Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV).

The env gene is an excellent candidate for inclusion in any DNA-based vaccine approach against equine infectious anemia virus (EIAV). Unfortunately, this gene is subjected to mutational pressure in E. coli resulting in the introduction of stop codons at the 5' terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors.

Derivation and characterization of a live attenuated equine influenza vaccine virus.

Objective: To develop and characterize a cold-adapted live attenuated equine-2 influenza virus effective as an intranasal vaccine.

Animals: 8 ponies approximately 18 months of age.

Procedures: A wild-type equine-2 virus, A/Equine/Kentucky/1/91 (H3N8), was serially passaged in embryonated chicken eggs at temperatures gradually reduced in a stepwise manner from 34 C to 30 C to 28 C to 26 C.

Safety, efficacy, and immunogenicity of a modified-live equine influenza virus vaccine in ponies after induction of exercise-induced immunosuppression.

Objective: To determine safety, efficacy, and immunogenicity of an intranasal cold-adapted modified-live equine influenza virus vaccine administered to ponies following induction of exercise-induced immunosuppression.

Design: Prospective study.

Animals: Fifteen 9- to 15-month old ponies that had not had influenza.

Transcutaneous immunization of domestic animals: opportunities and challenges.

Transcutaneous immunization (TCI), the topical application of antigen and adjuvant directly onto intact skin, can safely and effectively elicit systemic immune responses in mice and humans against a variety of antigens. This novel method of vaccine delivery has the potential to provide a safe and convenient method by which vaccines may be delivered to elicit protective immunity in domestic animals. To date, however, immune responses induced by TCI in companion and production animals has not been reported.

Tissue sites of persistent infection and active replication of equine infectious anemia virus during acute disease and asymptomatic infection in experimentally infected equids.

Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA.

Effects of long terminal repeat sequence variation on equine infectious anemia virus replication in vitro and in vivo.

The long terminal repeat (LTR) is reported to be one of the most variable portions of the equine infectious anemia virus (EIAV) genome. To date, however, no information is available on the effects of observed sequence variations on viral replication properties, despite a widespread assumption of the biological importance of EIAV LTR variation. EIAV LTR sequence variability is confined mostly to a small portion of the enhancer within the U3 segment of the LTR.

Immunization of dogs and cats with a DNA vaccine against rabies virus.

The applicability of DNA immunization technology for vaccine development in companion animals was investigated by immunizing dogs and cats by the intramuscular (i.m.) and intradermal (i.

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases.

Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR.

Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses.

Replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dUTPase activity.

As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells.

Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.

Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody.

Lentivirus cross-reactive determinants present in the capsid protein of equine infectious anaemia virus.

In this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80% of sera from long-term experimentally infected ponies or naturally infected horses react with human immunodeficiency virus type 1 CA in Western immunoblot assays.

Enhancement of EIAV replication and disease by immunization with a baculovirus-expressed recombinant envelope surface glycoprotein.

The potential for antibody-dependent enhancement of replication of macrophage/monocyte tropic viruses has posed a significant problem in the development of vaccines for several animal and human viruses and has raised significant concern in the design of potential AIDS vaccines. Using the previously described equine infectious anemia virus/Shetland pony system as a model for HIV-1 vaccine development, we have evaluated the efficacy of a recombinant subunit vaccine containing a baculovirus-expressed envelope surface glycoprotein (gp90) of EIAV. The results of these trials demonstrate not only that the recombinant vaccine failed to protect against infection by standard homologous and heterologous EIAV challenge strains, but that it resulted in a marked enhancement of virus replication and exacerbation of disease in immunized ponies exposed to the heterologous virus strain.

Characterization of infectious molecular clones of equine infectious anaemia virus.

We have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (EIAV). The clones were recovered from fetal equine kidney (FEK) cells infected with a virulent, cell culture-adapted virus stock (designated PV) and have been characterized at a molecular level. Each clone has unique envelope and long terminal repeat (LTR) sequences.

Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant.

The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells.

Lentivirus envelope sequences and proviral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors.

A promoter-selection vector (pKK232-8) was used to identify sequences with strong Escherichia coli promoter activity positioned near the start of the envelope-encoding genes (env) of two lentiviruses, simian immunodeficiency virus (SIV) and equine infectious anemia virus (EIAV). For EIAV, cloning the cryptic promoter sequences together with downstream sequences encoding the envelope glycoprotein (gp90) in moderate- to high-copy-number (hcn) plasmid vectors, such as pBR322 or pUC, resulted in rearrangements and point mutations in env when propagated in E. coli.

Equine infectious anemia virus gene expression: characterization of the RNA splicing pattern and the protein products encoded by open reading frames S1 and S2.

The utilization of predicted splice donor and acceptor sites in generating equine infectious anemia virus (EIAV) transcripts in fetal donkey dermal cells (FDD) was examined. A single splice donor site identified immediately upstream of the gag coding region joins the viral leader sequence to all downstream exons of spliced EIAV transcripts. The predominant 3.

Expression of the protease gene of equine infectious anemia virus in Escherichia coli: formation of the mature processed enzyme and specific cleavage of the gag precursor.

A 620-bp Bg/II restriction fragment containing the putative protease coding sequence from equine infectious anemia virus (EIAV) proviral DNA was cloned and expressed in E. coli as a Pol precursor protein. In contrast to the 25-kDa fusion protein predicted from the expressed pol sequence, a protein of approximately 10 kDa was generated by apparent autocatalytic processing of the Pol precursor.

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90.

Analysis of equine humoral immune responses to the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus.

Defined segments of the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus were expressed as TrpLE fusion proteins and examined for their reactivity in Western immunoblots against a diverse panel of equine immune sera. The most immunogenic region of gp45 was localized to its amino terminus, positioned between the hydrophobic fusion and the transmembrane domains. A series of overlapping synthetic peptides were used in enzyme-linked immunosorbent assays to define an immunodominant epitope within this region.

Characterization of the antigenic domains of the major core protein (p26) of equine infectious anemia virus.

A panel of recombinant trpLE-gag fusion proteins and synthetic peptides was used in Western immunoblot and enzyme-linked immunosorbent assays to identify segments of the major core protein (p26) of equine infectious anemia virus that are antigenic in horses during experimental and natural infections with the virus. The predominant humoral immune response was directed toward a highly immunogenic domain composed of 83 amino acids from the carboxy terminus of p26. The observed immunogenicity of p26 resembled that reported for p24 of human immunodeficiency virus type 1, suggesting the conservation of structural motifs in the lentiviral core proteins which are responsible for their observed immunogenicity during persistent lentivirus infections.

Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli.

Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion of gp45, indicating the highly conserved and immunodominant nature of these regions.

Lentivirus genomic organization: the complete nucleotide sequence of the env gene region of equine infectious anemia virus.

The nucleotide sequence of the envelope (env) gene region of equine infectious anemia virus (EIAV), a member of the lentivirus subfamily of retroviruses, has been determined from a clone of integrated proviral DNA for which the gag and pol sequences have been reported previously. The env gene is 859 codons in length and the sequence reported here is consistent with the published biochemical properties of EIAV glycoproteins. The env gene region of EIAV shares considerable structural similarities but negligible sequence homologies with the env genes of other members of the lentivirus subfamily, visna virus, and human T-lymphotropic virus (HTLV-III) or lymphadenopathy virus (LAV).