Journal of Medical Virology 36162-166 (1992)

Herpes Simplex Virus Infection of Cultured Human Term Trophoblast N. N0rskov-Lauritsen, G. Aboagye-Mathisen, C.B. Juhl, P.M. Petersen, V. Zachar, and P. Ebbesen Department of Virus and Cancer, Danish Cancer Society, &hus C, Denmark Mononuclear trophoblast cells were isolated from term placentas of uncomplicated pregnancies, purified to homogeneity by negative immunomagnetic separation using monoclonal antibodies to the major histocompatibility complex, and challenged with herpes simplex virus (HSV). The cultures were highly susceptible to virusinduced cytopathic effect as evidenced by cytopathic alteration and inhibition of human chorionic gonadotropin (hCG) secretion. Both HSV I and II underwent a full replicative cycle in the trophoblast, although the production of progeny virus was 10-100 times less than that obtained with placental fibroblasts or choriocarcinoma cells. The permissiveness was independent of in vitro syncytial differentiation of the trophoblast. The results suggest that the trophoblast layer may be involved in intrauterine HSV infection.

tion on pregnancy requires more data on HSV as a cause of spontaneous abortion. A possible pathogenesis in intrauterine HSV infection may consist of viral replication in the placenta with subsequent spreading to fetal tissues. For such a mechanism to be operating, viral interaction with the physical and biochemical barrier between mother and fetus-the placental trophoblast layer-would be decisive. The present investigation was undertaken to examine the susceptibility of primary cultures of human term trophoblast to HSV I and 11.

METHODS Cells Mononuclear cells were isolated from term placentas as described previously [Toth et al., 1990al. Following Percoll density gradient centrifugation, the cell suspensions consisted of 70-95% trophoblast cells and 5 3 0 % fibroblasts and macrophages combined. This was revealed by immunofluorescence staining using mouse monoclonal antibodies (mAb) specific for cytokeratin KEY WORDS: fetal infection, transplacental, (trophoblast [Jie et al., 19901), vimentin (fibroblasts), virus-transmission, vertical, imand CD68 (macrophages). Further purification of the mu nosorting cell preparation was carried out according to Douglas and King [1989]. In brief, cell suspensions were incubated with mAb to major histocompatibility complex (MHC)class I and 11. Cells were washed free of unbound INTRODUCTION antibody, magnetic microspheres coated with antiHerpes simplex virus (HSV)infection of the newborn mouse IgG antibodies were added to achieve a ratio of 5 infant is associated with significant morbidity and mor- beads per cell, and after a second incubation, cells tality. Some controversy exists with regard to the fre- bound to the spheres were removed from the solution by quency of the condition, with estimates ranging from 1 applying a n external magnetic field. The procedure rein 2,000 to 1 in 5,000 deliveries in the U.S.A. [Whitley, sulted in a reproducible reduction of the admixture of 19901 to less than 1in 10,000 births in the U.K. [Long- vimentin-, CD68- or MHC-positive cells to less than 1%. son, 19901. It is commonly accepted that the fetus usu- After purification, 200,000 trophoblast cells/cm2 were ally is infected when passing a n infected birth canal seeded in 48-well Costar tissue culture clusters (Costar, during delivery, but it has become increasingly clear MA, USA) and grown in RPMI 1640 medium supplethat these viruses may also infect the fetus in utero mented with 10% fetal calf serum and antibiotics at [Florman et al., 1973; Hutto et al, 19871. In addition to 37°C in a humidified atmosphere containing 5% CO,. the devastating effects of herpes infection of the newborn, congenital malformations such as microphthalmia, microcephaly, or hydranencephaly are relaAccepted for publication September 3, 1991. tively common in cases of intrauterine infections Address reprint requests to P. Ebbesen, Department of Virus [Hutto e t al., 19871. In most series of patients with and Cancer, Danish Cancer Society, Gustav Wiedsvej 10, DKneonatal herpes, congenital herpes now constitutes 8000 h h u s C, Denmark. 5-10% of the cases [Brown e t al., 1987; Hutto e t al., V. Zachar’s present address is the Institute of Virology, Slovak 19871, but the magnitude of the impact of herpes infec- Academy of Sciences, Bratislava, Czechoslovakia. 8 1992 WILEY-LISS, INC.

