Expression of regulatory T cell (Treg) activation markers in endometrial tissues from early and late pregnancy in the feline immunodeficiency virus (FIV)-infected cat.

Regulatory T cells (Tregs) support pregnancy maintenance by suppressing placental inflammation, while diminished Treg function may accompany reproductive failure. Experimental FIV infection frequently results in vertical transmission and increased pregnancy failure in the cat. The mechanism of reproductive compromise is unknown. We hypothesized that FIV infection alters endometrial Treg population dynamics and function, potentiating vertical transmission and reproductive failure. RNA collected from early and late gestation reproductive tissue and fetuses from FIV infected and control cats was probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. Frequent placental and fetal infection and reproductive failure were detected at early and late pregnancy. Expression of FOXP3 and CTLA4 was higher in early gestation tissues from control cats. FIV infection significantly reduced expression of FOXP3 and CTLA4 at early, but not late pregnancy. At late pregnancy, CTLA4 was expressed to higher levels in infected tissues. The number of tissues with decreased co-expression of FOXP3 and CTLA4 was significant in infected cats at early pregnancy. No significant changes in CD25 expression occurred between FIV-infected and control animals at early or late pregnancy. Differences in Treg marker expression were not significant between viable and non-viable pregnancies in infected cats. The detection of Treg markers in these feline tissues provides the first evidence of feline endometrial Tregs and suggests that such cells diminish as pregnancy progresses. These cells may be depleted or rendered less functional by viral infection, but understanding their role in pregnancy requires further study.

Placental immunopathology and pregnancy failure in the FIV-infected cat.

Placental HIV infections frequently result in infected babies or miscarriage. Aberrant placental cytokine expression during HIV infections may facilitate transplacental viral transmission or pregnancy perturbation. The feline immunodeficiency virus (FIV)-infected cat is a model for HIV infections due to similarities in biology and clinical disease. The purpose of this study was to evaluate placental immunomodulator expression and reproductive outcome using the FIV-infected cat model. Kittens were cesarean delivered from FIV-B-2542-infected and control queens near term; placental and fetal tissues were collected. Real-time RT-PCR was used to measure expression of representative placental Th1 cytokines, interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma), a Th2 cytokine, IL-10, and chemokine receptor CXCR4. On average, control queens delivered 3.8 kittens/litter; 1 of 31 kittens (3.2%) was non-viable. FIV-infected queens produced 2.7 kittens/litter; 15 of 25 concepti (60%) were non-viable. FIV was detected in 14 of 15 placentas (93%) and 21 of 22 fetuses (95%) using PCR. Placental immunomodulator expression did not differ significantly when placentas from infected cats were compared to those of control cats. However, elevated expression of Th1 cytokines and increased Th1/Th2 ratios (IL-1beta/IL-10) occurred in placentas from resorptions. Therefore, increased placental Th1 cytokine expression was associated with pregnancy failure in the FIV-infected cat.

Natural bovine lentivirus type 1 infection in Holstein dairy cattle. II. Lymphoid tissue lesions.

Bovine immunodeficiency virus (BIV) in Holstein cows was associated with morphologic evidence of lymphoid organ deficiency. Cows were subjected to normal management practices including parturition and lactation without adverse environmental stresses. During the clinical disease process there was marked weight loss and wasting with frequent and severe concurrent infections. Lymphoid follicular hyperplasia and dysplasia in lymph nodes, and hypertrophy and hyperplasia in hemal lymph nodes were characteristics of the lymphoid tissues. Atrophy of lymphoid cell compartments with depletion of lymphocytes and a lymphocytic lymphoid folliculitis were components of the lymphoid system pathology. The nodal tissue lesions resembled those observed in feline, simian, and human lentiviral disease. A functional correlation with immune system deficiency was the development of multiple bacterial infections which failed to resolve after appropriate therapy. The BIV-associated disease syndrome in dairy cows may be useful as a model system for investigation of the pathogenesis of the lymphoid organ changes that occur in humans and animals with lentiviral infection.

Natural bovine lentiviral type 1 infection in Holstein dairy cattle. I. Clinical, serological, and pathological observations.

Clinical, serological, and pathological abnormalities observed in Holstein cows naturally infected with bovine lentivirus 1 bovine immunodeficiency virus (BIV) and other infections were progressive and most commonly associated with weight loss, lymphoid system deficiency, and behavioral changes. Clinical evidence of meningoencephalitis was dullness, stupor, and occasional head or nose pressing postures. The polymerase chain reactions associated the BIV provirus with the lesions in the central nervous system and lymphoid tissues. Multiple concurrent infections developed in retrovirally infected cows undergoing normal stresses associated with parturition and lactation. A major functional correlate of the lymphoreticular alterations was the development of multiple secondary infections which failed to resolve after appropriate antibacterial therapy. The chronic disease syndrome in dairy cows associated with BIV may be useful as a model system for investigation of the pathogenesis of the nervous system lesions and lymphoid organ changes that occur in humans with lentiviral infection.

Bovine immunodeficiency virus expression in vitro is reduced in the presence of beta-chemokines, MIP-1alpha, MIP-1beta and RANTES.

