Department of Biology, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC 28223, USA1
Author for correspondence: Kenneth Bost. Fax +1 704 547 3128. e-mail klbost{at}email.uncc.edu
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Abstract |
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Introduction |
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Investigations using the HV-68 murine model have produced some surprising findings. For example, while B lymphocytes (Sunil-Chandra et al., 1992b
; Usherwood et al., 1996b
), and possibly macrophages (Weck et al., 1999
), are considered the major cellular compartments for splenic latency, lung epithelial cells (Stewart et al., 1998
) may also represent cells capable of serving as virus reservoirs. Other unexplained findings included the observation that despite being an intracellular pathogen, the production of IL-6 (Sarawar et al., 1996
) or interferon-
(Sarawar et al., 1997
) during the initial/acute phase of infection seems to provide limited benefit to the host. Furthermore, while a viral superantigen has been hypothesized to contribute to the selective expansion of the VB4+ CD8+ T lymphocyte population (Tripp et al., 1997
), there are most likely other, as yet unidentified, factors which augment this selective expansion (Doherty et al., 1997
). Clearly, investigations of
HV-68-infected mice have contributed significantly to our understanding of gammaherpesvirus infections, and have defined questions for future investigations (Doherty et al., 1997
; Nash & Sunil-Chandra, 1994
; Simas & Efstathiou, 1998
).
EBV initially infects epithelial cells of the nasopharynx, and this conclusion has been reached as a result of several lines of investigation. Scrapings of epithelial cells from acutely infected patients have demonstrated the presence of virus or viral genomes (Lemon et al., 1977 ; Sixbey et al., 1984
). Furthermore, epithelial cells derived from the nasopharynx have been shown to be infectable following exposure to EBV in vitro (Shapiro & Volsky, 1983
). The fact that cervical lymph nodes are often enlarged during mononucleosis also points to the nasopharynx as a site of infection. In addition, the high prevalence of nasopharyngeal carcinomas which are positive for EBV genomes or replicative virus has suggested that transformation can follow infection with this virus (Glaser et al., 1976
; Kaschka-Dierich et al., 1976
; Klein et al., 1974
; Nonoyama & Pagano, 1973
; Wolf et al., 1973
).
Interestingly, there is also a strong association of EBV with gastric carcinomas. EBV genomes or replicative virus (Gulley et al., 1996 ; Imai et al., 1994
; Selves et al., 1996
; Tokunaga et al., 1993
) have been found in a significant percentage of gastric carcinomas. There have been some recent studies suggesting that gastric or intestinal epithelial cells can be directly infected by EBV (Tajima et al., 1998
; Takasaka et al., 1998
; Yanai et al., 1997
). However, gastric or intestinal epithelial cells have not been considered as likely targets for EBV infection since the virus would have to survive the harsh environment of the gastrointestinal tract to infect these cell populations. If such cells were important targets for EBV infection, this finding would have important implications for carcinomas of the gastrointestinal tract.
In the present study we demonstrate that HV-68 readily induces splenic leukocytosis and a latent infection of splenic leukocytes following oral or gastric inoculation. This result was especially surprising since gastric instillation of the virus bypassed the oral cavity. Furthermore, intestinal epithelial cells were found to be lytically infected by
HV-68 and to maintain expression of viral RNA and DNA even 30 days post-infection. Together these results demonstrate that
HV-68 can survive passage through the gastrointestinal tract to infect epithelial cells, followed by dissemination of the initial infection into the periphery.
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Methods |
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Intranasal, oral and gastric inoculations with
HV-68.
BALB/c mice (Charles Rivers, Wilmington, MA, USA) were given food and water ad libitum, and were housed in isolation cages throughout the experimental period. Intranasal inoculations with HV-68 were performed as previously described (Cardin et al., 1996
; Sunil-Chandra et al., 1992a
). Briefly, mice were anaesthetized and allowed to aspirate, via the nasal passages, 20 µl of inoculum containing the indicated p.f.u. of
HV-68. Mice were allowed to recover from the anaesthesia prior to returning to their cages.
