FURTHER EVIDENCE OF FETAL INFECTION WITH HERPES SIMPLEX VIRUS

THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 37, No. 3, pp. 289-293 March, 1962 Copyright 1902 by The Williams & Wilkins Co. Printed in U.S.A. FURTHER EVIDENCE OF FETAL INFECTION WITH HERPES SIMPLEX VIRUS JOSEPH Z. BIEGELEISEN, JR., PH.D., L. VERNON SCOTT, Sc.D., AND WALTER JOEL, M.D. Departments of Microbiology and Pathology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma Herpes simplex virus has been isolated in the chick embryo from homogenates of fetuses from corneal ly infected pregnant rabbits. 2 With the viral isolation technics used, it was not possible to determine whether the virus isolated was from foci of infection in the fetal tissue, the circulating blood within the fetus, or from contamination with infectious material from the adult rabbit. Fluorescent antibody technics and standard staining methods with hematoxylin and eosin, therefore, were applied, in order to confirm the presence of foci of herpes simplex virus in fetal tissue. MATERIALS AND METHODS The HF strain of herpes simplex virus obtained from the Communicable Disease Center, Montgomery, Alabama, and propagated in this laboratory in embryonated hen's eggs, in white Swiss mice, and on the cornea of the rabbit was used as the seed virus. At varying times after breeding (7, 8, 11, 13, and 14 days), pregnant white rabbits were anesthetized lightly with ether, and 0.1 ml. of virus suspension, which had a titer of 10 4 LD50 for yolk sac per 0.5 ml., was massaged into the scarified cornea of the left eye; 0.1 ml. of buffered gelatin saline solution (BGS), as a control, was massaged into the right eye of each rabbit. Specimens of blood were obtained from the marginal ear vein of each rabbit prior to infection, at 24 and 48 hr. Received, May 19, 1961; revision received, August 22; accepted for publication November 3. Dr. Biegeleisen was Research Assistant; his present address is Special Research Unit, Communicable Disease Center, Atlanta, Georgia. Dr. Scott is Professor of Microbiology. Dr. Joel is Professor of Pathology. This investigation was supported in part by a research grant (B9G4C4) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, Department of Health, Education, and Welfare. 289 after corneal inoculation, and later at the time of uterine surgery. Penicillin and streptomycin were added to these specimens, winch then were inoculated onto chorioallantoic membranes (CAM) of developing chick embryos. 1 Eight embryos were used per specimen. After 72 hr. of incubation at 36.5 C, characteristic herpetic lesions were observed on the CAM, winch had been inoculated with specimens which contained herpes simplex virus. At 54 or 96 hr. after inoculation, the rabbits were anesthetized again, and surgical procedures were used, in order to obtain a gravid horn of the bifurcate uterus. The other gravid horn was left intact in the rabbit. The surgically removed fetuses of each rabbit were dissected carefully from the excised uterine horn and washed thoroughly with sterile physiologic saline solution. Several fetuses from each rabbit were quickfrozen and stored at 62 C. for fluorescent antibody studies at a later time. Homogenates of the remaining fetuses of each of the 11 animals were prepared in BGS. Antibiotics were added, and each of these specimens was inoculated onto 8 chorioallantoic membranes, in order to determine the presence of the virus. Fetuses from 2 uninfected rabbits were treated in a similar manner. In order to identify the viruses isolated from the blood and fetal homogenates, comparative infectivity titers from CAM were determined in the presence of specific immune serum and BGS. 9 The globulin fraction from herpes simplex virus-immune serum, which had been produced in rabbits in response to a series of subcutaneous injections of infectious allantoic fluid, was precipitated with ammonium sulfate at half saturation. It was conjugated with fluorescein isocyanate 3 and dialyzed, in order to remove excess fluorescein. This was adsorbed twice with acetone-extracted, dried mouse liver powder,

