Isolation and characterization of new wild-type isolates of bovine lentivirus.

Two new isolates of bovine lentivirus, also known as bovine immunodeficiency-like virus (BIV), were obtained from a seropositive cattle herd in Florida. This is the first report of new isolates of BIV since the original BIV strain, R29, was isolated in 1969. The two new BIV isolates were derived from blood buffy coat cells cocultivated in vitro with fetal bovine lung cell cultures. The new isolates differed in vitro from the original R29 isolate in replication and syncytium formation in fetal bovine lung cells. Both new isolates were confirmed as BIV by immunofluorescence assay, Western blotting (immunoblotting), and polymerase chain reaction. Sequence analyses of the polymerase chain reaction pol gene product showed 92.6 and 93.6% homology to the published nucleotide sequence of BIV R29-127, a molecular clone derived from BIV R29. Each of the new BIV isolates was inoculated into two calves, and virus was recovered between 5 and 10 days postinoculation (p.i.), with BIV seroconversion between 10 and 21 days p.i. Virus was recoverable and antibody was detectable for at least 4 months p.i. Two calves developed a transiently elevated mononuclear cell count, similar to what was reported for BIV R29 in the original experimental calf inoculations. No other clinical abnormalities were observed.     

Characterization of early pathogenic effects after experimental infection of calves with bovine immunodeficiency-like virus.

The early pathogenic effects of bovine immunodeficiency-like virus (BIV) were studied in calves experimentally inoculated with BIV. All animals inoculated with BIV R29-infected cells seroconverted by 6 weeks postinoculation, and BIV was recoverable from each animal at 2 weeks postinoculation. However, levels of BIV replication in vivo appeared to be low. In situ hybridization studies indicated that during peak periods of viral replication in vivo, less than 0.03% of peripheral blood mononuclear cells were expressing detectable levels of viral RNA. Moreover, the levels of viral RNA in these cells in vivo were less than 1/10 the levels observed in persistently infected cells in vitro. BIV-inoculated calves had significantly higher numbers of circulating lymphocytes, and follicular hyperplasia was observed in lymph nodes, hemal nodes, and spleen. The histopathological changes observed in BIV-infected calves were similar to changes found early after infection with the immunosuppressive lentiviruses, including human immunodeficiency virus type 1.     

Humoral immune response to the bovine immunodeficiency-like virus in experimentally and naturally infected cattle.

Calves inoculated with bovine immunodeficiency-like virus (BIV) produced virus-specific antibodies that could be detected from 2 weeks to 2.5 years postinoculation by using both indirect fluorescent-antibody and Western immunoblot assays. Antibodies were primarily to p26. Virus and BIV-specific antibodies were isolated from calves given BIV-infected blood. Antibodies to BIV proteins were found in sera from naturally infected cattle.     

CD16+ NK lymphoproliferative disorders: cellular and molecular characterization

We studied the mononuclear cells obtained from 2 patients with CD16+ lymphoproliferative disorders. In both subjects, over 80% of the circulating peripheral blood mononuclear cells were CD16+, CD2+, CD7+, CD3-, CD4-, and CD8-. In 1 patient, greater than 60% of the cells expressed HLA-DR and HLA-DQ gene products. Functional analysis of the natural killer (NK) cell activity of cells from this patient demonstrated 76% killing of K562 targets at ratios as low as 1:1 effector:targets. Karyotype analysis demonstrated a deletion on the long arm of chromosome 6, supporting the contention that the lymphocytosis in this patient was due to a clonally expanded population of cells. In additional studies, Southern blot analysis of DNA extracted from cells of this patient revealed that the beta-chain of the T cell receptor was of germ line configuration. This information supports the hypothesis that the clonally expanded NK population in this patient is of a lineage distinct from T cells and represents a true NK leukemia.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Serum Antibodies to the Catalase Antigen of Aspergillus fumigatus in Cattle

