Establishment of a cell line persistently infected with bovine herpesvirus-4 by use of a recombinant virus

Gaetano Donofrio1, Sandro Cavirani1 and Vicky L. van Santen2

Istituto di Malattie Infettive Veterinarie, Facoltà di Medicina Veterinaria, Università degli Studi di Parma, 43100 Parma, Italy1
Department of Pathobiology, 264 Greene Hall, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA2

Author for correspondence: Vicky van Santen. Fax +1 334 844 2652. e-mail vvsanten{at}mail.auburn.edu


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
Bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, productively infects multiple cell lines of various species and causes cell death. A human rhabdomyosarcoma cell line, RD-4, infected with BHV-4 produced low levels of early and late viral RNAs and infectious virus, but exhibited no cytopathic effect. Using a recombinant BHV-4 containing a neomycin-resistance gene, we established RD-4-derived cell lines persistently infected with BHV-4. The viral genome in these cells was predominantly circular. Because of drug selection, every cell contained a viral genome. In addition, all cells stained with a BHV-4-specific antiserum. Therefore, these cell lines are not carrier cultures. These cells produced infectious virus at all passages tested. Even though cells were selected and maintained at a concentration of geneticin at least 2·5 times that necessary to kill uninfected RD-4 cells, selected cells contained only approximately one viral genome per diploid host cell genome. Persistently infected cells grew more slowly than uninfected cells, even in the absence of drug. The slower growth of these cells suggests that any growth advantage conferred by multiple copies of the neomycin-gene-carrying viral genome might be offset by the detrimental effects of viral gene expression. This situation contrasts with other gammaherpesviruses, which are able to growth-transform cells.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
Bovine herpesvirus-4 (BHV-4) is a gammaherpesvirus with no clear disease association (reviewed in Bartha et al., 1987 ; Thiry et al., 1989 ). Like other herpesviruses, it establishes persistent infections in its natural host (Dubuisson et al., 1989b ; Krogman & McAdaragh, 1982 ; Osorio & Reed, 1983 ) and in an experimental host, the rabbit (Osorio et al., 1982 ). Although BHV-4 has been demonstrated in many tissues, accumulated evidence suggests that one site of persistence in both natural and experimental hosts is cells of the monocyte/macrophage lineage (Dubuisson et al., 1989b ; Egyed & Bartha, 1998 ; Naeem et al., 1993 ; Osorio & Reed, 1983 ; Osorio et al., 1985 ). However, nothing else is known about BHV-4 persistent infection. An in vitro model for persistent infection would be useful for study of viral genome maintenance and gene expression during persistent infection.

Cell lines persistently infected with the gammaherpesviruses Epstein–Barr virus (EBV), herpesvirus saimiri (HVS), human herpesvirus-8 (HHV-8), and murine gammaherpesvirus-68 (MHV-68) have been established from cells isolated from infected hosts (Cesarman et al., 1995 ; Jung et al., 1999 ; Nilsson, 1979 ; Usherwood et al., 1996 ). This process has been facilitated by the growth-transforming ability of these gammaherpesviruses (Flore et al., 1998 ; Jung et al., 1999 ; Miller, 1990 ; Moses et al., 1999 ). In contrast, no evidence for growth-transformation by BHV-4 has been obtained. None of the genes associated with transformation by other gammaherpesviruses, unique to each individual virus, are in the BHV-4 genome (Lomonte et al., 1996 ). In cell lines persistently infected with gammaherpesviruses, the infection is predominantly latent. In the vast majority of cells, viral gene expression is restricted to a specific subset of genes and the cells survive and replicate. In a small subset of cells, the virus is reactivated from latency, resulting in production of infectious virus and death of the cell (Kieff, 1996 ; Moses et al., 1999 ). In cell lines persistently infected with gammaherpesviruses, the viral genome is maintained as a circular episome (Cesarman et al., 1995 ; Decker et al., 1996 ; Jung et al., 1999 ; Kieff, 1996 ; Usherwood et al., 1996 ). Origins of replication for the circular viral genomes, oriP, distinct from the origins of replication used during lytic virus replication, have been identified (Ballestas et al., 1999 ; Kung & Medveczky, 1996 ; Yates et al., 1984 ). A viral gene product is required for episomal maintenance of DNA containing oriP (Ballestas et al., 1999 ; Kung & Medveczky, 1996 ; Lupton & Levine, 1985 ; Yates et al., 1985 ).