HSV Infection of Trophoblast Placental fibroblasts were prepared by incubating unpurified placental cell cultures until overgrowth of fibroblasts had taken place (10-20 days), before trypsinization and subcultivation. The cells were used for experiments after 5 rounds of subcultivation, at which time all cells were vimentin-positive. Malignant transformed trophoblast, the choriocarcinoma cell line JAR (HTB 144), was obtained from the American Type Culture Collection (ATCC),Rockville, MD, USA. Fibroblasts and JAR cells were seeded at 40,000 cells per well and infected after 2 days, at which time the cultures were subconfluent.

Viruses and Infection Human herpes virus I (strain McIntyre) and I1 (strain MS) were obtained from the ATCC. The viruses were propagated and plaque-titered on the cervix cancer cell line MS-751. Virus was added to placental cultures at a multiplicity of infection of approximately 1 (calculated as plaque-forming units (PFU)[titered on MS-751 cells] divided by the number of trophoblast cells seeded) and allowed to adsorb onto the cells for 1hour before withdrawal, wash, and addition of fresh medium. At 12 hour-intervals after infection, separate wells were processed to measure the contents of infectious virus or herpes antigens. For titration of virus, cultures with medium were made hypotonic by addition of 1vol H,O and subjected to one cycle of freeze-thawing to release cell-bound virus. Medium with cell debris was harvested, cleared by centrifugation at 2,OOOg for 5 minutes, and the supernatant was then added to MS-751 cells in 10-fold dilutions in serum-free RPMI. Virus was allowed to adsorb onto the target cells for 1hour before withdrawal, wash, and addition of fresh medium. Cells were incubated without solid overlay, and 24 hours later the number of plaques was counted by microscopic examination. Antibodies Antibodies from Dakopatts, Copenhagen, Denmark, were used in the following dilutions: mAb against human cytokeratin (M 717), 1:50; mAb against CD68 (M 718), 1:50; mAb against porcine vimentin (M 725), 1:lO; mAb against MHC class I (M 736), 1:50 (1p1/106 cells for purification); mAb against MHC class I1 (M 704), 1:50 (0.5 pl/106 cells for purification); FITC-labelled rabbit polyclonal antibody (pAb) against mouse immunoglobulin (F 261), 1:20; peroxidase-conjugated rabbit anti-mouse pAb (P 161),1:40; rabbit pAb against HSV I (B 114) and HSV-I1 (B-1151, 1:1,000 (for coating of wells for ELISA); peroxidase-conjugated rabbit pAb against HSV-I (P 175) and HSV-I1 (P 176), 1:400 (ELISA anti HSV-conjugate solutions). Immunofluorescence and Immunocytochemistry To examine the composition of cell suspensions, 5-10,000 cells were spotted on wells in multitest slides, air-dried, fixed with acetone, incubated for 1hour with primary antibody and 30 minutes with FITC-labelled rabbit antimouse pAb before counterstaining with

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Evans blue. Trophoblast cells in culture were fixed with 80% acetone and stained using peroxidase-conjugated rabbit anti-mouse as secondary antibody and 0.05% diaminobenzidine in PBS containing 0.035% H,Oz as substrate.

Other Methods Herpes proteins were assayed by a double-antibody sandwich enzyme-immunoassay (antigen-ELISA) as described [Vestergaard, 19861 after solubilization of proteins by addition of Nonidet p40 to a final concentration of 1%.Human chorionic gonadotropin (hCG) was measured in the medium at 24 hour-intervals by a twosite antigen-ELISA test against standards obtained from the National Institute for Biological Standards and Control, London, U.K. Medium from infected cells was inactivated by addition of Triton X-100 to a final concentration of 0.1% and heating at 65°C for 30 minutes, which did not interfere with the subsequent hormone analysis. The cellular content of hCG was measured after disruption of cells by one cycle of freezethawing in PBS containing 0.1% Triton X-100.

