Propagation of Korean Hemorrhagic Fever Virus in Laboratory Rats

INFECTION AND IMMUNITY, Jan. 1981, p. 334-338 0019-9567/81/010334-05$02.00/0 Vol. 31, No. 1 Propagation of Korean Hemorrhagic Fever Virus in Laboratory Rats PYUNG-WOO LEE,' HERBERT L. AMYX,' CLARENCE J. GIBBS, JR.,' D. CARLETON GAJDUSEK*,l AND HO-WANG LEE2 Laboratory of Central Nervous System Studies, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205,' and The Institute for Viral Diseases, Korea University Medical College, Seoul, Korea2 Korean hemorrhagic fever virus (KHFV) has been adapted to the Wistar and Fisher strains of rats. Infection was detected by the appearance of specific antigen in the lung tissue of the infected rats at 14 to 64 days after inoculation and by the appearance of circulating antibodies in the serum which reacted specifically with KHFV antigen in the lungs of infected Apodemus agrarius subsp. coreae 3 weeks after inoculation. Distribution of antigen in rat tissues as determined by immunofluorescent staining was the same as that in Apodemus mice except that antigen was present in the spleens of rats. Adaptation of KHFV to the laboratory rat provides an animal model that is free of wild rodent viruses and is readily available for use in studies on the characterization of KHFV. In Korea the reservoir of Korean hemorrhagic fever virus (KHFV) is the wild field mouse Apodemus agrarius subsp. coreae (11-13), whereas in Japan it appears to be the laboratory rat (28). Although the virus can be serially propagated in Apodemus mice, many attempts to propagate KHFV in other species of wild rodents and laboratory animals were without success. We have recently established KHFV infection in the Wistar and Fisher strains of rats and report here the results of our studies in Wistar rats showing antigenic homogeneity with the virus propagated in Apodemus mice. MATERIALS AND METHODS Rats. Female Wistar rats (outbred), 1 month of age, were obtained from Microbiological Associates, Walkersville, Md. Female Fisher F344/N rats (inbred), 1 month of age, were obtained from the Animal Production Area, Frederick Cancer Research Center, Fort Detrick, Frederick, Md. KHFV. The original virus stock was KHFV-infected lung tissue prepared from Apodemus mice inoculated with 10% lung suspension of KHFV 76/118/ AP-27 (Apodemus mouse passage 27). This stock had a titer of 1062 50% infective doses per ml in Apodemus mice inoculated intramuscularly (i.m.). Lung tissues of infected Apodemus mice were homogenized in glass tissue grinders and made up to a 20% suspension in minimum essential medium containing a final concentration of 1% bovine serum albumin (vol/vol), 500 U of penicillin, and 50,ug of dihydrostreptomycin per ml. This lung homogenate was centrifuged at 3,000 rpm for 10 min, and the supernatant was decanted and shown to have a titer of 1065 50% infective doses per ml i.m. in Apodemus mice. It was utilized as follows. Passage 1. Three rats were inoculated i.m. with 0.5 ml of the above-described supernatant. Tissues and 334 sera were harvested 21 days later. KHFV antigen in lung tissue and serum antibody were tested by the indirect immunofluorescent method of Lee and Lee (11). Further serial passages. Lung tissues of rats that were serologically positive for anti-khfv antibody but negative for antigen by the indirect immunofluorescent test were selected, and suspensions were prepared for subsequent i.m. inoculation into rats. After passage 2, an additional seven serial passages were carried out as described above at ca. 20-day intervals. Procedures for the indirect immunofluorescent test. The method employed for detection of KHFV antigen in infected tissues and cell cultures is that previously described in the Korean language by Lee and Lee (11). For this reason details of the test are presented in this paper as follows. (i) Preparation of specimens. Normal and KHFV-infected tissues embedded in Tissue-Tek II (Miles Laboratories, Naperville, Ill.) mounting medium were cut at a thickness of 4 pm in a Cryo-Cut II cryostat (American Optical Corp., Buffalo, N.Y.) at -300C. Individual sections on glass slides were air dried at ambient temperature and then fixed in cold absolute acetone for 