Immune responses of goats persistently infected with caprine arthritis-encephalitis virus.

Eight cesarean-derived goat kids were inoculated with caprine arthritis-encephalitis virus (CAEV), and proliferative responses of their peripheral blood mononuclear cells to mitogens and CAEV antigen were monitored for 9 months. Antibody specific for CAEV was measured by an enzyme-linked immunosorbent assay. Five cesarean-derived noninfected goats were tested simultaneously. Significant differences between the infected and control mononuclear cell proliferation reactions to CAEV began 14 days post-inoculation and continued in a fluctuant manner until 134 days post-inoculation. The magnitude of the proliferative reaction steadily increased in infected goats until the end of the experiment at 271 days post-inoculation. Responses to mitogens were not significantly different between infected and control goats. Virus-inoculated goats produced CAEV-specific antibody that reached a maximum level between 49 and 77 days post-inoculation and then declined to lower levels through 271 days post-inoculation. The virus-inoculated goats developed mild but characteristic clinical evidence of caprine arthritis-encephalitis, and CAEV was reisolated from four goats at 286 days post-inoculation. The five control goats developed neither an anti-CAEV immune response nor clinical disease, and CAEV could not be reisolated from them.     

Rapid evolution of two discrete regions of the caprine arthritis-encephalitis virus envelope surface glycoprotein during persistent infection

Five major regions of sequence diversity between strains (V1-V5) have been described in the caprine arthritis-encephalitis lentivirus (CAEV) envelope surface unit glycoprotein (SU). To determine which of these variable regions is important in persistent infection in vivo, we evaluated SU sequence diversity in five neutralization variants from two goats and proviral DNA from five additional goats infected with CAEV-63 for up to 7 years. Overall amino acid sequence divergence in the SU encoded by provirus and neutralization variants compared to parental CAEV-63 ranged from 1.1 to 4%. However, most of the amino acid substitutions and all of the deletions and insertions were present in two discrete regions designated HV1 and HV2. The HV2 region was variable in all neutralization variants and provirus sequences from most animals. This region overlapped the V4 domain of CAEV SU and the neutralization domain of the closely related ovine maedi-visna lentivirus. HV1 was located in a region of SU strictly conserved in all small ruminant lentivirus strains except CAEV-63. This region only varied in a subset of neutralization variants and proviruses, all derived from goats with arthritis. In contrast, sequences in the V1,V2,V3, and V5 regions were stable in neutralization variants and proviruses from infected goats, indicating that sequence diversity between strains in these regions is not due to selection of variants in persistently infected animals. Our results define two discrete regions of CAEV SU that undergo rapid sequence variation in persistently infected goats which may have important roles in virus-host interactions.     

Antigenic variants are not selected during persistent infection with Theiler's virus.

In this study, we demonstrated that non-neutralizable antigenic variants of Theiler's murine encephalomyelitis virus do not predominate during the course of chronic relapsing infection.     

Activation of caprine arthritis-encephalitis virus expression during maturation of monocytes to macrophages.

Lentiviruses, which cause arthritis-encephalitis and maedi-visna in goats and sheep, respectively, cause persistent infections in these animals. The viruses replicate productively at low levels in macrophages in diseased organs such as the "maedi lung" and nonproductively in other cell types such as leukocytes in peripheral blood. Nonproductive infections become productive during in vitro cultivation of the cells. This study showed that monocytes were the only cells in the peripheral blood leukocytes of an infected animal in which virus was detected and that virus activation occurred only when these cells matured into macrophages. Only a minute fraction of cultured monocytes matured into macrophages, and viral infectivity was associated exclusively with this fraction. Antiglobulin-coated glass wool fragments were lethal for monocyte macrophages because of toxic phagocytosis, but had no effect on B or T lymphocytes. The simultaneous addition of the glass fragments and leukocytes to culture dishes resulted in no macrophage maturation and no virus production. The addition of the fragments to virus-producing macrophages caused the death of the cells and a decline in virus production. Virus production in less avidly phagocytic cells was unaffected by the glass. Thus, although macrophages may be permissive for virus replication, one mechanism for restricted virus expression in vivo may be physiological factors controlling the maturation of these cells.     

Detection of caprine arthritis-encephalitis virus by polymerase chain reaction.