BHV-4 causes cytopathic effect (CPE) and replicates in a broad range of cell lines and primary cultures of various non-human animal species in culture (Peterson & Goyal, 1988 ; Truman et al., 1986 ). It also causes CPE and replicates in some human cell lines and primary cell cultures, but 14 of 17 human cell lines and primary cell cultures tested exhibited neither CPE nor virus replication (Egyed, 1998 ; Truman et al., 1986 ). In the work presented here, we examined the interaction of BHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although no CPE is noted, some infectious virus is produced. In addition, persistently infected cell lines that are not carrier cultures could be established.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Cell culture.
The human rhabdomyosarcoma cell line RD-4, obtained from David Derse, National Cancer Institute, Frederick, MD, USA, and cell lines established from it were cultured in DMEM containing 10% foetal bovine serum or 10% supplemented donor calf serum (DCS). Bovine turbinate (BT; ATCC CRL 1390) cells were cultured in DMEM containing 10% DCS.

{blacksquare} Probe labelling.
32P-labelled probes were labelled by random primer extension using the RadPrime kit (BRL).

{blacksquare} In situ lysis gel electrophoresis.
In situ lysis gel electrophoresis and gel processing were as previously described (Gardella et al., 1984 ), except that gels were blotted by capillary blotting.

{blacksquare} Quantification of DNA and RNA.
Amounts of DNA and RNA detected by Southern or Northern blot analysis were compared using ImageQuant software after detection of radioactivity by a Molecular Dynamics PhosphorImager IS.

{blacksquare} Construction of recombinant virus.
Recombination plasmid pR14neoI was used to construct recombinant BHV-4 using a protocol provided by Michael Goltz (personal communication). pR14neoI contains the 4·35 kb BHV-4 strain DN-599 EcoRI H fragment derived from pR14 (van Santen & Chang, 1992 ) and the 2 kb BamHI fragment containing a neomycin-resistance gene expression cassette derived from pRc/CMV (Invitrogen), cloned into the EcoRI and BamHI sites, respectively, of pTZ18U (see Fig. 3). Plasmid DNA (7·5 µg; linearized with SalI) and BHV-4 strain DN-599 DNA (0·3 µg), prepared as previously described (van Santen, 1991 ), were electroporated (Bio-Rad Gene Pulser, 270 V, 960 µF) in DMEM without serum into BT cells from a confluent 75 cm2 flask. Cells were returned to the flask, fed the next day, and split 1:2 when they reached confluence 2 days post-electroporation. When extensive CPE appeared, virus stock was prepared by freezing and thawing the cells three times and removing cell debris by low-speed centrifugation, and was screened for recombinant virus.



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Fig. 3. Diagram showing insertion of the neomycin-resistance gene by recombination into the BHV-4 genome. A restriction map of the entire BHV-4 (DN-599) genome is shown at the bottom, with the region containing the junction of unique and prDNA expanded above it. The dark bars indicate the portion of the BHV-4 EcoRI H fragment containing unique BHV-4 DNA and the white bars indicate prDNA. Although the diagram shows three copies at the left end and four copies at the right end, the number of copies of prDNA at each end of the linear genome varies, with approximately 15 copies total per genome (Ehlers et al., 1985 ). A diagram of the linearized recombination plasmid is shown at the top. The EcoRI site at the right end of the EcoRI H fragment is 226 bp to the right of the junction of unique and prDNA (in the BHV-4 strain 66-p-347 sequence; Broll et al., 1999 ), and lies within the first copy of prDNA. Abbreviations: Pr, SV40 promoter; neo, neomycin-resistance gene; PA, SV40 polyadenylation signal. The crossed dotted lines indicate the region in which homologous recombination between the recombination plasmid and the BHV-4 genome could occur. Recombination would result in a recombinant genome lacking prDNA at the right end. However, prDNA would be restored upon circularization of the linear genome.

