Horizontal Transmission of Murine Retroviruses

Transcription

1 JOURNAL OF VROLOGY, Apr. 1987, p X/87/ $02.00/0 Copyright C) 1987, American Society for Microbiology Vol. 61, No. 4 Horizontal Transmission of Murine Retroviruses JOHN L. PORTS,.* FRANK J. McATEE,1 AND STANLEY F. HAYES2 Laboratory of Persistent Viral Diseases1 and Laboratory of Pathobiology,2 National nstitute of Allergy and nfectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana Received 14 October 1986/Accepted 11 December 1986 Both a feral mouse ecotropic virus (WM-E) and Friend ecotropic virus (F-MuLV) were transmitted horizontally among adult mice. nfection resulted in the production of antiviral antibody in the recipients, with no evidence of viremia or clinical disease. However, persistent low-level virus replication was detectable in the spleens of these mice as long as 8 months after initial infection. External secretions, including saliva, semen, and uterine secretions from viremic mice contained high concentrations of infectious virus. Nevertheless, transmission occurred only from viremic males to either males or females. Male-to-male transmission appeared to occur by parenteral inoculation of infectious saliva during fighting behavior. Evidence is presented that infection of females was by the venereal route. Of four mouse strains examined, NFS/N, RW, and C57L females were all susceptible to venereal infection, whereas AKR mice were not. Since AKR mice are susceptible to infection by WM-E administered parenterally, this resistance appeared to be mediated by local viral interference due to the high-level expression of endogenous Akv gp7o within the female reproductive tract. Although both WM-E and F-MuLV were transmitted from viremic males to females, infection by WM-E was significantly more efficient than that by F-MuLV. This difference correlated with a distinct difference in cellular tropism of WM-E and F-MuLV within the epididymis of viremic males. F-MuLV gp7o was expressed only within stromal elements, whereas WM-E gp7o was seen largely within the epithelial lining cells and luminal contents of the duct. No evidence of virus expression within germ cells was observed. The possible influence of virus expression by epithelial cells of the female reproductive tract on infection of embryos is discussed. The transmission of murine retroviruses occurs primarily via germ line viral DNA integration or through congenital infection by infectious virus either in the birth canal or in mammary secretions. Horizontal transmission is generally not considered to be an important mode of infection, although definitive studies have not been reported. Gardner et al. (10) first presented evidence suggesting that murine retroviruses isolated from wild mice were transmissible horizontally, possibly by a venereal route. This mode of infection was reported to be restricted to transmission from feral male mice to one strain of laboratory mice, C57L. Neither NH Swiss nor feral female mice appeared to be susceptible to horizontal infection (10). The restriction thus appeared to be at the level of the female recipient, but the mechanism of this resistance has not been studied. Further examination of this phenomenon is of interest for two reasons. First, the possible use of retroviral vectors for introduction of exogenous genes into the germ line (31) requires an understanding of the factors (both host and viral) which determine cellular tropism of these viruses within the male and female reproductive tracts and the capacity of these viruses to infect germ cells. Second, the existence of mechanisms which restrict horizontal transmission should be of general interest to investigators studying other transmissible retroviruses. n the current study, we used type-specific monoclonal antibodies which react with gp7o only of the inoculated exogenous viruses, being nonreactive with endogenous viruses often expressed spontaneously by a variety of tissues including both male and female reproductive tract tissues (19). Evidence is presented that horizontal transmission of a neurotropic retrovirus from feral mice (WM-E) occurs with high frequency between several inbred strains of mice, * Corresponding author. although AKR mice were found to be completely resistant. Viremic male mice were the primary source of infection, and transmission by viremic females was infrequent. Mice