Detection of caprine arthritis-encephalitis virus (CAEV) infection in goats is currently limited to serologic testing or cell culture. We developed a polymerase chain reaction (PCR) assay to detect CAEV sequences in peripheral blood mononuclear cells (PBMC), synovial fluid cells (SFC), and milk cells (MC) obtained from infected goats. Results were positive for 18 of 20 PBMC, 8 of 8 MC, and 5 of 5 SFC samples from seropositive goats, whereas 3 of 33 PBMC samples and none of 8 MC or 5 SFC samples from seronegative goats were positive. Two of the PCR-positive and seronegative goats seroconverted upon follow-up testing 2 months later. This PCR assay provides a useful method for detecting CAEV infection in goats.     

Susceptibility of blood-derived monocytes and macrophages to caprine arthritis-encephalitis virus.

Permissiveness of blood-derived caprine monocytes to infection by caprine arthritis-encephalitis virus increased during in vitro cultivation and differentiation into macrophages, as evidenced by immunofluorescence and release of extracellular infectious virus. The degree of cell susceptibility to virus infection varied among individual goats, independent of age or breed. Caprine arthritis-encephalitis virus infection of macrophages in vitro resulted in no alteration of five characteristic functional activities.     

Development of an enzyme-linked immunosorbent assay for caprine arthritis-encephalitis virus.

Because relatively few caprine arthritis-encephalitis virus (CAEV)-infected animals exhibit clinical signs of illness, efforts to control and eradicate this virus will depend heavily on a sensitive diagnostic test that can be easily carried out. The currently utilized tests are of limited usefulness because of relatively low sensitivity or because of incomplete cross-reactivity of goat sera with heterologous test antigens. An enzyme-linked immunosorbent assay (ELISA) with purified CAEV antigen and biotin-avidin amplification steps was therefore developed and compared with a radioimmunoassay (RIA) against CAEV p28. Of over 500 sera tested, there was 99% concordance between the two tests. On the other hand, 23 of 24 sera obtained from animals with clinical signs of disease that were negative by agar gel immunodiffusion test (with ovine progressive pneumonia virus antigen) were positive by ELISA and RIA. These results suggest that an ELISA with CAEV antigen is superior to the agar gel immunodiffusion test and is easier and faster than an RIA, and therefore may be the method of choice for diagnosing CAEV infection.     

Proviral DNA in the brains of goats infected with caprine arthritis-encephalitis virus.

Archive paraffin wax-embedded sections of brain from goats and kids naturally infected with caprine arthritis-encephalitis virus (CAEV) were examined. Severe leucoencephalitis was present, with infiltration of lymphocytes, plasma cells and macrophages into the white matter, meninges and choroid plexus. On both CAEV-positive and negative (control) tissues, in-situ polymerase chain reactions were used to amplify a DNA sequence specific to the proviral Pol region. In the infected tissues, strong hybridization signals were observed, mainly located in macrophages, microglial cells, astrocytes and oligodendrocytes, and in the ependymal epithelium and choroid plexus. Positive areas were also found in the spinal cord in endothelial cells of small blood vessels. Some neurons showed a positive reaction.     

Characterization of the immunodominant cross-reacting epitope of visna maedi virus and caprine arthritis-encephalitis virus capsid antigen.

Maedi visna (MV) and caprine arthritis-encephalitis (CAE) are two retroviral infections distributed world wide. Antigenic cross reactions between the viruses have been demonstrated in gag and env encoded structural proteins. Antigens from ovine lentiviruses are easier to produce in cell culture systems and therefore have been used in the development of diagnostic tests for both infections. Antigenically relevant epitopes have been characterised in the transmembrane protein, but little information is available on the immunodominant and cross reacting epitopes in the major capsid antigen (p25). In this study four different recombinant subunits of ovine lentivirus p25 were tested against sera from infected goats and a detailed characterisation of the immunodominant subunit was carried out. Highest ELISA absorbances were obtained with a 29 amino acid subunit located in the N'-terminal half of p25. Through the analysis of overlapping peptides spanning this region we identified a 17 amino acid sequence that can be used in the development of a highly standardized synthetic peptide-based assay.     