For oral inoculations, mice were restrained, but not anaesthetized. Dilutions of HV-68 containing the indicated p.f.u. were diluted in a total of 50 µl of RPMI 1640. Mice readily consumed droplets of fluid from a pipette tip while being held, and the time required for administration was less than 1 min.
For intragastric inoculations, mice were restrained, but not anaesthetized. Dilutions of HV-68 containing the indicated p.f.u. were diluted in 500 µl of RPMI 1640 or saline and instilled directly into the stomach using a feeding tube connected to a 1 ml syringe. The feeding tube was cleaned before and after each instillation, and intragastric fluid was not aspirated during this procedure. It should be noted that these animals received no special treatment (e.g. neutralization of stomach acid, or withholding of food) prior to instillation.
To assure that gastric intubation completely bypassed the oral cavity, a mock intragastric intubation was also performed. A feeding tube connected to a 1 ml syringe containing 60000 p.f.u. of virus per 500 µl was inserted gastrically into groups of mice and removed without instilling any fluid. These mice were then used as negative controls to demonstrate that insertion and removal of the feeding tube did not result in infection.
Isolation of splenic leukocytes.
At the indicated times post-infection, spleens were removed and weighed. Single cell suspensions were made by pressing tissue through a 30 gauge wire-mesh screen, followed by centrifugation of cells on HypaqueFicoll gradients as previously described (Pascual et al., 1991 ). Mononuclear leukocytes were recovered from the interface, and counted.
Isolation of lung or intestinal epithelial cells.
Intestinal epithelial cells were isolated as previously described (Yamamoto et al., 1993 ). Briefly, following removal of the small intestines, 1 cm segments were placed in RPMI 1640 containing 2% FCS and gentamicin for 30 min with gentle agitation. Intestinal segments were then placed in fresh medium and vigorously agitated for 15 s. This process was repeated, and the resulting supernatants were passed over nylon wool columns to remove debris. Cells were then separated on discontinuous Percoll gradients (Pharmacia), and epithelial cells isolated from the 30% interface.
Lung epithelial cells were isolated as previously described (Stewart et al., 1998 ). Lung tissue was diced into 3 mm2 pieces and placed in RPMI 1640 containing 2% FCS and gentamicin for 10 min with gentle agitation. Tissue fragments were removed from the wash medium and incubated in RPMI 1640 containing gentamicin and 200 U/ml collagenase D (Boehringer Mannheim) for 60 min at 37 °C. Tissue was subsequently dissociated by passage through a 30 gauge wire-mesh screen, and cells were then washed in medium containing 2% FCS.
Purity of the isolated epithelial cells was assessed by immunofluorescent staining for cytokeratin using a fluorescein-conjugated monoclonal antibody (clone C-11, Sigma). Essentially all of the isolated epithelial cells stained positive for cytokeratin, indicating that these cells were of epithelial cell origin (Benya et al., 1991 ; Chandler et al., 1991
). Furthermore, to demonstrate the absence of contaminating B lymphocytes, a PCR to detect rearranged DJ regions was performed as previously described (Gu et al., 1991
) on aliquots of the same DNA samples that were used for PCR amplification of
HV-68 gp150. Primers specific for the 5' region of the immunoglobulin DH gene and the 3' region of the immunoglobulin JH4 gene (Gu et al., 1991
), ACAAGCTTCAAAGCACAATGCCTGGCT and GGGTCTAGACTCTCAGCCGGCTCCCTCAGGG, respectively, were used. DNA (10 ng) was amplified for 40 cycles using an annealing temperature of 56 °C; 10 % of the total PCR reaction was then electrophoresed on ethidium bromide-stained agarose gels, and visualized under UV illumination. DNA from epithelial cell preparations showed no amplified products for rearranged DJ regions, whereas splenic DNA amplified by PCR was strongly positive for amplified products.
Quantification of lytic virus.
