Bovine herpesvirus 4 is tropic for bovine endometrial cells and modulates endocrine function

in Reproduction

Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.

Abstract

Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.

Introduction

Bovine herpesvirus 4 (BoHV-4) was originally included in the β-herpesvirinae subfamily and referred to as bovine cytomegalovirus, primarily because its biological properties in tissue culture most closely resembled those of human cytomegaloviruses (Storz et al. 1984). However, molecular evidence has accumulated which indicate that BoHV-4 is genetically more closely related to members of the γ-herpesvirinae subfamily. This evidence includes large blocks of homologous genes arranged in the same order as two other γ-herpesviruses, Epstein–Barr virus and herpesvirus saimiri (Bublot et al. 1992). BoHV-4 replicates in a broad range of cell lines and primary cultures, causing a cytopathic effect (CPE; Truman et al. 1986, Peterson & Goyal 1988, Donofrio et al. 2002, Gillet et al. 2004). However, like other herpesviruses, BoHV-4 can establish persistent infections in its natural host (Krogman & McAdaragh 1982, Osorio & Reed 1983, Dubuisson et al. 1989, Dewals et al. 2005) and experimental hosts such as the rabbit (Osorio et al. 1982). Although the presence of BoHV-4 has been demonstrated in many tissues, the cells of the monocyte/macrophage lineage are a common site of persistence in natural and experimental hosts (Osorio & Reed 1983, Osorio et al. 1985a, 1985b, Dubuisson et al. 1988, 1989, Naeem et al. 1993). Cell lines persistently infected with γ-herpesviruses such as Epstein–Barr virus, herpesvirus saimiri, human herpesvirus 8 and murine γ-herpesvirus-68 have been established from cells isolated from infected hosts (Nilsson 1979, Ceserman et al. 1995, Usherwood et al. 1996, Jung et al. 1999). This process has likely been greatly facilitated by the growth-transforming ability of these γ-herpesviruses (Miller et al. 1997, Flore et al. 1998, Jung et al. 1999, Moses et al. 1999). In contrast, no evidence of growth transformation has been obtained for BoHV-4. Each of the genes associated with transformation by other γ-herpesviruses is unique to each virus, and a homologous gene is not found in BoHV-4 (Lomonte et al. 1996).

BoHV-4 was first isolated in Europe from animals with respiratory and ocular diseases by Bartha et al.(1966) and later in the United States by Mohanty et al.(1971). BoHV-4 has been isolated from a variety of samples and cells from healthy cattle and animals with metritis, abortion, pneumonia, diarrhoea, respiratory infection or mammary dermatitis (Bartha et al. 1966, Thiry et al. 1988, Egyed 1998). The pathogenic role of BoHV-4 remains unclear, and correlations between BoHV-4 and disease are unresolved even by experimental infection. Indeed, few investigators have successfully produced experimental disease (Thiry et al. 1988), and direct inoculation of the natural host only occasionally elicited respiratory and genital diseases including abortion (Wellemans et al. 1986, Castrucci et al. 1987).

Abortion may follow infection with a variety of α-, β- and γ-herpesvirus, but viral causes of uterine disease are seldom investigated in cattle. Although postpartum metritis affects up to 40% of cattle, causing considerable infertility and economic loss, it has been assumed that most diseases are of bacterial origin and virus isolation or serology is rarely considered (Sheldon & Dobson 2004). The first reported isolation of BoHV-4 from a case of bovine metritis was in Park & Kendrick (1973). Later, several other isolates were obtained from cows with reproductive disorders from different countries, including Italy (Castrucci et al. 1986) and India (Mehrotra et al. 1986). In Belgium, BoHV-4 seroprevalence was associated with postpartum metritis and chronic infertility of cattle (Czaplicki & Thiry 1998). Postpartum metritis has also been associated with BoHV-4 in the USA (Frazier et al. 2001, 2002), Spain (Monge et al. 2006) and Serbia (Nikolin et al. 2007). There is a lacuna in the knowledge about the direct correlation between viral infection and uterine pathology. Thus, the objectives of the present study were to determine the interaction, tropism and outcome of BoHV-4 challenge of endometrial epithelial and stromal cells.

Materials and Methods

Endometrial cell isolation and primary cultures

To better understand the interaction of BoHV-4 with the endometrium, it was important to use purified populations of epithelial and stromal cells without contamination by leukocytes, because BoHV-4 function can be influenced by macrophages (Donofrio & van Santen 2001). Bovine uteri from post-pubertal non-pregnant BoHV-4 serum-negative animals with no evidence of genital disease were collected at a local abattoir immediately after slaughter and kept on ice until further processing in the laboratory. The physiological stage of the reproductive cycle for each genital tract was determined by observation of the ovarian morphology (Ireland et al. 1980). Genital tracts with an ovarian stage I corpus luteum were selected for endometrial tissue and cell culture, and only the horn ipsilateral to the corpus luteum was used.

The endometrium was cut into strips and placed into serum-free RPMI-1640 (Sigma) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin and 2.5 μg/ml amphotericin B (Sigma), working under sterile conditions. The strips were then chopped into 1 mm3 pieces and placed into Hanks Balanced Salt Solution (HBSS; Sigma) and used as previously described (Fortier et al. 1988, Asselin et al. 1996), with the following modifications. Briefly, tissue was digested in 25 ml sterile filtered digestive solution, which was made by dissolving 50 mg trypsin III (Roche), 50 mg collagenase II (Sigma), 100 mg BSA (Sigma) and 10 mg DNase I (Sigma) in 100 ml phenol red-free HBSS. Following a 1.5 h incubation in a shaking water bath at 37 °C, the cell suspension was filtered through a 40 μm mesh (Fisher Scientific) to remove undigested material and the filtrate was resuspended in phenol red-free HBSS containing 10% fetal bovine serum (FBS; Sigma) and 3 μg/ml trypsin inhibitor (Sigma; washing medium). The suspension was centrifuged at 100 g for 10 min and following two further washes in washing medium the cells were resuspended in RPMI-1640 containing 10% FBS, 50 IU/ml penicillin, 50 μg/ml streptomycin and 2.5 μg/ml amphotericin B. The cells were plated at a density of 1 × 105 cells in 2 ml per well using 24-well plates (Nunc). To obtain separate stromal and epithelial cell populations, the cell suspension was removed 18 h after plating, which allowed selective attachment of stromal cells (Fortier et al. 1988). The removed cell suspension was then replated and incubated, allowing epithelial cells to adhere (Kim & Fortier 1995). Stromal and epithelial cell populations were distinguished by cell morphology as previously described (Fortier et al. 1988). The absence of immune cells in the uterine cell cultures was confirmed by RT-PCR for the CD45 pan-leukocyte marker as previously described (Herath et al. 2006). The culture media was changed every 48 h until the cells reached confluence. All cultures were maintained at 37 °C with 5% CO2 in air, in a humidified incubator.

Cell line cultures

To explore BoHV-4 tropism, the effect of viral challenge of the endometrial cells was compared with other cells, including Madin–Darby bovine kidney (MDBK, ATCC, CCL-22), and from M Ferrari (Istituto Zooprofilattico, Brescia, Italy) bovine embryo kidney (BEK), bovine embryo lung (BEL) and bovine turbinates (BT). The cells were maintained as monolayers in DMEM (Cambrex), containing 10% FBS, 2 mm l-glutamine, 100 IU/ml penicillin and 10 μg/ml streptomycin. The cells were incubated at 37 °C with 5% CO2 in air, in a humidified incubator and subcultured when growth reached 70–90% confluence every 3–5 days.

Virus

To test the susceptibility of cells to BoHV-4 infection, a recombinant BoHV-4 expressing enhanced green fluorescent protein was employed. Recombinant BoHV-4EGFPΔTK was obtained by insertion of the cytomegalovirus/enhanced green fluorescent protein (CMV/EGFP) expression cassette from the pEGFP-C1 plasmid into the thymidine kinase (TK) locus of the DN 599 BoHV-4 strain (Donofrio et al. 2002). BoHV-4EGFPΔTK and the NADL strain of bovine viral diarrhoea virus (BVDV) were propagated by infecting confluent monolayers of MDBK at a multiplicity of infection (m.o.i.) of 0.5 tissue cell infectious doses/50 (TCID50) per cell and maintained in minimum essential medium (MEM) with 2% FBS for 2 h. The medium was then removed and replaced by fresh MEM containing 10% FBS. The virus was purified when ~90% of the cell monolayer exhibited a CPE, at ~72 h post-infection (P.I.). Cell-associated virions were freed by three cycles of freezing the flasks at −80 °C and thawing. Cell debris was removed by low-speed centrifugation (1000 × g) and virions were pelleted through a 3 ml cushion of 30% sucrose in PBS, in a Beckman 70 Ti rotor at 35 000 r.p.m. (100 000 × g) for 90 min at 4 °C. Viral pellets were resuspended in cold MEM without FBS and TCID50 were determined on MDBK cells by limiting dilution (Vanderplasschen et al. 1995).

Infection of primary cell cultures with BoHV-4

Stromal and epithelial cells were challenged once confluence had been reached with BoHV-4EGFPTK at the concentrations indicated in results, or 1 μg/ml O55:B5 lipopolysaccharide (LPS, Sigma) as a positive control. Viral infection was monitored every 12 h by observation of cell fluorescence using a fluorescence microscope (Axiovert S100, Zeiss). The supernatants were harvested and frozen at −20 °C until used for prostaglandin RIA, and the endometrial cells were collected immediately for RNA isolation and further analysis.