infected in this way did not develop a viremia but instead exhibited a sustained antiviral antibody response, persistent low-level viral replication being detectable in the spleen. The propensity of this virus for horizontal transmission was found to correlate with the unique tropism of WM-E for epithelial cells lining the epididymis. MATERALS AND METHODS Mice. RW is an inbred mouse strain developed at the Rocky Mountain Laboratories (5). NFS/N mice were obtained from the National nstitutes of Health. C57L/J and AKR/J mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Mice used in all transmission experiments were aged 12 to 15 weeks. n one experiment, virgin female mice were subjected to a superovulation protocol (27) to synchronize ovulation and thereby enhance the frequency of coitus during a short exposure to male mice. Each female was given an intraperitoneal injection of 5 U of pregnant mare gonadotropin (Sigma Chemical Co., St. Louis, Mo.) at 4:00 p.m. and was then given an intraperitoneal injection of 5 U of human chorionic gonadotropin 48 h later. At the time of the second injection, males were placed with females for 18 h. This protocol was effective in inducing ovulation in 100% of the females. When the mice were paired two females per male, the impregnation frequency was approximately 50%. Saliva was collected with type 4 Calgiswabs (Spectrum Laboratories nc., Houston, Tex.). The swabs were dissolved by incubation in media at 4 C for 1 h, and the virus assayed by infectivity (see below). For larger volumes of saliva, mice were treated with pilocarpine hydrochloride (Sigma), 40,ug subcutaneously. Seminal fluid was obtained from males by sectioning the ductus deferans and allowing 1037

2 1038 PORTS ET AL. semen to be expressed by peristalsis into 0.05 ml of RPM 1640 (GBCO Laboratories, Grand sland, N.Y.)-10% fetal calf serum. Uteri were lavaged with 0.5 ml of media after section at the cervic and oviducts. Peripheral anosmia was induced in male mice by three intranasal instillations of 7.6% (wt/vol) ZnSO4 spaced 5 days apart (26). Virus and inoculations. The wild-mouse ecotropic virus (WM-E) was kindly supplied by Paul Jolicoeur, Montreal, Canada, and is a molecular clone designated PBR-NE-8 (17). Virus stocks were prepared as previously described (24) and contained 1 x 105 to 3 x 105 focus-forming units per ml (FFU/ml). Neonatal RW mice were inoculated with this virus intraperitoneally (ca. 5 x 103 FFU) at <24 h of age. Mice that reached the age of 12 weeks without evidence of neurologic deficit were used as sources of infectious virus in transmission experiments. F-MuLV strain B3 (28) was kindly provided by Marc Sitbon, Rocky Mountain Laboratories. This virus is highly infectious in vivo but produces less morbidity within the first 3 months after inoculation than do other strains of F-MuLV. Viruses were assayed by a focal immunofluorescence assay with anti-gp7o monoclonal antibody 667 for WM-E (21) and 48 for F-MuLV (7) followed by fluoresceinated goat anti-mouse immunoglobulin (Cappel Laboratories, Cochranville, Pa.), as described previously (29). ndicator cells were a tail skin cell line from Mus dunni (18). n assays of serum, saliva, and seminal fluid, Polybrene (8,ug/ml) was used to increase the sensitivity of the assay. Splenic infectious centers were assayed as described previously by using M. dunni cells without Polybrene (21). Assay of antiviral antibody. Antibody to WM-E was quantitated by an enzyme-linked immunosorbent assay. Polyvinyl plastic 96-well plates (Dynatech Laboratories, nc., Alexandria, Va.) were coated overnight at 4 C with WM-E, purified by sucrose density gradient (23), suspended in 10 mm Tris-150 mm NaCl-1 mm EDTA (protein concentration, 40,ug/ml). Antibody binding was detected by using alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulin (Sigma) with p-nitrophenyl phosphate as substrate. The reaction product was quantitated by using an automated enzyme-linked immunosorbent assay reader (Titertek Multiscan; Flow Laboratories, nc., McLean, Va.) at 405 nm. Titers are expressed as the serum dilution giving 50% maximal binding. mmunofluorescence. Mice were anesthetized with ether and exsanguinated via axillary incision. Organs were quickfrozen in liquid nitrogen, and 6-,um sections were made on a Histostat microtome (American Optical Corp., Buffalo, N.Y.). Sections were