Four Serotypes of Haemorrhagic Fever with Renal Syndrome Viruses Identified by Polyclonal and Monoclonal Antibodies

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1 J. gen. ViroL (1987), 68, Printed in Great Britain 979 Key words: HFRS virus/ serotyping/ MAbs Four Serotypes of Haemorrhagic Fever with Renal Syndrome Viruses Identified by Polyclonal and Monoclonal Antibodies By K. SUGIYAMA,I*t S. MORIKAWA, 1 Y. MATSUURA, 2 E. A. TKACHENKO, 4 C. MORITA, 2 T. KOMATSU, 1 Y. AKAO 3 AND T. KITAMURA 1 t Department of Enteroviruses, Z Department of Veterinary Science and 3Central Virus Diagnostic Laboratory, National Institute of Health, Gakuen 4-7-1, Musashimurayama, Tokyo , Japan and '*Institute of Poliomyelitis and Viral Encephalitides, Moscow, U.S.S.R. (Accepted 3 December 1986) SUMMARY Antigenic relationships among 20 strains of haemorrhagic fever with renal syndrome (HFRS) viruses isolated in Korea, China, U.S.S.R., Finland, Japan and U.S.A. were examined with rat immune sera, patient sera, eight monoclonal antibodies against the SR-11 strain and 10 monoclonal antibodies against the strain. Antigen analyses by indirect immunofluorescent antibody and immune adherence haemagglutination tests using polyclonal and monoclonal antibodies demonstrated that HRFS viruses may be divided into four serotypes, i.e. Apodemus (Type 1), Rattus (Type 2), Clethrionomys (Type 3) and Microtus (Type 4). Further, it was demonstrated that Type 1 could be divided into three subtypes and Type 3 into two subtypes. The two sets of monoclonal antibodies were useful for identification of the antigenic types of viruses isolated from patients in endemic areas. INTRODUCTION Haemorrhagic fever with renal syndrome (HFRS) viruses are widely distributed among various rodent species in the Eurasian continent, Japan, the Americas and Africa. Scandinavia, U.S.S.R., China and Korea are endemic areas of clinical human HFRS. In 1978, the causative virus of Korean haemorrhagic fever (KHF), Hantaan virus , was isolated from Apodemus agrarius (Lee et al., 1978). In Japan, since 1975 there have been a number of instances of HFRS associated with laboratory rats and in 1982 we isolated the aetiological viruses, named SR-11 and SR-14, from lung specimens of experimental rats associated with an outbreak of HFRS in Sapporo by direct inoculation onto Vero-E6 cells (Kitamura et al., 1983). Other isolations of HFRS virus or HFRS-related virus have been reported (Yamanishi et al., 1983; Yanagihara et al., 1984; Sugiyama et al., 1984 c; Arikawa et al., 1985). Recently, HFRS virus has been shown to be a member of the family Bunyaviridae (Schmaljohn & Dalrymple, 1983 : Schmaljohn et al., 1985). The main hosts of HFRS viruses in endemic areas of China and Korea have been shown to be species of the genera Apodemus and Rattus, and those of nephropathia epidemica (NE) virus in Scandinavia and HFRS in the European part of the U.S.S.R. belong to the species Clethrionomys glareolus. Although the clinical manifestations of NE are quite mild, those of HFRS in Korea and China are severe, and those of HFRS in the European part of the U.S.S.R. may be described as intermediate. These differences may depend on the pathogenic properties of the different viruses and it is presumed that the characteristics of the HFRS viruses in European regions are different from those of the HFRS viruses in the Far East. The serological relationships of KHF to NE viruses (Svedmyr et al., 1979, 1980; Lee et al., 1979a), and to HFRS virus in Japan (Lee et al, 1979 b) and China (Lee et al., 1980), have been compared. The serological relationships of t Present address: NERC Institute of Virology, Mansfield Road, Oxford OX1 3SR, U.K SGM

2 980 K. SUGIYAMA AND OTHERS Table 1. History of HFRS virus strains Related HFRS Virus strain Animal host Location outbreak SR-11 Rattus norvegicus Japan (Sapporo) Laboratory type TR-352 Rattus norvegicus Japan (Tokyo) Unknown Tchoupitoulas Rattus norvegicus U.S.A. (New Orleans) Unknown Girard Point Rattus norvegicus U.S.A. (Philadelphia) Unknown Prospect Hill-I Microtus pennsylvanicus U.S.A. (Frederick) Unknown Apodemus agrarius Korea KHF Apodemus agrarius Korea KHF KHF patient Korea KHF Rattus norvegicus Korea (Seoul) KHF A9 Apodemus agrarius China EHF R22 Rattus norvegicus China EHF Chen EHF* patient China EHF CG38-83 Clethrionomys glareolus U.S.S.R. (Bashkiria) HFRS CLS 1/452 Clethrionomys glareolus U.S.S.R. (Bashkiria) HFRS