MTT cell survival assay

CPE is the morphological change associated with the detrimental effects of viral replication on host cell homeostasis that ends with cell death. For epithelial cells, the CPE induced by BoHV-4 is characterised by swelling, whilst stromal cells shrink and detach from the surface of the culture flask. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell metabolic assay was used to measure the number of live cells. Briefly, 48 h after infection with BoHV-4EGFPΔTK, the cell cultures were incubated for 4 h with 100 μg/well MTT before addition of 100 μl solubilisation solution (10% SDS in HCl 0.01 M) and further incubation for 16 h at 37 °C. The yellow tetrazolium MTT salt is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilised by the addition of a detergent. The optical density was measured at 540 nm, using 690 nm as the reference wavelength in an SLT-Lab microreader (Salzburg, Austria); for each cell type, a linear relationship between cell number and optical density had already been established. Each experiment was done thrice and each treatment was performed with eight replicates. Statistical differences among treatments were tested by ANOVA.

Apoptosis assays and viral production

Apoptosis assays were performed because CPE by viruses such as BVDV is mediated by apoptosis. BVDV-or BoHV-4EGFPΔTK-infected confluent monolayers were gently scraped from 25 cm2 flasks with a sterile scraper in the presence of culture medium and the cells were pelleted by centrifugation at 12 000 g for 1 h at 4 °C. The cell pellet was gently resuspended in 500 μl extraction buffer (400 mM NaCl, 10 mM Tris–HCl, pH 7.8, 1 mM EDTA and 0.1% NP 40), transferred to a polypropylene microcentrifuge tube and kept on ice for 30 min. The solution was centrifuged at 12 000 g for 15 min, the supernatant recovered carefully in a fresh microfuge tube and extracted with phenol. The aqueous phase was transferred to a microfuge tube and a 0.1 volume of 10 M ammonium acetate and 1 volume of isopropanol added, mixed and centrifuged at 12 000 g for 5 min. The supernatant was removed and the white nucleic acid pellet washed with 500 μl of 70% ethanol and dissolved in 20 μl TE (10 mM Tris–HCl, 0.1 mM EDTA, pH 7.8) containing 20 μg/ml DNase-free pancreatic RNase (Sigma). Nuclear fragments were examined by electrophoresis in a 1.5% agarose gel and visualised by u.v. after staining with ethidium bromide.

For propidium iodide staining, cells were washed with PBS, stained with 400 ng/ml propidium iodine for 30 s in the darkness and fragmented nuclei were visualised by fluorescence microscopy.

Viral production by BT, BEL, MDBK, BEK, endometrial epithelial and stromal cells was tested after infecting cells with 1 m.o.i. of BoHV-4EGFPΔTK. The viral inoculums were removed after a 3-h absorption period and replaced with fresh media, and the viral titre quantified 48 h P.I.

Cell culture electroporation and viral reconstitution

To determine which step of the virus life cycle is important for the expression of the tropic phenotype, a reconstitution viral assay was performed. MDBK, BT and BEK cells from a sub-confluent 25 cm2 flask were electroporated (Equibio apparatus, 270 V, 960 μF) with 2 μg viral DNA purified as previously described (Gillet et al. 2005) in DMEM without serum and antibiotics. Electroporated cells were returned to new 25 cm2 flasks and fed with DMEM containing 10% FBS, 2 mm l-glutamine, 100 IU/ml penicillin and 10 μg/ml streptomycin. Endometrial stromal, endometrial epithelial and BEL cells from a sub-confluent 25 cm2 flask were electroporated (Equibio apparatus, Opty-Puls, 300 V, 25 μF, 240 V, 1050 μF and 481 R) with 2 μg viral DNA in DMEM with 10% FBS. Electroporated cells were returned to new 25 cm2 flasks and stromal and epithelia cells were fed with RPMI-1640 containing 10% FBS, 50 IU/ml of penicillin, 50 g/ml streptomycin and 2.5 g/ml amphotericin B and BEL cells with 90% DMEM containing 10% FBS, 2 mm l-glutamine, 100 IU/ml penicillin and 10 μg/ml streptomycin. The time necessary for the formation of viral plaques was monitored every 24 h by fluorescence microscopy.

Recombinant IE2 plasmid construction and transfection

To further investigate the mechanisms associated with endometrial tropism, a molecular switch involving the viral immediate early (IE) genes was investigated. The IE genes are expressed immediately during cell infection, do not require prior viral protein synthesis for their expression and their expression is mediated by the pool of transcription factors made by the cell, already present at the moment of infection and able to transactivate at the transcriptional level the IE promoters. BoHV-4 IE2 protein (replication and transcription activator homologous, Rta) encoded by open reading frame 50 (ORF 50) is well conserved among γ-herpesviruses (Zimmermann et al. 2001). Rta expression plays a primary role in initiating viral lytic replication, not only during reactivation of latently infected non-permissive cells but also during de novo infection of permissive cells (van Santen 1993, Sun et al. 1998, Song et al. 2002, Donofrio et al. 2004). The capability of endometrial cells to transactivate the BoHV-4 IE2 promoter was investigated by transient transfection of a fluorescent-labelled IE2 construct. A region of the BoHV-4 (DN599) genome corresponding to the region from nucleotide 61 391 to nucleotide 62 534 (GenBank accession number AF318571; Zimmermann et al. 2001) was generated by PCR using total DNA isolated from BoHV-4-infected MDBK cells as template and a pair of IE2 promoter primers (sense: 5′-gggaattccatatggccagtgccaagctttttaag-3′; antisense: 5′-gggaactagctagcctgttgttctgctccctttta-3′) containing an artificial NdeI site on the 5′ end and a NheI site on the 3′ end respectively. One microgram sample DNA was amplified over 35 cycles, each cycle consisting of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min and chain elongation with High Fidelity PCR Enzyme Mix (Fermentas) at 72 °C for 2 min. PCR amplification was carried out in a final volume of 50 μl, containing 0.2 mM deoxynucleoside triphosphate and 0.25 μM of each primer. In the first cycle, the samples were denatured at 94 °C for 5 min, and in the last cycle, the extension step was increased to 7 min. The amplicon was column purified, NdeI, NheI digested and subcloned in pEGFP-C1 vector (Clontech), excised with NdeI and NheI to remove the CMV promoter and generate the pIE2prom. EGFP construct. Transient transfection using 25 μg of the plasmid DNA construct was performed by electroporation, as described above. Endometrial epithelial and stromal cells, BT, BEL, MDBK and BEK cells were electroporated with the reporter construct and monitored every 24 h by fluorescence microscopy for green cells.

Prostaglandin RIA

Prostaglandins have a central role in many reproductive functions in mammals (Poyser 1995). Indeed, the duration of ovarian cycles and pregnancy depends on the presence of an active corpus luteum in the ovary, and the life span of the corpus luteum is regulated by the secretion of prostaglandin F (PGF) and prostaglandin estradiol (E2; PGE) from the uterine endometrium. Under physiological conditions, the uterine epithelial cells predominantly secrete PGF when stimulated with oxytocin, whereas the stromal cells produce PGE (Asselin et al. 1996). However, when epithelial or stromal cells are challenged with bacterial LPS, prostaglandin secretion is also stimulated (Herath et al. 2006). As BoHV-4 is isolated from animals with uterine disease, the effect of 24 h challenge with different amounts of BoHV-4 (10, 1 and 0.1 m.o.i. of virus) or a positive control of 1 μg/ml LPS on endometrial cell function was determined by measuring PGE and PGF in culture supernatants by RIA, as previously described (Cheng et al. 2001, Leung et al. 2001). Samples were diluted in 0.05 M Tris buffer containing 0.1% gelatine and 0.01% sodium azide. Standards and tritiated tracers for the PGs were purchased from Sigma and Amersham International PLC (Amersham) respectively. The antisera were a generous gift from Professor N L Poyser (University of Edinburgh, UK) and their cross-reactivity was: PGF antiserum, 34% with PGF, 25% with PGF and 0.54% with PGE2; PGE2 antiserum, 23% with PGE1, 15% with PGE3 and 0.47% with PGF (Poyser 1987). The limits of detection for PGE2 and PGF were 2 and 1 pg/tube respectively. The intra- and inter-assay coefficients of variation were 4.4 and 7.8% for PGE2 and 5.1 and 9.7% for PGF respectively.

Western blotting

PGF and PGE are synthesised from arachidonic acid under the regulation of cyclooxygenase 2 (COX-2) isoenzymes in the endometrium (Smith et al. 1996, Arosh et al. 2002). Stromal cells in 6-well culture plates were serum starved overnight, infected with 5 m.o.i. of BoHV-4 in the absence of serum and the cells were collected 1, 2, 4 and 8 h P.I. to measure COX-2 protein expression by western immunoblotting, with a positive control established by treating stromal cells with medium containing 20% FBS for 1 h. Cell extracts were prepared by adding 100 μl/well of cell extraction buffer (50 mM Tris–HCl, 150 mM NaCl and 1% NP-40; pH 8). Cell extracts containing 50 μg total protein were electrophoresed through 12% SDS–PAGE and transferred to nylon membranes by electroblotting. Membranes were incubated with rabbit anti-COX-2 polyclonal antibody (AB5118; Chemicon International Chandlers Ford, Hampshire, UK), probed with horseradish peroxidase-labelled goat anti-rabbit immunoglobulin G1 (IgG1) antibody (A0545, Sigma) and visualised by ECL (ECL Kit; Pierce, Rockford, IL, USA).

