Host Cell Specificity of Minute Virus of Mice in the Developing Mouse Embryo

Productive infection by the murine autonomous parvovirus minutevirus of mice (MVM) depends on a dividing cell population andits differentiation state. We have extended the in vivo analysisof the MVM host cell type range into the developing embryo byin utero inoculation followed by further gestation. The fibrotropicp strain (MVMp) and the lymphotropic i strain (MVMi) did notproductively infect the early mouse embryo but were able toinfect overlapping sets of cell types in the mid- or late-gestationembryo. Both MVMp and MVMi infected developing bone primordia,notochord, central nervous system, and dorsal root ganglia.MVMp exhibited extensive infection in fibroblasts, in the epitheliaof lung and developing nose, and, to a lesser extent, in thegut. MVMi also infected endothelium. The data indicated thatthe host ranges of the two MVM strains consist of overlappingsets of cell types that are broader than previously known fromneonate and in vitro infection experiments. The correlationbetween MVM host cell types and the cell types that activatethe transgenic P4 promoter is consistent with the hypothesisthat activation of the incoming viral P4 promoter by the hostcell is one of the host range determinants of MVM.

INTRODUCTION

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Introduction

The autonomously replicating parvoviruses are small, nonenveloped,icosahedral DNA viruses of vertebrates. They include severaleconomically important livestock viruses and the human-specificmember B19, which causes erythema infectiosum, a common childhooddisease. Phylogenetic analysis (23) suggests the existence oftwo major branches of the mammalian autonomous parvoviruses,with the bovine, chipmunk, and primate viruses in one lineageand the carnivore, pig, and other rodent viruses in the other.Minute virus of mice (MVM), the best studied of the rodent parvoviruses(18), contains a 5,149-nucleotide single-stranded genome. Theviral genome harbors two transcription units encoding two nonstructural(NS) and three structural (VP) proteins. Almost all aspectsof the viral life cycle are dependent upon virus-host interactions,and MVM has proven to be an excellent model system for studyingthese. Upon infection of a permissive host, the single-strandedviral genome is converted to a double-stranded replicative-formDNA, following which the viral nonstructural promoter, P4, isactivated by the host (11), leading to the synthesis of thenonstructural NS1 and NS2 proteins. Host and viral functionscooperate in the subsequent amplification and expression ofthe viral genome.

Whether a potential host cell is permissive has been shown todepend on a number of cellular characteristics. In vitro experimentsindicate an absolute requirement for a dividing cell populationand S-phase functions (36, 43). MVM infection also depends uponthe degree of differentiation of the host cell in vitro (16,37) and in vivo (4, 9). However, the nature of this dependencyhas not been elucidated; several tissues in the adult animalthat contain actively dividing cells are not targets for parvovirusreplication. Study of two MVM strains, the fibrotropic p strain(MVMp) (8, 36) and the lymphotropic i strain (MVMi) (25), indicatea cell type-dependent interaction between the host and the viruscapsid proteins. The two strains exhibit 97% sequence identityand are serologically indistinguishable, yet they differ radicallyin their tropisms in vitro, displaying reciprocal cell typespecificities. MVMp establishes productive infection in culturedmouse fibroblasts, whereas MVMi replicates lytically in mouseT lymphocytes (3). Growth of each is restricted in the other'spermissive cell types (16, 38). Entrance of MVMi and MVMp particlesinto the host cell is mediated by the same cell surface receptor(34), which was found in abundance on mouse permissive A9 cells(22). The host range variation is determined intracellularlyby a capsid determinant consisting of a limited number of aminoacids in VP2 (1, 24) and which is assumed to interact with astill-unknown host factor. Other than the VP2-host interaction,Rubio et al. have demonstrated cell-type-dependent interactionsbetween host cellular factors and the NS proteins (33). Activationof the viral P4 promoter by the host following infection isa critical early step in the viral life cycle (11) and is completelydependent on the host transcription machinery. Since promoteractivities often vary widely in different cell types and atdifferent developmental stages, it is possible that the initialactivation of P4 in the completed double-stranded viral genomeconstitutes a host range determinant. Indeed, we have recentlyshown that host activation of a transgenic P4 promoter is tissueand developmental stage specific and loosely correlates withtissue type susceptibility to MVM (9). Finally, NS2 was shownto be essential for productive infection in a cell-type-specificfashion in vitro (6) and in vivo (5).