RESULTS The mononuclear trophoblast cells underwent morphological and functional differentiation upon cultivation. The cells adhered to the wells in a clustering manner, and a gradual development into syncytia-like structures occurred. After 3 days in culture, cell boundaries between many of the original mononuclear cells could no longer be discerned by light microscopic examination of fixed cultures (Fig. 1A). Concomitant with the morphological development, the cells began to secrete hCG into the culture medium (Fig. 2). The total cellular content of hCG did not exceed 20 mU/106 cells at any time during the period of study, and the appearance of considerable quantities of hCG in the medium during cultivation could therefore not be attributed to a release from degenerating cells. Similar to the findings of others [Kliman et al., 1987; Bax et al., 19891, hormone production was maximal around the fourth day in culture, and then declined. Addition of HSV I or HSV I1 to the trophoblast cultures induced a pronounced cytopathic effect (CPE) with condensation of cytoplasm, cell rounding, and finally detachment. At unit multiplicity of infection, virus-induced cytopathicity was discernible within 15 hours, and a marked effect was prominent on all cells 48 hours after addition of virus (Fig. lB,C). No significant difference in induction of CPE was observed between the two subtypes of HSV. We examined whether the susceptibility of trophoblast to HSV was restricted to a particular state of differentiation by adding the virus to cells cultured for periods varying from 4 hours to 4 days. A CPE was induced in all cultures irrespective of in vitro maturation (results not shown). HSV infection of the trophoblast inhibited the production of hCG (Fig. 2). Again, the impact on hormone synthesis was evident both when infection was initiated after 1or 4 days of previous cultivation.

Nprrskov-Lauritsen et al.

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Fig. 1. Susceptibility of cultured trophoblast to cytopathic effect induced by HSV. Trophoblast cells were incubated for 24 hours before addition of virus at a n approximate multiplicity of infection of 1. Forty-eight hours after infection cultures were fixed and processed for immunocytochemical staining directed against cytokeratin. A Uninfected control; B: HSV I (strain McIntyre); C:HSV I1 (strain MS). Original magnification x 400.

c

2.0

n O 1.0

HSV-1

0.0

Days in culture Fig. 2. Hormone production by normal and HSV I1 infected trophoblast. Culture medium was changed at 24 hour-intervals and assayed for hCG content. t . , uninfected trophoblast; 0-0, trophoblast cultures infected with HSV 11. Numbers indicate days of cultivation before infection was carried out. Qualitatively similar results were obtained with HSV I.

0

2L

0

2L

46

Time after addition of virus(h1 Fig. 3. Kinetics of HSV replication. HSV I or HSV I1 (approximately

Figure 3 shows the kinetics of HSV replication in trophoblast, fibroblasts, and the choriocarcinoma cell line JAR measured by the antigen capture ELISA test (Fig. 3A,B) and plaque-titration (Fig. 3C,D) of medium plus cells combined. The synthesis of virus-specific proteins by the trophoblast resulted in a full replicative cycle with production of infectious progeny virus for both HSV I and 11. Compared with the growth in placental fibroblasts or malignant transformed trophoblast, HSV replication in normal trophoblast was slower and did not reach the same levels; no increase in PFUs over residual inoculum activity was observed 12 hours after addition of virus, and the total amount of infectious virus recovered was 10-100 times less at all assay time points.

DISCUSSION We isolated mononuclear cells from villous biopsies of term placentas and obtained essentially pure trophoblast cell preparations by negative immunosorting of

1 PFUicell) was added to 24-hour old trophoblast cultures (*), placental fibroblasts W, or malignant transformed trophoblast JAR (A).At indicated intervals separate wells were processed for antigen capture ELISA (A, B) or plaque-titration C, D) as described in the Methods section. ELISA values represent the absorption a t 492 nm of 25-fold dilutions of cells and medium combined.

MHC-expressing cells. Immunohistochemical examinations have shown that the only mononuclear cells devoid of MHC are confined to the villous cytotrophoblast [Sasagawa et al., 19871. Upon cultivation, the initial mononuclear cells developed into syncytial structures and began to synthesize hCG. Although we did not perform ultrastructural examinations of our cultures, transmission electron microscopic studies of cultured trophoblast have previously confirmed that the apparent formation of syncytia corresponds to ultrastructural changes representing true cellular fusion [Lobo et al., 1987; Bax et al., 19891.

