INTRODUCTION

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Studies of immunity to human cytomegalovirus (HCMV) are essential for the development of vaccines and adoptive transfer therapies for the populations most at risk for HCMV infection and disease. Although the vigorous antibody responses generated by the virus during infection may limit the spread of recurrent infection, the cell-mediated responses to the virus appear to play the dominant role in control of the acute infection and suppression of reactivation (46, 47, 52). Investigations into the viral gene products targeted by the host immune responses have demonstrated that the HCMV 65-kDa tegument phosphoprotein pp65 (UL83) is the target of strong antibody (30), cytotoxic T-lymphocyte (CTL) (9, 39, 63), and lymphoproliferative (5, 61) responses. Moreover, by limiting-dilution analysis of CTLs from seropositive individuals, it was found that the frequency of pp65-specific CTL precursors was between 1 in 12,000 and 1 in 28,000, thereby representing a significant fraction of the total HCMV-specific CTL precursor frequency, which ranges from 1 in 7,500 to 1 in 19,000 (9). Results of another such study measuring the lysis of fibroblasts infected with wild-type HCMV or a pp65 deletion mutant suggested that between 70 and 90% of HCMV-specific CTL precursors were pp65 specific (63). Although these results implicate pp65 as a major target of CTLs, its role in protective immunity following acute infection remains to be proven.

The function of pp65 in HCMV replication is not known. The pp65 phosphoprotein is an especially abundant component of dense bodies, but it is also found in the tegument of infectious virions and noninfectious enveloped particles (2). A unique bipartite nuclear localization signal causes virion-associated pp65 to be rapidly translocated to the nucleus upon infection (17, 55), suggesting that this protein may play a role in very early events in gene regulation. However, no transcriptional activity has been demonstrated for this protein. A kinase activity has been found associated with pp65 in several studies (10, 56), and although Polo-like kinase 1 was recently found to bind to pp65 in infected cells (18), pp65 itself has not been definitively shown to be a kinase.

An HCMV deletion mutant lacking the UL83 open reading frame (ORF) encoding pp65 has been successfully generated by Schmolke and coworkers (56). This UL83 mutant, designated RVAd65, shows wild-type growth in cultured fibroblasts, demonstrating that pp65 is not required for viral replication in cell culture. Maturation of mutant virions in infected human foreskin fibroblasts appeared to be delayed compared to the parent virus when examined at 6 days postinfection (p.i.), though both viruses found in the supernatant were equally infectious. When the virion-associated kinase activities of the parent and mutant viruses were examined in vitro, the lower-molecular-weight virion proteins of RVAd65 were found to be underphosphorylated compared to those of the wild-type virus.

Experiments examining HCMV antigen presentation supported the existence of a pp65-associated kinase activity and, more notably, suggested a novel role for pp65 in immune evasion (20). When primary fibroblasts were infected with HCMV AD169 or RVAd65 and subjected to in vitro lysis by immediate-early 1 (IE1)-specific CTL clones, only the cells infected with RVAd65 were specifically lysed. The data indicated that increased phosphorylation of IE1 threonine residues occurred in the presence of pp65 and that this was responsible for the lack of presentation of IE1. These results describe an immune evasion mechanism distinct from several others described for HCMV (for a review, see reference 26), and it will be important to determine whether this mechanism of CTL evasion occurs in vivo.

Because of the limits of examining HCMV pathogenesis in humans, the murine cytomegalovirus (MCMV) model of infection has provided an experimental model for pathogenesis and immunity studies (28). Due to the immunodominance of pp65 in HCMV infection, our laboratory has previously identified and described the homologs of pp65 in MCMV (13). We found that both M83, the positional homolog of UL83 in the MCMV genome, and the adjacent M84 ORF exhibit homology to UL83, with the deduced amino acid sequence of M84 showing slightly stronger UL83 homology. However, M84 also exhibits significant amino acid homology to its positional homolog, UL84, a nonstructural protein possibly involved in negative regulation of the IE2 transactivator (19) and implicated in promoting viral DNA replication (54). We also demonstrated that like pp65, the M83 protein is a late, virion-associated protein that is the target of humoral responses during the infection.