 
The first round of screening was accomplished by dot blot hybridization of cell lysates of BT cells infected with pools containing twenty 50% tissue culture infective doses (TCID50) in 96-well plates. Seven days post-infection (p.i.), cell lysates and dot blots were prepared by a modification of the second alternative procedure described by Galik et al. (1993) . Medium was removed from each well and stored at -80 °C. After cells were rinsed with DBSS, 40 µl of 0·4 M Tris pH 7·4, 0·15 M NaCl, 0·001 M EDTA, 1% SDS, 500 µg/ml Proteinase K was added to each well. Plates were incubated 3 h at 50 °C, and 200 µl 1x SSC, 0·5 M NaOH, 1 M NaCl was added to each well to denature the DNA. A portion of the denatured cell lysate (10 µl) was diluted into 190 µl 1x SSC, 0·5 M NaOH, 1 M NaCl. After 10 min at 20 °C, samples were applied to Genescreen Plus membrane using a dot blot apparatus and let stand 30 min. After application of vacuum, bound DNA was neutralized by filtering 200 µl 1 M Tris pH 7·4, 2 M NaCl through each well. Dot blots were hybridized and washed as previously described (Chang & van Santen, 1992 ), except that the most stringent wash was 0·5x SSC at 68 °C. Probe was the 2 kb fragment containing the neomycin-resistance gene, mentioned above. Recombinant virus from positive wells was amplified by inoculation of the reserved medium onto BT cells in 6-well plates. After appearance of extensive CPE, virus stock was prepared as described above. DNA was prepared from 100 µl of each virus stock using the QIAamp Blood Kit (Qiagen) following the Blood & Body Fluid Protocol provided with the kit. The presence of the neomycin-resistance gene in each virus stock was confirmed by PCR, using one-twentieth of the DNA as template.

The second round of screening for recombinant virus utilized PCR and Southern blot analysis of virus originating from single plaques derived from pools of virus selected after the first screening. Dilutions of each pool were added to BT cells in 96-well plates to obtain wells with single plaques. After 8 days, wells containing only one plaque were noted. Cells were disrupted by freezing and thawing three times, and one-fifth of the freeze–thaw lysate from each well containing one plaque was added to BT cells in a 6-well plate to amplify each plaque-purified virus. However, the cells became confluent before CPE appeared. Therefore, cells were transferred to a 25 cm2 flask and allowed to continue to grow until extensive CPE was apparent. Virus stock and DNA from 200 µl of the virus stock were prepared as described above. The presence of the neomycin-resistance gene was determined by PCR using one-fortieth of the DNA solution as template, and confirmed by Southern blot analysis of one-quarter of the DNA with a neomycin-resistance gene probe. Recombinant virus 26A3neo was propagated as described (van Santen, 1991 ).

{blacksquare} Establishment of geneticin-resistant cell lines.
Confluent RD-4 cells were infected with 26A3neo (50 TCID50 per cell). One day p.i., cells were split 1:5 and geneticin (400–1000 µg/ml) selection was applied. Medium and geneticin were replaced every 3 days. All doses of geneticin resulted in colonies. After colonies were well-established, cells were trypsinized, allowed to reattach to the same flask, grown to confluence, and tested for the presence of circular and linear viral genomes by in situ lysis gel electrophoresis. Pools of cells selected with and maintained with 1000 µg geneticin/ml were used for all experiments shown.