Infection studies with live animals have shown that the twoMVM strains differ in their pathogenicities. Infection of adultor neonatal mice with MVMp was asymptomatic (5, 21). Analysesof viral distribution revealed significant amounts of MVMp inthe gastrointestinal tract, frequently in fibroblasts (5). Incontrast, MVMi infection in neonates was lethal, probably dueto its affinity for the capillary endothelium of the renal medulla,cells which MVMp fails to infect (4). MVMi was shown to be ableto infect still-dividing neuroepithelial cells in the neonatebrain (31). Other mammalian parvoviruses have varied tropismsin vivo (17, 41), with an overall tropism for the hematopoetic/lymphoidtissues, gut, heart, and proliferative neuroepithelium (13,29).

Despite the dependence on a dividing cell population and sensitivityto differentiation status, MVM infection has rarely been studiedin the developing embryo, which consists largely of dividingcells in a wide range of differentiation states. Early studiesshowed that some rodent parvoviruses could induce fetal diseasein laboratory animals. Following maternal injection, transplacentalinfection of mid-gestation rat embryos with parvovirus occasionallytook place (20, 40), resulting in mongoloid deformities, craniofaciallesions, and microcephaly (39), without observable effect onthe mother. In embryonic tissues, parvovirus H1 inclusions wereidentified in developing bone, the basal layer of the dermis,the muscular and lamina propria layers of the gastrointestinaltract, tooth germ, and vascular endothelium. Early-gestationembryos were resistant to infection (40). At least the bovine,porcine, and canine parvoviruses have also been shown to causetransplacental infection of gestating embryos, infecting, inthe pig, the fetal myocardium and lung (2, 26).

In this study we report the susceptibilities of different mouseembryonic cell types to MVMi and MVMp infection following inutero injection of MVM and further gestation. We compare theseto the published cell type tropisms of the two viral strainsand to the distribution of embryonic activation of a transgenicMVM P4 promoter.

MATERIALS AND METHODS

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Materials and Methods

Virus preparation. MVMp and MVMi stocks were grown in suspension in A9 (peritonealtumor-derived Ehrlich ascites) and EL4 (mouse lymphoma) cellsand purified by CsCl or glycerol gradient centrifugation, andtiters were determined by plaque assay in NB324K indicator cellsas described previously (32).

Mouse care. Hsd:ICR mice, a locally maintained (Harlan, Rehovot, Israel)population of the outbred strain CD-1, were kept in the medicalfaculty animal care facility. All housing and experimental proceduresfollowed institution-approved protocols and guidelines. Femalesfed a high-fat (8%) breeder diet were mated to males and werechecked the following morning for vaginal plugs. The morningon which a vaginal plug was observed was considered to be 0.5day postconception (dpc). Females were housed individually throughoutpregnancy and experimentation.

Surgical procedure. Virus was injected at 9.5 or 12.5 dpc as described previously(17a). After abdominal incision, the number of embryos on eachuterine horn was recorded. At 9.5 dpc, the embryo itself isnot visible through the uterine wall and the embryonic organprimordia are very small; therefore, 2 µl of inoculumcontaining 10⁵ PFU of MVM was injected into the amniotic cavity.Four microliters of inoculum was injected into the livers of12.5-dpc embryos. No more than four embryos, all on one uterinehorn, were injected. Following 24, 48, or 96 h of further gestation,the females were killed by cervical dislocation. The abdominalwall was opened, and the injected embryos and at least two contralateraluninjected control embryos were taken for analysis. Any resorptionsites or dead embryos were noted.

Histology and immunohistochemistry. Only living, morphologically and histologically normal embryoswere chosen for histological analysis. Once triggered, embryonicresorption is morphologically distinguishable within a few hours,which is less than the life cycle of MVM. The observed MVM immunoreactivityis due to infection of normal, not resorbing, embryos.