7 min. Fixed sections were washed three times (15 min/wash) in cold 0.01 M phosphatebuffered normal physiological saline, ph 7.2 (PBS), rinsed in cold distilled water, and dried at ambient temperature. (ii) Primary antibody reaction. To each fixed section, 20 il of test serum (Accufol 90 micropipette, 1 drop) was added and incubated for 30 min at 37 C in a moist chamber. Slides were washed in three changes of 0.01 N PBS [ph 7.2] for 5 min each, rinsed in cold distilled water, and air dried. (iii) Secondary antibody reaction. Each section was covered with 20 ua of fluorescein isothiocyanatelabeled antiglobulin (Cappel Laboratories, Inc., Cochranville, Pa.) to the globulin of the serum used as the primary reagent, containing a final concentration of 1:

VOL. 31, 1981 2,000 Evans blue dye; slides were incubated in a moist chamber for 30 min at 37 C, washed as described above in PBS and distilled water, and dried at ambient temperature. Slides were mounted in a mixture of 9 parts glycerin and 1 part PBS (ph 7.2) and examined under a Leitz epi-fluorescence microscope with a 111- RS condensor. In this study specific KHFV antigen in rat and Apodemus mouse tissues was demonstrated by treatment of frozen and fixed 4-nAm sections with positive KHFV convalescent-phase human serum of known antibody titer; reference antisera were diluted to contain 16 U in 20 pl. Tests to determine comparative antigenic sensitivity of the Apodemus mouse and Wistar rat lung immunofluorescent systems employed human, mouse, and rat anti-khfv antibodies. RESULTS KHFV was adapted to Wistar and Fisher strains of rats, and in both strains it produced a persistent asymptomatic infection. The data presented in this paper, although similar for both species, are for the Wistar strain of rats. The first appearance of KHFV antigen in lung tissue of infected Wistar rats was not detected until the sixth serial blind passage of rat lung tissues (Table 1). Fluorescent-antibody (FA) staining of the intracellular antigen was weak, and antigen was not detected in tissues other than lung at this time. However, on the seventh and subsequent serial passages of the virus, intracellular KHFV antigen in rat lung reacted as strongly by FA staining as that observed in the lung tissues of Apodemus mice. In contrast to the absence of intracellular antigen in early passages (1 to 5) was the presence of specific antibodies, detected by the immunofluorescent test, in the sera of inoculated rats. Specific antibody to KHFV was detected in greater than 68% of the rats in the first three passages, although intracellular viral antigen was not detected in lung or other tissues (Table 1). By the fourth blind passage and in all subsequent passages of the virus, 100% of inoculated rats developed specific antibodies to KHFV in high titers ranging from 1:512 to 1:4,096 by immunofluorescence. Antibody was detected as early as 10 days after inoculation and persisted in a high concentration for more than 60 days (Fig. 1). Beginning with the seventh subinoculation of KHFV in Wistar rats, viral antigen was also detected in kidney, liver, spleen, and submaxillary glands, and to a lesser degree in parotid and lacrimal gland tissues. It should be noted, however, that maximum intensity of immunofluorescent staining occurred in infected lung tissues (++++) and in spleen tissues (++). These findings are similar to the distribution of the virus in Apodemus mice except that the antigen was not present in spleen tissues of the latter animals. KOREAN HEMORRHAGIC FEVER VIRUS 335 TABLE 1. Development offluorescent antigens in lung tissue and antibodies to KHFFV in the serum of Wistar rats on serial passage of the virus Fluores- Antibody in serumb cent anti- Days gen in Passage after in- lung tis- No. posino. ocula- sue (no. No- Mni tion' positive/ tested no. tested) 1 21 0/3 2/3 1:426 2 21 0/8 5/8 1:704 3 21 0/8 6/8 1:592 4 20 0/7 7/7 1:754 5 23 0/6 6/6 1:1,280 6 26 1/6 6/6 1:1,365 7 21 3/3 3/3 1:4,096 8 21 6/6 6/6 1:1,962 9 21 6/6 6/6 1:2,901 aanimals were inoculated serially i.m. with 0.5 ml of 20% suspensions of rat lung after the first passage of KHFV strain 76/118/AP-27 Apodemus mouse lung. On the 9th passage the concentration of the