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PubMed1971

Persistent cultural systems.

I have indicated here some features of a kind of entity which I have called a cultural identity system, and I have focused on a variety of this general type-the persistent system. In general terms it is best described as a system of beliefs and sentiments concerning historical events. I suggest using the term "a people" for the human beings who, at any given time, hold beliefs of this kind. These are phenomena with which we have been long familiar, but they have not been systematically studied by any but a few investigators. I have emphasized that a persistent system is a cumulative cultural phenomenon, an open-ended system that defines a course of action for the people believing in it. Such peoples are able to maintain continuity in their experience and their conception of themselves in a wide variety of sociocultural environments. I hold that certain kinds of identifiable conditions give rise to this type of cultural system. These may best be summarized as an oppositional process involving the interactions of individuals in the environment of a state or a similar large-scale organization. The oppositional process frequently produces intense collective consciousness and a high degree of internal solidarity. This is accompanied by a motivation for individuals to continue the kind of experience that is "stored" in the identity system in symbolic form. The persistent identity system is more stable as a cultural structure than are large-scale political organizations. When large-scale states disintegrate, they often appear to decompose into cultural systems of the persistent type. Large-scale organizations also give rise to the kind of environment that can result in the formation of new persistent systems. It is possible that, while being formed, states depend for their impetus on the accumulated energy of persistent peoples. A proposition for consideration is that states tend to dissipate the energy of peoples after transforming that energy into state-level integrations, and then regularly break down in the absence of mechanisms for maintaining human motivations in the large-scale organizations that they generate.

Anthropology, Cultural↗
PubMed1975

Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic bacteria on human skin.

The districution of Staphylococcus and Micrococcus species and associated coryneform bacteria, Acinetobacter, Klebsiella, Enterobacter, Bacillus, and Streptomyces on skin was determined during October 1971 from samples collected on persons living in North Carolina and New Jersey. Persistence of these organisms on skin was estimated in temporal studies conducted during the period from June 1971 to June 1972 on persons living in North Carolina. Staphylococci and coryneforms were the most predominant and persistent bacteria isolated from the nares and axillae. Staphylococci, coryneforms, micrococci, and Bacillus were the most predominant and persistent bacteria isolated from the head, legs, and arms. Acinetobacters were most frequently isolated during the warmer months of the years. Staphylococcus aureus and S. epidermidis were the most predominant and persistent staphylococci isolated from the nares, whereas S. epidermidis and S. hominis were the most predominant and persistent staphylocicci isolated from the axillae, head, legs, and arms. S. capitis was often isolated from the head and arms and S. haemolyticus was often isolated from the head, legs, and arms. S. simulans, S. xylosus, S. cohnii, S. saprophyticus, S. warneri, and an unclassified coagulase-positive species were only occasionally isolated from skin. Micrococcus luteus was the most predominant and persistent Micrococcus isolated from skin and preferred regions of the head, legs, and arms. M. varians was the second most frequent Micrococcus isolated. M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis were only occasionally isolated from skin. M. lylae was most frequently isolated during the colder months of the years.

Acinetobacter↗
PubMed1981

Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.

Almost all the cells in populations of mouse fibroblasts (L cells) persistently infected with the 6BC strain of Chlamydia psittaci were immune to superinfection with high multiplicities of C. psittaci, whether or not the L cells contained visible chlamydial inclusions. As ascertained by experiments with 14C-labeled C. psittaci, immunity to superinfection resulted from the failure of added chlamydiae to attach to persistently infected host cells. However, when exogenous C. psittaci was introduced into persistently infected L cells by centrifuging the inoculum onto host cell monolayers or by pretreating the monolayers with diethylaminoethyl-dextran, these chlamydiae produced expected numbers of infectious progeny. Persistently infected L cells were associated in an unknown way with a C. psittaci population that entered the host cells only with the aid of centrifugation or pretreatment with diethylaminoethyl-dextran. Inclusion-free, persistently infected L cells appeared to present at least two separate hindrances to chlamydial activity: blockage of the attachment of exogenous elementary bodies to persistently infected host cells and prevention of the initiation of chlamydial multiplication by means of a normal developmental cycle in the absence of added C. psittaci.