Injected and control embryos were embedded in OCT (TissueTek)and frozen by immersion into liquid nitrogen. Individual frozenembryos were serially sectioned at 16 µm in a cryomicrotome(IEC) and collected on poly-L-lysine coated slides. Air-driedsections were fixed for 10 min in 4% paraformaldehyde and rinsedfour times in Tris-buffered saline (TBS) for 12 min. Sectionswere blocked with 1% bovine serum albumin (BSA) in TBS and thenincubated for 1 h at room temperature with the primary antibody(polyclonal anti-MVM capsid protein purified from rabbit serum)diluted 1:500 in 1% BSA in TBS. The slides were then rinsedin TBS as after fixation and incubated for 1 h at room temperaturewith one of two equivalent, minimally mouse-cross-reactive secondaryantibodies, Alexa Fluor 546-conjugated goat anti-rabbit immunoglobulinG heavy plus light chains (Molecular Probes) or Cy3-conjugatedaffinity-pure goat anti-rabbit immunoglobulin G heavy plus lightchains (Jackson ImmunoResearch), diluted 1:250 in 1% BSA inTBS. Slides were then rinsed as before, and coverslips wereapplied in glycerol containing 1 mg of phenyldiamine per ml.Immunoreactivity or tissue was visualized on a Leica DMR compoundmicroscope equipped for immunofluorescence and differentialinterference contrast (DIC) and photographed with a Spot RTdigital camera (Diagnostic Instruments).

RESULTS

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Results

The injection procedure developed has been described in detail(17a) and was demonstrated to be effective and benign. Injectionof sterile saline at either embryonic stage did not significantlyperturb embryonic development. The efficacy was confirmed eitherby observing the healing needle wound in the injected embryonicliver after 24 h of further gestation or by injecting Indiaink into 9.5-dpc yolk sacs, dissecting immediately after, andobserving the distribution of the ink. We therefore used theprocedure to characterize the pattern and kinetics of MVM infectionin the developing mouse embryo. The times of injection and analysisrelative to the activity of a transgenic MVM P4 promoter atdifferent developmental stages are illustrated in Fig. 1.

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FIG. 1. MVM injection and analysis relative to activity of a transgenic MVM P4 promoter. (A) Gestation series showing activity of a transgenic MVM P4 promoter visualized by ß-galactosidase reporter activity (blue) at the indicated times (dpc). Green dots indicate inoculation with MVM; red dots indicate stage at time of analysis. (B to J) Tissue-specific activity of a transgenic MVM P4 promoter, as visualized by ß-galactosidase reporter activity at 12.5 or 13.5 dpc. (B) Forebrain neuroepithelium, mesenchyme, and skin. (C) Enlargement of skin region in panel B. (D) Section through vertebra primordium and notochord. (E) Section through spinal chord and dorsal root ganglion. (F) Dorsal view of 13.5-dpc whole-mount embryo, showing staining in CNS, developing bone primordium, and dorsal root ganglion. (G) Fold of nasal epithelium. (H) Section through developing limb bud. (I) Blood vessel. (J) Section through heart and liver. der, dermis; drg, dorsal root ganglion; end, endothelium; epi, epidermis; ery, erythrocytes; h, heart; lbp, limb bone primordium; liv, liver; nas, nasal epithelium; nc, notochord; ne, neuroepithelium; rbp, rib primordium; sc, spinal chord; vrp, vertebra primordium.

MVMi is more pathogenic than MVMp. The relative pathogenicities of MVMi and MVMp in mouse embryoswere assessed following intrauterine inoculation. Two microlitersof MVMi or MVMp (10⁵ PFU) was injected into the amniotic sacsof 9.5-dpc embryos. Four microliters was injected into embryoniclivers of 12.5-dpc embryos as described in Materials and Methods.Gestation was left to proceed for an additional 24, 48, or 96h. The patterns of surviving embryos and resorption sites werescored (Table 1). MVMi injection at 12.5 dpc was significantlymore likely to result in embryonic death than MVMp injection(P_null < 0.01) over the times shown. Injection did not resultin any significant increase in resorption among contralateralor uninjected adjacent embryos. To examine whether the greatersurvival of MVMp injected embryos indicated a recovery frominfection or a slower pathology, 20 embryos in five femaleswere injected and the pregnancies were left to continue for120 h, i.e., within 2 days of parturition. The females werethen killed, and the patterns of embryos and resorption siteson the uterine horns were determined. By this late stage, 17of the 20 injected embryos had resorbed, suggesting that MVMpmay ultimately be as lethal as MVMi but takes longer to killthe host embryo.

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TABLE 1. Embryo fatality following MVMp/ or MVMi injection^a

Distribution of infection. Injected embryos, uninjected neighboring embryos, and contralateraluninjected control embryos were removed and cryosectioned. Thedistribution of MVM capsid proteins in different tissues andcell types was determined by immunohistochemistry.