inoculum was 10%. Animals were killed by exsanguination on the day shown after inoculation at each passage level. b Antibody determinations were done by using the indirect immunofluorescent test and KHFV-infected lung of Apodemus mice as antigen, and serial twofold dilutions of sera prepared in PBS antibody titers are expressed as arithmetic mean titers of the highest dilution of serum giving + or greater fluorescence. C6h 409* 1024t 25* 6* 10 20 30 40 50 60 days after Inoculation FIG. 1. Development of antibody to KHFV in Wistar rats. Antibody titers are expressed as the reciprocal of the highest dilution of serum that produced a ++ or greater reaction in the indirect immunofluorescence of KHFV in rat lung tissue. The specificity of the immunofluorescent staining of antigen in KHFV-infected rat tissues and the efficacy of using infected rat lungs in immunofluorescent tests to diagnose KHFV infections in patients were determined by comparing reactions of KHFV-infected Apodemus mouse lung and Wistar rat lung tissues with the

336 LEE ET AL. same acute- and convalescent-phase sera from a patient with KHF. There was no difference in the intensity of the immunofluorescence between infected Apodemus lung and rat lung tissues. The two systems gave nearly identical antibody titers (Apodemus lung: acute serum, 1: 80 and convalescent serum, 1:5,120; Wistar rat lung: acute serum, 1:40 and convalescent serum, 1:5,120). Further evidence for the efficacy and specificity of KHFV-infected rat lungs for serological diagnosis of KHF was obtained by endpoint titrations of antibody in human serum. One pair of acute and convalescent sera and 11 additional convalescent sera known to contain imnmunofluorescent antibodies to KHFV were serially diluted and reacted with viral antigen in Apodemus lung and rat lungs. Both systems were equally sensitive for the detection of specific antibodies and produced comparable titers (Table 2). Moreover, background staining due to KHFV immune complexes, though present, was appreciably less in rat lungs than in Apodemus lungs. Illustrative data on the adaptation and serial passage of KHFV to rats, as determined by the detection of immunofluorescence of intracellular viral antigen in lung tissue, are presented in Table 3. Primary inoculation of KHFV-infected Apodemus mouse lung tissue into rats did not result in the appearance of virus antigen in rat lungs, and it was not until the sixth serial blind passage of rat lungs that intracellular KHFV was detected in one of six rats. However, subse- TABLE 2. Comparison of antigenicity offluorescent antigen in rat lung to KHFV antigen in Apodemus mouse lung Fluorescent antigen in Apode- KHFV antigen in Wistar rat Anti-KHFV serum mus mouse lung lung Reac- Serum Reac- Serum tiona titerb tion titera KHF-66-362 ++++ 1:2,048 ++++ 1:2,048 KHF-66-423 ++++ 1:1,024 ++++ 1:512 KHF-66-463 ++++ 1:256 ++++ 1:256 KHF-67-37-2 +++ 1:256 +++ 1:256 KHF-68-24-2 ++++ 1:2,048 ++++ 1:1,024 KHF-68-35-2 ++++ 1:2,048 ++++ 1:1,024 KHF-77-107-3 ++ 1:128 ++ 1:128 KHF-77-114-2 + 1:64 + 1:64 KHF-77-122 ++++ 1:4,096 ++++ 1:4,096 KHF-77-123 ++++ 1:2,048 ++++ 1:2,048 KHF-77-124 ++ 1:128 ++ 1:64 KHF-76-21 Acute phase ++ 1:80 ++ 1:40 Convalescent phase ++++ 1:5,120 ++++ 1:5,120 a Reaction shown is the intensity of the immunofluorescence observed with individual serum specimens at a dilution of 1:16. 'The highest dilution of serum which gave a + or greater fluorescence. INFECTr. IMMUN. TABLE 3. Primary adaptation and serial passage ofkhfva in the Wistar strain of rats Cumulative KHFV fluores- No. of virus Total days.logio dilu- cent antigen in passages in ratsb tion of orig- rat lung (no. inal inocu- positive/no. lum tested) 5 106 3.5 0/6 6 132 4.2 1/6 7 153 4.9 3/3 8 174 5.6 6/6 9 195 6.6 29/29 a The primary inoculum consisted of 20% lung suspension of Apodemus mice infected with the 118/AP- 27 strain of KHFV; this concentration was employed up until the 9th passage, after which a 10% lung suspension was employed. b Total number of days that KHFV was maintained in Wistar rats. Serial subinoculations of the virus contained in rat lung tissues were done at intervals of ca. 21 days. quent rat-to-rat passage resulted in infection of all