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C J Issel

Publications and source records attributed to C J Issel.

At least 37 records · Page 2Linked to original sources
PubMed1997

Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.

Equine infectious anemia virus (EIAV) provides a natural model system by which immunological control of lentivirus infections may be studied. To date, no detailed study addressing in parallel both the humoral and cellular immune responses induced in horses upon infection by EIAV has been conducted. Therefore, we initiated the first comprehensive characterization of the cellular and humoral immune responses during clinical progression from chronic disease to inapparent stages of EIAV infection. Using new analyses of antibody avidity and antibody epitope conformation dependence that had not been previously employed in this system, we observed that the humoral immune response to EIAV required a 6- to 8-month period in which to fully mature. During this time frame, EIAV-specific antibody evolved gradually from a population characterized by low-avidity, nonneutralizing, and predominantly linear epitope specificity to an antibody population with an avidity of moderate to high levels, neutralizing activity, and predominantly conformational epitope specificity. Analyses of the cell-mediated immune response to EIAV revealed CD4+ and CD8+ major histocompatibility complex-restricted, EIAV-specific cytotoxic T-lymphocyte (CTL) activity apparent within 3 to 4 weeks postinfection, temporally correlating with the resolution of the primary viremia. After resolution of the initial viremia, EIAV-specific CTL activity differed greatly among the experimentally infected ponies, with some animals having readily detectable CTL activity while others had little measurable CTL activity. Thus, in contrast to the initial viremia, it appeared that no single immune parameter correlated with the resolution of further viremic episodes. Instead, immune control of EIAV infection during the clinically inapparent stage of infection appears to rely on a complex combination of immune system mechanisms to suppress viral replication that effectively functions only after the immune system has evolved to a fully mature state 6 to 8 months postinfection. These findings strongly imply the necessity for candidate EIAV and other lentivirus vaccines to achieve this immune system maturation for efficacious immunological control of lentivirus challenge.

Animals↗
PubMed1996

Fine specificity of equine infectious anaemia virus gp90-specific antibodies associated with protective and enhancing immune responses in experimentally infected and immunized ponies.

Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological control of a persistent lentivirus infection and for evaluating the efficacy of various vaccine strategies. As an initial characterization of antibody responses associated with protective or enhancing immune responses elicited by experimental infections or vaccinations, we have utilized synthetic peptide ELISA to characterize the fine specificity of antibodies to linear determinants of the EIAV surface glycoprotein, gp90. The data indicated that serum antibodies associated with protective or enhancing immune responses differed quantitatively and qualitatively in their pattern of reactivity to gp90 peptides. Protective and enhancing EIAV vaccines could also be distinguished by their ability to evoke anamnestic antibody responses to gp90 peptides. These studies demonstrate for the first time definitive differences in the specificity of protective and enhancing antibody responses to EIAV and emphasize the importance of using native viral glycoprotein immunogens in lentivirus vaccines.

Animals↗
PubMed1996

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. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detected in 63 of 63 horses with EIAV antibody test-positive histories on approved serologic tests, demonstrating that RT-nPCR was probably directed against highly conserved sequences in the viral genome. The RT-nPCR assay, agar gel immunodiffusion test, and conventional virus isolation were compared for detection of early infection in 12 experimentally infected ponies. Viral gag sequences were detected in all 12 animals by 3 days postinfection (p.i.) by RT-nPCR, whereas virus could not be routinely isolated on cell culture until 9 to 13 days p.i. and EIAV antibodies could not be detected by agar gel immunodiffusion until 20 to 23 days p.i. Finally, specificity of the RT-nPCR assay was examined by testing plasma from 43 horses with serologic test-negative histories and no known contact with EIAV-infected animals. Viral gag sequences were not detectable in this control group. These data suggest that the EIAV RT-nPCR assay effectively detects EIAV and is more sensitive than current standard methods for detection of early stages of infection.

Animals↗
PubMed1996

Genomic quasispecies associated with the initiation of infection and disease in ponies experimentally infected with equine infectious anemia virus.

Equine infectious anemia virus (EIAV) provides a uniquely dynamic system in which to study the mechanism and role of genomic variation in lentiviral persistence and pathogenesis. We have used a Shetland pony model of infection to investigate the association of specific long terminal repeat (LTR) and env gene genomic sequences with the initiation of infection and the onset of disease. We analyzed viral RNA isolated from a pathogenic stock of virus (EIAV PV) and from plasma taken during the first disease episode from two ponies infected with EIAV PV. Overall sequence variation within gp90 was low in EIAV PV and only slightly higher in plasma virus samples isolated from ponies during the first disease episode. However, a high proportion of mutations were localized to the principal neutralizing domain in EIAV PV and to the principal neutralizing domain and the gp90 hypervariable region in the two pony-derived samples. The rate of fixation of mutations was analyzed and determined to be approximately 4 x 10(-2) mutations per site per year. Sequence diversity within the U3 region of the LTR was extremely low, which suggested that the previously reported hypervariability of this region may be a consequence of selection for replication of EIAV in different host cells. The predominant EIAV PV env and LTR sequences were used to construct chimeric viruses so that the contribution of these sequences to viral pathogenicity could be examined. The chimeras replicated in cultured equine monocytes to the same extent as the parental nonpathogenic virus and did not cause disease in Shetland ponies by 120 days postinfection, suggesting that the EIAV genomic determinants of pathogenesis are complex.