{blacksquare} Immunostaining.
Monolayers were washed three times with PBS, fixed with 4% paraformaldehyde for 10–15 min at 37 °C, washed twice with PBS, 0·1% BSA, and incubated 5 min at 20 °C with the same solution. Cells were incubated 5 min at 20 °C with PBS, 0·3% Triton X-100, washed two or three times with PBS, and incubated 10 min at 37 °C with 0·15% H2O2 in PBS. Rabbit anti-BHV-4 hyperimmune serum, diluted 1/500 in PBS, or monoclonal antibody 29 (Dubuisson et al., 1989a ), diluted 1/50 in PBS, was incubated with the cells for 2 h at 37 °C. After three washes with PBS, cells were incubated with peroxidase-conjugated secondary antibody, diluted 1/500 in PBS, for 1 h at 37 °C, and washed three times with PBS. Secondary antibody was detected by development in 250 µg/ml DAB (Sigma), 0·015% H2O2, 50 mM Tris pH 7·4 for approximately 10 min at 20 °C.


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
RD-4 cells can be infected with BHV-4, exhibit low-level viral gene expression and virus production, but survive infection
When human rhabdomyosarcoma cell line RD-4 was infected with BHV-4, even at an m.o.i. of 50 TCID50 per cell, no CPE was apparent, even after passage of the cells. To begin to examine the nature of this resistance to CPE, we determined whether virus entered RD-4 cells. A very early event after entry of herpesviruses into cells is circularization of the linear genome (Poffenberger & Roizman, 1985 ). Therefore, circular viral genomes indicate that virus has entered the cells and released its genome. We used Southern blot analysis after in situ lysis agarose gel electrophoresis to distinguish between circular and linear viral DNA (Gardella et al., 1984 ). Circular viral DNA was readily detectable in infected RD-4 cells 6–9 days p.i. (Fig. 1). Circular viral DNA in RD-4 cells 6 h p.i. was only approximately 2-fold less abundant than in permissive BT cells 6 h p.i., prior to the onset of viral DNA replication in BT cells. The linear viral DNA detectable 6 h p.i. in both BT and RD-4 cells was likely input virion DNA rather than replicated, packaged viral DNA, because viral DNA replication in permissive cells does not begin until after 8 h p.i. (Chang & van Santen, 1992 ). The approximately 2-fold decrease in amount of circular DNA in BT cells between 6 and 12 h p.i. may be due to conversion of circular monomers to replicative intermediates, which do not migrate into the gel (Bataille & Epstein, 1994 ; Severini et al., 1994 ; Zhang et al., 1994 ). An approximately 30-fold increase in the amount of linear viral DNA occurred in BT cells between 12 and 24 h p.i. This likely represents cleavage and packaging of replicated viral DNA. In contrast, the amount of linear viral DNA in RD-4 cells did not increase between 12 and 24 h p.i. An approximately 2-fold increase in linear viral DNA occurred between 24 and 48 h p.i. in RD-4 cells. This likely represents limited viral DNA replication and packaging (see below). Thereafter, the amount of linear viral DNA in RD-4 cells decreased at a rate faster than the rate of decrease of circular viral DNA. The amount of circular viral DNA in RD-4 cells increased slightly between 6 and 12 h p.i., and then remained relatively constant until the cells were passaged. Because equivalent numbers of cells were loaded onto each lane of the gel, the decrease in the amount of viral DNA after passage of infected RD-4 cells was likely due to ‘dilution’ of viral DNA as the cells divided. This suggests that the circular viral genome persisted in RD-4 cells, but was not replicated, at least not at a rate sufficient for maintenance at a constant level in dividing cells.