mounted on microscope slides, fixed in 4% neutral buffered Formalin, and incubated with anti-gp7o monoclonal antibodies. For detection of Friend ecotropic virus, antibody 500 (4) was used. This antibody has the gg3 isotype and was detected with heavy-chain-specific fluorescein isothiocyanate-conjugated anti-gg3 (Southern Biotechnology Associates, Birmingham, Ala.). WM-E gp7o was localized with antibody 667 (21) conjugated with biotin (12) followed by fluorescein isothiocyanate-avidin (Beckton Dickinson and Co., Mountain View, Calif.). Endogenous Akv ecotropic viral gp7o was detected with rat monoclonal antibody 83A25', kindly provided by Leonard Evans (Rocky Mountain Laboratories), followed by a fluorescein isothiocyanate-conjugated monoclonal anti-rat K light chain (Becton Dickinson). Sections were examined by using an Orthoplan fluorescence microscope (E. Leitz nc., Rockleigh, N.J.) with a K2 fluorescein filter. Electron microscopy. Tissues were fixed at 4 C for 48 h in w 80 >640- Cu o 640._E C. 320 a) t WM-E Viremic RW df /st mating x RWNFS 2nd mating RW NFS C57L AKR AKR *0@ * 00 *- ::::::0 0* *00000:00 x *---- *00-- FG. 1. Horizontal transmission of WM-E from males to females. Three viremic male RW mice were housed with three or four normal females for 10 days. Plasma was collected 3 weeks after removal of the males and assayed for anti-wm-e antibody by ELSA. AKR females were reexposed to the same male mice but remained seronegative. 2.5% glutaraldehyde-2% paraformaldehyde-0.1% phosphotungstic acid-0.1 M HEPES (N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid) buffer-1% NaCl-2 mm CaC M tri-nitrocresol (Eastman Kodak Co., Rochester, N.Y.) in 0.1 M sodium cacodylate buffer (ph 7.2). Fixative was exchanged for a 50:50 mix of minimal essential mediummedium 199 (GBCO) and NaCl (8 mg/ml) to which glutamine was added (final concentration, 400,ug/ml), and incubation was carried out at room temperature for 1 h. Tissues were treated with 0.1% tannic acid for 1 h and washed twice in 0.1 M phosphate buffer (ph 6.9) for 1 h each. After a tap water rinse, the specimens were prestained with 1% aqueous uranyl acetate (ph 3.9) and dehydrated by acetone exchange (4 C). After the samples were incubated for 4 h at room temperature in Spurr low-viscosity resin (30), the resin was replaced with fresh resin and samples were incubated for another 16 h. Polymerization was carried out with fresh resin at 55 C for 24 h. Sectioning and photography were carried out as described previously (11). RESULTS J. VROL. Horizontal transmission of WM-E from adult males to female mice. Three viremic male RW mice inoculated as newborns with WM-E were used as sources of infectious virus and were rotated at 10-day intervals through cages housing 3 to 4 sexually mature uninfected female mice of different strains. This period corresponds to two estrous cycles, which was an interval found empirically to ensure a high frequency of pregnancy (70 to 90%). The male mice exhibited a consistent level of viremia ranging from 5 x 102 to 1 x 103 FFU/ml of serum and had no detectable serum antibody. Female mice were bled 2 to 3 weeks after removal of the viremic males (around the time of delivery),and their sera were tested for infectious virus and for antiviral antibody. Although all female mice were consistently nonviremic, ca. 50% of female RW, NFS/N, and C57L/J mice housed with the viremic males developed antiviral

3 VOL. 61, 1987 HORZONTAL TRANSMSSON OF MURNE RETROVRUSES 1039 TABLE 1. Asymmetric transmission of WM-E Transmission routea: % Seroconversion' Female to female... 8 (n = 12) Female to malec... <10 (n = 10) Male to femaled (n = 29) Male to male (n = 12) aviremic adult RW mice were housed with two or three normal RW mice for 10 days and then separated. Plasma was collected 3 weeks after removal of viremic mice, and anti-wm-e antibody was quantitated by ELSA. Seroconversion is defined as a titer of >1/10. n, Number of mice used. The frequency of pregnancy was 90%. c d The frequency of pregnancy was 72%. antibody ranging in titer between 1/40 and 1/640 (Fig. 1). Controls consisted of uninfected RW males housed with normal females of each strain (10 to 12 females per group) under the same protocol. There was no evidence of serum antibody reactive with WM-E in any of these females (data not shown). These data suggested that horizontal transmission