Aspergillus fumigatus is an important agent of mycotic infection in cattle and a potent source of antigens. However, the efficacy of serological diagnosis of aspergillosis in cattle remains controversial. Corbel (1972) and Knudtson et al. (1974) considered a precipitin assay useful as a supplementary test in the diagnosis of mycotic abortion, whereas Wiseman et al. (1984) found the specificity too low to justify its routine use. We have studied 1) the antibody response to the catalase antigen of A. fumigatus in experimentally infected cattle and 2) the prevalence of catalase antibodies and A. fumigatus precipitins in healthy and diseased cattle. The aim was to ascertain how far detection of antibodies to a defined fungal antigen can contribute to the often difficult diagnosis of mycosis.     

Alteration of Immune Responses of Rabbits Infected with Bovine Immunodeficiency-Like Virus

Nine 3-month-old rabbits were inoculated with bovine immunodeficiency-like virus (BIV) to study the pathogenesis of BIV and alteration of the immune responses in experimentally infected rabbits. BIV proviral DNA and anti-BIV antibodies were detected from all rabbits inoculated with BIV-infected bovine embryo spleen (BESP) cells. Rabbits inoculated with spleen cells of the BIV-infected rabbit also converted to proviral DNA-positive and BIV-antibody-positive. The blastogenic responses to concanavalin A of peripheral blood mononuclear cells prepared from BIV-infected rabbits were not significantly different from those from uninfected controls at 2 and 4 months post-inoculation (PI). The humoral immune responses against bovine serum albumin (BSA) were depressed in two of four BIV-infected rabbits at 1 to 3 months PI. The antibody responses against sheep red blood cells (SRBCs) were significantly depressed in all BIV-infected rabbits at 2 to 4 months PI. BIV was rescued by cocultivation of spleen cells of infected rabbits with BESP cells. Distinct development of lymphoid follicle was observed in lymph nodes and spleens of uninfected rabbits which received BSA and SRBCs. In contrast, moderate lymphoid cell depletion was observed in BIV-infected rabbits which received the same immunogens.     

牛免疫缺陷病毒（BIV）92044毒株的分离及鉴定

从一头标号为９２０４４４的进口奶牛分离外周血淋巴细胞,将此淋巴细胞与正常胎牛肺细胞进行共培养。一个月后共培养物出现合胸体。此外电镜观察可见病毒的出芽过程。免疫染色显示,此培养物可与牛免疫缺陷病毒外膜蛋白的单克隆抗体特异性结合。     

Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.     

Adoptive transfer of splenocytes in SCID mice implicates CD4+ T cells in apoptosis and epithelial proliferation associated with Helicobacter pylori-induced gastritis

Helicobacter pylori causes severe, rapidly progressive gastritis in severe combined immunodeficient (SCID) mouse recipients of congenic splenocytes. The H. pylori-infected and uninfected C57BL/6J and recipient SCID mice were evaluated to detect CD4+ and CD8+ T cells, B cells, apoptotic epithelial cells, and epithelial cell proliferation at postinoculation weeks 5, 6, 8, and 12. Serum was evaluated for anti-H. pylori IgG and IgM. In all H. pylori-infected mice, gastric CD4+ cell scores were increased, compared with scores for uninfected controls. Recipient mice differed, however, according to the source of the transferred CD4+ cells. The CD4+ cell scores for recipients of splenocytes from H. pylori-infected (immune) donors were indistinguishable from those for wild-type donor mice at all time points. In contrast, gastric mucosal CD4+ cell scores did not become significantly high until two weeks after transfer (postinoculation week 6) in recipients of cells from uninfected (na?ve) donors. Gastric epithelial apoptosis and the gastric epithelial proliferation zone were significantly (P < 0.05) increased in infected recipient and donor, compared with non-recipient and uninfected mice at postinoculation week 12. Results indicated that CD4 cells are sensitized in vivo and migrate to the gastric mucosa where they induce gastritis in response to H. pylori antigens. Influx of CD4 cells and gastritis are correlated with epithelial proliferation and apoptosis, and suggest that CD4-dependent H. pylori gastritis leads to epithelial damage with attendant proliferative and metaplastic responses.     