Pronounced production of polyclonal immunoglobulin G1 in the synovial fluid of goats with caprine arthritis-encephalitis virus infection

Infection of goats with caprine arthritis-encephalitis virus, a lentivirus, resulted in arthritis characterized by the production of intrasynovial immunoglobulin G1 concentrations that were 2 to 5.3 times the serum concentrations in the inoculated carpi at 6 months postinoculation. The intrasynovial immunoglobulin was polyclonal, and its presence was accompanied by increased albumin leakage into the joints. Synovial fluid immunoglobulin levels fluctuated temporally but remained elevated compared with medium-inoculated controls for 38 months after infection. Elevated immunoglobulin G1 concentrations correlated with focal sublumenal plasmacytic infiltrates in the synovia of inoculated carpi at 5 months postinoculation. Inflammation in the uninoculated joints of infected goats was also accompanied by increased intrasynovial immunoglobulin G1 levels. Antibody to systemically administered antigens was a greater proportion of the immunoglobulin population in sera than in synovial fluids of infected goats, suggesting that antibody production to local antigens was responsible for increased intrasynovial immunoglobulin G1 levels.     

Bovine viral diarrhea virus quasispecies during persistent infection.

Analysis of viral genome sequences from two calves persistently infected with bovine viral diarrhea virus revealed a quasispecies distribution. The sequences encoding the glycoprotein E2 were variable, translating to a number of changes in predicted amino acid sequences. The NS3 region was found to be highly conserved in both animals. The number of E2 clones showing variant amino acids increased with the age of the animal and comparison of the consensus sequences at the different time points confirmed differences in the predicted E2 sequences over time. The immune tolerance that allows the lifelong persistence of this viral infection is highly specific. It is likely that some of the variant viruses generated within these animals will differ antigenically from the persisting virus and be recognized by the immune system. Evidence of an immune response to persisting virus infection was gathered from a larger sample of cattle. Serum neutralizing antibodies were found in 4 of 21 persistently infected animals. Accumulations of viral RNA in the lymph nodes of all animals examined, particularly in the germinal center light zone, may represent antigenic variants held in the form of immune complexes on the processes of follicular dendritic cells.     

Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.

The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.     

Recombinant gp135 envelope glycoproteins of caprine arthritis-encephalitis lentivirus variants inhibit homologous and heterologous variant-specific neutralizing antibodies.

The envelope (env) genes of two antigenic variants of caprine arthritis-encephalitis virus (CAEV), defined by serum neutralization, were expressed in vaccinia virus. Recombinant gp135 envelope glycoprotein competitively inhibited neutralizing activity of serum from CAEV-infected goats, indicating gp135 is a dominant target antigen of CAEV neutralizing antibody. In addition, type-specific neutralizing activity of goat serum directed against one variant was inhibited by both homologous and heterologous variant recombinant gp135. Hence, a CAEV variant env gene encodes type-specific neutralization epitopes of both variants. The results indicate that antigenic variation of CAEV involves env gene mutations encoding amino acid differences outside conserved neutralization epitopes affecting epitope exposure to the immune system.     

Detection of antibodies to caprine arthritis-encephalitis virus by protein G enzyme-linked immunosorbent assay and immunoblotting.

Sera from goats suffering from caprine arthritis-encephalitis contained antibodies to virus proteins of 15, 17, 28, 40, and 130 kilodaltons in immunoblots of maedi-visna virus. We propose to use immunoblotting as a validation test for enzyme-linked immunosorbent assay and demonstrate that the specificity of indirect enzyme-linked immunosorbent assay can be improved by replacing second antibody by a protein G-avidin-biotin conjugate.     

Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants

Summary.  Rabbit haemorrhagic disease virus (RHDV) isolates were obtained from several animals previously vaccinated with an inactivated vaccine. Seven isolates were analyzed by immunological and molecular biological methods and compared to reference strains. Antigenic characterization with monoclonal antibodies as well as haemagglutination assays demonstrated considerable differences between individual isolates. However, sequencing of the capsid protein genes revealed a high degree of homology between five of these isolates and the reference strain FRG. In contrast, two isolates specified remarkably different capsid proteins with a degree of variation not observed so far in RHDV. Amino acid alterations were found clustered between residues 301 and 328 (region C), 344 and 434 (region E) and also in the 3′ region of the capsid protein gene. Interestingly, experimental vaccination of rabbits followed by challenge with the heterologous variant strains showed restricted cross-protection against one of the strains. In summary, we found a level of antigenic variation not detected in RHDV so far, and describe two distinct new antigenic variants. Received November 3, 1997 Accepted October 19, 1998     