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Fig. 1. In situ lysis gel analysis of viral DNA in BHV-4 infected BT and RD-4 cells. Cells were infected with 50 TCID50 per cell and subjected to in situ lysis gel analysis (1x106 cells per lane) at the times p.i. indicated. Times indicated by d are in days. RD-4 cells harvested 3, 6, and 9 days p.i. had been passaged (1:3 split every 3 days) one, two and three times, respectively. U, uninfected. The probe was a 32P-labelled cloned cDNA of BHV-4 L1.7 RNA (Bermudez-Cruz et al., 1997 ). Circular (C) and linear (L) viral genomes are indicated.

 
We next examined viral gene expression in infected RD-4 cells. Total RNA prepared 24 h p.i. was assayed by Northern blot analysis for expression of representative early (BHV-4 homologue of herpes simplex virus 1 major DNA binding protein; Chang & van Santen, 1992 ) and late (L1.7; Bermudez-Cruz et al., 1997 ) RNAs. Early RNA was detected in RD-4 cells at approximately one-fifth the level detected in BT cells (not shown). Late RNA was also detectable in infected RD-4 cells (Fig. 2a), but only at about one-thirtieth the level found in infected BT cells. In an independent experiment, L1.7 RNA was still readily detectable 7 days p.i. by Northern blot analysis of cytoplasmic polyadenylated RNA prepared from RD-4 cells infected with 10 TCID50 per cell and split 1:15 3 days p.i. (not shown).



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Fig. 2. Northern blot analysis of late gene expression in BHV-4-infected RD-4 cells. Cells were infected with approximately 20 TCID50 per cell and total RNA was prepared 24 h p.i. using the TRIzol reagent (BRL) following instructions supplied with the reagent. RNA was denatured, electrophoresed on agarose gels containing 2·2 M formaldehyde, blotted onto nitrocellulose and hybridized by standard methods (Sambrook et al., 1989 ). (a) Comparison of levels of L1.7 RNA in BT and RD-4 cells. Lanes: BT, 2 µg total RNA isolated from BT cells; RD-4, 20 µg total RNA isolated from RD-4 cells; U, uninfected; I, infected. Note that RD-4 lanes contained ten times the amount of total RNA as BT lanes. (b) Effect of PAA on L1.7 RNA levels in RD-4 cells. Lanes: U, uninfected RD-4 cell RNA; P, RNA from RD-4 cells infected in the presence of 100 µg/ml PAA; I, RNA from RD-4 cells infected in the absence of inhibitor. Each lane contained 10 µg total RNA.

 
To determine whether expression of L1.7 RNA in RD-4 cells is dependent on viral DNA replication, we infected RD-4 cells in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus DNA polymerases (Huang, 1975 ), and compared the amount of L1.7 RNA to the amount produced in the absence of inhibitor (Fig. 2b). PAA decreased the level of L1.7 RNA in infected RD-4 cells nearly 10-fold, (Fig. 2b), while having no effect on early gene expression (not shown). This result suggests that most of the late RNA expressed in RD-4 cells is indeed dependent on viral DNA replication. Furthermore, this result implies that viral DNA replication occurs in RD-4 cells. However, this viral DNA replication appears to be extremely limited, as noted above (Fig. 1).

Viral DNA-replication-dependent late RNA synthesis in RD-4 cells suggested that infectious virus might be produced. Therefore, we determined the titre of BHV-4 8, 24 and 48 h p.i. and found that it increased approximately 2-fold between 8 and 24 h p.i., and a further 2-fold between 24 and 48 h p.i., to a titre of approximately 2x104 p.f.u./ml. This modest virus replication is consistent with the small increase of linear viral DNA in cells between 24 and 48 h p.i.