Immunologic abnormalities in pathogen-free cats experimentally infected with feline immunodeficiency virus.

Blood mononuclear cells from 47 cats experimentally infected with feline immunodeficiency virus (FIV) were examined by using monoclonal antibodies directed against feline CD4 and CD8 homologs, a pan-T-cell antigen, and cell surface immunoglobulin. Significant inversion of the CD4+/CD8+ T-cell ratio was observed only in cats that were infected for 18 months or more. This inversion was associated with a decrease in the absolute numbers of CD4+ T cells and a concomitant increase in CD8+ cells. However, the total numbers of circulating T and B cells were not significantly reduced. Cats infected with FIV for 24 to 28 months also had significantly elevated levels of serum immunoglobulin G (IgG), but normal levels of IgA and IgM. The long-term decline in CD4+ T cells and hypergammaglobulinemia observed in FIV-infected cats resemble the abnormalities occurring in humans after human immunodeficiency virus infection.     

Caryocyst-like host cell formation by Caryospora duszynskii (Apicomplexa: Eimeriidae) in human fetal lung cell cultures

Sporozoites of the coccidium, Caryospora duszynskii, penetrated human fetal lung cell cultures but did not undergo asexual or sexual multiplication during a 29-day observation period. Beginning three days postinoculation (PI), infected host cells lost their normal elongated fibroblast-like shape and became ellipsoidal in appearance and resembled caryocysts. These caryocyst-like infected cells were observed from 3 through 29 days PI. Sporozoites remained viable throughout the study as evidenced by motility of extracellular sporozoites in infected human fetal lung cell cultures. Results of this in vitro study suggest that some species of Caryospora may form caryocysts in secondary hosts without undergoing asexual or sexual multiplication in these hosts.     

Persistent infection of rabbits with bovine immunodeficiency-like virus.

Chronic infection of rabbits was induced by a single intraperitoneal injection of bovine immunodeficiency-like virus (BIV)-infected cells. Ten BIV-infected animals were monitored serologically for up to 2 years. Results of serologic and virus rescue assays indicated that all animals became infected and demonstrated a rapid and sustained BIV-specific humoral response. BIV was rescued by cocultivation from spleen, lymph nodes, and peripheral blood leukocytes of infected animals. Viral DNA in immune tissues was confirmed by polymerase chain reaction amplification of BIV sequences. These data and specific immunohistochemical staining of mononuclear cells of the spleen for BIV antigen suggest that the infection is targeted to immune system cells.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Study of the immunological profile towards Mycobacterium bovis antigens in naturally infected cattle

A number of studies have determined the contribution of Th1 and Th2 responses to the protective immunity and pathology of Mycobacterium bovis infection. However, much of that information is derived from experimentally infecting cattle with M. bovis and few data from naturally infected animals are available. The aim of this study was to characterize the immunological profile towards M. bovis antigens of naturally infected cattle by measurement of cytokine mRNA expression in PBMC, and to determine which lymphocyte subsets are involved in recall responses of PBMC from M. bovis infected cattle to M. bovis antigens. Consistent with data from cattle experimentally infected with M. bovis , naturally infected animals were found to display a Th1 cytokine profile in response to M. bovis PPDB stimulation. Production of IFN-γ mRNA by PBMC after PPDB stimulation statistically distinguishes between infected and healthy herds, suggesting that this molecule is usable as an M. bovis -infection marker. As happens in experimentally infected cows, CD4, CD8 and γδTCR cells from a herd naturally infected with M . bovis are the predominant T cell subsets expanded in response to PPDB.     