In this study, we began to assess the roles of M83 and M84 during MCMV infection in order to provide some insights into the role of HCMV pp65. We constructed two MCMV mutants in which one of these ORFs was deleted and replaced with a selectable marker cassette. The viruses in which M83 and M84 were deleted, designated M83 and M84, respectively, were found to grow in cultured fibroblasts with kinetics equivalent to those of the wild-type virus, demonstrating that each of these genes is dispensable for growth in culture. However, when either virus was inoculated into three inbred mouse strains by either the intraperitoneal (i.p.) or intranasal (i.n.) route, its replication was found to be attenuated. M83, in particular, was severely restricted for growth in the salivary glands and lungs. This phenotype was reversed upon restoration of M83 expression. Both mutants were found to establish latency in the spleen, salivary glands, and lungs of BALB/c mice, where latency is defined as the presence of viral DNA detectable by PCR in the absence of detectable infectious virus. We also examined the immunity generated against the mutant viruses by using them to immunize BALB/c mice prior to lethal challenge with the virulent MCMV strain K181. We found that responses to both mutant viruses provided protection, equal to that of the parent strain, against subsequent lethal challenge. An i.p. dose of 200 PFU of M83 was fully protective against replication of the challenge virus in the spleen and salivary glands, but this dose was still sufficient to allow the immunizing M83 virus as well as the challenge virus to establish latency.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture and virus preparation. NIH 3T3 cells (ATCC CRL 1658) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated calf serum (CS) and (per milliliter) 0.29 mg of L-glutamine, 200 U of penicillin, 0.2 mg of streptomycin, 0.05 mg of gentamicin, and 1.5 µg of amphotericin B (DMEM + 10% CS). Mouse embryonic fibroblasts (MEFs) were grown in the above-described medium with 10% heat-inactivated fetal bovine serum being substituted for the CS. The preparation of salivary gland-derived MCMV strain K181 and tissue culture-derived MCMV has been previously described (13, 15). Virus was stored at 80°C until use. For analysis of virion proteins by Western blotting, MCMV was purified by density gradient centrifugation of tissue culture supernatants as previously described (12).

Mice, in vivo infections, organ harvests, and MCMV plaque assay. Female BALB/c (H-2^d) mice were obtained from Harlan Sprague Dawley, Inc., The Jackson Laboratory, or Simonsen Laboratories, Inc., at 5 to 6 weeks of age. Female C3H/HeN (H-2^k) mice were obtained from Simonsen Laboratories at 5 to 6 weeks of age. Female 129/J (H-2^b) mice were obtained from The Jackson Laboratory at 6 to 9 weeks of age. Mice were housed in microisolator-covered cages in a vivarium (University of California, San Diego) and given food and water ad libitum.

Plasmid constructions. Restriction endonucleases, T4 DNA ligase, T4 DNA polymerase, calf intestinal alkaline phosphatase (CIP), and the Klenow fragment of DNA polymerase were purchased from BRL Life Technologies, Inc. (Bethesda, Md.). Phosphorylated oligonucleotide linkers and Escherichia coli SCS110 competent cells were purchased from Stratagene (La Jolla, Calif.). DNA fragments were gel purified by using either GeneClean (Bio101) or Ultrafree-MC 0.45-µm centrifugal filter devices (Millipore). The subcloning methods employed below were carried out as described by Sambrook et al. (53). Unless otherwise specified, plasmids were propagated in Escherichia coli DH5 and purified by anion-exchange chromatography, using Qiagen Maxiprep or Miniprep kits. Construction of the lacZ/gpt plasmid pON855 was previously described (62).

Electroporation and mutant MCMV selection. All mutant MCMVs were generated in NIH 3T3 cells by homologous recombination between linearized plasmids containing selectable markers, and the K181 genome was introduced by infection as described by Vieira and colleagues (62). To generate the M83, M83-2, and M84 viruses, 30 µg of linearized plasmid was electroporated into 4 × 10⁶ NIH 3T3 cells by using a Gene Pulser II apparatus (Bio-Rad). DNA and cells were added in 0.4 ml to a 4-mm-gap cuvette and pulsed with 0.22 kV and 975 µF, using the measure capacitance function. After being pulsed, cells were seeded in 10 ml of DMEM + 10% CS in a 10-cm-diameter tissue culture dish and incubated overnight. The following day, the electroporated cells were infected with MCMV K181 at a multiplicity of infection (MOI) of 3 for 6 h, the inoculum was removed, and 10 ml of fresh medium was added. Three days p.i., the supernatant from the infected cells was harvested and clarified by low-speed centrifugation, and 50 to 500 µl of the clarified supernatant was used to infect 75% confluent NIH 3T3 cells in a T-75 flask. At 3 to 4 h p.i., virus was selected as described elsewhere (62), except that 150 µg of xanthine per ml was used. Recombinant viruses were propagated two to three times under gpt selection conditions and then subjected to three rounds of limiting dilution and plaque purification until virus homogeneity was achieved.