from viremic males to females had occurred and had resulted in seroconversion. The antibody titers in sera of the antibody-positive female mice remained at high levels for at least 8 months after removal of the viremic males. A group of C57L female mice was sacrificed at that time, and spleen cells were assayed for WM-E-specific infectious centers. Five of eight of these mice expressed low but detectable levels of virus replication (range, 4 to 40 infectious centers per 106 spleen cells). As mentioned previously, no infectious virus was found in the serum, but in addition we assayed washed peripheral blood leukocytes both by infectious center assay in vitro and by inoculation of neonatal mice in vivo. There was no evidence of infectivity in 5 x 106 peripheral blood leukocytes. This suggested that the cells expressing infectious virus in the spleen were not in the recirculating pool. Thus, horizontal transmission of WM-E from adult male to susceptible female mice was manifested by a persistent but low-level productive infection accompanied by the production of serum antibody, which presumably limited the spread of the virus. None of these females has shown evidence of clinical disease (paralysis or lymphoma) in up to 1 year of observation. Seroconversion was not observed among AKR females (Fig. 1), and infectious centers were not detectable in the spleens of these mice. This apparent resistance of AKR mice is addressed below. Sites of retrovirus replication in organs producing external secretions. Although the above data are consistent with a venereal route of horizontal transmission, two observations indicated that the problem was more complex. (i) There was no correlation between pregnancy rate and frequency of seroconversion. Data accumulated from studies of the three susceptible mouse strains indicated that seroconversion occurred in 21 of 44 (48%) pregnant and 8 of 14 (57%) nonpregnant females exposed to viremic males. (ii) Although transmission from viremic females to either normal males or females was very infrequent, male-to-male transmission occurred with high frequency (Table 1). These observations indicated that the transmission of this virus is clearly linked to social behavior but that more than one mode of transmission is likely. To further study the routes of horizontal transmission of WM-E, various external secretions were titrated for infectious virus. Saliva, seminal fluid, and uterine washings of viremic RW mice were found to contain high levels of Uterus(Endometrial gland) FG. 2. Cellular localization of WM-E gp70 within organs producing external secretions. RW mice had been inoculated as neonates and were persistently viremic. Frozen sections were obtained from sexually mature adults and stained with biotinylated monoclonal antibody 667 followed by fluorescein isothiocyanateavidin. Viral gp7o was detected in the epithelial (acinar) cells of the submaxillary salivary gland and in Leydig cells and stromal cells in the testes. The epididymis contained abundant gp70 within columnar epithelial cells and in the luminal contents. n the female reproductive tract, gp7o was observed in the squamous epithelium of the cervix and vagina as well as in the secretory epithelium of the endometrium. infectious virus (Table 2). When male and female RW mice were compared for the titers of virus in saliva, no apparent differences were observed (data not shown). Frozen sections of submaxillary salivary gland, testis, epididymis, and uterus from viremic adult RW mice inoculated as neonates were examined for expression of WM-E gp70. Since cells of the reproductive tracts of both males and females as well as those of the salivary gland are known to express endogenous gp7o (19), we used a monoclonal antibody specific for WM-E gp70 which was unreactive with gp70 of endogenous viruses (21). WM-E gp7o was localized within acinar epithelial cells of the submaxillary salivary gland (Fig. 2). Within the testis, the only cells expressing viral gp70 were cells in the inter- TABLE 2. Titration of WM-E in external secretions Fluid FFU/ml" (n = 6) Serum x 103 Salivab x 103 Semenb... 2 x 104 Uterine washb... 1 X 103 " Virus was assayed by a focal immunofluorescence assay with monoclonal antibody 667 which is specific for WM-E gp7o. n, Number of mice examined. b Saliva, semen, and uterine washes were assayed without removal of cells.