The presence of lytic virus was quantified as previously described (Stevenson et al., 1999 ; Stewart et al., 1998
) using a plaque-forming assay. Briefly, isolated splenic leukocytes, or lung or intestinal epithelial cells, were pulse sonicated (Vibra Cell) to release intracellular virus. After sonication, lysates were centrifuged at 2500 r.p.m. to remove cellular debris. Limiting dilutions of the lysates were then placed on NIH-3T3 monolayers for 1 h followed by washing and overlaying with 0·15% agar (Difco) in RPMI 1640 with 30% FCS. After 5 days, overlays were removed and cell monolayers stained with crystal violet. The number of p.f.u. was quantified in duplicate at several serial dilutions of lysate to assure accuracy.
Infective centres assay.
The presence of latent virus was quantified using an infective centres assay as previously described (Cardin et al., 1996 ; Sunil-Chandra et al., 1992b
). For quantification of latent virus, limiting dilutions of isolated splenic leukocytes or lung or intestinal epithelial cells were placed onto monolayers of NIH-3T3 cells. After 24 h, an agar overlay supplemented with media and FCS was added and allowed to incubate for 5 days in 5% CO2. The monolayers were then fixed and stained with crystal violet and the number of infective centres counted in duplicate for several dilutions of cells for each experimental condition.
PCR amplification of
HV-68 gp150 DNA.
To demonstrate the presence of HV-68 DNA in cells or tissues, a sensitive and specific PCR amplifying the DNA encoding
HV-68 gp150 was developed. DNA was extracted from cells or tissues with a QIAamp DNA Mini Kit, according to the manufacturers instructions (Qiagen), quantified on DNA Dipsticks, (Invitrogen), denatured, and precipitated. A nested PCR procedure was developed which consisted of 20 cycles of amplification using the positive and negative strand primers, CCATCTAGCGGTGCAACATTTTCATTAC and TTTACTGGGTCATCCTCTTGTTTGGG, respectively; 10% of this PCR amplification was then amplified for 20 cycles using the positive and negative strand primers, CGAACAACAATCCCACTACAATTATGCG and GTATCTGATGTGTCAGCAGGAGCGTC, respectively, which were derived from the published sequence for
HV-68 gp150 (Stewart et al., 1996
). The annealing temperature for both PCR amplifications was 63 °C. 10% of the second PCR amplification was then electrophoresed on ethidium bromide-stained agarose gels, and amplified product was visualized under UV illumination. Amplified
HV-68 gp150 DNAs were compared to size standards (Promega) electrophoresed on the same gel to assure that a 462 bp fragment was being amplified. Amplified fragments were also isolated (Prep-a-gene, Bio-Rad) and subjected to direct DNA sequencing (fmol Sequencing, Promega) as previously described (Bost et al., 1995
; Bost & Mason, 1995
) to assure the identity of the
HV-68 gp150 coding sequence previously reported (Stewart et al., 1996
).
Images of ethidium bromide-stained gels were obtained with a Direct Scan Instant Image Polaroid camera. The images were then scanned into Adobe Photoshop 4.0 using an UMAX Astra 1200 scanner.
Cloning of
HV-68 gp150.
The DNA encoding HV-68 gp150 was amplified by PCR from viral stocks using the positive and negative strand primers derived from the published sequence (Stewart et al., 1996
), TTTCTGGGGAATCACAACTTAGTATGG and AGGTTCTGGCTTTGAAGGTTCAGC, respectively. Amplified material was isolated from agarose gels (Prep-a-gene, Bio-Rad), and cloned into the pNoTA/T7 vector using instructions supplied by the manufacturer (5 Prime-3 Prime). Plasmid expressing the cloned fragment was isolated (Del Sal et al., 1988
) from E. coli for use as a positive control in PCR and for use in determining the sensitivity of the nested PCR amplification described above.
RTPCR to detect RNA encoding
HV-68 gB.