Production and purification of antisera. A rabbit antiserum against GST-M84 purified from E. coli inclusion bodies was generated in a naive seronegative male New Zealand White rabbit by previously described methods (13). The resultant GST-M84 antiserum was subjected to caprylic acid precipitation and ammonium sulfate precipitation as described by Harlow and Lane (23). To further purify M84-specific antibodies from the serum, portions of serum were affinity purified with nitrocellulose membrane-immobilized M84 produced by COS-7 cells transiently expressing pcDNA3-M84.

Southern and Western blot analyses. Genomic DNA from NIH 3T3 cells infected with wild-type or plaque-purified recombinant viruses was prepared by using a commercial kit (Qiagen Blood Kit) in accordance with the manufacturer's recommendations. Restriction digestion and Southern blot analysis were performed on these DNAs by standard procedures (53). MCMV genomic probes were isolated from restriction enzyme-digested HindIII C plasmid (40), ³²P labeled by random priming (Prime-It II; Stratagene), purified by Sephadex G-50 chromatography, and hybridized to UV-cross-linked DNA blots in Rapid-Hyb buffer (Amersham) according to the manufacturer's recommendations.

Growth kinetics of MCMV in cell culture. For single-cycle growth analysis, triplicate cultures of NIH 3T3 cells in 24-well dishes were infected at an MOI of 3 with tissue culture-derived MCMV isolates. After adsorption, cells were washed twice with PBS and then fed with DMEM + 10% CS. On each day p.i., supernatants were harvested, cleared of cellular debris as described above, and stored at 80°C in 1% (vol/vol) dimethyl sulfoxide. For multicycle growth analysis, triplicate cultures were infected at an MOI of 0.05 and supernatants were harvested and stored as described above. Viral titers for entire growth experiments were determined together in plaque assays on NIH 3T3 cells as described above.

PCR detection of latent MCMV DNA. BALB/c mice were infected i.p. with tissue culture-derived wild-type or mutant MCMV and then housed for 5 to 10 months to allow the resolution of the acute infection. To confirm the absence of persistent infectious virus, portions of the spleens of these mice were homogenized, sonicated, and diluted prior to incubation with MEFs or NIH 3T3 cells as previously described (44). Cells were observed for cytopathic effects (CPE) for 10 to 14 days, and the establishment of latency was defined as the absence of infectious virus concomitant with the presence of MCMV DNA sequences as determined by PCR. To detect latent MCMV genomes, DNA was extracted from the tissues of uninfected or latently infected mice by using a commercial kit (Qiagen Tissue Kit) according to the manufacturer's instructions for preparation of RNA-free DNA. To prevent cross-contamination of viral DNA, dissections were performed with autoclaved instruments and in a BioGuard hood not used for the handling of MCMV or acutely infected mice. In addition, the groups of mice were sacrificed on consecutive days. Spleen DNA was extracted from only stromal fractions prepared by sedimentation as previously described (41), whereas lung and salivary gland DNAs were prepared from minced whole tissues. DNAs were quantified by measurement of the optical density at 260 nm, and agarose gel electrophoresis was used to confirm DNA concentrations and integrity.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