4 1040 PORTS ET AL. r71 vis.> sp.ermatozo '#' stermatozoonl V./ J. VROL. w \.. -, t virus '13 """',J, * stereocilis Sf..,. ". t....n.. LJ1On FG. 3. Transmission electron micrographs of epididymis from a viremic male RW mouse. (A) Clumps of virions associated with amorphous material in the lumen of the duct. (B) Virions associated with spermatozoon. stitium between seminiferous tubules, primarily Leydig cells (testosterone-secreting cells) (Fig. 2). No viral antigen was localized in germinal cells or supporting cells (Sertoli cells) of the seminiferous tubules. n contrast to the lack of viral gp7o in the germinal epithelium of the testis, a high level of gp7o expression was found within the columnar epithelial cells of the epididymis (Fig. 2) as well as within the stromal cells surrounding these ducts (presumably smooth-muscle cells). Viral antigen was also apparent within the luminal contents of these ducts. Electron micrographs of epididymis revealed a high frequency of free virus particles in the lumen in virtually every field. Virus was arranged in clumps, often associated with amorphous material (Fig. 3A) and spermatozoa (Fig. 3B). Consistent with the absence of detectable gp7o within germinal cells of the testis, there was no evidence of virus budding from spermatozoa. Within the epididymis of normal adult RW males could be seen rare extracellular retroviruslike particles, but there was no evidence of WM-E gp7o as detected by immunofluorescence (not shown). n the reprodutive tracts of neonatally inoculated viremic female mice, gp7o was localized within smooth-muscle cells of the uterus as well as both columnar epithelial cells of the endometrium and oviducts and the squamous epithelium of the cervix (Fig. 2). Uninfected controls were negative for WM-E gp7o. Portal of entry of horizontally transmitted WM-E. From the preceding observations, it is clear that abundant infectious virus was excreted both from the salivary glands and from the male and female reproductive tracts. Yet transmission occurred primarily in one direction, from viremic males to either males or females (Table 1). This would appear to rule out infection by the oral route, and we tested this hypothesis by feeding mice infectious saliva pooled from several viremic mice. None of the 12 normal RW recipients of 5 x 102 FFU of WM-E developed antiviral antibody in serum during the 1 month of observation. Saliva, however, may transmit the virus parenterally during fighting behavior among males. When male mice were treated intranasally with ZnSO4, which is effective in reducing fighting by the induction of anosmia (26), male-to-male transmission was eliminated (Fig. 4). The possibility that females were infected through the female reproductive tract during coitus was examined initially by the infusion of WM-E into the vaginas of superovulated females (5 x 103 FFU per mouse), with co C.0 Q a) - 6. LL 80 Viremic dt dv Viremic d' dv Anosmia 40.:: _ FG. 4. Elimination of horizontal male-to-male transmission of WM-E by induction of peripheral anosmia. Viremic and normal males were housed together for 10 days. Both viremic and normal mice in one group received ZnSO4 intranasally; mice in the other group remained untreated. Serum was collected from the normal cagemates 3 weeks later, and anti-wm-e antibody was assayed by ELSA.

