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.     

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.     

Experimental collection and transfer of embryos from bovine immunodeficiency virus (BIV) infected cattle

Three experiments were conducted to determine whether the lentivirus, bovine immunodeficiency virus (BIV) is likely to be transmitted via embryo transfer. In the first experiment, embryos collected from BIV-negative heifers were exposed in vitro to BIV for 24 h, washed and then tested for the presence of the provirus. In the second experiment, embryos obtained from BIV-negative heifers were transferred to the uterine horns of BIV-infected heifers; 24 h later these embryos were recovered and tested for the presence of BIV. In the third experiment, embryos were collected from heifers experimentally infected with BIV and then transferred to BIV-negative recipients. In all three experiments, (BIV) proviral DNA was not detected by PCR in association with any oocytes, embryos, follicular fluid, oviductal or uterine washes. Twelve single embryos collected from BIV experimentally infected donors were transferred to BIV-negative recipients resulting in the birth of 7 calves all of which were also negative for BIV; the recipients remained BIV-negative throughout the experiment. In conclusion, this study demonstrates that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are unlikely to transmit this agent either to the recipients or the resulting offspring.     

Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat.

Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.     

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the B...     

Bovine lentivirus induces early transient B-cell proliferation in experimentally inoculated cattle and appears to be pantropic.

Bovine immunodeficiency-like virus (BIV) was first isolated in 1972 (M. J. VanDerMaaten et al., J. Natl. Cancer Inst. 49:1649-1657, 1972). Much work has been done on the molecular characterization of BIV in studies using the original BIV R29 isolate; however, R29 is believed to be attenuated since it no longer causes either mononuclear cell number increases or detectable enlargement of lymphatic nodules in experimentally infected cattle. The host cell tropism and changes in host peripheral blood lymphocyte populations following infection with BIV are unknown. Recently, we isolated and characterized a field isolate of BIV, FL112 (D. L. Suarez et al., J. Virol. 67:5051-5055, 1993) that causes a transient, mononuclear cell lymphocytosis in experimentally infected cattle. In the present study, cattle were inoculated with BIV FL112, and data from flow cytometry showed that BIV causes a B-cell lymphocytosis with no consistent, significant changes in other mononuclear cell populations, including CD3+, CD4+, and CD8+ cells. Cell sorting and PCR amplification were used to show that BIV may be pantropic. Proviral DNA was present in CD3+, CD4+, CD8+, and B-cells, monocytes, and WC1 cells (gamma/delta T cells, null cells) by 3 to 6 days postinoculation and also at 2.5 years postinoculation.     

牛泡沫病毒中国株感染兔的研究

将牛泡沫病毒(BFV3026)感染的细胞经耳缘静脉注射兔子,并以正常细胞注射的兔为对照。1年后处死,病毒挽救实验及PCR检测显示：兔经一次注射即可被BFV3026感染,病毒广泛分布于感染兔的多种脏器中,通过共培养可从感染兔血、肝、脾、肺、肾中拯救出相应感染性病毒颗粒,并在脑、骨髓、心、胰、肠系膜中检到高拷贝BFV原病毒DNA存在。同时,血清学检测表明：感染兔在接受注射一个月后即产生高滴度抗病毒蛋白抗体,并维持该滴度水平直至实验终止,兔未表现任何可观病变。     

牛泡沫病毒中国株感染兔的研究

将牛泡沫病毒(BFV3026)感染的细胞经耳缘静脉注射兔子,并以正常细胞注射的兔为对照.1年后处死,病毒挽救实验及PCR检测显示兔经一次注射即可被BFV3026感染,病毒广泛分布于感染兔的多种脏器中,通过共培养可从感染兔血、肝、脾、肺、肾中拯救出相应感染性病毒颗粒,并在脑、骨髓、心、胰、肠系膜中检到高拷贝BFV原病毒DNA存在.同时,血清学检测表明感染兔在接受注射一个月后即产生高滴度抗病毒蛋白抗体,并维持该滴度水平直至实验终止,兔未表现任何可观病变.     

Immunological characterization of the gag gene products of bovine immunodeficiency virus.

The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.     

Bovine abortion and neonatal death associated with Ureaplasma diversum

Ureaplasma diversum was isolated from the lungs and/or stomach fluid and placentas of five aborted bovine fetuses and four newborn calves. All isolates were serotype D48. Placentitis was observed in all instances in which the placenta was examined. Gross lesions consisted of focal or diffuse reddening of the chorioallantois and amnion and thickening of the amnion. Microscopically there were fibrosis, edema and inflammation of the amnion. Microscopic lesions in the lung consisted of diffuse pneumonitis with thickening of the alveolar walls and in some cases peribronchiolar lymphoid accumulations. Macrophages and granulocytes were present in the alveoli. Inoculation of the vulva of a virgin heifer with one of the isolates from a fetal lung produced hyperemia and profuse purulent discharge with slight granularity.     