Animals↗
PubMed1981

Characterization of herpes simplex virus persistence in a human T lymphoblastoid cell line.

Persistent, dynamic-state infection with herpes simplex virus (HSV) type 1 has been maintained in human T lymphoblastoid (CEM) cells for many months after initial infection with the wild-type virus (HSV0) (input virus/cell multiplicity of 1.0). Persistently infected cells grew as well as uninfected cells, except during occasional periods of crisis (increased viral replication and cytopathic effect). Cells could survive the crisis when they were maintained for twice the usual time interval (8 to 10 rather than 4 to 5 days) before subculture. Interferon was not detectable in the cultures. HSV0 was compared with HSVp1, a small plaque-forming isolate from persistently infected CEM cells. Primary infection of CEM cells with HSV0 at a low input multiplicity (0.01) led to abortive replication, whereas infection with HSVp1 at the same multiplicity resulted in either rapidly lytic or persistent infection depending upon the time interval of subculture. Approximately 55% of plaque-purified clones of HSVp1, as compared with only 5% of HSV0 clones, displayed temperature-sensitive growth in Vero cells. Defective interfering virus was not detectable in uncloned HSVp1 by interference assay. Persistently infected cultures "cured" by treatment with HSV antiserum or incubation at 39 degrees C were resistant to reinfection with HSV but permissive for vesicular stomatitis virus replication, suggesting that these treatments modulated a shift from the dynamic-state of the static-state, latent infection. These studies provide a model for characterization of HSV persistence and latency in a highly differentiated human cell line.

Antibodies, Viral↗
PubMed1983

Inhibition of onset of overt multiplication of Chlamydia psittaci in persistently infected mouse fibroblasts (L cells).

When monolayers of mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci (strain 6BC) were dispersed in medium 199 and plated out in new flasks, the monolayers that grew out consisted almost exclusively of inclusion-free host cells that retained full resistance to superinfection with C. psittaci (covert infection). After a delay that was inversely proportional to the initial density of the newly transferred L cell population, the percentage of host cells containing visible chlamydial inclusions increased rapidly (overt infection), and most of the L cells were destroyed by extensive chlamydial multiplication (wipeout), leaving only a few survivors to start new persistently infected monolayers. When persistently infected L cell populations grown in medium 199 were transferred to Eagle minimal essential medium, the onset of overt multiplication was strongly suppressed although covert multiplication of C. psittaci continued unabated, as shown by host cell retention of resistance to superinfection and the prompt resumption of overt multiplication after transfer back into medium 199. The difference(s) between the two media responsible for the different expression of the persistently infected state was not determined. A single dose of 100 U of penicillin G per ml of medium 199 given at the time persistently infected monolayers were divided almost completely suppressed the appearance of visible signs of chlamydial infection for several weeks, although resistance to superinfection was retained at all times. The same amount of penicillin given 7 days after replating did not prevent the occurrence of the first expected wipeout, but there was a long period of inclusion-free L cell growth between the first wipeout and the second. It was concluded that covert multiplication of C. psittaci in persistently infected L cells may continue indefinitely without the appearance of visible signs of infection. The transition between covert and overt chlamydial multiplication appears to be a penicillin-sensitive, multistep process that is regulated, at least in part, by the host cell density and the composition of the growth medium.

Animals↗
PubMed1984

Acquired resistance to facultative intracellular bacteria: relationship between persistence, cross-reactivity at the T-cell level, and capacity to stimulate cellular immunity of different Listeria strains.