RNA was extracted from isolated epithelial cells using TRIZOL as previously described (Bost & Clements, 1995 ), treated with DNase, and the RNA re-isolated. To detect the presence of
HV-68 RNA encoding the viral gB protein, RNA was reverse transcribed as previously described (Bost & Clements, 1995
), and the cDNA was subjected to nested PCR. The gB RNA transcript was selected for analysis since this viral RNA has been shown to be expressed only during the replicative phase of virus infection and not during virus latency (Virgin et al., 1999
). The nested PCR for
HV-68 gB has been previously described (Virgin et al., 1999
) and included amplification for 25 cycles with the positive and negative strand primers, CTGTTCGAACCACCGTTAAC and TGTTTTCCAGTGCACCAGGTC, respectively, followed by reamplification of 10% of the first PCR reaction with the positive and negative strand primers, ATTGTAGACATGGTGGCACGC and TCTGGTGGCTGTTTTCCAGG. Amplified products were electrophoresed on ethidium bromide-stained agarose gels to show the presence of a 261 bp fragment of
HV-68 gB.
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Results |
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It should also be noted that the results in Fig. 1(b) were due to the presence of latent virus and not the presence of replicating virus. Plaque assays were performed at the same time as infective centre assays using lysates of isolated splenic leukocytes to quantify the presence of replicating virus. Such assays were repeatedly negative (i.e. less than 1 p.f.u.), as is consistent with previous reports for studies using intranasal inoculations of virus (Cardin et al., 1996
; Sunil-Chandra et al., 1992a
).
The presence of HV-68 gp150 DNA in splenic lymphocytes following oral and intragastric inoculation of virus was detected using a sensitive, nested PCR. This PCR was approximately 1000 times more sensitive than the infectious centres assay, and could detect the presence of viral DNA using dilutions of
HV-68 preparations which represented 10-3 p.f.u. Furthermore, addition of limiting amounts of cloned
HV-68 gp150 DNA to aliquots of 10 ng of uninfected splenic DNA demonstrated that this nested PCR procedure could detect as few as 10 copies of gp150 DNA. Taken together, these results demonstrated the specificity and sensitivity of this nested PCR procedure.
Using this nested PCR, mice gastrically or orally inoculated with various dilutions of HV-68 clearly expressed viral DNA in splenic lymphocytes (Fig. 2
). Viral DNA was always detected in the spleens of more than 50 mice which were intragastrically or orally inoculated with doses of
HV-68 as low as 60 p.f.u. Conversely, uninfected mice, or mice receiving a mock gastric inoculation, had no detectable viral DNA present in their splenic lymphocytes (Fig. 2
).
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Discussion |
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It would be unlikely that HV-68 could so readily infect mice via the gut epithelium if this virus was not adapted to do so. Therefore, these results suggest the possibility that a normal and common route of transmission for this virus might be via a productive infection of the intestinal epithelium. One question which remains unanswered from the present studies is the predominant site for infection of epithelial cells following oral inoculation with
HV-68. Since epithelial cells within the oral cavity as well as the gut were exposed to the virus following oral inoculations, it will be important in future studies to identify sites of productive virus replication.
The ability of HV-68 to efficiently infect intestinal epithelial cells provides an intriguing model for the study of viral pathogenesis and the host response at a mucosal surface. Several in vitro studies using human intestinal epithelial cells have suggested that these cells can be directly infected by a human gammaherpesvirus (Tajima et al., 1998
; Takasaka et al., 1998
; Yanai et al., 1997
), and direct infection of murine intestinal epithelial cells following gastric intubation with
HV-68 has been demonstrated here. However, it is also possible that the spread of virus during the replicative phase occurs predominately by virus entry across basolateral surfaces of gut epithelial cells, as has been suggested by Imai et al. (1998)
. The spread of virus via basolateral surfaces of epithelial cells would likely involve interaction with intraepithelial lymphocytes, which are predominantly CD8+ T lymphocytes. Since this T lymphocyte subset is dysregulated during the systemic mononucleosis phase (Cardin et al., 1996
; Tripp et al., 1997
), it will be important to determine what effect this viral infection has on the controlled immune response to normal gut flora, and on the maintenance of oral tolerance to most ingested antigens.