Based on results described thus far, we concluded that BHV-4 infection of RD-4 cells is predominantly non-permissive and non-cytopathic. The virus enters the cell and the genome is released. Extremely low levels of early and late gene expression and viral DNA replication occur. Some infectious virus is produced, but the RD-4 cells continue to grow without CPE. Dependence of BHV-4 DNA replication on the S phase of the cell cycle (Vanderplasschen et al., 1995 ) is unlikely to be responsible for the limited viral DNA replication and lack of CPE observed in RD-4 cells. Because RD-4 cells do not exhibit contact inhibition, cells in S phase are present at all times. Furthermore, even when infected RD-4 cells were passaged 24 h p.i., no CPE was observed. In contrast, when BT cells infected at an unknown very low m.o.i., such that they reached confluence before plaques formed, were passaged, extensive CPE developed, indicating that the ‘arrest’ of virus replication due to lack of S phase is readily reversible in BT cells. Therefore RD-4 cells had an outcome of infection distinct from BT cells that was not merely a function of the cell cycle phase at which initial infection occurred.

Persistently infected cells can be selected
Because the amount of circular viral DNA present in infected RD-4 cells steadily declined with passage of the cells, but was still detectable 9 days p.i., after three passages, we reasoned that it might be possible to select cells maintaining the viral genome if the viral genome contained a selectable marker. Therefore, we constructed a recombinant BHV-4 containing a neomycin-resistance gene. We chose to insert the neomycin-resistance gene near the junction of unique and terminally repeated DNA [polyrepetitive DNA (prDNA)]. Insertion of foreign DNA into this region in the HVS genome is more efficient than insertion into the central portion of the genome, presumably because only one recombination event rather than two is required (Desrosiers et al., 1985 ; Grassmann & Fleckenstein, 1989 ).

Furthermore, others have demonstrated that insertion of DNA into this region in BHV-4 has no detectable effect on BHV-4 replication in culture (Keil et al., 1990 ). Accordingly, a recombination plasmid was constructed that contains a neomycin-resistance gene following the BHV-4 EcoRI H fragment, the last EcoRI fragment containing unique DNA at the right end of the genome (Fig. 3). Recombinant virus 26A3neo, generated by homologous recombination following electroporation of BT cells with recombination plasmid and wt DN-599 DNA, was isolated from a well of a 96-well plate containing a single plaque. PCR and Southern blot analysis (not shown) confirmed that the virus contained the neomycin-resistance gene. As expected, recombinant virus grew to the same titre and produced plaques with the same kinetics and morphology as wt (not shown).

To establish cell lines containing the BHV-4 genome, RD-4 cells were infected with 26A3neo, and split and subjected to geneticin selection (400–1000 µg/ml) 1 day p.i. Although RD-4 cells are sensitive to 400 µg/ml geneticin, higher doses were used in an attempt to select cells with the highest copy number of recombinant virus. Pools of cells selected with 1000 µg/ml geneticin (RD4BHV4neo1000) were used for all experiments shown. However, pools of cells selected with lower doses behaved similarly whenever tested. Seven days p.i., microscopic colonies began to appear and were macroscopic by 14 days p.i. The efficiency of establishment of geneticin-resistant colonies selected with 1000 µg/ml geneticin was 5x10-5. Colonies were trypsinized, pooled, and allowed to continue to grow in the presence of geneticin.

Selected cells contain predominantly circular viral genomes
Twenty-four days p.i., selected RD4BHV4neo1000 cells were assayed for circular and linear viral DNA by in situ lysis gel electrophoresis (Fig. 4). Selected cells contained both circular and linear viral DNA. Circular DNA was approximately eight times more abundant than linear. This predominance of circular DNA continued in subsequent passages tested. In contrast, the viral genome was rapidly lost in the absence of geneticin selection and was undetectable 7 days p.i., after two passages (Fig. 4). The selected cells contained approximately 30-fold more viral DNA 24 days p.i. than they did 1 day p.i., before selection was applied. This result suggests that initially not all cells were infected or that the viral genome was amplified during selection. However, RD4BHV4neo1000 cells (passage 5) contained only approximately one viral genome per diploid genome, as estimated by Southern blot analysis of total DNA (not shown). This result suggests that the viral genome was not amplified during selection, and that therefore initially not all cells were infected. The conclusion that initially not all cells were infected conflicts with the observation (Fig. 1) that RD-4 cells contain only approximately 2-fold less viral DNA 6 h p.i. than BT cells and suggests that perhaps the m.o.i. was not as high as intended. Nevertheless, it is surprising that only one recombinant viral genome per host cell genome is present in cells selected with a high dose of drug. However, maintenance of a higher number of viral genomes might be incompatible with cell survival and/or multiplication.