C57BL/6 mice were infected with different strains of Listeria sp., and bacterial survival in spleens was assessed. Six strains (EGD, NCTC 5348, ATCC 19113, ATCC 19114, NCTC 10527, and ATCC 19116) were able to persist in spleens (persistent strains), whereas with five other strains (ATCC 19111, ATCC 19119, ATCC 33090, ATCC 33091, and ATCC 14870), only few if any bacteria were demonstrable after infection with up to 10(8) organisms (nonpersistent strains). Immunization of mice with persistent listeriae induced strong immune responses as determined in vitro (antigen-induced proliferation and interleukin production) and in vivo (protection and delayed-type hypersensitivity), whereas immunization with nonpersistent bacteria resulted in weaker responses. On the other hand, T lymphocytes from mice immunized with live organisms of the persistent strain EGD were stimulated equally well by heat-killed listeriae of all strains. Furthermore, three T-cell clones which were able to adoptively mediate antibacterial protection in vivo could be stimulated by heat-killed organisms of persistent as well as nonpersistent Listeria strains. It is concluded that both persistent and nonpersistent listeriae express antigenic epitopes which are recognized by protective T cells, although nonpersistent strains are not effective in inducing cellular immune responses due to rapid elimination in the host.

Animals↗
PubMed1985

Persistent infection of L cells with an ovine abortion strain of Chlamydia psittaci.

L cells inoculated at multiplicities of infection greater than or equal to 1 inclusion-forming unit of the abortigenic chlamydial strain B577 were destroyed within 10 to 15 days. Upon continued incubation in fresh medium, a few surviving cells repopulated the flasks, and the reemerging cultures remained persistently infected. The persistent state was characterized by cycles of repopulation with a low ratio of infected cells and cycles of extensive cytopathic changes in which greater than 90% of the cells had chlamydial inclusions and which could be delayed or even terminated by penicillin treatment. Immunofluorescence and superinfection during the period of repopulation revealed that the persistently infected cells could adsorb chlamydiae but their multiplication was arrested. This nonpermissive state could be terminated by the specific action of cycloheximide. L cells spontaneously cured from a persistent infection exhibited no change in susceptibility to chlamydiae when compared with normal L cells. However, chlamydiae derived from L cells after 7.5 months of persistence destroyed L-cell monolayers more rapidly and at lower multiplicities of infection than the wild type. This state of chlamydia-host cell interaction could not be established with the arthropathogenic strain LW613 because chlamydial infectivity was lost after the first cytolytic burst of infection in the cell cultures. The persistence described for the strain B577-L-cell system appears to differ from previously described models involving other chlamydial strains.

Abortion, Septic↗
PubMed1973

Persistent Newcastle disease virus infection in embryonic chicken tracheal organ cultures.

The persistent infection of embryonic chicken tracheal organ cultures with Newcastle disease virus (NDV) is described. Tracheal explants remained morphologically intact and were able to support the replication of NDV for 6 months. Peak titers of released virus occurred at 1 week postinfection, whereas maximal immunofluorescence was not observed until 30 days postinfection. The inoculum titer was not critical, and viral persistence resulted with either of two strains of NDV tested. Serum was not required in the medium for explant viability or to maintain the persistent infection. The presence of a contaminating virus morphologically resembling a leukovirus neither altered the course of infection nor affected the survivability of explants. Although interferon was not detected in the culture medium, persistently infected explants were resistant to heterologous viral challenge, and a similar resistant state could be induced in uninfected explants with exogenous interferon or ultraviolet light-inactivated NDV. No evidence was found to implicate antibody as a regulatory factor in the establishment or maintenance of persistence. The results from electron microscopy and immunofluorescence suggest the cells of the subepithelial connective tissue as the site of NDV persistence.

Animals↗
PubMed1977

Persistent infections in L cells with temperature-sensitive mutants of reovirus.