At 9.5 dpc, 13 embryos were injected with MVMp or MVMi. After48 h of further gestation, we observed resorption of five embryosand retarded development of one. There was no significant differencebetween MVMp and MVMi. Five MVMp- and three MVMi-injected embryosand their placentas were removed and sectioned, and the distributionof MVM coat proteins was determined by immunohistochemistry.A widespread but diffuse and weak increase in signal comparedto uninjected control samples was apparent in all tissues ofembryos injected with either virus strain. The signal was strongerin the central nervous systems (CNS), the dorsal aortas, andthe hearts of MVMp-inoculated embryos (Fig. 2) and was strongerin the CNS of MVMi-inoculated embryos. In contrast to the resultsfor the embryos, the developing placentas exhibited a much strongervirus coat protein signal distributed in regional foci on onlythe embryonic side of the placenta, with increased signal observedaround blood vessels (Fig. 2C and D). Since the placenta itselfwas not injected, the route of infection must have been througheither the amniotic fluid or, more likely, embryonic blood circulationto the placenta.

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FIG. 2. MVM immunoreactivity following inoculation at 9.5 dpc and 48 h of further gestation. This and subsequent figures follow a similar format. Embryo diagrams (after reference 19) at the top left illustrate the embryonic stage and position of histological cuts in the figure elements. Low-magnification images of the entire embryo section are included on the left of the figure panels if available, and rectangular insets labeled with a capital letter at the top or bottom indicate the area covered by the corresponding figure element. Double images bearing a single-letter label are identical fields of view under different illuminations, with DIC on the left and immunofluorescence showing the distribution of MVM coat proteins on the right. (A) Diagram of 11.5-dpc embryo, showing section levels in panels E and H. (B) Section through an 11.5-dpc placenta associated with an MVMp-injected embryo. (C) Enlargement of region in panel B showing MVM infection in embryonic chorionic villus but not surrounding maternal tissue. (D) DIC (left) and immunofluorescence (right) detail of region of villus in panel C showing individual MVM-infected cells. (E to J) Sections through 11.5-dpc embryo (E and H) showing weak and scattered anti-MVM immunoreactivity in the dorsal aorta (F), heart (G), and neuroepithelium (I). (J) Detail of heart showing scattered punctate staining. da, dorsal aorta; h, heart; lum, CNS lumen; ne, neuroepithelium; ov, otic vesicle; sc, spinal chord; vil, placental villus.

By 12.5 dpc embryonic organogenesis is well under way, and agreater variety of more fully differentiated cell types arefound in the embryo than at 9.5 dpc. By this stage the transgenicMVM P4 promoter was active in a wide variety of different celltypes (9) (Fig. 1). We therefore examined the course and extentof MVM infection in the developing embryos by injecting thevirus directly into the embryonic livers of 12.5-dpc embryosand examining the distribution of MVM coat proteins after afurther 24, 48, or 96 h of gestation following inoculation.

At 24 h following inoculation, 7 embryos out of 13 injectedembryos from three females were serially sectioned and stainedfor MVM viral capsid proteins. The wound due to the injectioninto the liver was clearly visible and showed intense localimmunoreactivity (not shown). Outside the injection site itself,liver tissue was completely negative for viral capsid proteinsin both MVMp- and MVMi-injected embryos. In the rest of theembryo, localized staining was observed only in small patchesin chondrocytes of some bone primordia and in the notochord(Fig. 3B and C).

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FIG. 3. Distribution of MVM coat proteins in embryos inoculated at 12.5 dpc after a further 24 or 48 h of gestation. For format, see the legend to Fig. 2. (A and D) Approximate planes of sections in panels B, C, E, and G. (B) MVMi coat protein distribution in forelimb bone primordium 24 h after inoculation. (C) MVMp coat protein in the notochord 24 h after inoculation. (E and G) Whole sections showing locations of field in panels F and H. (F and H) Distribution of MVMi coat proteins in bone primordia and notochord 48 h after inoculation. (I and J) Distribution of MVMp coat proteins in bone primordia and notochord 48 h after inoculation. bp, bone primordium; nc, notochord; ne, neuroepithelium; sc, spinal chord.

At 48 h after inoculation, the initial weak and patchy stainingobserved in bone primordia became somewhat more extensive andintense (Fig. 3F, H, I, and J). The bone primordium stainingwas restricted to the developing axial skeletal elements, thelimb and mandibular bones of the head. Each of the infectedcells showed punctate signal distributed mainly in the cytoplasm.No staining was observed in any other tissues, including thearea where the flat bones of the skull will form.