5 VOL. 61, 1987 HORZONTAL TRANSMSSON OF MURNE RETROVRUSES 1041 negative results. However, it was not possible, without causing trauma to the mucosa, to introduce virus through the cervix into the uterine horns. After copulation, the uterine horns become virtually engorged with semen. n an attempt to assess the fate of WM-E within the female reproductive tract after copulation, superovulated virgin RW female mice were caged with viremic RW males for 18 h and then sacrificed. The contents of the uterine horns were assayed for infectivity. n seven mice which had been inseminated, infectious WM-E was detected only when motile spermatozoa were observed. The uteri of two of the seven mice contained nonmotile spermatozoa, but there was no evidence of infectivity. Thus, infectivity appeared to decay in parallel with the loss of viability of spermatozoa which occurs rapidly in the mouse (half-life, ca. 6 h). However, early after insemination, the endometrium was exposed to high concentrations of infectious virus in the ejaculate (see above). The susceptibility of these cells to infection by WM-E (Fig. 2) indicates their potential as initial sites of infection. However, we have been unable, using immunofluorescence, to detect WM-E gp70 in frozen sections of uteri or oviducts taken from RW females 24 to 72 h after insemination by viremic males. AKR female mice were resistant to horizontal transmission of WM-E. n contrast to the high frequency of infection of RW, NFS/N, and C57L females (Fig. 1), 0 of 12 AKR/J females bred to viremic RW males expressed WM-E antibody in serum, despite a pregnancy rate comparable to that of the three susceptible strains. Even after reexposure of these females to the viremic males, seroconversion was not observed (Fig. 1) and no WM-E specific infectious centers were detected in 5 x 106 spleen cells (not shown). We have previously shown that intraperitoneal or intravenous inoculation of neonatal AKR/J mice with WM-E results in levels of virus replication comparable to that in RW mice (21). n addition, adult AKR mice responded to parenteral inoculation of WM-E with an antibody response quantitatively comparable to that of RW mice (21), indicating that AKR mice are not immunologically tolerant to WM-E. Thus, the lack of an antibody response among AKRJ females appeared to reflect resistance of this mouse strain to horizontal infection. Examination of uteri from normal sexually mature virgin as well as multiparous AKR mice revealed high levels of Akv ecotropic gp70 (not shown). This gp70, detected in frozen sections with an Akv-reactive monoclonal antibody, was seen in virtually all cellular elements, including smoothmuscle cells and the endometrium. Since Akv belongs to the same interference group as WM-E (21), the resistance to horizontal transmission exhibited by AKR female mice may be due to local viral interference at the level of the female reproductive tract. Horizontal transmission of F-MuLV. To determine whether horizontal transmission was restricted to the feral mouse viruses, viremic male RW mice which had been neonatally inoculated with F-MuLV were bred to adult female RW mice. Of 24 females, only 1 (4%) developed F-MuLV antibody (Fig. 5). The frequency of seroconversion, however, increased to 48% after a second exposure to the viremic males. The increased frequency of seroconversion on reexposure was independent of parity, since seroconversion among multiparous females exposed once to viremic males was comparable to that among nulliparous females (Fig. 5). Thus, F-MuLV was transmissible horizontally, albeit with lower efficiency than WM-E. mmunofluorescence studies of the viremic male mice used in the F-MuLV transmission studies offered an explanation of the decreased efficiency of horizontal transmission compared with that of WM-E. Like WM-E, F-MuLV gp7o was expressed in epithelial cells of the salivary gland and Leydig cells in the testis (not shown), but there was no evidence of viral antigen within epithelial cells of the epididymis (Fig. 6). nstead, antigen was seen only within stromal cells surrounding these ducts, and there was only scant evidence of viral antigen within the luminal contents. These findings imply that the lack of virus replication in epithelial cells of the epididymis limited the amount of virus released into the seminal fluid, requiring multiple exposures for successful transmission to females. DSCUSSON Gardner et al. (10) first reported that indigenous retroviruses from feral mice were transmissible horizontally. n their study, transmission appeared to be highly mouse strain specific; neither NH Swiss nor feral female mice were susceptible. n fact, the apparent lack of transmission to feral female mice indicated that in the wild, horizontal transmission was not an important means of virus spread. However, horizontal infection was determined by the occurrence of viremia. Since antiviral antibody was not assayed, it was not possible to assess the true prevalence of this mode of infection. Although Gardner et al. (10) suggested that transmission had occurred by the venereal route, the presence of virus in external secretions was not examined and the requirement for physical contact was not established. Finally, since the feral male mice used in the transmission studies of Gardner et al. harbored at least two types of retroviruses, amphotropic and ecotropic, it is not clear which of these was transmitted or whether both were required for horizontal infection to occur. n the current study, we have attempted to clarify these questions. When neonatal mice are inoculated with WM-E, persistent viremia is observed (1, 22) and a proportion of these mice develop clinical disease (hind-limb paralysis or, rarely, lymphoma) (2). These mice excreted abundant infectious virus in saliva, semen, and uterine secretions, and co 0 0 a -) -J LL 6 X lst mating F-MuLV Viremic RW dv x 2nd mating x 1St mating Multiparous t's FG. 5. Horizontal transmission of F-MuLV from males to females. Mice were handled as described in the legend to Fig. 1. One group of RW females was nulliparous at the time of initial exposure to the viremic RW males. n addition, multiparous females which had been bred to normal RW males and had delivered one to three litters were exposed once to the same viremic males. Anti-WM-E antibody was assayed by ELSA