Serial passage of reovirus temperature-sensitive (ts) mutant C(447) produced by passage 9 (P9) a heavily defective population of virus from which the double-stranded RNA genomic segments L(1), L(3), and M(1) were largely missing. Viral cores obtained from this P9 population were heterogeneous with respect to buoyant density in CsCl gradients, suggesting that particles were present with different combinations of deleted segments. Similar observations were made with the E(320) ts mutant of reovirus. By serial passage P15, 90% of the E(320) viral population was defective and the major missing genomic segments were L(1) and L(3). Persistent infections were readily established in monolayer cultures of L cells with P9 of C(447) virus and P15 of E(320) virus and in Vero cells with P9 of C(447) virus. Under similar conditions persistent infections could not be initiated with defective-free populations of C(447) or E(320) viruses. The greater the capacity of defective virus in the population to interfere with viral growth, the more readily persistent infection was initiated. During their maintenance persistently infected cells were subcultured approximately twice a week. More than 80% of the cells continuously produced virus. By subculture 6 the original ts infectious viral component had been replaced by a small-plaque mutant with a ts(+) phenotype. Defective virus was always present in the carrier cells. In addition to the more commonly observed defectives whose cores banded at approximately rho = 1.40 to 1.415 g/ml in CsCl gradients, a new class of defective core was seen banding in the region of 1.34 to 1.36 g/ml. This latter particle, which has not been thoroughly characterized as yet, is termed "light defective." Persistently infected cells underwent periodic crises during their maintenance, during which the cultures partially lysed and then rapidly grew to confluence. Crises corresponded to a burst of infectious virus from the cells and a relatively low concentration of light defectives. During quiescent periods the concentration of light defectives amounted to as much as 98% of the total viral population. The function of light defectives is not yet clear, but it seems essential to assign major importance to defective virus in maintaining persistent infections in this system.

Defective Viruses↗
PubMed1982

Novel phenotype of RNA synthesis expressed by vesicular stomatitis virus isolated from persistent infection.

Vesicular stomatitis virus (VSV) stocks isolated from two persistently infected mouse L-cell lines (designated VSV-PI stocks) express an altered phenotype of RNA synthesis. This phenotype is different from the RNA synthesis phenotype expressed by the viruses used to initiate the persistently infected lines, wild-type VSV and VSV ts-0-23 (a group III, ts-, RNA+ mutant). At 34 and 37 degrees C in L cells productively infected with VSV-PI stocks derived from the two cell lines, transcription of virus mRNA was significantly reduced, whereas replication of the 40S genomic RNA species was enhanced compared with wild-type VSV or ts-0-23. At 34 and 37 degrees C, both VSV-PI stocks replicated with equal or greater efficiency than wild-type VSV; 37 degrees C was the temperature at which the persistently infected cultures were maintained. At 40 degrees C, both VSV-PI stocks were temperature sensitive, and clonal VSV-PI isolates from both cell lines belong to complementation group I (RNA-). Standard ts- mutants (derived by mutagenesis of wild-type VSV) belonging to RNA- complementation groups I, II, and IV do not express the VSV-PI RNA synthesis phenotype at the permissive temperature, making this phenotype distinctive to persistent infection. Since the two VSV-PI populations from persistently infected cell lines initiated with different viruses both evolved this unique phenotype of RNA synthesis, the expression of this phenotype may play an important role in the maintenance of persistence.

Animals↗
PubMed1988

Molecular analysis of viral RNAs in mice persistently infected with lymphocytic choriomeningitis virus.

Infection of newborn mice with lymphocytic choriomeningitis virus (LCMV) results in a lifelong persistent infection. Persistently infected animals continuously produce low levels of infectious virus and accumulate large amounts of intracellular viral nucleic acid (P. J. Southern, P. Blount, and M. B. A. Oldstone, Nature [London] 312:555-558, 1984). We have used gel electrophoresis and hybridization techniques to analyze viral RNAs that appear during the establishment and maintenance of a persistent LCMV infection in vivo to identify any role for defective and/or defective interfering RNAs. We have found a complex, heterogeneously sized population of viral RNAs in multiple independent tissues that is uniquely associated with persistent infections in vivo, but we have not yet established whether these RNAs have a causal or a consequential association with persistent infection by LCMV. Within the complex virus RNA population, full-length genomic L and S RNAs were readily detectable and represented the most abundant individual viral RNA species. RNAs apparently corresponding in size to the viral nucleoprotein and glycoprotein mRNAs could also be detected in these tissue RNA samples. The presence of glycoprotein mRNA indicates a potential mechanism of posttranscriptional regulation to account for the previously documented restriction in viral glycoprotein expression in persistently infected mice (M. B. A. Oldstone and M. J. Buchmeier, Nature (London) 300:360-362, 1982).