The infection pattern seen after 96 h of gestation followinginoculation was much more extensive than that observed at shorterincubation times. Individual positive cells were observed scatteredthroughout the dorsal root ganglia of both MVMp- and MVMi-inoculatedembryos (Fig. 4C and E), although positive cells in the MVMp-inoculatedembryos were more likely to be found at the periphery of thedorsal root ganglia. Similarly, scattered positive cells wereobserved in the developing CNS of both MVMp- and MVMi-inoculatedembryos (Fig. 4F, G, and I). Positive cells were more frequentlyobserved in the CNS of MVMi-inoculated embryos, in which immunoreactivityappeared more frequently near the interface between the activelydividing cells of the ventricular zone and the overlying marginalzone, which contains the postmitotic neurons (Fig. 4F).

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FIG. 4. Distribution of MVM coat proteins in neural tissues of embryos inoculated at 12.5 dpc after a further 96 h of gestation. For format, see the legend to Fig. 2. (A) Approximate planes of sections in panels B, D, and H. (B) Whole section showing location of field in panel C. (C and E) Embryonic dorsal root ganglia and parts of spinal chord 96 h after inoculation with MVMi (C) or MVMp (E), showing occasional immunoreactive cells in the ganglia. (H) Whole section showing field of view in panel F. (F, G, and I) Embryonic spinal chord 96 h after inoculation with MVMi (F and G) or MVMp (I), showing occasional immunoreactive cells in the spinal chord. bp, bone primordium; drg, dorsal root ganglion; mz, marginal zone; ne, neuroepithelium; sc, spinal chord.

By 96 h, aspects of MVM strain tropisms were apparent in thepatterns of embryonic infection. MVMi infection was found inthe endothelia of some blood vessels (Fig. 5C), notably in theliver, lung, spleen, dermis, and branch vessels in bone, andin a few scattered cells that were judged to be lymphocyteson the basis of morphology (not shown). We never observed signalin the endothelia of MVMp-inoculated embryos. However, extensiveand intense MVMp staining was observed in fibroblastic tissuesthroughout the embryo, including the dermis (Fig. 6B), connectivetissues (Fig. 6D), head mesenchyme (Fig. 5J), developing boneperiost (Fig. 6F), heart (Fig. 6H and I), and lung epithelium(Fig. 5H), and to a lesser extent in the developing gut (datanot shown). Both MVMp and MVMi were observed in the developingnasal epithelium (Fig. 5J and data not shown). Some foci ofinfection were seen in the subarachnoid space, mainly in thearea of the blood vessels passing anterior to the spinal cord.MVMi continued to infect some developing bone, notably the developingmandible and long bones of the limb (Fig. 5E).

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FIG. 5. Distribution of MVM coat proteins in nonneural tissues of embryos inoculated at 12.5 dpc after a further 96 h of gestation. For format, see the legend to Fig. 2. (A) Approximate planes of sections in panels B, D, G, and I. (B, D, G, and I) Whole sections showing fields of view in indicated figure panels. (B to F) Embryonic endothelium (C) and developing bone (E and F), showing distribution of MVMi coat proteins 96 h after inoculation. (G to J) Embryonic lung (H) and nasal (J) epithelium, showing large amounts of MVMp coat proteins 96 h after inoculation. bep, bronchi epithelium; bp, bone primordium; end, endothelium; liv, liver; lng, lung; mbp, mandible primordium; nas, nasal epithelium; sc, spinal chord.

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FIG. 6. Distribution of MVMp coat proteins in fibroblastic tissues of embryos inoculated at 12.5 dpc after a further 96 h of gestation. For format, see the legend to Fig. 2. (A) Approximate planes of sections in panels C, E, and G. (C, E, and G) Whole sections showing fields of view in indicated figure panels. (B) Section through developing skin, showing large amounts of MVMp in fibroblastic dermal layer but not in ectoderm-derived epidermal layer. (D) Section through mesenchymal tissue showing single intensely labeled fibroblast. (F) Section through developing bone showing MVMp coat proteins in fibroblast-derived periost. (H) Composite of heart section showing scattered distribution of intensely labeled putative fibroblast cells. (I) Single intensely labeled fibroblast in the heart myocardium. der, dermis; epi, epidermis; fib, fibroblast; lim, limb bud; liv, liver; lng, lung; per, periost; rbp, rib primordium; sc, spinal chord.