HSV Infection of Trophoblast

The trophoblast cultures were susceptible to cytopathic infection with HSV I and I1 both before and after in vitro cellular differentiation had taken place. The replication was slower and less intense in normal trophoblast than in placental fibroblasts and malignant transformed trophoblast, but this more likely reflects differences in cellular growth rather than different levels of infection permissiveness. Normal term trophoblast does not proliferate in vitro [Bierings et al., 1987; Bax et al., 19891, and although the cellular density of the different cell types was roughly equivalent at the time of infection, virus regularly replicates to higher titers in actively growing cells. Our results strengthen the hypothesis that HSV can infect the fetus via the transplacental route. Clinical data on placental involvement in intrauterine HSV infections are scarce. Case reports have been presented both in favor of a hematogenous transplacental infection (grossly unremarkable placentas with areas of villous necrosis) and of ascending infections (chorioamnionitis without signs of villitis), as recently reviewed by Benirsche and Kaufmann [1990], and both forms may occur. Evaluation of their relative importance awaits further studies from the clinic. HSV represents several types of risk to the fetus. HSV has been detected in abortion material [Benirsche and Kaufmann, 1990; Brown et al., 19871and primary symptomatic genital HSV infection prior to week 20 of gestation has been associated with an increased rate of spontaneous abortion [Nahmias et al., 19711. Another complication of maternal infection is preterm labor and fetal growth retardation [Nahmias et al., 19711. In a recent study by Brown et al., [19871, primary infection in the third trimester appeared particularly morbid with premature labor within 2 weeks after onset of infection in 4 out of the 5 women. Two babies developed neonatal herpes. The etiology of the association between primary HSV infection and spontaneous abortion, fetal growth retardation, or preterm labor remains undefined [Baldwin and Whitley, 19891, but in light of the present findings, a possible explanation is that the virus destroys the trophoblast layer. Finally, the fetus can be infected in utero by both HSV I and 11. Herpetic lesions or malformations present a t birth represent clear evidence of antepartum acquisition of the virus, but in other cases the precise time of infection can be difficult to establish. Witzleben and Driscoll [19651 found intranuclear inclusion bodies in necrotic syncytiotrophoblast in the placenta of a newborn who did not have systemic disease until the sixth day of life, and Gagnon [1968] isolated HSV from the cord blood and placenta of a child who developed symptoms a t the fifth day of life. The relevance of such slowly progressing intrauterine infections for our understanding of virus transmission in neonatal herpes remains to be established, and the diagnosis of intrauterine acquisition of HSV is presently restricted to culture-confirmed cases with evidence of disease within 48 hours of life [Hutto et al., 1987;Whitley, 19901. Using these criteria, the clinical manifestations of intrauterine infections have already been characterized [Hutto et al., 1987;

165 Baldwin and Whitley, 19891. The growing number of cases has, however, not resulted in identification of factors responsible for or associated with this rare transmission. In the study of 13 babies by Hutto et al. [1987], 4 mothers had experienced an apparent primary episode of genital HSV, 2 in the first and 2 in the third trimester, 1 had had a recurrent episode in the first trimester, and 1 had a past history of recurrent orolabial vesicular lesions without manifestations in the studied pregnancy. The rest of the women denied a history of HSV infection. Primary HSV infection is associated with viremia [Longson, 19901and is thus compatible with transplacental infection. Viremia during recurrent episodes of herpes remains to be documented in. the immunocompetent host, but immunological mechanisms associated with the state of pregnancy may influence activation and replication of latent HSV as suggested by the widely disseminated and lifethreatening HSV infections which can occur during pregnancy [Baldwin and Whitley, 19891. In conclusion, HSV represents a rare but serious and poorly understood danger to the fetus. The present results have shown the permissiveness of cultured trophoblast to these viruses, and the results support the assumption that a hematogenous placental and transplacental infection may occur. Furthermore they present fruitful ground for further experimental studies of certain aspects of placental virology. Work in progress centers on the interaction of immune cells with infected trophoblast and the modulatory action of the trophoblast interferons recently described from this laboratory [Toth et al., 1990b; Aboagye-Mathiesen et al., 19901.