The effect of stromal cells on a macrophage cell line persistently infected with BoHV-4

The persistently infected macrophage cell line (BOMAC/ BoHV-4EGFPΔTK) was established as previously described (Donofrio & van Santen 2001) by infecting BOMAC cells, a cell line established from peritoneal macrophages by transformation with simian virus 40 DNA (Stabel & Stabel 1995). Confluent monolayers of BOMAC cells were inoculated m.o.i. of 1 TCID50/cell) with recombinant BoHV-4 (BoHV-4EGFPΔTK); by day 3 after inoculation, more than 95% of cells had detached from the monolayer, leaving behind a small number of cells that did not exhibit CPE. Confluent monolayers established from surviving cells showed 100% infection, as indicated by the strong fluorescent signal, but without apparent signs of CPE. Also consistent with our previous observations (Donofrio & van Santen 2001), the persistently infected macrophages produced infectious BoHV-4; medium recovered from BOMAC/BoHV-4EGFPΔTK cells inoculated onto BEK cells produced green plaques typical of BoHV-4EGFPΔTK. BOMAC/BoHV-4EGFPΔTK cells were subcultured at a dilution of 1:2 every 3 days and their growth medium was stored at −80 °C for viral titration. The yield of virus in the culture medium on the day the cells were subcultured remained in the range of 4 × 102 TCID50/ml throughout the first 20 passages. The macrophages were co-cultured with uninfected stromal cells or control BEK cells, using a transwell approach. After 48 h of co-culture, the stromal cells or BEK cells in the lower well were analysed under fluorescence microscope for EGFP expression. In addition, the viral titre was measured in the medium from the upper well, where the persistently infected macrophages were located.

Results

Isolation of pure populations of bovine endometrial stromal and epithelial cells

The bovine endometrium principally comprises of epithelial cells and stromal cells, but there are also sporadic leukocytes. To study the interaction of BoHV-4 with the endometrium, it was important to use purified populations of epithelial and stromal cells without leukocytes, which can influence BoHV-4 function (Donofrio & van Santen 2001). Endometrial cell cultures were established by the fractional enzyme dissociation method described in Materials and Methods, and there was no mRNA expression of the CD45 pan-leukocyte marker in epithelial or stromal cells (data not shown) as previously described (Herath et al. 2006). The epithelial and stromal cell purity was >95% as determined by microscopy (Fig. 1a and b) and the preferential secretion of PGF and PGE respectively as described previously (Asselin et al. 1996, Herath et al. 2006).

BoHV-4 infects bovine endometrial cells and induces cell death

To test the susceptibility of bovine endometrial cells to BoHV-4 infection, epithelia and stromal cells were infected with 1 m.o.i. of a recombinant BoHV-4 (BoHV-4EGFPΔTK) expressing EGFP (Donofrio et al. 2002). A few epithelial cells showed fluorescence 12 h P.I. (Fig. 2a) and this became generalised by 24 h, with the CPE starting 48 h P.I. and complete by 72 h. In contrast, most stromal cells were infected by 12 h P.I. (Fig. 2b), and the CPE started at 24 h P.I. and was complete by 48 h. To quantify the CPE of BoHV-4 infection, cell survival after challenge with different viral doses was measured using a MTT assay. Epithelial and stromal cells were killed in a dose-dependent manner 48 h after infection with BoHV-4 and the CPE was greater for stromal than epithelial cells (Fig. 2c). Thus, it appears that BoHV-4 infects both epithelial and stromal cells leading to a CPE, but the stromal cell infection seems to be more efficient.

CPE induced by BoHV-4 in bovine endometrial epithelial and stromal cells is not mediated by apoptosis, but is associated with a productive infection

BoHV-4 replicates in cell lines or primary cell cultures from a broad spectrum of host species. The infection in some permissive cells leads to viral progeny and a CPE; in other cells, there is a CPE even though no viral progeny are produced, whereas in some non-permissive cells, there is persistent BoHV-4 infection with no effect on cell survival (Donofrio et al. 2000, Gillet et al. 2004). As BoHV-4 infection induced a CPE in endometrial cells, the nature of the cell death and virus production was investigated.

Endometrial epithelial and stromal cells were infected with 5 m.o.i. of BoHV-4 and compared with 5 m.o.i. of BVDV, which is an established apoptotic virus (Schweizer & Peterhans 2001). The CPE induced by BoHV-4 was not mediated by cell apoptosis as determined by intranucleosomal DNA fragmentation (Fig. 3a) and propidium iodine staining (data not shown), in contrast to the cells infected with BVDV where there was clear DNA fragmentation (Fig. 3a).

Endometrial epithelial and stromal cell viral production was compared with other fully permissive cell types (BT, BEL, MDBK and BEK cells) infected with 1 m.o.i. of BoHV-4EGFPΔTK. The viral titres 48 h P.I. were approximately one log higher for epithelial and two logs higher for stromal cells when compared with the other cell types (Fig. 3b).

Post-viral entry is a determinant step for BoHV-4 replication in endometrial cells

From the above data, it appears that BoHV-4 has a striking tropism for endometrial cells or that endometrial cells are highly susceptible to BoHV-4 replication. However, viral attachment and penetration do not appear to be critical because many cell types were infected by BoHV-4. To determine which step of the virus life cycle is important for the expression of the tropic phenotype, a reconstitution viral assay was performed. Endometrial epithelial and stromal cells, BT, BEL, MDBK and BEK cells were electroporated with purified BoHV-4EGFP DNA, and the time necessary for the formation of viral plaques was monitored by fluorescence microscopy. Green plaques started to appear by 24 h after electroporation in epithelial and stromal cells, in contrast to the other cell types where visible green plaques formed between 3 and 5 days after electroporation (Fig. 4a and b).

BoHV-4 IE2 gene promoter is strongly transactivated in endometrial cells

To further investigate the mechanisms associated with endometrial tropism, a molecular switch involving the viral IE was investigated using an EGFP-labelled construct containing the IE2 gene (Fig. 5a) electroporated into endometrial epithelial and stromal cells, BT, BEL, MDBK and BEK cells. EGFP started to accumulate robustly as soon as 24 h after electroporation in the cytoplasm of stromal cells (Fig. 5b) and epithelial cells, in contrast to the other cell types where weak visible green cells appeared not before than 3 days post-electroporation (Fig. 5c).

Low-dose BoHV-4 infection stimulates PGE production in stromal cells

Under physiological conditions, the uterine epithelial cells predominantly secrete PGF when stimulated with oxytocin, whereas the stromal cells produce PGE (Asselin et al. 1996). The capacity of the endometrial cells to produce PGs in response to BoHV-4 was investigated as BoHV-4 is frequently isolated from the uterus. The cells were capable of responding to pathophysiological challenge as LPS stimulated PGF (Fig. 6a) and PGE (Fig. 6b) secretion from the epithelial and stromal cells respectively as reported previously (Herath et al. 2006). Epithelial cells did not produce PGF in response to BoHV-4 (Fig. 6a). However, stromal cells challenged with 0.1 m.o.i. produced more PGE than untreated controls (Fig. 6b). In contrast, higher viral doses suppressed PGE production, which is likely a consequence of the greater CPE induced by the virus replication.

The rate-limiting enzyme for PGE synthesis in the endometrium is COX-2 (Smith et al. 1996, Arosh et al. 2002). So, as BoHV-4 stimulated PGE secretion by endometrial stromal cells, the induction of COX-2 by BoHV-4 was examined by western blotting and COX-2 was up-regulated as early as 1 h P.I. (Fig. 6c).

Endometrial stromal cells enhance BoHV-4 replication in persistently infected macrophages

As BoHV-4 stimulated stromal cell PGE secretion, a scenario whereby PGE produced by stromal cells could activate BoHV-4 lytic replication in persistently infected macrophages was envisioned: i) Macrophages are the site of persistency of BoHV-4 (Osorio et al. 1985a, 1985b, Lopez et al. 1996, Donofrio & van Santen 2001); ii) persistently infected macrophages can easily reach the endometrium through the bloodstream and iii) the small amount of BoHV-4 viral particles could infect stromal cells, which will release PGE and reactivate BoHV-4 in persistently infected macrophages. To test the above scenario, stromal cells were co-cultivated with a bovine macrophage cell line persistently infected with BoHV-4EGFPΔTK, producing small amount of infectious viral particles (Donofrio et al. 2005). The effect of stromal cells on BoHV-4 replication in persistently infected macrophages was tested using a transwell approach (Fig. 7a). After 48 h of co-culture, the stromal cells had a higher level of infection and a higher viral titre in the upper well than the BEK cells (Fig. 7a and b). These data support the concept of a symbiotic relationship between the stromal cells and persistently infected macrophages.

Discussion

Bos taurus is particularly prone to uterine infection and metritis after parturition, which causes considerable infertility and economic loss (Sheldon & Dobson 2004). The most commonly recognised uterine pathogens are Arcanobacterium pyogenes and Escherichia coli (Griffin et al. 1974, Ruder et al. 1981, Olson et al. 1984, Bonnet et al. 1993, Sheldon & Dobson 2004). Although viral isolation or serology is not routinely performed in animals with uterine disease, BoHV-4 has been implicated in cases of bovine metritis (Park & Kendrick 1973, Nilsson 1979, Fortier et al. 1988, Czaplicki & Thiry 1998, Frazier et al. 2002, Monge et al. 2006). The present study demonstrated a viral tropism by BoHV-4 for the endometrium. The high efficiency of BoHV-4 replication in endometrial cells was associated with strong transactivation of viral IE genes. Furthermore, BoHV-4 increased stromal cell COX-2 protein and stimulated PGE secretion. This viral-induced stromal cell PGE secretion may be a mechanism by which viral replication is stimulated in macrophages, which are the usual repository for persistent bovine infections.