DISCUSSION

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Discussion

Experimental data have shown that productive MVM infection requiresa dividing cell population and depends upon the differentiationstate of the potential host cell. Although the embryo consistslargely of dividing cells in a range of differentiation states,the susceptibility of the developing embryo to infection byeither virus strain has never been properly assessed. Also,our initial analysis of a transgenic MVM P4 promoter activityin the developing embryo suggested that the ability of the hostcell to activate the promoter may constitute one of the hostrange determinants of MVM. By infecting the embryo, we couldmore directly compare the pattern of transgenic MVM P4 promoteractivation with the pattern of MVM infection.

MVMi is more pathogenic than MVMp in the developing embryo. Early studies on the infection of neonates showed that MVMiadministered oronasally mounted a lethal infection and was founddisseminated throughout the animal, whereas similarly administeredMVMp was innocuous. It was not clear whether this was due toan inability of MVMp to disseminate from the inoculation site(21) or to an inability to mount an infection in the host animal(4). The in utero infections described here show that both arecapable of mounting an aggressive infection, disseminating totissues throughout the embryo, and that both can kill the embryo.The kinetics of early infection are similar; between 24 and48 h, both virus strains infected bone primordia at the samerate and to the same extent. At later times, MVMp spread rapidlyto fibroblastic cells throughout the embryo, resulting in widespreadinfection and a high viral burden. In contrast, infection withMVMi remained restricted largely to the developing bone andlater endothelium, with scattered infected cells in the CNSand a much lower viral burden. Despite the more restricted viralinfection, MVMi was significantly more pathogenic, resultingin more and faster embryonic death than with MVMp. The developingembryo is quite resistant to tissue damage. Loss of functionor structure often results in continued development and deathat birth. The most critical exception is the embryonic cardiovascularsystem, where loss of function results in rapid embryonic deathand resorption. It is most likely that the increased pathogenicityof MVMi compared to MVMp, despite the lower viral burden, isdue to its observed infection of endothelium, as was proposedfor MVMi-infected neonates (4). However, unlike infection inthe neonates, where infection of the renal capillaries was judgedthe most likely cause of death, we did not observe any histologicaldamage to infected endothelium anywhere in the embryo, and socannot yet confirm the immediate cause of death.

Patterns of infection. Three distinct patterns of signal above background were observedin different embryonic tissues. (We exclude the strong signalat the injection site in the liver, which is certainly due toincoming virus.) In many tissues (bone, mesenchyme, endothelium,some epithelial tissues, and the developing placenta) strongimmunoreactivity was observed over localized regions (foci)covering hundreds of cells, with extensive involvement of cellswithin the region (see, e.g., Fig. 5H). This pattern suggestsa rapid and local production and spread of virus. The secondpattern was occasional strongly positive cells in some tissuetypes (see, e.g., Fig. 4F). This pattern was typical of bothMVMp and MVMi in the developing CNS and the dorsal root gangliaand perhaps of MVMp in the developing heart. Although not quantitative,the staining intensity in individual infected cells was similarto the staining intensity within the cells of infection foci,suggesting that the per-cell viral burden was similar. Sucha pattern might be expected if the viral life cycle is partiallyor temporarily blocked in these cells, resulting in poor localcell-cell transmission of the virus but still resulting in theeventual accumulation of similar quantities of viral coat proteinsin infected cells. Finally, an embryo-wide weak signal, whichvaried somewhat in strength in different tissues, was observedin the embryos infected at 9.5 dpc (see, e.g., Fig. 2I); thismost probably represents an accumulation in the embryo of virusshed by the observed strong placental infection. The possibilitythat some cell types hosted productive infection but were notobserved must also be considered. Since MVM infection rapidlykills some cultured cell types, perhaps some cells died beforewe observed their infection. We cannot exclude this possibility,but we can place a boundary on its frequency. Recent experimentsin which we have injected other MVM variant viruses indicatethat we are able to identify MVM-mediated cell killing in infectionfoci involving fewer than 50 cells (data not shown).