290 BIEGELEISEN ET AL. Vol. 37 and the coupled antibody with Merthiolate (1:10,000 final concentration), added as a preservative, was refrigerated until it was used. Microscopic slide preparations were made, when possible, of whole fetuses from infected and uninfected rabbits. These were stained with labeled antibody for 30 min., after which time the slides were washed with buffered saline solution and mounted with buffered glycerol. After examination with fluorescence microscopy, some of the fluorescent antibodystained sections were washed in buffered physiologic saline solution and restained with hematoxylin and eosin, in order to be examined for the presence of herpes simplex virus inclusion bodies. Controls that were stained with the conjugated antiserum consisted of frozen tissue sections of fetuses from uninfected rabbits, uninfected and herpes simplex virus-infected mouse brain, vaccinia virus-infected CAM, and uninfected and herpes simplex virus-infected CAM. Sections of fetuses from infected rabbits were treated, also, with unconjugated herpes simplex-immune serum. RESULTS The left eye of each of the rabbits manifested redness and swelling, with multiple vesi dilation of the cornea and nictitating membrane, as early as 24 hr. after inoculation with the virus. The right eye of each animal, as a control, remained normal in gross appearance. Typical herpetic lesions were observed on the CAM after inoculation with blood and fetal homogenate specimens from pregnant rabbits that had been experimentally inoculated. The agent responsible for the production of the lesions on the CAM was identified as herpes simplex virus by means of neutralization of infectivity in the presence of specific immune serum. The herpes simplex virus was recovered from the 48- and 54-hr. specimens of blood and from the fetal homogenates of the 9 animals on which surgical procedures were performed 54 hr. after the experimental infection with the virus. The virus was demonstrated, also, in the 24-, 48-, and 96-hr. specimens of blood and fetal homogenates of the 2 rabbits from which the gravid horn was removed 96 hr. after corneal inoculation. The fact that the experimental corneal inoculations were performed at varying times after breeding of the rabbits and the performance of surgery at either 54 or 96 hr. after inoculation did not seem to influence either the establishment of the infection in the mother and fetuses or the demonstration of the virus. Yellow-green to green areas of specific fluorescence were observed when sections of fetuses from the experimentally infected rabbits that had been stained with labeled antibody were studied by means of fluorescence microscopy. The bright white spots (Figs. 1 and 2) are pictures of areas where antigen-antibody (labeled) complexes were formed. The figures are low- and high-power photomicrographs, respectively, of a section of a fetus from an infected rabbit. These photomicrographs are representative of results obtained from all animals studied. Figure 3 is a photomicrograph of a microtome section of a portion of a fetus from another herpes virus-infected rabbit in which areas of specific fluorescence were noted. After this same section was stained with hematoxylin and eosin, a multinucleated cell with intranuclear inclusion bodies, typical of herpes simplex virus, was observed (Fig. 4). Inclusion bodies were seen in all sections of fetuses from infected rabbits in which specific fluorescence had been noted prior to staining with hematoxylin and eosin. No specific fluorescence was observed in a fluorescent antibody-treated section of a fetus from an uninfected rabbit or in an untagged antiserum-stained section of a fetus from herpes simplex virus-infected rabbit. Other controls included herpes simplex virusinfected mouse brain and chorioallantoic membranes of chick embryos, both of which manifested specific fluorescence after staining with the specific fluor. Uninfected mouse brain and chorioallantoic membranes and vaccinia-infected CAM, when stained with the fluor, did not fluoresce. DISCUSSION The observation of areas of yellow-green to green fluorescence in specific fluorescent

March 1962 FETAL HERPETIC INFECTION FIGS. 1 to 3 (upper left, upper right, and lower left, respectively). These illustrations are photomicrographs of fetal sections stained with specific fluorescent antibody. Figure 1 was prepared from the fetus of a rabbit that was infected 13 days after breeding, and Figure 2 is a higlier magnification of the portion indicated by arroius in Figure 1. Figure 3 was prepared from a rabbit that was infected 7 days after breeding. Magnifications, respectively, X 120, X 510, and X 120. FIG. 4 (lower right). A section from the same tissue illustrated in Figure 3. A multinucleated cell and intranuclear inclusion bodies can be seen. Hematoxylin and eosin. X 950.