ACKNOWLEDGMENTS Frode EngbEk, Aarhus Municipal Hospital, is thanked for performing the hCG measurements. REFERENCES Aboagye-Mathiesen G, Toth FD, Juhl C, N6rskov-Lauritsen N, Petersen PM, Ebbesen P (1990):Purification and initial characterization of human placental trophoblast interferon induced hy polyriboinosinic polyribocytidylic acid. Journal of General Virology 71:3061-3066. Baldwin S, Whitley R J (1989):Teratogen update: intrauterine herpes simplex virus infections. Teratology 39:1-10. Bax CMR, Ryder TA, Mobberley MA, Tyms AS, Taylor DL, Bloxam DL (1989): Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short term culture. Placenta 10:179-194. Benirsche K, Kaufmann P (1990): Infectious Diseases. In: “Pathology of the Human Placenta, 2nd ed.” New York: Springer, Chapter 24, pp 542-632. Bierings MB, Adriaansen HJ, van Dijk JP (1987):The appearance of transferrin receptors on cultured human cytotrophoblast and in vitro-formed syncytiotrophoblast. Placenta 9:387-396. Brown ZA, Vontver LA, Benedetti J , Critchlow CW, Sells CJ, Berry S, Corey L (1987): Effects on infants on a first episode of genital herpes during pregnancy. New England Journal of Medicine 317:124&1251. Douglas GC, King BF (1989):Isolation of pure villous cytotrophoblast from human term placenta using immunomagnetic microspheres. Journal of Immunological Methods 119:259-268. Florman AL, Gershon AA, Blackett PR, Nahmias AJ (1973): Intrauterine infection with herpes simplex virus. Resultant congenital malformations. Journal of the American Medical Association 225129-132.

166 Gagnon RA (1968):Transplacental inoculation of fatal herpes simplex in the newborn. Obstetrics and Gynecology 31:682-684. Hutto C, Arvin A, Jacobs R, Steele R, Stagno S, Lyrene R, Willett L, Powell D, Andersen R, Werthammer J , Ratcliff G, Nahmias A, Christy C, Whitley R (1987):Intrauterine herpes simplex virus infections. Journal of Pediatrics 110:97-101. Jie 2, Fey ST,Hager H, Hbllsberg P, Ebbesen P, Larsen PM (1990): Markers for human placental trophoblasts in two-dimensional gel electrophoresis. In Vitro 26:937-943. Kliman HJ, Feinberg R, Straus J A (1987): Differentiation of human cytotrophoblast into syncytiotrophoblast in culture. Trophoblast Research 2:407421. Lob0 JO, Bellino FL, Siege1A (1987):Characterization of isolated cells in primary culture from human term placenta by electron microscopy and immunohistochemistry. Trophoblast Research 2:447460. Longson M (1990):Herpes simplex. In Zuckerman AJ, Banatvala J E , Pattison J R (eds): “Principles and Practice of Clinical Virology.” Chichester, John Wiley, pp 3-42. Nahrnias AL, Josey WE, Naib ZL, Freeman MG, Fernandez RJ, Wheeler J H (1971):Perinatal risk associated with maternal genital herpes simplex virus infection. American Journal of Obstetrics and Gynecology 110:825-837.

Norskov-Lauritsen et al. Sasagawa M, Yamazaki T, Endo M, Kanazawa K, Takeuchi S (1987): Immunohistochemical localization of HLA antigens and placental proteins (crhCG, PhCG, CTP, hPL, and SP,) in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast. Placenta 8:515528. Toth FD, Juhl CB, NGrskov-Lauritsen N, Petersen PM, Ebbesen P (1990a):Interferon production of cultured human trophoblast induced with double stranded polyribonucleotide. Journal of Reproductive Immunology 17:217-227. Toth FD, Nbrskov-Lauritsen N, Juhl CB, Aboagye-Mathiesen G, Ebbesen P (1990b):Interferon production by cultured human trophoblasts and choriocarcinoma cell lines induced by Sendai virus. Journal of General Virology 71:3067-3069. Vestergaard BF (1986):Herpes simplex virus. In Bergmeyer HU (ed): “Methods of Enzymatic Analysis Vol 10: Antigens and Antibodies.” Weinheim, FRG: VCA Publishers, pp 226242. Whitley RJ (1990):Herpes simplex virus infections. In Remington JS, Klein J O (eds):“Infectious Diseases of the Newborn Infant.” Philadelphia: WB Saunders, pp 282-318. Witzleben CL, Driscoll SG (1965): Possible transplacental transmission of herpes simplex infection. Pediatrics 36:192-198.

Herpes simplex virus infection of cultured human term trophoblast.

Mononuclear trophoblast cells were isolated from term placentas of uncomplicated pregnancies, purified to homogeneity by negative immunomagnetic separ...
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