Expansion of the host range of MVMi and MVMp into overlapping sets of cell types. Some cell type tropisms observed in neonate or tissue culturestudies were not observed in the embryos. Neither infectionin the stomach (21) nor hematopoietic infections (4) (whetherin the blood or liver) were seen in utero, although rare positivecells tentatively identified as lymphocytes were observed inMVMi-infected embryos. Some of this difference is perhaps dueto the stages of embryonic development examined. The matureT-cell lineages known to be infected by MVMi appear late indevelopment. The T-cell progenitors do not arrive in the thymusuntil between 12 and 14 dpc (12), and the Notch1-dependent T-celllineage differentiation events are postnatal (30), whereas thelatest embryonic stage we examined was 16.5 dpc.

The immunohistochemical data indicate that a number of tissuetypes in the developing embryo that had not been identifiedpreviously as MVM host cell types were capable of supportingproductive MVM infection. Certainly the MVMi/MVMp reciprocaltropism observed in tissue culture (14, 15, 32, 38) and in neonatestudies (4, 21) is still discernible in the embryo. MVMp showedextensive infection of fibroblasts, whereas MVMi infection wasprominent in endothelium of blood vessels; however, both virusstrains exhibited productive infection in bone primordia, notochord,CNS, and dorsal root ganglia (summarized in Fig. 7). This expansionof the host range and its overlap are not surprising. The embryopresents a richer supply of different populations of dividingcells in various differentiation states than either the neonateor the adult animal. With respect to the overlap, it has alreadybeen shown that cultured NB324K cells (simian virus 40-transformedhuman kidney cells) and TM4 cells (derived from mouse Sertolicells) support lytic infection by both viruses (7, 16, 28).

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FIG. 7. Relative distribution of MVMp and MVMi infection following inoculation of the mouse embryo in utero. An image of an approximately 15.5-dpc mouse embryo in which the vasculature and peripheral nervous system has been highlighted is shown. The site of inoculation into the embryonic liver is indicated by a large filled circle. The fore- and hindlimbs of the right side of the embryo are outlined. Satellite diagrams of sections through the embryo are marked with symbols representing loci of MVMi infection (filled triangles) and MVMp infection (filled circles). The approximate placements of the diagrams within the embryo are indicated by the transecting planes. a, aorta; b, bone primordium; btu, base of tongue; drg, dorsal root ganglion; e, eye; eso, esophagus; gm, grey matter; h, heart; k, kidney; liv, liver; lng, lung; nas, nasal epithelium; nc, notochord; ns, nasal septum; st, stomach; ver, vertebrae.

The data also indicate a diverging host range in the developingbone. At 24 and 48 h after development, similar MVMi and MVMpinfection was evident in bone primordia. After 96 h of furthergestation, only MVMi infected bone; however MVMp was observedin the periost. The time interval when embryonic MVM infectionwas characterized is a very active one with respect to bonedevelopment. The axial and limb and jaw bone primordia resultfrom mesenchymal condensation followed by differentiation intochondrocytes and then into hyperproliferative chondrocytes.This process begins at approximately 11.5 dpc, and the laterprocess of differentiation into hyperproliferative chondrocytescontinues throughout late embryogenesis. The chondrocytes laydown a cartilaginous model of the later bone. At approximately14.0 dpc other mesenchymal cells of the periost differentiateinto osteoblasts and invade these bone primordia. In contrast,the flat skull bones (calvaria) are produced by intramembranousossification, a process which does not involve chondrocytesor a cartilaginous template (10, 42). Since we observe MVM infectionin the bone primordia prior to the appearance of active osteoblastsand do not observe infection in the developing calvaria, theprimary cell type infected by MVM in the developing bone isundoubtedly chondrocytes. The later infection of cells in theperiost raises the possibility that early osteoblasts may alsobe susceptible to MVMp, but not MVMi, infection.

The MVMp infection of fibroblasts is interesting from a developmentalviewpoint. The early embryonic mesenchyme that forms followinggastrulation later gives rise to chondrocytes, osteoblasts,endothelium, adipocytes, smooth muscle, and fibroblasts. Thedifferentiation mechanisms which give rise to all of these havebeen successfully studied at the molecular level, except forfibroblasts, which remain an ambiguous collection of later mesenchymalcells. There are very few fibroblast-specific markers (35).The MVMp infection of late-development fibroblasts, but notof the abundant early-development mesenchyme, confirms the existenceof a so-far very poorly characterized developmental pathway.