M84 protein is expressed at early times p.i. and is not detectable in the virion. We previously showed by Northern blot analysis that the M83 gene product was likely encoded by a 5-kb mRNA that was detectable at 24 to 48 h p.i. and that M83 protein was expressed with late gene kinetics and phosphorylated in vivo (13). We also showed that the M84 gene product was likely encoded by a 6.9-kb transcript that was directed by either of the TATA boxes located just 5' of the M84 ORF (13). This transcript was detectable at 8 h p.i., suggesting that it belonged to the early class of genes. To determine the kinetics of M84 protein expression, we used Western blot analysis and a rabbit polyclonal antiserum to GST-M84 to examine whole-cell lysates prepared from NIH 3T3 cells infected with MCMV at a high MOI and harvested at 8, 24, and 48 h p.i. We found that solubilization of cell lysates at 100°C in Laemmli buffer resulted in aggregation of more than 99% of the M84 protein into an insoluble mass (data not shown). The M83 protein was also found to aggregate somewhat upon boiling, resulting in M83-immunoreactive species migrating to positions corresponding to 125, 105, and 70 kDa and often to greater than 200 kDa (13). However, when lysates were solubilized at 42°C, single immunoreactive 105- and 65-kDa species were detected with the M83- and M84-specific antisera, respectively (Fig. 1).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Kinetics of M84 protein expression and absence of virion association. NIH 3T3 cells were infected with K181 at an MOI of 3, and at 8, 24, and 48 h p.i., cells were harvested and whole-cell lysates were prepared as described in Materials and Methods. Cells were also treated for the first 8 h of infection with CHX before the 12-h-p.i. harvest or were infected and incubated in the presence of PAA until the 48-h-p.i. harvest. Western blots of lysates and purified virions (Vir.) were probed with an affinity-purified rabbit antiserum to GST-M84 or a GST-M83-specific antiserum. Each panel depicts the same blot, and lanes are numbered at the bottom. Lanes 8 to 10 depict lanes 5 to 7 after prolonged exposure, and lanes 11 to 13 show lanes 5 to 7 after the blot was stripped and reprobed with the M83 antiserum. Un., uninfected. Positions of molecular mass markers are shown on the left.

Construction of MCMV deletion mutants M83 and M84 and rescued virus rM83. To begin characterizing the functions of M83 and M84 in vivo, single-deletion MCMV mutants were constructed. Insertional mutagenesis of the MCMV HindIII C region (Fig. 2A) was performed by replacing the M83 or M84 ORF with the lacZ/gpt cassette (Fig. 2B and C, respectively). As described in detail in Materials and Methods, recombinant plasmids were constructed such that the marker cassette was flanked by 0.9 to 1.4 kbp of genomic sequence surrounding the region of the genome to be deleted. Following restriction digestion, these plasmids were electroporated into NIH 3T3 fibroblasts, which were subsequently infected with wild-type MCMV K181. Viruses that underwent homologous recombination between genome and plasmid sequences were isolated by mycophenolic acid selection and plaque purification.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Construction of the M83, M83-2, and M84 deletion mutants of MCMV. (A) HindIII restriction map of MCMV K181 and location of ORFs M82 to M86 in the HindIII C region. Arrows indicate the transcription direction and sizes of the ORFs. All ORF lengths are to scale, with the scale (in base pairs) indicated on the right. Also shown are the positions of restriction sites important for plasmid construction. Abbreviations: Hind, HindIII; Eco, EcoRI; RV, EcoRV; Pst, PstI; Not, NotI. (B) Construction of M83 and M83-2 by homologous recombination between either the pM83KO8 or pM83-2 insert, respectively, and the K181 genome. Identical shading and cross-hatching indicate homologous regions, while dashed lines indicate continuation of genome sequences. Indicated by a horizontal bar above pM83-2 is the 28 bp provided as a NotI-to-PstI oligonucleotide adapter as described in Materials and Methods. (C) Construction of M84 by recombination of homologous sequences of K181 and the insert from pM83KOT-5. The restriction sites at the borders of some homologous regions have been changed in the plasmid to facilitate subcloning.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Construction of the M83 revertant rM83, using the EGFP-puro cassette. (A) DNA sequences of the M83 genome were recombined in NIH 3T3 cells with homologous sequences (indicated by identical shading and cross-hatching) in the rescue plasmid cassette pM83Res13.6 (B). Selected restriction sites used for subcloning or for reference are indicated with abbreviations as in Fig. 2. Bam, BamHI. (C) Recombination between both homologous regions yields an rM83 intermediate virus in which the EGFP-puro cassette and full-length M83 ORF have been inserted into the M83 genome as indicated. Intramolecular recombination between M84 direct-repeat sequences in the rM83 intermediate would be expected to delete intervening marker gene sequences (bracketed in panel C), resulting in an EGFP LacZ virus with a wild-type (wt) HindIII C region (D).