It is also clear from the studies presented here that gastric intubation is an efficient route of inoculation for establishment of a systemic leukocytosis. Dissemination of virus to lymph nodes draining the small intestines could occur following infection of epithelial cells, but could also occur via virus entry into the Peyers patches as M cells sample gut immunogens. Intranasal infection does not result in significant numbers of latently infected B lymphocytes within the mesenteric lymph nodes (Sunil-Chandra et al., 1992a ), suggesting that the route of inoculation may influence the compartment of B lymphocytes, and possibly macrophages, which harbour the virus. It is not clear at present whether the number of latently infected cells present in the Peyers patches or mesenteric lymph nodes will be significantly increased if the route of exposure to the virus is via the gut.
The recent observation that epithelial cells may serve as a reservoir for persistent virus (Stewart et al., 1998 ) is supported by the studies performed here. At 30 days post-infection following intragastric inoculation, intestinal epithelial cells contained viral DNA and RNA (Fig. 5b
, c
) in the absence of any detectable replicating virus (Fig. 5a
). The possibility of persistently infected intestinal epithelial cells may have implications for human disease. For example, gammaherpesvirus-related gastrointestinal diseases (Shinohara et al., 1998
; Tokunaga et al., 1993
; Yanai et al., 1997
) may occur preferentially in patients with persistent viral infection of intestinal epithelial cells.
The presence of populations of gut epithelial cells which are persistently infected with HV-68 may also have important clinical implications for the development of virus-associated cancers (Tokunaga et al., 1993
). Intranasal and intravenous administration of
HV-68 to mice resulted in a significant portion of these animals developing lymphoproliferative disease (Sunil-Chandra et al., 1994
). Gammaherpesvirus-associated lymphoproliferative diseases in humans are also well documented (Young et al., 1989
), and this correlation supports the use of
HV-68 as a model for investigating the mechanisms associated with gammaherpesvirus-induced cellular transformation. In addition, EBV is known to associate with cancers of epithelial cell origin, including nasopharyngeal (Glaser et al., 1976
; Klein et al., 1974
; Nonoyama et al., 1973
; Wolf et al., 1973
) and gastric (Gulley et al., 1996
; Selves et al., 1996
; Tokunaga et al., 1993
) carcinomas. In the light of our studies (Fig. 5
) and recent investigations demonstrating persistent, or possibly latent, infection of lung epithelial cells (Stewart et al., 1998
), it is tempting to speculate that cells (i.e. B lymphocytes or epithelial cells) which can be latently infected with a gammaherpesvirus have an increased susceptibility for transformation. Correlations of the role of EBV in gastric carcinomas have been supported by studies demonstrating that EBV can infect intestinal epithelial cells (Gulley et al., 1996
; Imai et al., 1994
; Tajima et al., 1998
; Takasaka et al., 1998
; Yanai et al., 1997
), and that EBV is associated with some inflammatory bowel diseases (Grotsky et al., 1971
; Ruther et al., 1998
; Wakefield et al., 1992
). Studies to assess whether the development of gastric carcinomas or inflammatory bowel diseases can be augmented following introduction of
HV-68 directly into the gut can now be performed.
Taken together, the studies presented here demonstrate a surprising ability of HV-68 to survive the acidic and proteolytic environment of the upper gastrointestinal tract, and to productively infect intestinal epithelial cells. The rapid development of systemic mononucleosis-like disease following intragastric administration suggests a normal route of infection for this pathogen in these rodents. Based on our studies,
HV-68 infection of the gut mucosa should be an intriguing model system for investigating viral pathogenesis at a mucosal surface and for exploring the possibility of cellular transformation of intestinal epithelial cells.
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Received 7 July 1999;
accepted 3 November 1999.