CG-1820 Clethrionomys glareolus U.S.S.R. (Bashkiria) HFRS Apodemus agrarius U.S.S.R. (Far East) HFRS 4590 Apodemus peninsulae U.S.S.R. (Far East) HFRS 5302 Microtusfortis U.S.S.R. (Far East) HFRS 4605 HFRS patient U.S.S.R. (Far East) HFRS Sotkamo Clethrionomys glareolus Finland NE * EHF, epidemic haemorrhagic fever. HFRS viruses in the U.S.S.R. have also been reported (Tkachenko et al., 1982). Studies on the serotyping of HFRS viruses have been reported by Goldgaber et al. (1985) and Lee et al. (1985 b). We have developed an immune adherence haemagglutination (IAHA) test for the detection of HFRS virus antigen and antibody (Sugiyama et al., 1984b), and demonstrated that it discriminates Rattus virus strains from the Apodemus strain (Sugiyama et al., 1984a). In the present study, further serological analyses of 20 representative strains of HFRS virus isolated in Korea, China, Japan, the U.S.S.R., Finland and the U.S.A. from different rodent species were carried out using polyclonal and monoclonal antibodies to clarify the antigenic relationships of HFRS viruses. METHODS Virus strains. Twenty strains of HFRS virus were used. Strains , , and were isolated in Korea (Lee et al., 1978; Schmaljohn et al., 1985), A9, Chen, R22 in China (Song et al., 1982a, b), 23328, 4590, 4605, CG-1820, 5302, CG38-83 and CLS 1/452 in the U.S.S.R. (Tkachenko et al., 1984), Sotkamo in Finland (Brummer- Korvenkontio et al., 1980; Schmaljohn et al., 1985), SR-11 (Kitamura et al, 1983) and TR-352 (Sugiyama et al., 1984c) in Japan, and Tchoupitoulas (TP) (Tsai et al., 1985), Girard Point (GP) (LeDuc et al., 1984) and Prospect Hill-I (PH-I) (Lee et al., 1985a) in the U.S.A. Their rodent hosts, isolation sites and epidemiological backgrounds are summarized in Table 1. We greatly appreciate the provision of the Korean, CLS 1/452 and Sotkamo strains by Dr H. W. Lee (Korea University, College of Medicine, Seoul, South Korea), the Chinese strains by Dr Song Gan (China National Center for Preventive Medicine, Beijing, China), the TP strain by Dr J. B. McCormick (CDC, Atlanta, Ga., U.S.A.), the GP strain by Dr K. M. Johnson (USAMRIID, Frederick, Md., U.S.A.), the PH-I strain by Dr D. C. Gajdusek (NIH, Bethesda, Md., U.S.A.) and the CG38-83 strain by Dr G. van der Groen (PLITM, Antwerp, Belgium). All these strains were propagated in Veto-E6 ceils in Eagle's MEM supplemented with 2~ foetal bovine serum. Rat immune sera and HFRSpatient sera. Eight-week-old female Wistar rats (specific pathogen-free) were used for immunization against the SR-11 and strains and 3-week-old female Wistar rats (specific pathogen-free) were used for the other 18 strains. Virus preparations propagated in Vero-E6 cells were inoculated subcutaneously and sera were isolated by bleeding 4 weeks after virus inoculation. Two convalescent sera from human HFRS cases in the European and far-eastern regions of the U.S.S.R. were examined. Serological tests. The immune fluorescent antibody (IFA) test was used (Kitamura et al., 1983). Fluorescein isothiocyanate conjugates of antibodies to human IgG, IgM and IgA (Hyland Laboratories, Deerfield, IU., U.S.A.), rat IgG and mouse IgG, IgM and IgA (Cappel Laboratories) were used. IAHA tests were carried out in

3 Serotyping of HFRS viruses 981 U-bottomed polystyrene microplates by methods described previously (Inouye et al., 1981; Sugiyama et al., 1984b). All reagents were diluted with veronal-buffered NaCI (ph 7.3) with Ca 2+, Mg 2, 0.1 ~ bovine serum albumin, 0.002~ gelatin, and 0.02~ NAN3. Aliquots (25 ktl) of antigen and antibody were incubated at room temperature (25 C) for 1 h; 25 ktl of diluted (1 : 100) fresh guinea-pig serum was then added. After incubation at 37 C for 40 min, 25 ~tl of dithiothreitol (3 mg/ml) and 50 ~tl of 0.4~ type O human erythrocyte suspension were added. The HA pattern was read after incubation at room temperature for 2 h or more. For both serological tests master-diluted sera were distributed to antigen slides or microplates. In comparative titrations, sera were titrated against homologous antigens to check the efficiency of antigenic materials and sera in every test. Back titration of the antigens used in the IAHA tests was carried out routinely in each test. In both tests, standard sera showed reproducible titres. Production ofhybridoma cell lines. BALB/c mice were immunized by intr aperitoneal injection of 1.0 ml of culture fluid of Vero-E6 cells