Viruses are restricted to using the metabolic and biosynthetic pathways of the cells that they infect. These pathways vary between cell types, lineage, stage of differentiation and with the state of cell activation. There are many examples of viruses that replicate in specific cells and at particular stages of cell growth, differentiation or activation. This includes the reactivation of cytomegalovirus when host cells differentiate into macrophages; initiation of papillomavirus replication by keratinocytes and replication of minute virus in testicular cells. The key mechanism mediating these effects is the regulation of viral gene expression at the transcriptional level by host cell factors. The present study identified a striking tropism of BoHV-4 for endometrial cells. BoHV-4 efficiently infected purified populations of bovine endometrial stromal and epithelial cells, leading to a non-apoptotic cell death and de novo viral production, which could be an important mechanism underlying the metritis associated with BoHV-4 infection in cattle. The lack of BoHV-4 apoptogenicity found in endometrial cells where BoHV-4 infection was highly productive fits well with the BoHV-4 pro-apoptotic behaviours found in some cell types (Gillet et al. 2005) where BoHV-4 infection is completely unproductive. In a complex multicellular organism, rapid apoptotic cell suicide on virus infection can be seen as an altruist response (Allsopp & Fazakerley 2000). If it occurs before complete virus replication and assembly, it will be highly effective in limiting viral production. For many viruses, tropism and successful replication are determined by specific cellular receptors that must be engaged for virus binding and entry. However, BoHV-4 can enter many cell types from different species (Donofrio et al. 2002). Furthermore, successful replication of BoHV-4 seems to be governed primarily by post-entry events as shown by the fast viral reconstitution following the electroporation of nude viral DNA into the cells. The ORF 50 gene product, also known as the replication and transcription activator (Rta), is an IE gene which is well conserved among all γ-2 herpesviruses. BoHV-4 IE2 RNA is the less abundant, spliced, 1.8 kb RNA, which is transcribed from the left to the right on the restriction map of the BoHV-4 genome and contained in the 8.3 kb Hind III fragment F (van Santen 1993). The predicted amino acid sequence of the protein encoded by IE2 RNA reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein–Barr virus transactivator R and its homologue including herpesvirus saimiri, equine herpesvirus 2, murine herpesvirus 68 and Kaposi’s sarcoma-associated herpesvirus. Transactivation studies have shown that BoHV-4 IE2 protein specifically transactivates expression of a reporter gene linked to the promoter regulatory region of the BoHV-4 early (E) gene encoding the BoHV-4 homologue of herpes simplex virus type 1 major DNA-binding protein (van Santen 1991). The BoHV-4 ORF 50 homologues have been shown to play a pivotal role in regulating the lytic switch in a non-permissive cell line following their overexpression (Donofrio et al. 2004). Indeed, in the present study, IE2 promoter transactivation in endometrial cells triggered the BoHV-4 phenotype in those cells, as shown by the fast up-regulation of EGFP reporter gene expression driven by IE2 promoter. Interestingly, a preliminary analysis of the BoHV-4 IE2 promoter by Tfsitescan/ dynamicPlus server reveals five positive regulatory elements that are also found in the rabbit uteroglobin promoter and apparently specific for endometrial cells. The most surprising similarity was found for the responsive element close to the TATA box, which may indicate the existence of an endometrial-specific TATA box (Misseyanni et al. 1991). A comparison with the promoters of two other endometrium-specific genes, the rat homologous to rabbit uteroglobin (Hagen et al. 1990) and the pig uteroferrin gene (Simmen et al. 1989) indicates that this TATA box element is well conserved. Further studies are needed to identify the nature of the factors in bovine endometrial cells and how they interact with BoHV-4 IE2 promoter.

Specific enhancers and transcriptional activators produced by host cells are important to initiate and maintain viral replication. In persistently infected macrophages, BoHV-4 replication is stimulated by the addition of exogenous PGE (Donofrio et al. 2005). Bovine endometrial cells induce COX-2 and PGE production in response to a number of cell activators and inflammatory signals, such as LPS (Herath et al. 2006). Taken together, these observations support a scenario where viral particles infecting the endometrium could stimulate PGE production and establish a positive-feedback loop between PGE production and viral replication (Donofrio et al. 2005). That model of BoHV-4-induced metritis seems to be supported in the present ex vivo study by the observations that bovine endometrial stromal cells produced PGE following BoHV-4 infection and cells persistently infected with BoHV-4 were reactivated after co-cultivation with endometrial stromal cells using a transwell approach. Infections by several viruses, including many herpesviruses, such as herpes simplex virus, human cytomegalovirus, Epstein–Barr virus and murine γ-herpesvirus 68 have been reported to alter COX-2 expression (Reynolds & Enquist 2006). In fact, rhesus cytomegalovirus even encodes a COX-2 homologue in its genome, emphasising the importance of this enzyme (Hansen et al. 2003). In addition, many studies have examined the regulation of COX-2 expression and PGE2 production during viral infection as well as the effect of PGE2 production on viral replication and virulence (Steer & Corbett 2003). Prostaglandins are potent mediators of many critical physiological and inflammatory responses, and they modulate the host defence against various pathogens. They suppress some innate immune factors, including nitric oxide (NO) production, and have effects on the acquired immune response, specifically by suppressing the Th1 response. For instance, PGE2 can inhibit the production of γ-interferon by activated human T cells in vitro (Snijdewint et al. 1993) and that of Th1 cytokines such as interleukin-12 in vivo (Newberry et al. 1999, Kuroda et al. 2000). In addition to inhibiting the production of Th1 cytokines, PGE2 switches the immune response towards a Th2 response, which is less effective in mounting an antiviral response (Betz & Fox 1991, Kuroda et al. 2000). PGE is one of the most potent and abundant PGs present during inflammatory reactions (Appleton et al. 1996). The very early host responses to viral infections are usually non-specific and include the induction of cytokines such as interferons and tumour necrosis factor-α. NO synthase (NOS) is an interferon-inducible protein that is activated during innate immune responses (Reiss & Komatsu 1998). When present at high concentrations after the expression of the inducible isoform of NOS (iNOS), NO functions as a cytotoxic molecule, reacting with proteins or H2O2 to form a highly toxic compound called peroxynitrite (ONOO; Reiss & Komatsu 1998). NO is also thought to participate in the antiviral response to infection by attenuating the replication of both DNA and RNA viruses (Reiss & Komatsu 1998). The products of COX and NOS enzymes, PGs and NO, have been shown to share an antagonistic relationship with one another. The inhibition of COX activity in vesicular stomatitis virus (VSV)-infected cells causes a reduction inviral propagation and a concordant increase in extracellular NO levels. Treatment with an iNOS inhibitor, l-NAME or exogenous PGE2 in the presence of COX inhibitors can restore VSV growth and decrease NO production, underscoring a role for PGs in counteracting the antiviral effects of NO (Chen et al. 2000). Besides their role in immunomodulation and counteraction of the antiviral effects of NO, PGs have been shown to be involved in modulating transcription from viral promoters. The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains sequences that are important for DNA integration, as well as signals, such as an internal polymerase II promoter, which are necessary for the transcription of the integrated retroviral DNA. PGE2 can increase transcription driven by the HIV-1 LTR in T lymphocytes (Dumais et al. 1998). Transcription of one of the IE (IE2) of HCMV was reduced in cells that were treated with COX-2 inhibitors. Therefore, a potential role for COX induction in the context of a virus infection is the activation of transcription from viral promoters via PGs.

In the case of BoHV-4 persistently infected animals, stromal cell PGE secretion may reactivate viral replication, which in turn would lead to endometrial inflammation. Bacterially induced metritis in cattle persistently infected with BoHV-4 could possibly be exacerbated or become chronic following the recruitment from the bloodstream to the site of inflammation of macrophages persistently infected with BoHV-4. This theory could explain the fact that BoHV-4 can be isolated from a healthy animal too, where in the absence of inflammation, the pathogenic potential of BoHV-4 is ameliorated.

In conclusion, the present study indicates that BoHV-4 is a likely pathogen associated with uterine disease. The virus is tropic for endometrial epithelial and stromal cells, causing a rapid CPE. It appears that the endometrial cells rapidly induce IE gene expression and viral replication. Furthermore, there was a symbiotic relationship between stromal cell PGE secretion in response to BoHV-4 and reactivation of viral replication in macrophages. These data provide evidence of cellular and molecular mechanisms underlying the observation of uterine disease in animals with BoHV-4.

Figure 1

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Figure 1

Representative phase contrast images (10×) of pure population of (a) bovine endometrial epithelial and (b) stromal cells.

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Figure 2

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Figure 2

Representative fluorescence (green) and phase contrast images (10×) of (a) bovine endometrial epithelial (b) and stromal cells at different times (12, 24, 48 and 72 h) P.I. with 1 m.o.i. of BoHV-4 EGFPΔTK and the respective phase contrast images of uninfected control. Spreading of the infection can be observed by the green colour invading the field during the time and the CPE is morphologically appreciable by the change of the cell shape, where the cells tend to shrink, becoming rounder and detaching from the flask surface. The experiment was repeated thrice, giving the same result. (c) Assessment of cell survival measured by the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) at 48 h P.I. Hundred percent of survival was considered the uninfected control. Values are the mean ± s.d. of three independent experiments (n = 8 per treatment, P ≥ 0.009).