Equine infectious anemia virus (EIAV) Humoral responses of recipient ponies and antigenic variation during persistent infection

Summary Three ponies were inoculated with plasma containing 10^4.8 TCID₅₀ of equine infectious anemia virus (EIAV) and observed for 165 to 440 days. Each pony developed a febrile response within 3 weeks of infection during which a plasma viremia 10^3.5 TCID₅₀/ml was observed. Analyses of four isolates from sequential febrile episodes in a single pony were conducted by two-dimensional tryptic peptide maps and with monoclonal antibodies in immunoblots. Structural and antigenic alterations were observed in the envelope glycoproteins gp90 and gp45, with greatest variation in gp90. Specific IgG to EIAV gp90, gp45, and p26 of homologous and heterologous isolates was detectable by immunoblots within one month after infection although IgG levels to gp45 at this time were relatively low. The group-specific determinants of gp90 and gp45 were more antigenic than those of p26; however, binding of IgG to these determinants did not correlate with neutralization of EIAV as assayed in fetal equine kidney cells. Neutralizing antibodies were first detectable within two months of infection and only neutralized viruses isolated prior to serum collection. Neutralizing activity of sera collected later in the infection was broadly reactive regardless of the number of clinical episodes the donor had suffered.     

Human respiratory syncytial virus genomic and antigenic variants isolated in two hospitals during one epidemic, in Santiago, Chile.

BACKGROUND: Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract infection (LRI) in children. Distinct variants of the viruses have been described. OBJECTIVE: The objective was to compare the antigenic and genetic variability of HRSV strains recovered from infants admitted to two hospitals during one epidemic in a big city. STUDY DESIGN: We analyzed nasopharyngeal aspirates from 201 infants admitted for LRI to two hospitals during 2002 in Santiago, Chile. The analyses were carried out using a panel of monoclonal antibodies against G glycoprotein epitopes (EIA) and RFLP for N and G genes. RESULTS: No differences in HRSV groups A/B and in N patterns distribution were observed among both hospitals. On the contrary, antigenic and genetic G patterns displayed a wide diversity of strains circulating during one epidemic, in one big city. CONCLUSIONS: RSV variability assessment depended rather on the tool used for analysis than on the geographical location.     

Molecular and antigenic characterization of rabbit hemorrhagic disease virus isolated in Cuba indicates a distinct antigenic subtype

Summary. Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004–2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region. First three authors contributed equally to this work.     

Macrophage functions during dengue virus infection: antigenic stimulation of B cells.

This study was undertaken to investigate the function of dengue type 2 virus (DV)-infected mouse peritoneal macrophages (M phi) regarding the antigenic stimulation of B lymphocytes of the spleen. It was observed that a variable proportion of M luminal diameter show DV-specific immunofluorescent antigen, which depended upon the route of administration of the virus, being higher in i.p.-inoculated mice and in vitro-infected M luminal diameter monolayers. The DV-infected M luminal diameter presented the DV antigen to B cells in vitro and in vivo, leading to their clonal expansion as shown by counting the virus-specific IgM antibody plaque-forming cells (PFC). The PFC response depended upon the number of DV-infected M luminal diameter. The antigen was presented equally well both by I-A-negative and I-A-positive M luminal diameter. Superimposition of a heterologous antigen (Coxsackie B4 virus) in a Mackaness type of experiment depressed the capacity of M luminal diameter to present both the homologous as well as heterologous antigen.     

Small plaque variants of simian virus 40 from a persistent infection of rhesus monkey kidney cells.

Seven small-plaque variants from a simian virus 40-rhesus monkey kidney cell carrier system were compared with the standard large plaque strain by restriction endonuclease analysis. One of these variants, SP1, contains an insertion of about 50 base pairs between the Bgl1 (map location 0.67) and Hpa11(0.73) cleavage sites. Another, SSP2, contains a deletion of about six base pairs in Hind 11 + 111 fragment K. The rates of replication and release of both SP1 and SP2 are similar to that of wild-type virus at 37°, although SP1 is slightly temperature sensitive at 41° and SP2 is slightly cold sensitive at 33°. Both SP1 and SP2 effectively compete with wild-type virus at 37° under conditions of mixed infection. Competition by SP1 is expressed, at least in part, at the level of DNA synthesis.