Amino Acid Sequence↗
PubMed1996

Stability of equine infectious anemia virus in Aedes aegypti (Diptera: Culicidae), Stomoxys calcitrans (Diptera:Muscidae), and Tabanus fuscicostatus (Diptera:Tabanidae) stored at -70 degrees C.

Equine infectious anemia virus (EIAV) was injected intrathoracically into Aedes aegypti, Stomoxys calcitrans, and Tabanus fuscicostatus, and fed to Ae. aegypti in suspensions of either artificial blood of Eagle's Minimum Essential Medium. Insects were stored at -70 degrees C for up to 9 months before testing for the presence of EIAV. The viral tissue culture titers detected from stored insects were similar to those from insects tested at time 0.

Aedes↗
PubMed1995

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. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were significantly more sensitive to neutralizing antibodies than viruses from the parental (PR) molecular clone. These results demonstrated that sensitivity to neutralizing antibodies in EIAV can be conferred by amino acid residues in the SU protein. However, such amino acid substitutions were not sufficient to enhance cytopathogenicity, as the chimeric viruses did not cause excessive degenererative effects in FDD cells, as was observed with the parental FDD virus strain.

Amino Acid Sequence↗
PubMed1995

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. In FEK cells, which are permissive for EIAV replication, the mutant virus replicated as well as the parental virus. In primary cultures of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infection decreased, the difference between the parental and mutant viruses increased, such that at the lowest multiplicity of infection tested, there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shetland ponies that were infected with the parental and mutant viruses showed transient virus RNA levels in plasma approximately 5 to 10 days postinfection. The peak virus levels in plasma (as measured by a quantitative reverse transcriptase PCR assay) were 10- to 100-fold lower in the mutant virus-infected animals than in the animals infected with the parental virus. However, ponies infected with the mutant virus mounted similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for the efficient replication of the virus in macrophages in vitro and in vivo and suggests that variations in the DU sequence could markedly affect the biological and pathogenic properties of EIAV.

Amino Acid Sequence↗
PubMed1994

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. Thus, the recombinant EIAV gp90 vaccine provides a novel in vivo model for examining in detail the mechanisms of immune enhancement of a lentivirus infection and for evaluating strategies to avoid the production of deleterious immune responses in AIDS vaccine design.

AIDS Vaccines↗
PubMed1994

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. In addition, the cross-reactive determinants on the EIAV CA were localized within the immunodominant carboxyl terminus of the protein (residues 277 to 367). However, the cross-reactive determinants recognized by the equine sera do not appear to correlate with linear peptides from the carboxyl terminus of the EIAV CA, including the MHR. These results suggest cross-reactivity between more distant lentiviruses is associated with non-linear determinants. In contrast, MHR-specific MAbs did react with linear peptides by ELISA and distinguished the primate lentiviruses from EIAV and feline immunodeficiency virus. These data support the concept of a highly conserved structural and antigenic organization among the CA proteins of lentiviruses from different species.

Amino Acid Sequence↗
PubMed1994

Application of cycle dideoxy fingerprinting to screening heterogeneous populations of the equine infectious anemia virus.

Nucleotide sequence heterogeneity in a population of the equine infectious anemia virus (EIAV) was investigated using a modification of the dideoxy fingerprinting (ddF) technique. PCR-amplified regions of the gag gene from EIAV isolates were ligated into plasmid vectors and used to transform bacteria. The single dideoxynucleotide sequencing step was performed using plasmid DNA prepared from individual bacterial colonies using an 35S end-labeled primer and Taq DNA polymerase. Analysis of the products of this reaction was conducted using non-denaturing polyacrylamide gel electrophoresis. Polymorphism within this gene was suggested by the presence of several distinct electrophoretic profiles. Significantly, each profile could be correlated with variations in nucleotide sequence, which demonstrates that cycle ddF (CddF) offers a rapid and sensitive approach to identify polymorphism in PCR-amplified products.

Animals↗
PubMed1993

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.

Animals↗
PubMed1993

Insect blood meal studies using radiosodium 24Na and 22Na.