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Figure 3

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Figure 3

(a) At 72 h P.I, 1.5% agarose gel electrophoresis of DNA extracts from bovine endometrial epithelial, stromal and control BEK cells, infected with 5 m.o.i. of BoHV-4 and BVDV control virus. (b) Titres of BoHV-4 particles released by different cell types at 48 h P.I. Values are the mean ± s.d. of three independent experiments (n = 3 per treatment, P ≥ 0.05).

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Figure 4

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Figure 4

(a) BoHV-4 plaques obtained at different times (1, 2, 3, 4 and 5 days) post-electroporation (P.E.) of nude BoHV-4EGFPΔTK DNA in different cell types. Images are representative of bovine epithelial, stromal and BEK cells. (b) Graph bars summarising the time necessary for plaques formation in different cell types (endometrial epithelial cells, endometrial stromal cells, BT, BEL, MDBK and BEK cells). Values are the mean ± s.d. of three independent experiments (n = 3 per treatment, P ≥ 0.05).

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Figure 5

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Figure 5

(a) Diagram showing the 1143 bp IE2 promoter sequence containing the putative TATA box (underlined in black) and the first 15 non-coding nucleotides of the first exon. Sense and antisense primers used for the PCR amplification are in red and contain the NdeI and NheI restriction sites (underlined in red) respectively for sub-cloning of the amplicon in front of the EGFP ORF (green box) followed by the bovine growth hormone polyadenylation signal (pA (grey box)). (b) Representative fluorescence and phase contrast images of transfected endometrial stromal cells with the above-described construct and expressing EGFP 24 h post-transfection. (c) Summarising schema of the time necessary for EGFP accumulation into the different cell types (endometrial epithelial cells, endometrial stromal cells, BT, BEL, MDBK and BEK cells), following transfection with the above-described construct. Values are the mean of three independent experiments (n = 3 and P ≥ 0.05).

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Figure 6

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Figure 6

Prostaglandin production by (a) epithelial and (b) stromal cells after challenge with LPS (1 μg/ml) or BoHV-4 at the different m.o.i. indicated. After 24 h in culture, supernatants were harvested and prostaglandin production was measured by RIA. Values are presented as the mean ± s.d. of three independent experiments. Differences were statistically different at P < 0.05 when compared with a media control. (c) Western immunoblotting of endometrial stromal cell extracts and infection with BoHV-4 EGFPΔTK. Positive control was established treating cells with medium containing 20% of FBS. β-Actin as the loading control. The same experiment was repeated thrice, giving the same results.

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Figure 7

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Figure 7

(a) Schematic illustration of the transwell model used for co-culturing BoHV-4EGFPΔTK persistently infected bovine macrophage cell line (in green in the upper well), producing low amount of infectious virus (grey dots) with bovine endometrial stromal or control BEK cells (in yellow) seated in the lower well. The transwell consists of a permeable membrane and allows diffusion of the viral particles. (b) Fluorescence and phase contrast images of bovine endometrial stromal and control BEK cells seated in the lower well at 48 h post co-cultivation. (c) Graph bar of the viral titre in the upper well. Values are the mean ± s.d. of three independent experiments (n = 3 and P ≥ 0.05).

Citation: Reproduction 134, 1; 10.1530/REP-07-0065

Received 7 February 2007
 First decision 6 March 2007
 Revised manuscript received 13 March 2007
 Accepted 29 March 2007

We thank the Italian Minister of Science (Italian National Grant MIUR, PRIN 2005078885) and BBSRC (Grant 48/S19795) for financial support. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Allsopp TE & Fazakerley JK2000 Altruistic cell suicide and the specialized case of the virus-infected nervous system. Trends in Neurosciences23284–290.

  • Appleton I Tomlinson A & Willoughby DA1996 Induction of cyclooxygenase and nitric oxide synthase in inflammation. Advances in Pharmacology3527–78.

  • Arosh JA Parent J Chapdelaine P Sirois J & Fortier MA2002 Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biology of Reproduction67161–169.

  • Asselin E Goff AK Bergeron H & Fortier MA1996 Influence of sex steroids on the production of prostaglandins F2 alpha and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biology of Reproduction54371–379.

  • Bartha A Juhasz M & Liebermann H1966 Isolation of a bovine herpesvirus from calves with respiratory disease and keratoconjuntivitis. Acta Veterinaria Academiae Scientiarum Hungaricae16357–358.

  • Betz M & Fox BS1991 Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines. Journal of Immunology146108–113.

  • Bonnett BN Martin SW & Meek AH1993 Associations of clinical findings bacteriological and histological results of endometrial biopsy with reproductive performance of postpartum dairy cows. Preventive Veterinary Medicine15205–220.

  • Bublot M Lomonte P Lequarre AS Albrecht JC Nicholas J Fleckenstein B Pastoret PP & Thiry E1992 Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses Epstein–Barr virus and herpesvirus saimiri. Virology190654–655.

  • Castrucci G Frigeri F Cilli V Donelli G Ferrari M Chicchinri U & Bordoni E1986 A study of herpesvirus isolated from dairy cattle with a history of reproductive disorders. Comparative Immunology Microbiology and Infectious Diseases913–21.

  • Castrucci G Frigeri F Ferrari M Ranucci S Aldrovandi V Cilli V Rampichini L & Gatti R1987 Experimental infection of calves with strain of bovid herpesvirus-4. Comparative Immunology Microbiology and Infectious Diseases1041–49.

  • Ceserman E Moore PS Rao PH Inghirami G Knowles DM & Chang Y1995In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood862708–2714.

  • Chen N Warner JL & Reiss CS2000 NSAID treatment suppresses VSV propagation in mouse CNS. Virology27644–51.

  • Cheng Z Robinson RS Pushpakumara PG Mansbridge RJ & Wathes DC2001 Effect of dietary polyunsaturated fatty acids on uterine prostaglandin synthesis in the cow. Journal of Endocrinology171463–473.

  • Czaplicki G & Thiry E1998 An association exists between bovine herpesvirus-4 seropositivity and abortion in cows. Preventive Veterinary Medicine33235–240.

  • Dewals B Gillet L Gerdes T Taracha EL Thiry E & Vanderplasschen A2005 Antibodies against bovine herpesvirus 4 are highly prevalent in wild African buffaloes throughout eastern and southern Africa. Veterinary Microbiology110209–220.

  • Donofrio G & van Santen VL2001 A bovine macrophage cell line supports bovine herpesvirus 4 persistent infection. Journal of General Virology821181–1185.

  • Donofrio G Cavirani S & van Santen VL2000 Establishment of a cell line persistently infected with bovine herpesvirus 4 using a recombinant virus. Journal of General Virology811807–1814.

  • Donofrio G Cavirani S Taddei S & van Santen VL2002 Bovine herpesvirus 4 as a gene delivery vector. Journal of Virological Methods10149–61.

  • Donofrio G Cavirani S Taddei S & Flammini CF2004 Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by over expression of BoHV-4 immediate early (IE) 2 gene. Journal of Virological Methods116203–207.

  • Donofrio G Cavirani S van Santen VL & Flammini CF2005 Potential secondary pathogenic role for bovine herpesvirus 4. Journal of Clinical Microbiology433421–3426.

  • Dubuisson J Thiry E Thalasso F Bublot M & Pastoret PP1988 Biological and biochemical comparison of bovid herpesvirus-4 strains. Veterinary Microbiology16339–349.

  • Dubuisson J Thiry E Bublot M Thomas I van Bressem MF Coignoul F & Pastoret PP1989 Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone. Veterinary Microbiology2197–114.

  • Dumais N Barbeau B Olivier M & Tremblay MJ1998 Prostaglandin E2 up-regulates HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways. Journal of Biological Chemistry27327306–27314.

  • Egyed L1998 Replication of bovine herpesvirus type 4 in human cells in vitro. Journal of Clinical Microbiology362109–2111.

  • Flore O Rafii S Ely S O’Leary JJ Hyjek EM & Cesarman E1998 Transformation of primary human endothelial cells by Kaposi’s sarcoma-associated herpesvirus. Nature394588–592.

  • Fortier MA Guilbault LA & Grasso F1988 Specific properties of epithelial and stromal cells from the endometrium of cows. Journal of Reproduction and Fertility83239–248.

  • Frazier K Pence M Mauel MJ Liggett A Hines ME Sangster L Lehmkuhl HD Miller D Styer E West J & Baldwin CA2001 Endometritis in postparturient cattle associated with bovine herpesvirus-4 infection: 15 cases. Journal of Veterinary Diagnostic Investigation13502–508.

  • Frazier KS Baldwin CA Pence M West J Bernard J Liggett A Miller D & Hines ME2002 Seroprevalence and comparison of isolates of endometriotropic bovine herpesvirus-4. Journal of Veterinary Diagnostic Investigation14457–462.

  • Gillet L Minner F Detry B Farnir F Willems L Lambot M Thiry E Pastoret PP Schynts F & Vanderplasschen A2004 Investigation of the susceptibility of human cell lines to bovine herpesvirus 4 infection: demonstration that human cells can support a nonpermissive persistent infection which protects them against tumor necrosis factor α-induced apoptosis. Journal of Virology782336–2347.