inoculated animals. After nine such passages, the original Apodemus virus had undergone a cumulative dilution of 10-66 over 195 days. DISCUSSION KHF was recognized for the first time in 1951 as a major medical problem in United Nations troops serving in Korea (21). Subsequently it was recognized that KHF was undoubtedly the same disease as hemorrhagic fever with renal syndrome or hemorrhagic nephrosonephritis in the Soviet Union (22, 23), as epidemic hemorrhagic fever in Manchuria (7-9), and as nephropathia epidemica in Scandinavia (5, 6, 10). The disease presents as an acute infectious febrile, often fatal, otherwise self-limited illness of virus etiology characterized by severe toxemia, widespread capillary damage, hemorrhagic phenomena, and renal insufficiency (20). More recently it has also been recognized in Japan (26), China (3), Hungary (1, 27), Czechoslovakia (19), Yugoslavia (29), and Romania (18). Serological evidence linking these geographically distinct foci of both endemic and epidemic disease has until recently been lacking. After the isolation of the etiological virus of KHF (13), a positive antigenic relationship between KHF and nephropathia epidemica (14, 24), epidemic hemorrhagic fever in Japan (15), hemorrhagic nephrosonephritis in the Soviet Union (H. W. Lee, M. P. Chumakov, and M. Brummer-Korvenkontio, personal communication; C. J. Gibbs, Jr., P. W. Lee, M. S. Balayan, and D. C. Gajdusek, unpublished data), and hemorrhagic fever with renal syndrome in the People's Republic of China (16, 17) was found by the indirect immunofluorescent test as described by Lee and Lee (11). Recently, using

VOL. 31, 1981 both KHF and nephropathia epidemica viruses we have demonstrated antigenic differences between these viruses in their homologous and heterologous reactions in the immunofluorescent-antibody test (25). In Korea the only known reservoir of KHF is Apodemus agrarius subsp. coreae, whereas in Scandinavia the only reservoir for the closely related virus of nephropathia epidemica appears to be the bank vole Clethryonomys glareolus (2). In Japan outbreaks of KHF have occurred among researchers and technicians who, in several universities, were exposed to asymptomatic KHF-infected laboratory rats purchased from commercial breeders (28). In the sera of rats trapped in the city of Seoul, Korea, high titers of antibodies specific to KHFV have been detected (H. W. Lee, unpublished data). The in vivo propagation of KHFV has until recently been possible only in wild Apodemus mice, a system which has certain intrinsic problems; Apodemus mice are difficult to breed in the laboratory, and litters are often small with high mortality (4, 30). Thus, establishment of an experimental animal host has always been a high priority of KHF research. With adaptation to the Wistar and Fisher strains of rats, it is now possible to carry out experiments in a clean, well-defined, and easily bred and handled laboratory animal for which most biological parameters are known. The fluorescent antigen in rat tissues has been shown to be identical to the antigen in Apodemus tissue by using the previously described techniques. KHFV-positive rat lung tissue is superior to Apodemus lung tissue for the detection of KHFV antibody by the fluorescent-antibody technique because although the intensity is the same, rat lungs give fewer background reactions due to the presence of immune complexes. LITERATURE CITED 1. B'enyei, M. 1962. The incidence of hemorrhagic fever with renal syndrome in Szolnok County. Orv. Hetil. 113:209-210. 2. Brummer-Korvenkontio, M., A. Vaheri, T. Itovi, C. H. von Bonsdorff, J. Vuorimies, T. Manni, K. Penttinen, N. Oker-Blom, and J. Lahdevirta. 1980. Nephropathica Epidemica: detection of antigen in bank voles and serologic diagnosis of human infections. J. Infect. Dis. 141:131-134. 3. Ch'iu Fu-Hdi, Hou Teung-Ch'ang, Huang Ping-shan, Liu Wei-Ch'ang, Sung Hsi-Yu, and Li Ying. 1959 Epidemic hemorrhagic fever: investigations in Tuliho area, inner Mongolia and clinical study of 104 cases. Chin. Med. J. 78:515-531. 4. Filonov, K. P. 1975. Reproduction of the common field mouse. Ekologiya 6:92-95. 