Size variation within the second hypervariable region of the surface envelope gene of the bovine lentivirus BIV in experimentally and naturally infected cattle.

The bovine lentivirus also known as the bovine immunodeficiency-like virus (BIV) has conserved and hypervariable regions in the surface envelope (SU) gene. Size variation between isolates can be as large as 200 bp, mostly occurring in the second hypervariable (V2) gene region of the SU gene. The V2 region was cloned and sequenced from both experimentally and naturally infected cattle. Temporal evaluation of provirus from an experimentally inoculated cow showed two different-sized variants that appeared over time. The variation appeared to result from a recombinational event resulting in an apparent direct repeat. Cloned proviral nucleotide sequence diversity increased over time. Virus that was cultured and then cloned and sequenced showed progressive change from the inoculum virus, but culturing reduced the diversity of the clones as compared with direct amplification of provirus from leukocyte samples from the cow. The quasispecies phenomenon was evident in clones sequenced from a cow naturally infected with BIV. Of 10 clones examined from the V2 region, 6 different-size clones were present with nine different patterns of sequence rearrangement. Sequence length of different clones varied by as much as 43 amino acids (aa), with 21- and 15-aa direct repeats accounting for most of the size variation. Similar to other lentiviruses, BIV appears to mutate rapidly, which may be important in viral persistence and pathogenesis.     

Isolation and characterization of recombinants between attenuated and virulent aphthovirus strains.

A guanidine-resistant mutant of the attenuated strain of aphthovirus type 01 strain Campos and the original wild-type strain were crossed to generate recombinant viruses. Two independently derived recombinant viruses were isolated. One isolate (RI) contained the P1 (structural proteins) gene region of attenuated strain and P3 (polymerase precursor) gene region of the wild-type strain. The other isolate (RII) had a genomic structure complementary to that of RI, this is, P1 of the wild-type strain and P3 of the attenuated virus. Recombinant RII inherited some in vitro phenotypic markers that were characteristic of the attenuated strain, whereas the RI recombinant had in vitro behavior that was similar to that of the wild-type strain. The data obtained suggest that the polymerase precursor (P3) of the attenuated strain (01 Campos) could be involved in the determination of the attenuated phenotype for fetal bovine kidney cells and, eventually, for cattle.     

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。     

The bovine immunodeficiency virus Rev protein: identification of a novel nuclear import pathway and nuclear export signal among retroviral Rev/Rev-like proteins

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.     

Identification and characterization of the bovine immunodeficiency-like virus tat gene.

A cDNA clone of the bovine immunodeficiency-like virus (BIV) trans-activator gene (tat) was identified and characterized. The tat cDNA clone was generated by splicing, and on the basis of sequence analysis, the Tat protein was found to be encoded entirely by the first exon. It is 103 amino acids in size and shares sequence homology with the human immunodeficiency virus (HIV) Tat. The BIV tat clone can trans activate the BIV promoter effectively, as measured by the expression of the bacterial chloramphenicol acetyltransferase gene, when transfected into bovine cells. Besides activating the BIV promoter, the BIV Tat can also trans activate the HIV promoter effectively. It is possible that BIV Tat and HIV Tat employ similar mechanisms in trans activation of the viral long terminal repeat-directed gene expression.     

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.     

牛免疫缺陷病毒反式激活因子功能域的研究

牛免疫缺陷病毒 (Ｂｏｖｉｎｅｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙｖｉｒｕｓ,ＢＩＶ )与人免疫缺陷病毒 (Ｈｕｍａｎｉｍ ｍｕｎｏｄｅｆｉｃｉｅｎｃｙｖｉｒｕｓ,ＨＩＶ)同属反转录病毒科慢病毒属[1] 。ＢＩＶ基因组 5′端的长末端重复序列 (ＬＴＲ)起始病毒结构基因和非结构基因的转录[2 ] ,因而许多细胞因子和病毒编码的调节蛋白作用于ＬＴＲ ,以调节ＢＩＶ的基因表达。其中Ｔａｔ蛋白是ＢＩＶ的反式激活因子 ,可大大提高ＬＴＲ的转录水平 ,在ＢＩＶ的基因表达及基因组复制的调节中起重要作用[3 ] 。ＨＩＶ、马传染性贫血病毒 (Ｅｑｕｉ…