6 1042 PORTS ET AL. FG. 6. Frozen sections of epididymis taken from adult RW mice which had been inoculated as neonates with either F-MuLV (A) or WM-E (B). Sections were stained with monoclonal antibody 500 or antibody 667 for F-MuLV and WM-E gp7o, respectively. F-MuLV gp7o was distributed only within stromal elements surrounding the duct whereas WM-E gp7o was seen within stromal cells as well as epithelial cells of the duct. because of the relatively long latency of disease, some mice attained sexual maturity before onset of clinical signs. When viremic male mice were housed with normal females, we found no evidence of viremia among any of over 100 female mice examined. However, ca. 50% of females from susceptible mouse strains (NFS/N, RW, and C57L) developed antiviral antibody that was detectable within 3 weeks after exposure to viremic RW males and that persisted for at least 8 months. This antibody response was a manifestation of active infection, since low levels of WM-E-specific infectious centers were detected in the spleens of these mice. Thus, horizontal transmission resulted in a persistent infection which was well controlled by the immune system and was not accompanied by any clinical signs of disease. t should be mentioned that the detection of WM-E in the spleens of infected mice was facilitated by the type-specific monoclonal antibodies used in this study. Although RW, NFS, and C57L mice lack endogenous ecotropic virus, they do occasionally express low levels of endogenous xenotropic viruses, some of which replicate in the M. dunni cells used as However, monoclonal antibody 667 used in indicators (24). the infectious center assay reacts only with WM-E and is nonreactive with xenotropic viruses (21). Despite the presence of infectious virus in secretions of both viremic male and female mice, only males consistently J. VROL. transmitted the virus horizontally. Male-to-male infection occurred with high frequency but was prevented by the induction of peripheral anosmia, a procedure which decreases fighting behavior. t is thus likely that transmission of WM-E among males was the result of parenteral inoculation of infected saliva or blood. Several findings support the hypothesis that sexual contact is the primary mode of infection of females. Oral inoculation from infected saliva was ruled out, since peroral feeding of virus did not induce seroconversion. The endometrium was exposed to high concentrations of infectious virus in the ejaculate and was shown to be susceptible to infection by WM-E (Fig. 2). Our inability to demonstrate by immunofluorescence the initial site of infection in the uterus may be the result of the limited sample size of frozen sections. Another piece of evidence supporting the sexual transmission of WM-E was the resistance to horizontal infection exhibited by AKR females. This strain is susceptible to infection by WM-E administered parenterally and responds with the sustained production of antiviral antibody (21). The resistance of this strain to horizontal infection thus implies a nonparenteral portal of entry. n addition, the finding of high levels of endogenous Akv gp70 in most of the cellular elements of the uterus, including the endometrium, suggests that resistance in this strain is mediated by viral interference expressed locally in the female reproductive tract. WM-E is not unique in its predilection for horizontal transmission. F-MuLV was also transmissible from viremic males to females, although the frequency of infection was considerably lower than that of WM-E and required repeated contact of female mice with viremic males. Comparison of the sites of F-MuLV and WM-E replication within the reproductive tract of viremic male mice revealed a clear difference in the distribution of the respective viral gp7o. WM-E was expressed diffusely within the epithelial cells lining the epididymis, and abundant viral antigen was seen within the lumen of these ducts. n contrast, F-MuLV was expressed only within stromal cells, and only minimal amounts of gp7o were detectable in the lumen. Thus, the low efficiency of male-to-female transmission of F-MuLV can be explained by lower levels of virus exported into the seminal fluid. The lack of expression of F-MuLV by ductal epithelial cells of the male reproductive tract was not a manifestation of a restriction of this virus by epithelial cells in general. Both WM-E and F-MuLV infected secretory epithelial cells of the salivary gland. The restriction thus appeared to be highly specific for certain epithelial cell types. Cellular tropism is influenced by at least two regions of the viral genome, the envelope gene (25) and the viral long terminal repeat (3, 9). The viral envelope has a major influence on the central nervous system pathogenicity of WM-E (8), but it is not known whether this is the result of its influence on cellular tropism or posttranslational processing of the viral envelope precursor (32). The viral envelope gene is known to influence species specificity by determining the capacity of the virus to bind to as yet uncharacterized cell membrane receptors. Although receptor heterogeneity in mouse cell lines has been demonstrated in vitro (6), the heterogeneity of receptors in vivo is unknown, as is the role of the envelope in determining sites of virus replication. Since tissues like the male and female reproductive tracts present a welldefined morphological segregation of cell types, we are currently mapping the viral sequences which determine the cellular tropisms within these organs.

