parasite strains recognized an epitope in a non-dimorphic region of RAP-1. These data indicate that dimorphic regions of RAP-1 encode unique B and T helper lymphocyte epitopes and may be required for enhanced protective immune responses in cattle.

Differentiation of classical swine fever virus from ruminant pestiviruses by reverse transcription and polymerase chain reaction (RT-PCR).

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was designed to allow the differentiation of pestiviruses by the expected size of the amplified fragments. One oligonucleotide primer, conserved amongst pestiviruses, and two others specific for either classical swine fever virus (CSFV) or bovine viral diarrhea virus (BVDV), were designed from the 5' non-coding region of the genome. CSFV infected cultures (10 strains) amplified a fragment of an expected size of 200 bp; BVDV cultures (23 strains) or border disease virus (BDV) (2 strains) amplified a fragment of an expected size of 260 bp. The specificity of the amplified fragments was confirmed by restriction enzyme analysis. The threshold of sensitivity was 100 TCID50 for CSFV and 1 TCID50 for BVDV. The RT-PCR described here provides a rapid and sensitive diagnostic tool for the detection and differentiation of CSFV from ruminant pestiviruses.

Small extrachromosomal nucleic acid segments in protozoan parasites.

Viruses have been described in the following protozoa: Babesia spp., Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis and Eimeria spp. In order to study the Babesia bovis virus, merozoites have been prepared from the blood of infected cattle. Agarose gel electrophoresis of nucleic extracts from the bovine protozoa B. bovis and Babesia bigemina were separated into genomic DNA and at least two additional nucleic acids. One molecule with a relative mobility of 5.5 kilobase pairs (kbp) was identified as a double-stranded RNA virus-like particle. Another 6.2 kbp DNA molecule had sequences related to mitochondrial genome.

Caprine arthritis-encephalitis virus: isolation and identification in Rio Grande do Sul, Brazil.

We describe the first report of caprine arthritis-encephalitis virus (CAEV) isolation in Brazil. In December 1989 a four-year old anglonubian arthritic doe was submitted to euthanasia and its synovial membranes were cultured by explantation. This doe was positive for antibodies to the caprine arthritis-encephalitis virus (CAEV). The reverse transcriptase activity detected in culture supernatants, the characteristic cytopathic effect of lentivirus in these cultures, the detection of viral antigens in concentrated supernatants by the agar gel immunodiffusion test, and the results of the fluorescent antibody test and of Northern blot analyses are consistent with the isolation of the CAEV.

Caprine arthritis-encephalitis virus: isolation and identification in Rio Grande do Sul, Brazil

We describe the first report of caprine arthritis-encephalitis virus (CAEV) isolation in Brazil. In December 1989 a four-year old anglonubian arthritic doe was submitted to euthanasia and its synovial membranes were cultured by explantation. This doe was positive for antibodies to the caprine arthritis-encephalitis virus (CAEV). The reverse transcriptase activity detected in culture supernatants, the characteristic cytopathic effect of lentivirus in these cultures, the detection of viral antigens in concentrated supernatants by the agar gel immunodiffusion test, and the results of the fluorescent antibody test and of Northern blot analyses are consistent with the isolation of the CAEV

Extrachromosomal nucleic acids in bovine Babesia.

Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.