The inhibition of HIV expression in vitro by a cocktail of the beta-chemokines MIP-1alpha, MIP-1beta and RANTES provided the initial evidence that HIV utilizes chemokine receptors as co-receptors for infection of cells. Bovine immunodeficiency virus (BIV), a lentivirus, infects a wide variety of leukocyte populations, but the cellular receptor(s) utilized by this virus for infection of cells is not known. The purpose of this study was to determine whether MIP-1alpha, MIP-1beta and RANTES affect BIV expression in vitro, as a prelude to identifying the cellular receptors utilized by this virus. Fetal bovine lung (FBL) cells were pretreated with serial dilutions of a cocktail of the chemokines, and then the cells were infected with BIV. Virus expression in these cells was determined by counting the syncytia that had developed in the cultures by five days after infection. A significant decrease in syncytium formation, corresponding to increasing concentrations of the chemokines, was the result. Reacting the chemokines with chemokine-specific neutralizing antibodies prior to treatment of the cells neutralized the effect of the chemokines on virus replication in a dose-dependent manner, restoring viral expression to a level similar to that of untreated cells. The presence of a CCR5 homologue on the surface of FBL cells was confirmed using an anti-CCR5 monoclonal antibody and FACS analysis. Collectively, these data provide preliminary evidence that BIV may utilize the CCR5 receptor for infection of cells in vitro, but additional studies are necessary to confirm this.

Failure to detect bovine immunodeficiency virus contamination of stud bull spermatozoa, blood leukocytes, or semen leukocytes in samples supplied by artificial insemination centers.

OBJECTIVE: To determine whether bovine immunodeficiency virus (BIV) infection could be detected in spermatozoa, blood leukocytes, or semen leukocytes from stud bulls in artificial insemination centers. ANIMALS: 30 bulls at 3 artificial insemination centers. PROCEDURE: Polymerase chain reaction testing that used 3 sets of primer pairs targeting pol and env regions of the BIV proviral genome was performed on DNA extracted from semen leukocytes, spermatozoa, and blood leukocytes from each bull. Southern blot analysis was performed to increase sensitivity of detection. Western blot analysis of plasma samples was used to detect antibodies against BIV. RESULTS: BIV provirus was not detected in DNA samples obtained from semen leukocytes, spermatozoa, or blood leukocytes, and antibodies against BIV were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to our report of high point prevalence of BIV contamination of semen from a single artificial insemination center, bulls of the study reported here did not appear to be infected. Maximum risk of BIV infection in similar bulls was estimated at 10% with a confidence level of 95%.

Natural selection of the Pol gene of bovine immunodeficiency virus.

Genetic variability is a salient feature of lentiviruses, contributing to the pathogenesis of these viruses by enabling them to persist in the host and to resist anti-retroviral treatment. Bovine immunodeficiency virus (BIV), a lentivirus of unknown pathology, infects cattle in the United States and worldwide. Genetic diversity of BIV that is associated with naturally infected cattle is not well studied. We examined the genetic diversity and natural selection of a segment of the BIV pol gene amplified from the leukocyte DNA of naturally infected cattle. A portion of the reverse transcriptase domain (183 bp) of the pol region was targeted for amplification by PCR. PCR products were sequenced directly and aligned. When compared to the sequences of BIV R29-127, a molecular clone of the original BIV R29 isolate, all isolates were greater than 91% identical in nucleotide sequences and 77% identical in amino acid sequences. Pol genotypes were polymorphic at 14% of the nucleotide sites. The ratio of nonsynonymous to synonymous nucleotide substitutions (relative to the number of respective sites, Ka/Ks) was 0.16, indicating that this region of the BIV genome, like that of HIV-1, is subject to purifying selection. Based on the McDonald-Kreitman analysis, this region also was under positive Darwinian selection as HIV-1 and BIV diverged from a common progenitor. Phylogenetic analysis revealed that genotypes were geographically distinct, possibly indicating a common source of infection for animals within a herd.

Natural and experimental bovine immunodeficiency virus infection in cattle.

Since 1989, the LSU dairy herd, with its high seroprevalence of BIV, was recognized to have a high incidence of common diseases that reduced the economic viability of the dairy. The herd had a high percentage of cows with encephalitis associated with depression and stupor, alteration of the immune system associated with secondary bacterial infections, and chronic inflammatory lesions of the feet and legs. The occurrence of disease problems was associated with the stresses of parturition and early lactation and/or with unusual environmental stress cofactors.

Thermal inactivation of bovine immunodeficiency virus.

Cell-associated bovine immunodeficiency virus (BIV) and cell-free BIV were subjected to increasing temperatures, including pasteurization conditions. To determine the effect of heat treatment on BIV viability, reverse transcriptase activity and infectivity of the heat-treated virus were assessed. BIV was inactivated by heating to 47 degrees C for 30 min and by low- and high-temperature pasteurization conditions.

Structural proteins of bovine coronavirus strain L 9: effects of the host cell and trypsin treatment.

The polypeptide profile of the cell-adapted strain of bovine coronavirus (Mebus BCV-L 9) is remarkably affected by the host cell and trypsin. We compared the structural proteins of virus purified from different cell lines and found cell-dependent differences in the virus structure. BCV was purified from four clones of human rectal tumour cells (HRT-18): 3F3, D2, 3E3, and 4B3. The structural profiles of BCV propagated in clones 3E3 and 3F3 were identical, consisting of proteins with molecular weights of 185, 160, 140, 125, 110, 100, 52, 46, 37, 31-34, and 26-28 kilodaltons (kd). BCV purified from clone D2 lacked the 100 kd species, and clone 4B3 yielded virus lacking the 46 kd protein. We compared the structures of BCV propagated in HRT-18 cells [BCV(HRT-18)] and virus raised in bovine fetal spleen cells [BCV(D2 BFS)]. The concentration of the 185 kd protein was higher in BCV (D2BFS), and it also contained a 200 kd species. Protein profiles of in vitro trypsin treated and untreated BCV(HRT-18) differed only under reducing conditions, suggesting that trypsin cleavage sites are located within disulfide-linked regions of affected proteins. Propagation of BCV in D2 BFS cells in the presence of trypsin resulted in cleavage of the 185 kd protein and a concomitant increase of the 100 kd protein. Activation of the fusion function probably depends on this cleavage process because fusion of BCV-infected D2 BFS cells is trypsin dependent.