7 VOL. 61, 1987 HORZONTAL TRANSMSSON OF MURNE RETROVRUSES 1043 n the testes of viremic mice, both WM-E and F-MuLV were expressed by cells in the interstitium between the seminiferous tubules (particularly Leydig cells). The unique susceptibility of Leydig cells (which produce testosterone) to infection by these viruses is curious but unexplained. t is noteworthy that although Leydig cells in the testes, as well as cells lining the ducts transporting the spermatozoa, were uniformly infected by WM-E, this appeared to have no deleterious effect on fertility. n their study on the expression of endogenous retroviruses, Lerner et al. (19) specifically noted the absence of virus expression in the testes. This apparent difference in the expression of endogenous and exogenous viruses in the testes may relate to the cell specificity of the respective viral enhancer sequences or perhaps to the genomic location of the respective integrated proviruses. The capacity of WM-E to infect the male and female reproductive tracts suggests the possibility that this virus infects germ cells. n contrast to interstitial cells in the testes, we found no evidence for expression of virus by primordial germ cells within the seminiferous tubules or by mature spermatozoa in the epididymis. Jahner et al. (14) have demonstrated variable levels of de novo DNA methylation associated with Moloney murine leukemia virus germ line integration. The degree of methylation appeared to depend on the site of integration but was seen in sperm as well as embryonic tissues. Since transcription appears to be down-regulated by de novo methylation (15), the lack of expression of WM-E in seminiferous tubules does not necessarily rule out the possibility that germ cells were infected. The association of WM-E virions in the semen with mature spermatozoa suggests another possible way for this virus to gain access to germ cells. After insemination by a viremic male, infectious WM-E was found in the oviduct, accompanied by motile spermatozoa (not shown), suggesting the possibility that virus is carried actively to the site of ovulation. This hypothesis was suggested previously by Levy et al. (20), who found that endogenous viruses in the semen were also associated with spermatozoa. Recently, Jenkins and Copeland (16) reported unusually high-frequency germ line integration of endogenous ecotropic viruses from RF/J mice. Genetic experiments indicated that the germ line acquisition of these sequences was due to infection of either the ovum or the preimplantation embryo. This phenomenon appeared to be influenced both by host factors and by viral factors, the nature of which have not been elucidated. nfection of preimplantation embryos by retroviruses in vitro requires high concentrations of virus on the order 5 x 105 FFU/ml (13). Since virus titers as high as this in the blood are rarely attained, hematogenous infection of the early embryo in vivo would be expected to be inefficient. On the other hand, local production of virus within the female reproductive tract, especially by the epithelial lining cells of the oviduct or uterus, might provide sufficient local concentrations of virus to enhance the frequency of infection. f this hypothesis is correct, one would predict that the progeny of WM-E viremic females (which express high levels of virus in the endometrium and tubule epithelium) might exhibit a high frequency of new proviral acquisition. Studies are in progress to test this prediction. ACKNOWLEDGMENTS We thank Ann Kiessling, Department of Obstetrics and Gynecology, Harvard Medical School, and Richard Buller, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, for very helpful discussions during the course of these studies. 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