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Fig. 4. In situ lysis gel analysis of cells infected with recombinant virus and grown in the absence or presence of geneticin. RD-4 cells were infected with 50 TCID50 per cell recombinant BHV-4 26A3neo. Cells were subjected to in situlysis gel analysis at the days p.i. indicated. U, uninfected. Each day that cells were harvested for in situ lysis gel analysis, they were also passaged 1:5. The probe was a 32P-labelled clonedEco RI L fragment of the polyrepetitive DNA of the BHV-4 termini (van Santen & Chang, 1992 ). Lane 11 is a shorter exposure of lane 10. Circular (C) and linear (L) viral genomes are indicated.

 
Persistently infected cells express viral antigens
To determine whether viral genes, other than the neomycin-resistance gene, were expressed in RD4BHV4neo1000 cells, cells were immunostained using a monoclonal antibody recognizing a late BHV-4 glycoprotein (gp11/VP24; Dubuisson et al., 1991 ) or a rabbit anti-BHV-4 hyperimmune serum. The monoclonal antibody reacted poorly with RD4BHV4neo1000 cells, resulting in faint staining of only approximately 1 in 300 cells (not shown). The faint staining might be due to the previously noted low affinity of this monoclonal antibody (Dubuisson et al., 1989a ). Our results do not allow distinction between the possibilities that the remaining cells do not produce late viral proteins or that remaining cells produce late viral proteins at a level below sensitivity of detection with this monoclonal antibody. In contrast, the rabbit antiserum reacted uniformly with all RD4BHV4neo1000 cells, showing a predominantly cytoplasmic staining pattern (Fig. 5a). Cells reacted uniformly with the rabbit antiserum at all passages tested, through 20 passages. Detection of viral antigens is further evidence that the BHV-4 genome is present in all cells and that expression of viral genes is compatible with long-term survival of the cells.



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Fig. 5. Immunohistochemical staining of RD-4 cells with rabbit hyperimmune anti-BHV-4 serum. (a) RD4BHV4neo1000 cells. (b) Uninfected RD-4 cells.

 
Persistently infected cells produce infectious virus
The presence of linear viral DNA in RD4BHV4neo1000 cells suggested that they produced virus. Alternatively, linear viral DNA could be a result of cleavage of circular DNA by non-specific endonucleases. To determine whether RD4BHV4neo1000 cells produced infectious BHV-4, medium recovered from RD4BHV4neo1000 cells was inoculated onto susceptible cells. Plaques typical of BHV-4 were visible after 5 days and reacted with BHV-4 antiserum (not shown). Virus was produced by all passages of persistently infected RD-4 cells, through 20 passages. Production of infectious virus by cells persistently infected with selectable BHV-4 contrasts them with a variety of other human cell lines persistently infected with selectable HVS. Most cell lines selected for maintenance of a recombinant HVS genome exhibited only circular viral DNA and did not produce infectious virus (Simmer et al., 1991 ).