  • Gillet L Dewals B Farnir F de Leval L & Vanderplasschen A2005 Bovine herpesvirus 4 induces apoptosis of human carcinoma cell lines in vitro and in vivo. Cancer Research659463–9472.

  • Griffin JF Hartigan PJ & Nunn WR1974 Non-specific uterine infection and bovine fertility. I. Infection patterns and endometritis during the first seven weeks postpartum. Theriogenology191–106.

  • Hagen G Wolf M Katyal SL Singh G Beato M & Suske G1990 Tissue-specific expression hormonal regulation and 5′-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobi. Nucleic Acids Research182939–2946.

  • Hansen SG Strelow LI Franchi DC Anders DG & Wong SW2003 Complete sequence and genomic analysis of rhesus cytomegalovirus. Journal of Virology776620–6636.

  • Herath S Fischer DF Werling D Williams EJ Lilly ST Dobson H Bryant CE & Sheldon IM2006 Expression and function of toll-like receptor 4 in the endometrial cells of the uterus. Endocrinology147562–570.

  • Ireland JJ Murphee RL & Coulson PB1980 Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. Journal of Dairy Science63155–160.

  • Jung JU Choi JK Ensser A & Biesinger B1999 Herpesvirus saimiri as a model for gammaherpesvirus oncogenesis. Seminars in Cancer Biology9231–239.

  • Kim JJ & Fortier MA1995 Cell type specificity and protein kinase C dependency on the stimulation of prostaglandin E2 and prostaglandin F2 alpha production by oxytocin and platelet-activating factor in bovine endometrial cells. Journal of Reproduction and Fertility103239–247.

  • Krogman LA & McAdaragh JP1982 Recrudescence of bovine herpesvirus-5 in experimentally infected calves. American Journal of Veterinary Research43336–338.

  • Kuroda E Sugiura T Zeki K Yoshida Y & Yamashita U2000 Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice. Journal of Immunology1642386–2395.

  • Leung ST Cheng Z Sheldrick EL Derecka K Flint AP & Wathes DC2001 The effects of lipopolysaccharide and interleukins-1alpha -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium. Journal of Endocrinology168497–508.

  • Lomonte P Bublot M vanSanten VL Keil G Pastoret PP & Thiry E1996 Bovine herpesvirus 4: genomic organization and relationship with two other gammaherpesviruses Epstein–Barr virus and herpesvirus saimiri. Veterinary Microbiology5379–89.

  • Lopez OJ Galeotta J & Osorio FA1996 Bovine herpesvirus type-4 (BHV-4) persistently infects cells of the marginal zone of spleen in cattle. Microbial Pathogenesis2147–58.

  • Mehrotra ML Shucla DC & Srivastava NC1986 Isolation of a new herpesvirus from cases of reproductive disorders in cow. Indian Journal of Animal Sciences561196–1199.

  • Miller G Heston L Grogan E Gradoville L Rigsby M Sun R Shedd D Kushnaryov VM Grossberg S & Chang Y1997 Selective switch between latency and lytic replication of Kaposi’s sarcoma herpesvirus and Epstein–Barr virus in dually infected body cavity lymphoma cells. Journal of Virology71314–324.

  • Misseyanni A Klug J Suske G & Beato M1991 Novel upstream elements and the TATA-box region mediate preferential transcription from the uteroglobin promoter in endometrial cells. Nucleic Acids Research192849–2859.

  • Mohanty SB Hammond RC & Lillie MG1971 A new bovine herpesvirus and its effect on experimentally infected calves. Archiv Für Die Gesamte Virusforschung34394–395.

  • Monge A Elvira L Gonzalez JV Astiz S & Wellenberg GJ2006 Bovine herpesvirus 4-associated postpartum metritis in a Spanish dairy herd. Research in Veterinary Science80120–125.

  • Moses AV Fish KN Ruhl R Smith PP Strussenberg JG Zhu L Chandran B & Nelson JA1999 Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8. Journal of Virology736892–6902.

  • Naeem K Caywood DD Goyal SM Werdin RE & Murtaugh MP1993 Persistence of bovid herpesvirus-4 in experimentally inoculated pregnant rabbits. New Microbiologica1687–93.

  • Newberry RD Stenson WF & Lorenz RG1999 Cyclooxygenase-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigen. Nature Medicine5900–906.

  • Nikolin VDonofrio GMilošević BTaddei SRadosavljević V & Milićević V2007 First Serbian isolates of bovine herpesvirus 4 (BoHV-4) from a herd with history of postpartum metritis. New Micribiologica3053–57.

  • Nilsson K1979 The nature of lymphoid cell lines and their relationship to the virus. In The Epstein–Barr Virus pp 225–281. Eds MA Epstein & BG Achong. Berlin: Verlag.

  • Olson JD Ball L Mortimer RG Farin PW Adney WS & Huffman EM1984 Aspects of bacteriology and endocrinology of cows with pyometra and retained foetal membranes. American Journal of Veterinary Research452251–2255.

  • Osorio FA & Reed DE1983 Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection. American Journal of Veterinary Research44975–980.

  • Osorio FA Reed DE & Rock DL1982 Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection. Veterinary Microbiology7503–513.

  • Osorio FA Reed DE Van Der Maaten MJ & Metz CA1985a Comparison of the herpesviruses of cattle by restriction endonuclease analysis and serologic analysis. American Journal of Veterinary Research462104–2109.

  • Osorio FA Rock DL & Reed DE1985b Studies on the pathogenesis of a bovine cytomegalo-like virus in an experimental host. Journal of General Virology661941–1951.

  • Park JB & Kendrick JW1973 The isolation and partila characterization of a herpesvirus from a case of bovine metritis. Archiv Für Die Gesamte Virusforschung41211–215.

  • Peterson RB & Goyal SM1988 Propagation and quantitation of animal herpesviruses in eight cell culture systems. Comparative Immunology Microbiology and Infectious Diseases1193–98.

  • Poyser NL1987 Effects of various factors on prostaglandin synthesis by the guinea-pig uterus. Journal of Reproduction and Fertility81269–276.

  • Poyser NL1995 The control of prostaglandin production by the endometrium in relation to luteolysis and menstruation. Prostaglandins Leukotriens and Essential Fatty Acids53147–195.

  • Reiss CS & Komatsu T1998 Does nitric oxide play a critical role in viral infections? Journal of Virology724547–4551.

  • Reynolds AE & Enquist LW2006 Biological interactions between herpesviruses and cyclooxygenase enzymes. Reviews in Medical Virology16393–403.

  • Ruder CA Sasser RG Williams RJ Ely JK Bull RC & Butler JE1981 Uterine infections in the postpartum cow. II. Possible synergistic effect of Fusobacterium necrophorum and Corynebacterium pyogenes. Theriogenology15573–580.

  • van Santen VL1991 Characterization of the bovine herpesvirus 4 major immediate-early transcript. Journal of Virology655211–5224.

  • van Santen VL1993 Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein–Barr virus R transactivator. Journal of Virology67773–784.

  • Schweizer M & Peterhans E2001 Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis. Journal of Virology754692–4698.

  • Sheldon IM & Dobson H2004 Postpartum uterine health in cattle. Animal Reproduction Science82–83295–306.

  • Simmen RC Srinivas V & Roberts RM1989 cDNA sequence gene organization and progesterone induction of mRNA for uteroferrin a porcine uterine iron transport protein. DNA8543–554.

  • Smith WL Garavito RM & DeWitt DL1996 Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. Journal of Biological Chemistry27133157–33160.

  • Snijdewint FG Kalinski P Wierenga EA Bos JD & Kapsenberg ML1993 Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. Journal of Immunology1505321–5329.

  • Song MJ Li X Brown HJ & Sun R2002 Characterization of interactions between R.T.A. and the promoter of polyadenylated nuclear RNA in Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8. Journal of Virology765000–5013.

  • Stabel JR & Stabel TJ1995 Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA. Veterinary Immunology and Immunopathology45211–220.

  • Steer SA & Corbett JA2003 The role and regulation of COX-2 during viral infection. Viral Immunology16447–460.

  • Storz J Ehlers B Todd VJ & Ludwig H1984 Bovine cytomegaloviruses: identification and differential properties. Journal of General Virology65697–706.

  • Sun R Lin SF Gradoville L Yuan Y Zhu F & Miller G1998 A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus. PNAS9510866–10871.

  • Thiry E Meersschaert C & Pastoret PP1988 Epizootiology of herpesvirus infections in wild ruminants. I. Infectious bovine rhinotracheitis virus and antigenically related viruses. Revue D’élevage et De Médecine Vétérinaire Des Pays Tropicaux41113–120.

  • Truman D Ludwig H & Storz J1986 Bovine herpesvirus type 4: studies on the biology and spread in cattle herds and in insemination bulls. Journal of Veteterinary Medicine (Series B)33485–501.

  • Usherwood EJ Stewart JP & Nash AA1996 Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice. Journal of Virology706516–6518.

  • Vanderplasschen A Goltz M Lyaku J Benarafa C Buhk HJ Thiry E & Pastoret PP1995 The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle. Virology213328–340.

  • Wellemans G Van Opdenbosh E & Mammerickx M1986 Inoculation expérimental du virus LVR 140 (herpes bovin IV) à des vaches gestantes et non-gestantes. Annales de Recherches Vétérinaires1789–94.