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Genomic analysis of M83, M83-2, and rM83 viruses by restriction digestion and Southern blotting. (Left panels) The restriction maps of the portions of the HindIII C regions under analysis are shown for the K181, M83, and M83-2 viruses, with arrows indicating the positions, lengths, and directions of transcription of ORFs. B, BamHI; St, StuI; X, XhoI. Above each restriction map are shown the lengths (in kilobase pairs) and positions of restriction fragments detected by the genomic probe (indicated by the shaded regions). (Right panels) Genomic DNA was purified from uninfected cells or cells infected with the virus indicated. Genomic and HindIII C plasmid DNAs were digested with BamHI, electrophoresed, and blotted. The 4.4-kbp StuI-to-XhoI genomic probe was isolated from the HindIII C plasmid, 32P labeled by random priming, and used to probe the blot. The expected 0.52-kbp band in both panels was visible upon prolonged exposure of the blots.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Genomic analysis of the M84 and rM83 viruses by restriction digestion and Southern blotting. Details of genomic maps and DNA preparation are as described in the legend to Fig. 4. Ss, SstI; RV, EcoRV; N, NotI; X, XhoI. Genomic and HindIII C plasmid DNAs were digested with SstI and XhoI prior to electrophoresis, blotting, and detection with the 7.6-kbp EcoRV-to-NotI genomic probe.

Replication of M83 and M84 in cell culture. The roles of M83 and M84 proteins in MCMV replication were examined by single- and multicycle replication experiments using M83 and M84. Single-cycle replication experiments measured the accumulation of extracellular tissue culture virus in NIH 3T3 fibroblasts infected with K181, M83, or M84, or rM83. We found that the M84 (Fig. 6A), M83 (Fig. 6B), and rM83 (Fig. 6B and C) viruses replicated with wild-type kinetics and that their peak titers were comparable. When the combined levels of cell-associated and extracellular virus were measured in a separate experiment, the growth kinetics and final titers of all three viruses were also indistinguishable (data not shown). NIH 3T3 cells were also infected at a low MOI in order to examine the replication efficiency and cell-to-cell spread of the mutant viruses. Extracellular virus titers measured after infection with either M83 or M84 at an MOI of 0.05 were identical to those of the parent strain (Fig. 6D). This finding was consistent with the similar CPE, plaque sizes, plaque morphologies, and final viral titers obtained during several simultaneous preparations of K181, M83, and M84 stocks (data not shown). Taken together, these data indicate that the M83 and M84 ORFs are dispensable for replication in cultured fibroblasts.

View larger version (29K): [in this window] [in a new window]   FIG. 6.   Growth of mutant and revertant MCMVs in NIH 3T3 cells. All titers presented are the means of the log10 PFU/ml of the extracellular tissue culture medium of triplicate cultures, with error bars indicating the standard deviations (SD). (A) Single-cycle growth of K181 and M84 after infection at an MOI of 3. (B) Single-cycle growth of K181, M83, and rM83 in NIH 3T3 cells as described for panel A. (C) Single-cycle growth of K181, M83, M83-2, and rM83 as described for panel A. (D) Multicycle growth of K181, M83, and M84 in NIH 3T3 cells infected at an MOI of 0.05.

Protein expression from mutant and revertant MCMVs. The kinetics of protein expression from the various MCMV isolates were analyzed to determine whether expression of selected proteins representing the three temporal classes of gene products was grossly affected in any of the mutants as well as to confirm that the appropriate MCMV gene products were not expressed in their respective mutants. For these experiments, NIH 3T3 cells were infected with either K181, M83, M84, or rM83 at an MOI of 3.5, and at 8, 24, and 48 h p.i., cells were harvested and whole-cell extracts were prepared as described above.

View larger version (34K): [in this window] [in a new window]   FIG. 7.   Western blot analysis of mutant and revertant MCMVs. Whole-cell lysates were prepared from uninfected or MCMV-infected NIH 3T3 cells harvested at 8, 24, or 48 h p.i. Lysates were electrophoresed on 7.5% polyacrylamide-SDS gels, and separated proteins were electroblotted onto nitrocellulose. M83, M84, and M32 proteins were detected with rabbit polyclonal antisera as described in Materials and Methods. pp89 protein was detected with an antiserum from BALB/c mice immunized with intradermal injections of pcDNA3-pp89. Bound antibodies were detected with horseradish peroxidase-coupled anti-mouse or anti-rabbit antibodies as in Fig. 1. (A) Expression of M83, M84, M32, and pp89 proteins in NIH 3T3 cells after infection with K181, rM83, M83, or M84. (B) An independent experiment showing M83 and M84 expression 8 or 48 h after infection with K181, M83, or M83-2. uninf., uninfected. The positions of molecular mass markers are shown on the right.