Animals↗
PubMed1992

Enhancer dependence of polyomavirus persistence in mouse kidneys.

We previously showed that alterations in the enhancer sequence of polyomavirus DNA can alter both the level and the organ specificity of viral DNA replication during the acute phase of infection of newborn mice (R. Rochford, B. A. Campbell, and L. P. Villarreal, J. Virol. 64:476-485, 1990). In this study, we examined whether these enhancer sequence alterations can also affect polyomavirus replication during the persistent phase of infection in vivo. After infection of newborn mice with a mixture of three enhancer variants, the individual organs could select for enhancer-specific viral DNA replication during both the acute and the persistent phases of infection. Contrary to expectations, the ability of some variants to establish a high-level acute infection in some organs (e.g., the pancreas) did not necessarily lead to a persistent infection in those organs. Thus, enhancers can affect acute and persistent infections differently. In addition, some enhancer variants tended to establish a high-level persistent infection in the kidneys immediately following an acute infection; however, in all cases considerable histopathology was associated with these elevated long-term infections, and these mice were always runty. A persistent infection in the kidneys thus appears able to exist in two distinguishable states, a high-level pathological state and a low-level nonpathological state, which can be affected by the viral enhancer sequence.

Acute Disease↗
PubMed1993

Cells and viruses with mutations affecting viral entry are selected during persistent infections of L cells with mammalian reoviruses.

Previous studies demonstrated that both cellular and viral mutants are selected during maintenance of persistent infections established in murine L cells with high-passage stocks of mammalian reoviruses. In particular, when one culture was cured of persistent infection, the resulting cells were found to support the growth of viruses isolated from persistently infected cultures (termed PI viruses here) better than that of wild-type (wt) viruses (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25:325-332, 1981). To address the nature of cellular and viral mutations selected during maintenance of persistent reovirus infections, we established independent, persistently infected cultures with L cells and high-passage stocks of wt reovirus. These cultures served as sources of new PI viruses and cured cells for study. We found that although wt viruses grew poorly in cured cells when infection was initiated with intact virions, they grew well in cured cells when infection was initiated with infectious subvirion particles generated from virions by in vitro treatment with chymotrypsin. This finding indicates that the block to growth of wt viruses in cured cells involves an early step that is unique to infection by virions, such as proteolytic processing in an endocytic compartment. We also found that PI viruses grew better than wt viruses in L cells treated with ammonium chloride, a weak base that inhibits the pH decrease in endosomes and lysosomes. Because ammonium chloride blocks an early step in infection by intact virions, probably the proteolytic processing of viral outer capsid proteins by acid-dependent cellular proteases in late endosomes or lysosomes, this finding indicates that PI viruses differ from wt viruses with respect to viral entry into cells. Therefore, these results indicate that both cells and viruses evolve mutations that affect one or more early steps in the viral growth cycle during maintenance of L-cell cultures persistently infected with reoviruses.

Ammonium Chloride↗
PubMed1993

Vaccination to prevent persistent viral infection.