Caryocyst-Like Host Cell Formation by Caryospora duszynskii (Apicomplexa: Eimeriidae) in Human Fetal Lung Cell Cultures1

ABSTRACT. Sporozoites of the coccidium, Caryospora duszynskii, penetrated human fetal lung cell cultures but did not undergo asexual or sexual multiplication during a 29-day observation period. Beginning three days postinoculation (PI), infected host cells lost their normal elongated fibroblast-like shape and became ellipsoidal in appearance and resembled caryocysts. These caryocyst-like infected cells were observed from 3 through 29 days PI. Sporozoites remained viable throughout the study as evidenced by motility of extracellular sporozoites in infected human fetal lung cell cultures. Results of this in vitro study suggest that some species of Caryospora may form caryocysts in secondary hosts without undergoing asexual or sexual multiplication in these hosts.     

Induction of feline immunodeficiency virus-specific cytolytic T-cell responses from experimentally infected cats.

We have examined the in vitro induction and activity of feline immunodeficiency virus (FIV)-specific cytolytic T cells obtained from cats experimentally infected for 7 to 17 weeks or 20 to 22 months with the Petaluma isolate of FIV. Normal or FIV-infected autologous and allogeneic T lymphoblastoid cells were used as target cells in chromium-51 or indium-111 release assays. When effector cells consisted of either fresh peripheral blood mononuclear cells or concanavalin A- and interleukin-2-stimulated cells, only low levels of cytotoxicity were observed. However, the levels of FIV-specific cytotoxicity were consistently higher in both groups of cats following in vitro stimulation of the effector cells with irradiated, FIV-infected autologous T lymphoblastoid cells and interleukin-2. The effector cells lysed autologous but not allogeneic FIV-infected target cells and were composed predominantly of CD8+ T cells, indicating that the FIV-specific cytotoxicity measured in this system is mediated by CD8+, major histocompatibility complex class I-restricted T cells. These studies show that FIV-specific cytolytic T cells can be detected as early as 7 to 9 weeks postinfection, and they define a system to identify virus-encoded epitopes important in the induction of protective immunity against lentiviruses.     

Molecular evolution of human immunodeficiency virus env in humans and monkeys: similar patterns occur during natural disease progression or rapid virus passage

Neonatal rhesus macaque 95-3 was inoculated with nonpassaged simian-human immunodeficiency virus strain SHIV-vpu(+), which encodes env of the laboratory-adapted human immunodeficiency virus (HIV) strain IIIB and is considered nonpathogenic. CD4(+) T-cell counts dropped to <200 cells/microl within 4.6 years, and monkey 95-3 died with opportunistic infections 5.9 years postinoculation. Transfer of blood from 95-3 to two naive adult macaques resulted in high peak viral loads and rapid, persistent T-cell depletion. Progeny virus evolved in 95-3 despite high SHIV-vpu(+) neutralizing antibody titers and still used CXCR4 but, in contrast to parental SHIV-vpu(+), productively infected macrophages and resisted neutralization. Sequence analysis revealed three new potential glycosylation sites in gp120; another two were lost. Strikingly similar mutations were detected in a laboratory worker who progressed to AIDS after accidental HIV-IIIB infection (T. Beaumont et al., J. Virol. 75:2246-2252, 2001), thus supporting the SHIV-vpu(+)/rhesus macaque system as a relevant model. Similar mutations were also described after rapid passage of chimeric viruses encoding IIIB env in rhesus and pig-tailed macaques (M. Cayabyab et al., J. Virol. 73:976-984, 1999; Z. Q. Liu et al., Virology 260:295-307, 1999; S. V. Narayan et al., Virology 256:54-63, 1999; R. Raghavan et al., Brain Pathol. 7:851-861, 1997; E. B. Stephens et al., Virology 231:313-321, 1997). Thus, HIV-IIIB env evolved similarly in three different species; this selection occurred in chronically infected individuals during disease progression as well as after rapid virus passage. We postulate that evolutionary pressure led to the outgrowth of more aggressive viral variants in all three species.