M83 and M84 are both attenuated for replication in BALB/c mice, with M83 showing a specific defect for growth in salivary glands and lungs. The roles of M83 and M84 in viral replication in the host were examined by infection of inbred strains of mice with the mutant viruses by two different inoculation routes. We first examined the replication of the mutant viruses in the susceptible BALB/c strain following i.p. infection. Five mice per group were infected with 5 × 10⁵ PFU of tissue culture-derived K181, M83, or M84, and on day 4 p.i. the livers, spleens, and kidneys were harvested. At the time of sacrifice, both the K181- and, to a lesser degree, the M84-infected mice exhibited ruffled fur but no hunching or lethargy, while the M83-infected mice displayed no morbidity. As seen in Fig. 8A, titers of M83 and M84 in the spleen were reduced more than 11- and 12-fold, respectively, relative to that of the parent strain. Because virus was sometimes undetectable in one or more organs of a particular group (as denoted by the subscript 0 in Fig. 8 and 10), the titers of those organs were arbitrarily set to the limit of assay sensitivity, generally 10² PFU/organ, for mean calculations. Thus, fold reductions of titers in those groups are likely underestimates. In the liver, similar 11- to 12-fold reductions relative to wild-type titers were seen for both mutant viruses (Fig. 8A). The titer of M84 in the kidneys was 6-fold lower than that of K181, while M83 titers were at least 13-fold below the wild-type level. Taken together, compared to the wild type, tissue culture-passaged MCMV, both M83 and M84 appear to be growth attenuated in the abdominal target organs.

View larger version (47K): [in this window] [in a new window]   FIG. 8.   Growth of mutant and revertant MCMVs in BALB/c mice following i.p. or i.n. inoculation. (A) Five BALB/c mice per group were inoculated i.p. with 5 × 105 PFU of tissue culture-derived virus, and resultant viral titers in the livers, kidneys, and spleens 4 days postinoculation, or in the salivary glands 10 days postinoculation, are shown. Titers represent the means of the log10 PFU/organ values, with the standard deviations (SD) being shown by error bars. Values above each bar indicate the fold reductions of the nonlogarithmic mean titers (i.e., PFU/organ) of that group relative to the corresponding K181-infected controls. The subscript 0 indicates that in at least one organ in that group, virus was undetectable, and that the titer of that organ was set to the limit of detection for calculation purposes. (B) Day 4 spleen and day 10 salivary gland titers of four mice per group inoculated i.p. with K181, rM83, M83, or M83-2 as described for panel A. (C) MCMV titers in the lungs and salivary glands of four mice per group 14 days following inoculation i.n. with 1.5 × 105 PFU of virus.

M83 and M84 show patterns of restricted replication in C3H/HeN and 129/J mice similar to those seen in BALB/c mice. The ability of MCMV to replicate in vivo has been found to depend on the strain of mouse examined (22). We therefore tested the growth of the MCMV mutants in an inbred mouse strain highly resistant to MCMV. Four C3H/HeN female mice per group were infected with M83 or M84 by inoculation of either 1.5 × 10⁶ PFU (i.p.) or 1.5 × 10⁵ PFU (i.n.) of tissue culture-derived viruses. Because C3H/HeN mice are able to effectively limit MCMV replication in the abdominal organs, and because only tissue culture-derived virus was used for these experiments, salivary glands were the only tissue examined for viral replication in this strain.

View larger version (30K): [in this window] [in a new window]   FIG. 9.   Growth of mutant MCMV in the salivary glands of C3H/HeN mice. Shown are MCMV titers 10 days following i.p. inoculation with 1.5 × 106 PFU of tissue culture-derived virus and 14 days following i.n. inoculation with 1.5 × 105 PFU of virus. Means, standard deviations (SD), and fold-reduction values above each bar are as described in the legend to Fig. 8.

View larger version (31K): [in this window] [in a new window]   FIG. 10.   Growth of mutant MCMV in 129/J mice following i.p. or i.n. inoculation. (A) MCMV titers of four 129/J mice per group following i.p. inoculation with 5 × 105 PFU of tissue culture-derived virus. Organ harvest days, titer value calculations, and fold reduction values above each bar are as described in the legend to Fig. 8. (B) MCMV titers in the lungs and salivary glands of four mice per group 14 days following i.n. inoculation with 2.5 × 105 PFU of virus. SD, standard deviation.