Persistent virus infections are increasingly being recognized as a significant cause of human morbidity and mortality. To establish persistence, a virus must establish infection and evade eradication by the host immune response, in particular by cytotoxic T lymphocytes (CTL). We have studied a virus that establishes persistence in part by suppressing the CTL response of the infected host. The virus persists in many cell types, including lymphocytes and macrophages. We show that prior immunization with a vaccine designed to induce CTL (in the absence of antiviral antibody) confers complete protection against subsequent establishment of persistence in all tissues analyzed. The vaccine can be designed to express as few as 10 amino acids of a viral protein that comprise the CTL epitope. Further, two CTL epitopes for two discrete MHC haplotypes can be successfully used in a single vaccine that protects both strains of mice. Hence, a "string of CTL epitopes" (beads) concept for vaccination is feasible. Finally, the CTL vaccine provided protection against the establishment of persistence by an immunosuppressive virus.

Amino Acid Sequence↗
PubMed1993

Identification of a region of the poliovirus genome involved in persistent infection of HEp-2 cells.

Poliovirus mutants were selected during the persistent infection of human neuroblastoma cells. These viruses could establish secondary persistent infections in HEp-2 nonneural cells. We report the identification of a region of the genome of a persistent virus (S11) that was sufficient to confer to a recombinant virus the phenotype that causes persistent infection in HEp-2 cells. This region, between nucleotides 1148 and 3481, contained 11 missense mutations mapping exclusively in the genes of capsid proteins VP1 and VP2. Because recombinant viruses carrying only one of these two mutated genes were not able to cause persistent infection, it seems very probable that two or more mutations in these genes are required for expression of the phenotype that causes persistent infection.

Capsid↗
PubMed1995

Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.

Animals↗
PubMed1995

Regulation of I kappa B alpha and p105 in monocytes and macrophages persistently infected with human immunodeficiency virus.

The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in NF-kappa B activation. Whether virus-induced NF-kappa B activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of NF-kappa B in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF-kappa B not only in monocytic cells but also in macrophages. In HIV-infected cells, I kappa B alpha protein levels are decreased secondary to enhanced protein degradation. This parallels the increased I kappa B alpha synthesis secondary to increased I kappa B alpha gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, p105, is also modified in HIV-infected cells: p105 and p50 steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of p105. Transcriptional activity of p105 is also increased in infected cells and is also mediated by NF-kappa B through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, p105 and p50, and MAD3, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules.

Base Sequence↗
PubMed1995

Latency, without persistence, of murine cytomegalovirus in the spleen and kidney.

It is not known if murine cytomegalovirus (MCMV) establishes a state of molecular latency independent of low-level persistent infection. The presence of low levels of infectious MCMV distinguishes persistence from molecular latency. Thus, the distinction between persistence and latency has depended on the sensitivity of plaque assays for detecting low levels of infectious virus in tissue of previously infected mice. To determine whether MCMV establishes molecular latency or remains persistent, we developed two assays for detecting low levels of MCMV in tissue. Using prolonged in vitro culture of virus with either mouse embryonic fibroblasts or the murine 3T12 fibroblast cell line, we reproducibly detected a single PFU of MCMV. Inclusion of undiluted sonicated tissue in this assay decreased sensitivity by up to 100-fold. However, sensitivity was improved to 1 PFU of MCMV when sonicated tissue was appropriately diluted. Severe combined immunodeficient (SCID) mice were also used to detect MCMV in sonicated tissue. Infection of SCID mice with a single PFU of MCMV killed two of eight SCID mice, and the 50% lethal dose of MCMV in SCID mice was 2 to 3 PFU. Applying these two methods, we detected infectious virus in 0 of 34 spleens, 1 of 34 kidneys, and 0 of 37 salivary glands from latently infected mice. Spleens and kidneys assessed for persistent virus contained MCMV DNA by PCR and reactivated after 10 to 50 days in explant cultures. Latently infected kidney cells reactivated after adoptive transfer to SCID mice. Quantitation of the MCMV genome by PCR showed that latently infected spleens without detectable infectious MCMV contained about 3,000,000 copies of the MCMV genome. These results demonstrate that MCMV latency in spleen and kidney exists in the absence of low-level persistent infection. Use of assays with defined sensitivity for detection of MCMV in tissue provides a basis for evaluation of cytomegalovirus gene expression in the spleen and kidney during molecular latency.

Animals↗