Rabbits were infused with 3.7 GBq (100 mCi) of [24Na]Na ion in a 100-ml sodium carbonate solution. Beta particles were detected using a Tennelec Counting System; background counts were 1.6 +/- 1 counts per minute (cpm). Counts for one nanoliter of blood ranged from 22 to 30 cpm. Blood volumes on the mouthparts of tabanids following a 15-sec interrupted feeding were estimated to be 12.5 nl for Tabanus fuscicostatus, 10.8 nl for T. nigrovittatus and 6.12 nl for Chrysops fuliginosus. Estimates of the quantity of blood adhering to 22-gauge needles and insect pins (size 2) following a percutaneous intramuscular needle-stick were 8.8 +/- 1.0 nl and 5.7 +/- 1.8 nl, respectively. Mosquitoes, Aedes aegypti females, were fed to repletion with a 22Na-artificial diet, and radioactivity was measured using a Packard Autogamma 5650. The estimated average blood meal size was 2.80 +/- 0.94 microliters.

Aedes↗
PubMed1992

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. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.

Amino Acid Sequence↗
PubMed1992

Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus.

We report here on a series of vaccine trials to evaluate the effectiveness of an inactivated equine infectious anemia virus (EIAV) whole-virus vaccine and of a subunit vaccine enriched in EIAV envelope glycoproteins. The inactivated vaccine protected 14 of 15 immunized ponies from infection after challenge with at least 10(5) 50% tissue culture-infective doses of the homologous prototype strain of EIAV. In contrast, it failed to prevent infection in any of 15 immunized ponies that were challenged with the heterologous PV strain. Levels of PV virus replication and the development of disease, however, were significantly reduced in 12 of the 15 ponies so challenged. The subunit vaccine prevented infection from homologous challenge in four of four ponies tested but failed to prevent infection in all four challenged with the PV strain. Two of the four subunit vaccinates had more severe symptoms of equine infectious anemia than nonimmunized ponies infected in parallel. Both vaccines stimulated EIAV-specific cell-mediated immunity. The in vitro lymphoproliferative response was shown to be mediated by T lymphocytes and appeared to be indistinguishable from that induced by EIAV infection. Significant differences were observed in the in vivo lymphocyte responses following challenge with the two virus strains. While peripheral blood mononuclear cells from the inactivated virus vaccinates were equally stimulated by both the prototype and PV strains, the subunit vaccinates challenged with PV exhibited lower levels of spontaneous proliferation and serine esterase activity. This diminished cellular response to PV was correlated with more severe clinical disease in the same ponies. These studies demonstrate for the first time that both an EIAV inactivated whole-virus vaccine and a viral envelope glycoprotein-based subunit vaccine can provide protection against rigorous challenge levels of homologous virus but are unable to protect against similar challenge levels of a heterologous virus. Moreover, the data demonstrate that protection can be achieved in the absence of detectable levels of virus-specific neutralizing antibody in the vaccine recipients at the time of virus challenge. While vaccine-induced virus-specific cell-mediated immune responses were detected, their role in conferring protection was not obvious. Nevertheless, protection from disease appeared to be correlated with the induction of high levels of serine esterase activity following challenge. A significant observation is that while the whole-virus vaccine was usually capable of preventing or markedly moderating disease in the PV-infected ponies, the subunit vaccine appeared to have a high potential to enhance the disease induced by PV infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

A nonradioactive micro-assay for released reverse transcriptase activity of a lentivirus.

A nonradioactive micro-assay procedure for detection of released reverse transcriptase activity from cells infected with equine infectious anemia virus is described. This procedure utilizes biotinylated-dUTP in conjunction with a streptavidin-alkaline phosphatase conjugate. Detection of alkaline phosphatase is by autoradiography of the chemiluminescence produced during enzymatic dephosphorylation of Lumi Phos 530. This method, as with reverse transcriptase micro-assays employing 32P-labeled nucleotides, is suited to the processing of numerous samples, while having the advantages of safety and stability normally associated with nonradioactive methods of detection. Sensitivity is comparable to a reverse transcriptase micro-assay using 32P-dTTP.

Alkaline Phosphatase↗
PubMed1991

Transient suppression of equine immune responses by equine infectious anemia virus (EIAV).

Suppression of the immune system is a common aspect of the disease pathogenesis associated with retroviral infections in both man and animals. We have measured transient suppression of the equine immune system as a loss or decrease in antigen-specific and polyclonal lymphocyte proliferation following experimental infection of ponies with three variants of equine infectious anemia virus (EIAV) with difference virulence characteristics. The transient suppression of proliferative responses was temporally associated with recurrent febrile episodes, which are the hallmark symptom of EIAV-induced disease. Decreased proliferative responses occurred at all times when EIAV viremia was identified, based on the detection of an infectious virus in plasma or viral proteins on peripheral blood mononuclear cells. The immunosuppression was observed most frequently in ponies infected with virulent variants of EIAV which suggested that this effect may contribute to disease pathogenesis. Suppression of polyclonal proliferative responses was induced in vitro by the addition of either infectious or heat-inactivated EIAV to cultures, demonstrating that the viral structural proteins were immunosuppressive in the absence of infection. These studies indicated that EIAV is similar to other retroviruses in that it has the ability to suppress the immune system.

Animals↗