The overall distribution of embryonic infection raises againthe question of the determinants of susceptibility to the virus.Host cell components are important at all steps in the virallife cycle, but very few have been identified. The observationthat strain-specific variation in host cell type maps to theviral capsid proteins certainly indicates that some unknownintracellular factors interact differentially with the viralcoat proteins. However, the role of other viral components indetermining the host cell type range cannot be excluded. Thereare many other sequence variations between MVMi and MVMp, includingthree nucleotide changes in the P4 promoter and 15 amino acidchanges in the NS proteins, which may not play a role in specifyingthe previously observed host range differences between the virusstrains but which may be involved in specifying the more completehost range determined here. On the host side of the interaction,there are no obvious cell-type-specific variations in molecularcomponents that could account for the differential responseto the virus strains.

Infection versus transgenic P4 promoter activity. We have shown that a transgenic MVM P4 promoter exhibits a dynamictissue- and stage-specific pattern of activation (9) (Fig. 1),and we have hypothesized that the ability of the host cell toactivate the P4 promoter may constitute a host cell type determinantof MVM. An initial comparison of the transgenic promoter activityin the embryo or adult and the host cell type range in neonatemice was consistent with the suggestion but was weakened bythe disparate developmental stages involved. A closer comparisonof the patterns of infection and transgenic P4 activity is madepossible by this in utero infection study. Indeed, the comparisonbetween P4 activity (9) and VP expression may be incomplete,because P4 activity by itself does not necessarily assure capsidformation. We are currently studying early events (NS1 and NS2production) in embryonic infections.

Numerous cell types that were shown to activate the transgenicpromoter were also susceptible to either MVMi or MVMp infection(Table 2), including the previously uncharacterized host cellsof the bone primordia, the notochord, and the dorsal root ganglia.Embryos at 9.5 dpc, which did not activate the P4 promoter,also failed to develop clear infections, although they containcell types, such as neuroepithelium, endothelium, and mesenchyme,which would later both exhibit activation of the P4 promoterand support MVM infection. This finding is consistent with theobservation that undifferentiated embryonic carcinoma cellsin culture were resistant to infection, whereas differentiatedderivatives were sensitive (27).

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TABLE 2. Tissue distribution of MVM following in utero infection at 12.5 dpc and subsequent gestation compared to transgenic P4 promoter activity

Since there are other host range-determining interactions betweenthe virus and potential host cell, it is to be expected thatsome cell types will express the promoter but remain resistantto infection. Certainly in the embryo, the developing CNS activatedthe transgenic P4 promoter relatively strongly (Fig. 1). Nevertheless,infection in the embryonic CNS was restricted to scattered cells.Similarly, transgene activation was observed in the cardiomyocytes,but the MVM infection that we observed in the heart is likelyfibroblastic or, at most, some scattered cardiomyocytes. Weobserved one tissue, the embryonic bronchus, in which the transgenicP4 promoter was not active yet was susceptible to infection.There is a possibility that this discrepancy is due to the differencein time of analysis, since the bronchi of the adult lung wereshown to express the transgene whereas the embryonic bronchiwere established to be negative at 13.5 dpc (9). Although thetransgene expression studies were done with BALB/c mice andthe in utero infections were done with CD-1 mice, we do notthink that mouse strain differences are responsible for theobserved discrepancy. The transgene-bearing BALB/c mice werecrossed with CD-1 mice, and staining in the first- and second-generationembryos was the same as that in the BALB/c mice (data not shown).The transgene was irreversibly and completely shut off in subsequentCD-1 generations. We were unable to regain expression by backcrossingto BALB/c mice, suggesting that the observed attenuation wasdue to strain-specific transgene inactivation, a familiar phenomenon,rather than to strain-specific variations in promoter activity(C. Davis and J. Tal, unpublished data).

The observed correlation between infected cell type and expressionof the transgene in the embryo strengthens the hypothesis thatactivation of the P4 promoter by the host cell is a host rangedeterminant. Further experiments to address the hypothesis morerigorously are in progress.

ACKNOWLEDGMENTS

This work was supported by grants from the Israel-USA BinationalScience Foundation (to J.T.) and from the Israel Science Foundation(to C.D. and J.T.).

We thank Peter Tatersall for the original MVMi and MVMp stocksand Michal Mincberg for virus preparations.

FOOTNOTES

* Corresponding author. Mailing address: Department of Developmental Molecular Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel. Phone: 972 8 647 9956. Fax: 972 8 627 6215. E-mail: clay{at}bgumail.bgu.ac.il.

REFERENCES

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