5. Gajdusek, D. C. 1953. Acute infectious hemorrhagic fevers and mycotoxicoses in the Union of Soviet Socialistic Republics. Med. Sci. Publ. Walter Reed Army Inst. Res. 2:140. KOREAN HEMORRHAGIC FEVER VIRUS 337 6. Gaidusek, D. C. 1962. Virus hemorrhagic fevers: special reference to hemorrhagic fever with renal syndrome (epidemic hemorrhagic fever). J. Pediatr. 60:841-857. 7. Ishii, S. 1942. Studies on Song-go fever. Jpn. Army Med. J. 355:1755-1758. 8. Kasahara, S., and M. Kitano. 1943. Studies on pathogen of epidemic hemorrhagic fever. Jpn. J. Pathol. 33:476-483. 9. Kitano, M. 1944. A study of epidemic hemorrhagic fever. Jpn. Army Med. J. 370:269-282. 10. Lahdevirta, J. 1971. Nephropathia epidemica in Finland: a clinical histological and epidemiological study. Ann. Clin. Res. 3:12-17. 11. Lee, H. W., and P. W. Lee. 1976. Korean hemorrhagic fever. L. Demonstration of causative antigen and antibodies. Korean J. Intern. Med. 19:371-383. (In Korean.) 12. Lee, H. W., and P. W. Lee. 1977. Korean hemorrhagic fever. m. Natural reservoir. Korean J. Virol. 7:31-39. (In Korean.) 13. Lee, H. W., P. W. Lee, and K. M. Johnson. 1978. Isolation of the etiologic agent of Korean hemorrhagic fever. J. Infect. Dis. 137:298-308. 14. Lee, H. W., P. W. Lee, J. Lahdevirta, and M. Brummer-Korvenkontio. 1979. Etiological relation between Korean hemorrhagic fever and nephropathia epidemica. Lancet i:186-187. 15. Lee, H. W., P. W. Lee, M. Tamura, T. Tamura, and Y. Okuno. 1979. Etiological relation between Korean hemorrhagic fever and epidemic hemorrhagic fever in Japan. Biken J. 22:4145. 16. Lee, P. W., D. C. Gajdusek, C. J. Gibbs, Jr., and Z. Y. Xu. 1980. Etiological relation between Korean hemorrhagic fever and epidemic hemorrhagic fever with renal syndrome in the People's Republic of China. Lancet i: 819-820. 17. Lee, P. W., C. J. Gibbs, Jr., D. C. Gajdusek, C. M. Hsiang, and G. D. Hsiung. 1980. Identification of epidemic hemorrhagic fever with renal syndrome in China with Korean hemorrhagic fever. LAncet i:1025-1026. 18. Manasia, M., N. Olinic, I. Z'agreanu, and A. Serban. 1977. Hemorrhagic fever with renal syndrome; report of 11 observations. Int. Urol. Nephrol. 9:177-184. 19. Plank, J., et al. 1955. Prve dva diagnostikovane pripady hemoragickej nefrozo nefritidy na uzemi nasej republiky. Virova nefrozo negritida Dalekeho vychodu. Cas. Lek. Cesk Prague 94:1078. 20. Sheedy, J. A., et al. 1954. The clinical course of epidemic hemorrhagic fever. Am. J. Med. 16:619-628. 21. Smadel, J. E. 1953. Epidemic hemorrhagic fever. Am. J. Public Health 43:1327-1330. 22. Smorodintsev, A. A., I. S. Altshuller, M. I. Dunaevskii, K. A. Kokhreidze, V. 0. Neustroev, and A. V. Churilov. 1944. Etiology and clinics of hemorrhagic nephroso-nephritia, p. 2647. Moscow Medgiz. 23. Smorodintsev, A. A., V. G. Chudakov, and A. V. Churilov. 1959. Hemorrhagic Nephroso-nephritis, p. 124. Pergamon Press, London. 24. Svedmyr, A., H. W. Lee, A. Berglund, B. Hoorn, K. Nystrom, and D. C. Gajdusek. 1979. Epidemic nephropathy in Scandinavia is related to Korean hemorrhagic fever. Lancet i:100. 25. Svedmyr, A., P. W. Lee, D. C. Gajudsek, C. J. Gibbs, Jr., and K. Nystrom. 1980. Antigenic differentiation of the viruses causing Korean hemorrhagic fever and epidemic (endemic) nephropathy of Scandinavia. Lancet ii:315-316. 26. Tamura, M. 1964. Occurrence of epidemic hemorrhagic fever in Osaka City: first cases found in Japan with characteristic feature of marked proteinuria. Biken J. 7:79-94. 27. Trencseni, T., and B. Keleti. 1971. Clinical aspects and epidemiology of hemorrhagic fever with renal syn-

338 LEE ET AL. drome. In Analysis of the clinical and epidemiological experience in Hungary. Akademiai Kiado, Budapest. 28. Umenai, T., P. W. Lee, T. Toyoda, K. Yoshinaga, T. Horiuchi, H. W. Lee, T. Saito, M. Hongo, T. Nobunaga, and N. Ishida. 1979. Korean hemorrhagic fever in staff in an animal laboratory. Lancet i:1314-1316. 29. Vesenjak-Hirjan, J., A. Hrabar, V. Vince-Ribaric, B. INFECT. IMMUN. Bor'ci'c, and Z. Brudnjak. 1971. An outbreak of hemorrhagic fever with a renal syndrome in the Plitvice Lakes area (preliminary report). Folia Parasitol. (Prague) 18:275-279. 30. Will, U., and H. Reichstein. 1972. Successful breeding of Apodemus agrarius in captivity. Z. Saeugetierk. 37: 359-362.