  • Zimmermann W Broll H Ehlers B Buhk HJ Rosenthal A & Goltz M2001 Genome sequence of bovine herpesvirus 4 a bovine Rhadinovirus and identification of an origin of DNA replication. Journal of Virology751186–1194.

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    Representative phase contrast images (10×) of pure population of (a) bovine endometrial epithelial and (b) stromal cells.

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    Representative fluorescence (green) and phase contrast images (10×) of (a) bovine endometrial epithelial (b) and stromal cells at different times (12, 24, 48 and 72 h) P.I. with 1 m.o.i. of BoHV-4 EGFPΔTK and the respective phase contrast images of uninfected control. Spreading of the infection can be observed by the green colour invading the field during the time and the CPE is morphologically appreciable by the change of the cell shape, where the cells tend to shrink, becoming rounder and detaching from the flask surface. The experiment was repeated thrice, giving the same result. (c) Assessment of cell survival measured by the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) at 48 h P.I. Hundred percent of survival was considered the uninfected control. Values are the mean ± s.d. of three independent experiments (n = 8 per treatment, P ≥ 0.009).

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    (a) At 72 h P.I, 1.5% agarose gel electrophoresis of DNA extracts from bovine endometrial epithelial, stromal and control BEK cells, infected with 5 m.o.i. of BoHV-4 and BVDV control virus. (b) Titres of BoHV-4 particles released by different cell types at 48 h P.I. Values are the mean ± s.d. of three independent experiments (n = 3 per treatment, P ≥ 0.05).

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    (a) BoHV-4 plaques obtained at different times (1, 2, 3, 4 and 5 days) post-electroporation (P.E.) of nude BoHV-4EGFPΔTK DNA in different cell types. Images are representative of bovine epithelial, stromal and BEK cells. (b) Graph bars summarising the time necessary for plaques formation in different cell types (endometrial epithelial cells, endometrial stromal cells, BT, BEL, MDBK and BEK cells). Values are the mean ± s.d. of three independent experiments (n = 3 per treatment, P ≥ 0.05).

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    (a) Diagram showing the 1143 bp IE2 promoter sequence containing the putative TATA box (underlined in black) and the first 15 non-coding nucleotides of the first exon. Sense and antisense primers used for the PCR amplification are in red and contain the NdeI and NheI restriction sites (underlined in red) respectively for sub-cloning of the amplicon in front of the EGFP ORF (green box) followed by the bovine growth hormone polyadenylation signal (pA (grey box)). (b) Representative fluorescence and phase contrast images of transfected endometrial stromal cells with the above-described construct and expressing EGFP 24 h post-transfection. (c) Summarising schema of the time necessary for EGFP accumulation into the different cell types (endometrial epithelial cells, endometrial stromal cells, BT, BEL, MDBK and BEK cells), following transfection with the above-described construct. Values are the mean of three independent experiments (n = 3 and P ≥ 0.05).

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    Prostaglandin production by (a) epithelial and (b) stromal cells after challenge with LPS (1 μg/ml) or BoHV-4 at the different m.o.i. indicated. After 24 h in culture, supernatants were harvested and prostaglandin production was measured by RIA. Values are presented as the mean ± s.d. of three independent experiments. Differences were statistically different at P < 0.05 when compared with a media control. (c) Western immunoblotting of endometrial stromal cell extracts and infection with BoHV-4 EGFPΔTK. Positive control was established treating cells with medium containing 20% of FBS. β-Actin as the loading control. The same experiment was repeated thrice, giving the same results.

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    (a) Schematic illustration of the transwell model used for co-culturing BoHV-4EGFPΔTK persistently infected bovine macrophage cell line (in green in the upper well), producing low amount of infectious virus (grey dots) with bovine endometrial stromal or control BEK cells (in yellow) seated in the lower well. The transwell consists of a permeable membrane and allows diffusion of the viral particles. (b) Fluorescence and phase contrast images of bovine endometrial stromal and control BEK cells seated in the lower well at 48 h post co-cultivation. (c) Graph bar of the viral titre in the upper well. Values are the mean ± s.d. of three independent experiments (n = 3 and P ≥ 0.05).

References

Allsopp TE & Fazakerley JK2000 Altruistic cell suicide and the specialized case of the virus-infected nervous system. Trends in Neurosciences23284–290.

Appleton I Tomlinson A & Willoughby DA1996 Induction of cyclooxygenase and nitric oxide synthase in inflammation. Advances in Pharmacology3527–78.

Arosh JA Parent J Chapdelaine P Sirois J & Fortier MA2002 Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biology of Reproduction67161–169.

Asselin E Goff AK Bergeron H & Fortier MA1996 Influence of sex steroids on the production of prostaglandins F2 alpha and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biology of Reproduction54371–379.

Bartha A Juhasz M & Liebermann H1966 Isolation of a bovine herpesvirus from calves with respiratory disease and keratoconjuntivitis. Acta Veterinaria Academiae Scientiarum Hungaricae16357–358.

Betz M & Fox BS1991 Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines. Journal of Immunology146108–113.

Bonnett BN Martin SW & Meek AH1993 Associations of clinical findings bacteriological and histological results of endometrial biopsy with reproductive performance of postpartum dairy cows. Preventive Veterinary Medicine15205–220.

Bublot M Lomonte P Lequarre AS Albrecht JC Nicholas J Fleckenstein B Pastoret PP & Thiry E1992 Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses Epstein–Barr virus and herpesvirus saimiri. Virology190654–655.

Castrucci G Frigeri F Cilli V Donelli G Ferrari M Chicchinri U & Bordoni E1986 A study of herpesvirus isolated from dairy cattle with a history of reproductive disorders. Comparative Immunology Microbiology and Infectious Diseases913–21.

Castrucci G Frigeri F Ferrari M Ranucci S Aldrovandi V Cilli V Rampichini L & Gatti R1987 Experimental infection of calves with strain of bovid herpesvirus-4. Comparative Immunology Microbiology and Infectious Diseases1041–49.

Ceserman E Moore PS Rao PH Inghirami G Knowles DM & Chang Y1995In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood862708–2714.

Chen N Warner JL & Reiss CS2000 NSAID treatment suppresses VSV propagation in mouse CNS. Virology27644–51.

Cheng Z Robinson RS Pushpakumara PG Mansbridge RJ & Wathes DC2001 Effect of dietary polyunsaturated fatty acids on uterine prostaglandin synthesis in the cow. Journal of Endocrinology171463–473.

Czaplicki G & Thiry E1998 An association exists between bovine herpesvirus-4 seropositivity and abortion in cows. Preventive Veterinary Medicine33235–240.

Dewals B Gillet L Gerdes T Taracha EL Thiry E & Vanderplasschen A2005 Antibodies against bovine herpesvirus 4 are highly prevalent in wild African buffaloes throughout eastern and southern Africa. Veterinary Microbiology110209–220.

Donofrio G & van Santen VL2001 A bovine macrophage cell line supports bovine herpesvirus 4 persistent infection. Journal of General Virology821181–1185.

Donofrio G Cavirani S & van Santen VL2000 Establishment of a cell line persistently infected with bovine herpesvirus 4 using a recombinant virus. Journal of General Virology811807–1814.

Donofrio G Cavirani S Taddei S & van Santen VL2002 Bovine herpesvirus 4 as a gene delivery vector. Journal of Virological Methods10149–61.

Donofrio G Cavirani S Taddei S & Flammini CF2004 Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by over expression of BoHV-4 immediate early (IE) 2 gene. Journal of Virological Methods116203–207.

Donofrio G Cavirani S van Santen VL & Flammini CF2005 Potential secondary pathogenic role for bovine herpesvirus 4. Journal of Clinical Microbiology433421–3426.

Dubuisson J Thiry E Thalasso F Bublot M & Pastoret PP1988 Biological and biochemical comparison of bovid herpesvirus-4 strains. Veterinary Microbiology16339–349.

Dubuisson J Thiry E Bublot M Thomas I van Bressem MF Coignoul F & Pastoret PP1989 Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone. Veterinary Microbiology2197–114.

Dumais N Barbeau B Olivier M & Tremblay MJ1998 Prostaglandin E2 up-regulates HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways. Journal of Biological Chemistry27327306–27314.

Egyed L1998 Replication of bovine herpesvirus type 4 in human cells in vitro. Journal of Clinical Microbiology362109–2111.

Flore O Rafii S Ely S O’Leary JJ Hyjek EM & Cesarman E1998 Transformation of primary human endothelial cells by Kaposi’s sarcoma-associated herpesvirus. Nature394588–592.

Fortier MA Guilbault LA & Grasso F1988 Specific properties of epithelial and stromal cells from the endometrium of cows. Journal of Reproduction and Fertility83239–248.

Frazier K Pence M Mauel MJ Liggett A Hines ME Sangster L Lehmkuhl HD Miller D Styer E West J & Baldwin CA2001 Endometritis in postparturient cattle associated with bovine herpesvirus-4 infection: 15 cases. Journal of Veterinary Diagnostic Investigation13502–508.

Frazier KS Baldwin CA Pence M West J Bernard J Liggett A Miller D & Hines ME2002 Seroprevalence and comparison of isolates of endometriotropic bovine herpesvirus-4. Journal of Veterinary Diagnostic Investigation14457–462.

Gillet L Minner F Detry B Farnir F Willems L Lambot M Thiry E Pastoret PP Schynts F & Vanderplasschen A2004 Investigation of the susceptibility of human cell lines to bovine herpesvirus 4 infection: demonstration that human cells can support a nonpermissive persistent infection which protects them against tumor necrosis factor α-induced apoptosis. Journal of Virology782336–2347.