292 BIBGELEISEN:- r Ah. Vol. 87 antibody-stained microscopic preparations of fetuses from infected adult rabbits and the absence of this reaction with control preparations indicate that foci of herpetic infections were localized within the fetal tissues. The fluorescent staining areas were not limited to the inclusion bodies. Masses of viral particles, as well as inclusion bodies, can be stained specifically. The demonstration of intranuclear inclusion bodies in tissues of these fetuses is further proof of viral proliferation in fetal cells, as well as proof that the fluorescent staining procedure was functioning properly. It may be assumed that the inclusion bodies and other foci of localization were involved in the immunofluorescent antigen-antibody complex, as depicted in the photographs. These findings confirm earlier reports of isolation of herpes virus. 2 Other workers have used fluorescent antibody technics in order to demonstrate herpes simplex virus in preparations of infected tissue culture. 6,7 Inasmuch as this virus is primarily a pathogen of human beings, and is found only in rabbits which have been experimentally infected, the observation of inclusion bodies and specific fluorescence is proof that the virus was probably transmitted from the experimental corneal lesion of the adult where foci of infections were produced. Likewise, the detection of virus in the blood of the infected adult rabbits is an indication that the viremia played a role in fetal infection. Identification of the specific tissues involved has not been successful at present. Attempted studies in very young fetal tissues have presented many problems. Many infected pregnant animals abort. Studies are being continued, in order to determine specific tissues or organs which are involyed. There is increasing evidence that viruses may affect the fetus in utero, and bring about congenital malformations, abortion, and death. 4 Herpes simplex virus has been isolated from fatal human neonatal infections. 0 8 These reports and the results presented herein indicate that herpes simplex virus may be involved in infection in utero of fetuses when the expectant mother suffers from primary or recurrent infections with this virus. SUMMARY After corneal inoculation of 11 pregnant rabbits, foci of infection by herpes simplex virus within the tissues of the fetuses were.demonstrated by means of the fluorescent ' antibody technic, and confirmed by the observation of herpetic intranuclear inclusion bodies in the same sections of tissue. The viral agent was isolated from fetal homogenates by means of the chorioallantoic membrane method, and was identified as herpes simplex virus by means of the neutralization of viral infectivity. STJMMARIO IN INTERLINGTJA Post le inoculation corneal de virus de herpete simplice in 11 rattas pregnante, focos de infection per ille virus esseva demonstrate intra le tissus fetal per medio del technica a anticorpore fluorescente. Le presentia del focos esseva confirmate per le observation de herpetic corpores de inclusion intranucleari in le mesme sectiones de tissu. Le agente virusal esseva isolate ab homogenatos fetal per medio del methodo a membrana chorioallantoic. Illo esseva identificate como virus de herpete simplice per medio del neutralisation del infectivitate virusal. Acknowledgments. The authors wish to thank Mr. Bernard A. Brown and Mrs. Julia Brewster for their technical assistance. REFERENCES 1. BEVBRIDGB, W. I. D., AND BURNET, F. M.: The cultivation of viruses and rickettsiae in the chick embryo. In Special Report of the London Medical Research Council, Series 256. London: His Majesty's Stationery Office, 1946, pp. 16, 30-33. 2. BlEGELEISEN, J. Z., JR., AND SCOTT, L. V.: Transplacental infection of fetuses of rabbits with herpes simplex virus. Proc. Soc. Exper. Biol. & Med., 97: 411-412, 1958. 3. COONS, A. H., AND KAPLAN, M. C: Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent antibody. J. Exper. Med., 91: 1-13, 1950. 4. GREGG, N. M.: Congenital cataract following German measles in the mother. Tr. Ophth. Soc. Australia, 3: 35-36, 1941.

March 1962 FETAL HERPETIC INFECTION 293 5. LEBBUN, J.: Cellular localization of herpes simplex virus by means of fluorescent antibody. Virology, 2: 496-510, 1956. 6. MCDOUGAL, R. A., BEAMER, P. R., AND HELLERSTEIN, S.: Fatal herpes simplex hepatitis in a newborn infant. Am. J. Clin. Path., 24: 1250-1258, 1954. 7. O'DEA, J. F., AND DINEEN, J. K.: Fluorescent antibody studies with herpes simplex virus in unfixed preparations of trypsinized tissue culture. J. Gen. Microbiol., 17: 19-24, 1957. 8. QUILTJGAN, J. J., JR., AND WILSON, J. L.: Fatal herpes simplex infection in a newborn infant. J. Lab. & Clin. Med., 38: 742-74C, 1951. 9. SCOTT, T. F. MCN.: Diagnostic Procedures for Virus and Rickettsial Diseases, Ed. 2. New York: American Public Health Association, 1956, pp. 313-340.