Persistently infected cells grow more slowly than uninfected cells
RD-4 cells persistently infected with BHV-4 took longer to reach confluence than uninfected RD-4 cells, even in the absence of geneticin. An MTT proliferation assay (Fig. 6) confirmed that persistently infected RD-4 cells grew more slowly than uninfected RD-4 cells. This observation suggests why only approximately one viral genome per host cell diploid genome is maintained in RD-4 cells even though the cells were selected and maintained in a concentration of geneticin at least 2·5-fold that necessary to kill uninfected RD-4 cells. The presence of the viral genome, and presumably expression of viral proteins, as indicated by immunostaining, is apparently detrimental to cell growth, as indicated by the slower growth of persistently infected cells. Therefore, any growth advantage to cells containing multiple copies of the neomycin-resistance-gene-containing viral genome might be offset by the disadvantage of containing multiple copies of the viral genome. This is in contrast to the situation for primate T cells infected with recombinant HVS carrying a neomycin-resistance gene, in which 100 copies of viral DNA per cell are maintained (Grassmann & Fleckenstein, 1989 ). T cells are the cells normally latently infected and transformed by HVS (Jung et al., 1999 ). Cell lines persistently infected with gammaherpesviruses (Epstein–Barr virus, HHV-8, MHV-68, HVS), which have been established without the need for recombinant virus and drug selection, because viral genes contribute to growth-transformation (Jung et al., 1999 ; Kieff, 1996 ; Moore & Chang, 1998 ), also contain numbers of viral genomes much greater than the approximately one copy per host cell diploid genome in RD-4 cells selected for maintenance of recombinant BHV-4 (Drexler et al., 1998 ; Sugden et al., 1979 ; Tsygankov & Romano, 1999 ; Usherwood et al., 1996 ). For these other gammaherpesviruses, expression of viral genes promoting cell growth presumably leads to selection of cells with higher numbers of viral genomes. BHV-4 is unique among well-characterized gammaherpesviruses in its lack of association with growth-transformation and malignancy.



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Fig. 6. MTT proliferation assay comparing uninfected and persistently infected RD-4 cells. Cells (3000 in 0·1 ml medium per well) were added to 96-well plates. At various times, the relative number of metabolically active cells was assessed by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) following the protocol supplied with the Boehringer Mannheim Cell Proliferation Kit I. The following second order polynomial equations were fitted to the data. Uninfected, OD=275·6-0·365t + 0·065t2, R2=0·99; persistently infected, OD=270·0+0·158t+0·023t2, R2=0·99. The quadratic terms in the two equations are significantly different from each other (P=0·01).

 
We have established cell lines persistently infected with BHV-4. Because cells were selected for maintenance of a recombinant viral genome containing a drug-resistance gene, we know that all cells contain the viral genome. In addition, all cells express viral antigens. Therefore, RD4BHV4neo1000 cells are not a carrier culture. Our results do not indicate whether most of the cells are latently infected and reactivation in a small proportion of cells is responsible for production of virus. Selection of cells transcribing the SV40 promoter-driven neomycin-resistance gene from the viral genome might have precluded establishment of latency, because this promoter might not be active during latency. Therefore, we call these cells persistently infected. However, this designation does not exclude latency. It has been suggested that the dependence of BHV-4 DNA replication on the S phase of the cell cycle might play a role in latency establishment, maintenance, and reactivation in the host animal (Vanderplasschen et al., 1995 ). Thus, BHV-4 might establish latency in non-dividing cells and be reactivated if those cells re-enter the cell cycle. However, our results show that, at least in our highly artificial system, persistent, possibly latent, BHV-4 infection compatible with cell survival can occur in dividing cells. The recombinant BHV-4 used to select persistently infected RD-4 cells might be useful for establishment of other persistently infected cell lines more relevant to understanding BHV-4 persistence in cattle.


   Acknowledgments
 
We gratefully acknowledge the technical assistance of Patricia DeInnocentes. We thank David Derse for providing RD-4 cells, Alain Vanderplasschen for monoclonal antibody and Michael Goltz for providing his unpublished protocol for electroporation for construction of recombinant BHV-4. This work was partially supported by a grant from the Food Animal Health and Disease Research Line Item, College of Veterinary Medicine, Auburn University.

This is Auburn University College of Veterinary Medicine Publication no. 2576.


   References
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 8 November 1999; accepted 16 March 2000.