Gillet L Dewals B Farnir F de Leval L & Vanderplasschen A2005 Bovine herpesvirus 4 induces apoptosis of human carcinoma cell lines in vitro and in vivo. Cancer Research659463–9472.

Griffin JF Hartigan PJ & Nunn WR1974 Non-specific uterine infection and bovine fertility. I. Infection patterns and endometritis during the first seven weeks postpartum. Theriogenology191–106.

Hagen G Wolf M Katyal SL Singh G Beato M & Suske G1990 Tissue-specific expression hormonal regulation and 5′-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobi. Nucleic Acids Research182939–2946.

Hansen SG Strelow LI Franchi DC Anders DG & Wong SW2003 Complete sequence and genomic analysis of rhesus cytomegalovirus. Journal of Virology776620–6636.

Herath S Fischer DF Werling D Williams EJ Lilly ST Dobson H Bryant CE & Sheldon IM2006 Expression and function of toll-like receptor 4 in the endometrial cells of the uterus. Endocrinology147562–570.

Ireland JJ Murphee RL & Coulson PB1980 Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. Journal of Dairy Science63155–160.

Jung JU Choi JK Ensser A & Biesinger B1999 Herpesvirus saimiri as a model for gammaherpesvirus oncogenesis. Seminars in Cancer Biology9231–239.

Kim JJ & Fortier MA1995 Cell type specificity and protein kinase C dependency on the stimulation of prostaglandin E2 and prostaglandin F2 alpha production by oxytocin and platelet-activating factor in bovine endometrial cells. Journal of Reproduction and Fertility103239–247.

Krogman LA & McAdaragh JP1982 Recrudescence of bovine herpesvirus-5 in experimentally infected calves. American Journal of Veterinary Research43336–338.

Kuroda E Sugiura T Zeki K Yoshida Y & Yamashita U2000 Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice. Journal of Immunology1642386–2395.

Leung ST Cheng Z Sheldrick EL Derecka K Flint AP & Wathes DC2001 The effects of lipopolysaccharide and interleukins-1alpha -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium. Journal of Endocrinology168497–508.

Lomonte P Bublot M vanSanten VL Keil G Pastoret PP & Thiry E1996 Bovine herpesvirus 4: genomic organization and relationship with two other gammaherpesviruses Epstein–Barr virus and herpesvirus saimiri. Veterinary Microbiology5379–89.

Lopez OJ Galeotta J & Osorio FA1996 Bovine herpesvirus type-4 (BHV-4) persistently infects cells of the marginal zone of spleen in cattle. Microbial Pathogenesis2147–58.

Mehrotra ML Shucla DC & Srivastava NC1986 Isolation of a new herpesvirus from cases of reproductive disorders in cow. Indian Journal of Animal Sciences561196–1199.

Miller G Heston L Grogan E Gradoville L Rigsby M Sun R Shedd D Kushnaryov VM Grossberg S & Chang Y1997 Selective switch between latency and lytic replication of Kaposi’s sarcoma herpesvirus and Epstein–Barr virus in dually infected body cavity lymphoma cells. Journal of Virology71314–324.

Misseyanni A Klug J Suske G & Beato M1991 Novel upstream elements and the TATA-box region mediate preferential transcription from the uteroglobin promoter in endometrial cells. Nucleic Acids Research192849–2859.

Mohanty SB Hammond RC & Lillie MG1971 A new bovine herpesvirus and its effect on experimentally infected calves. Archiv Für Die Gesamte Virusforschung34394–395.

Monge A Elvira L Gonzalez JV Astiz S & Wellenberg GJ2006 Bovine herpesvirus 4-associated postpartum metritis in a Spanish dairy herd. Research in Veterinary Science80120–125.

Moses AV Fish KN Ruhl R Smith PP Strussenberg JG Zhu L Chandran B & Nelson JA1999 Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8. Journal of Virology736892–6902.

Naeem K Caywood DD Goyal SM Werdin RE & Murtaugh MP1993 Persistence of bovid herpesvirus-4 in experimentally inoculated pregnant rabbits. New Microbiologica1687–93.

Newberry RD Stenson WF & Lorenz RG1999 Cyclooxygenase-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigen. Nature Medicine5900–906.

Nikolin VDonofrio GMilošević BTaddei SRadosavljević V & Milićević V2007 First Serbian isolates of bovine herpesvirus 4 (BoHV-4) from a herd with history of postpartum metritis. New Micribiologica3053–57.

Nilsson K1979 The nature of lymphoid cell lines and their relationship to the virus. In The Epstein–Barr Virus pp 225–281. Eds MA Epstein & BG Achong. Berlin: Verlag.

Olson JD Ball L Mortimer RG Farin PW Adney WS & Huffman EM1984 Aspects of bacteriology and endocrinology of cows with pyometra and retained foetal membranes. American Journal of Veterinary Research452251–2255.

Osorio FA & Reed DE1983 Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection. American Journal of Veterinary Research44975–980.

Osorio FA Reed DE & Rock DL1982 Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection. Veterinary Microbiology7503–513.

Osorio FA Reed DE Van Der Maaten MJ & Metz CA1985a Comparison of the herpesviruses of cattle by restriction endonuclease analysis and serologic analysis. American Journal of Veterinary Research462104–2109.

Osorio FA Rock DL & Reed DE1985b Studies on the pathogenesis of a bovine cytomegalo-like virus in an experimental host. Journal of General Virology661941–1951.

Park JB & Kendrick JW1973 The isolation and partila characterization of a herpesvirus from a case of bovine metritis. Archiv Für Die Gesamte Virusforschung41211–215.

Peterson RB & Goyal SM1988 Propagation and quantitation of animal herpesviruses in eight cell culture systems. Comparative Immunology Microbiology and Infectious Diseases1193–98.

Poyser NL1987 Effects of various factors on prostaglandin synthesis by the guinea-pig uterus. Journal of Reproduction and Fertility81269–276.

Poyser NL1995 The control of prostaglandin production by the endometrium in relation to luteolysis and menstruation. Prostaglandins Leukotriens and Essential Fatty Acids53147–195.

Reiss CS & Komatsu T1998 Does nitric oxide play a critical role in viral infections? Journal of Virology724547–4551.

Reynolds AE & Enquist LW2006 Biological interactions between herpesviruses and cyclooxygenase enzymes. Reviews in Medical Virology16393–403.

Ruder CA Sasser RG Williams RJ Ely JK Bull RC & Butler JE1981 Uterine infections in the postpartum cow. II. Possible synergistic effect of Fusobacterium necrophorum and Corynebacterium pyogenes. Theriogenology15573–580.

van Santen VL1991 Characterization of the bovine herpesvirus 4 major immediate-early transcript. Journal of Virology655211–5224.

van Santen VL1993 Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein–Barr virus R transactivator. Journal of Virology67773–784.

Schweizer M & Peterhans E2001 Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis. Journal of Virology754692–4698.

Sheldon IM & Dobson H2004 Postpartum uterine health in cattle. Animal Reproduction Science82–83295–306.

Simmen RC Srinivas V & Roberts RM1989 cDNA sequence gene organization and progesterone induction of mRNA for uteroferrin a porcine uterine iron transport protein. DNA8543–554.

Smith WL Garavito RM & DeWitt DL1996 Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. Journal of Biological Chemistry27133157–33160.

Snijdewint FG Kalinski P Wierenga EA Bos JD & Kapsenberg ML1993 Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. Journal of Immunology1505321–5329.

Song MJ Li X Brown HJ & Sun R2002 Characterization of interactions between R.T.A. and the promoter of polyadenylated nuclear RNA in Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8. Journal of Virology765000–5013.

Stabel JR & Stabel TJ1995 Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA. Veterinary Immunology and Immunopathology45211–220.

Steer SA & Corbett JA2003 The role and regulation of COX-2 during viral infection. Viral Immunology16447–460.

Storz J Ehlers B Todd VJ & Ludwig H1984 Bovine cytomegaloviruses: identification and differential properties. Journal of General Virology65697–706.

Sun R Lin SF Gradoville L Yuan Y Zhu F & Miller G1998 A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus. PNAS9510866–10871.

Thiry E Meersschaert C & Pastoret PP1988 Epizootiology of herpesvirus infections in wild ruminants. I. Infectious bovine rhinotracheitis virus and antigenically related viruses. Revue D’élevage et De Médecine Vétérinaire Des Pays Tropicaux41113–120.

Truman D Ludwig H & Storz J1986 Bovine herpesvirus type 4: studies on the biology and spread in cattle herds and in insemination bulls. Journal of Veteterinary Medicine (Series B)33485–501.

Usherwood EJ Stewart JP & Nash AA1996 Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice. Journal of Virology706516–6518.

Vanderplasschen A Goltz M Lyaku J Benarafa C Buhk HJ Thiry E & Pastoret PP1995 The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle. Virology213328–340.

Wellemans G Van Opdenbosh E & Mammerickx M1986 Inoculation expérimental du virus LVR 140 (herpes bovin IV) à des vaches gestantes et non-gestantes. Annales de Recherches Vétérinaires1789–94.

Zimmermann W Broll H Ehlers B Buhk HJ Rosenthal A & Goltz M2001 Genome sequence of bovine herpesvirus 4 a bovine Rhadinovirus and identification of an origin of DNA replication. Journal of Virology751186–1194.

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