infected with the SR- 11 or strain emulsified in the same volume of Freund's complete adjuvant. A booster inoculation was given intravenously 4 weeks later with the same material without adjuvant, and the mice were sacrificed 3 days after the second injection. Spleen cells were fused with SP2/O-Ag-14 myeloma cells as described by KShler & Milstein (1975) and modified by Oi & Herzenberg (1980). After cultivation for 7 to 14 days, culture fluids were screened for antibody by IFA using E6 cells infected with the homologous strain. Antibody-positive hybridoma cell lines were subjected to two cycles of cloning by limiting dilution. After propagation of the hybridoma cells, they were finally inoculated intraperitoneally into pristane-primed BALB/c mice. Ascitic fluid was collected 2 to 4 weeks later. The isotype of the antibody was determined by testing a 10-fold concentrate of hybridoma culture fluid by immunodiffusion against a set of rabbit antisera against mouse immunoglobulin (Nordic Immunological Laboratories, Tilburg, The Netherlands). RESULTS Cross IFA test using rat immune sera and patient sera Fifteen rat immune sera against HFRS virus strains, a convalescent serum from an HFRS patient in the European part of the U.S.S.R. (Bashkiria) and a convalescent serum from an HFRS patient in the far-eastern U.S.S.R. were tested against 20 strains by the IFA test (Table 2). Immune sera against the CG38-83 or CLS 1/452 strains were cross-reactive with the rat and Apodemus strains, but not against the Sotkamo or PH-I strains. However, convalescent serum from a case in the European part of the U.S.S.R., and the anti-sotkamo and PH-I sera were cross-reactive with the CG-1820, Sotkamo, PH-I and 5302 strains, but were clearly less crossreactive against the Apodemus, rat, CG38-83 and CLS 1/452 strains. IFA tests indicated that the CG-1820, Sotkamo, 5302 and PH-I strains were distinct from the Apodemus, rat, CG38-83 and CLS 1/452 strains. These results suggested that there are two subtypes of Clethrionomys-borne HFRS virus in Europe, i.e. CG38-83 and CLS 1/452 form one type, and CG-1820 and Sotkamo another. Serological relationships between the Microtus strains and the CG-1820 and Sotkamo strains were virtually identical by cross IFA tests. Cross IAHA test Rat sera against the and Chen strains reacted with Apodemus strains eight- to 32-fold more than with the rat strains, and anti-a9 rat serum also reacted with the Apodemus strains fourfold more than with the rat strains (Table 3). Anti and rat sera, however, were reactive with the rat strains at the same level. There was a close antigenic relationship between the CG38-83 and strains, and some antigenic difference between the CG38-83 and CLS 1/452 strains. The IAHA test indicated that there are two subtypes among the Apodemus strains, i.e. one formed by the , Chen and A9 strains and the other by the and strains. Further, antigenic differences between the Microtus strains and the Clethrionomys strains were revealed by this test. Cross IFA test using monoclonal antibodies against SR-11 Eight monoclonal antibodies against the SR-11 strain were tested against 20 strains by an IFA test (Table 4). The reaction patterns of two Chinese strains, A9 and Chen isolated from A. agrarius and a human patient, respectively, and two Russian strains, and 4605 also isolated from Apodemus and a patient, respectively, were similar to that of the strain. The Korean strains and were slightly different from the strain. However, strain

4 OO to Z o,-] Table 2. Cross IFA tests using rat immune sera and patient sera A, agrarius Human R norvegicus C. glareolus A ~ A.p.~ r - - ~ ' ~ A, ~" ~ M~(Jf M,p/f 'KOR KOR CHI USSR USSR KOR CH1 USSR JPN JPN USA USA KOR CH1 USSR USSR USSR FIN USSR USA Serum A Chen 4605 SR-II TR-352 TP GP R22 CG38-83CLSI/452CG-1820Sotkamo 5302 PH-I rat rat Nl't NT rcr A9rat Nr NT lwr rat Chen rat +++ _1_}_-1.-1, -1,++ NT NT +-1-t ,+ NT -~- q , ,++ -1,-] q-q- U.S.S.R. far-east patient NT NT , -1, q- -1. NT -1, -1, -1, -1, -~- + NT NT NT NT bit NT N'l" SR-llrat TR-352rat TP-rat GP-rat rat R22rat CG38-83 rat CLS 1/452 rat _ + U.S.S.R. European patient + + Sotkamo rat + PH-1 rat -1. Reciprocal IFA titre* against NT + NT + NT + r, rr NT + NT NT NT +q-q- q-q-+ NT q-+ +q-q- +++ q-++ NT q- NT +q- NT NT ++q NT q q-q- NT q- NT +q- NT NT +++ +q-q- NT q q-+ + q- -t- +++ q-++ NT q-q- NT ++ NT NT NT q NT 4.+ NT +4. NT NT NT q-q NT -[-4- NT -~-+ lift NT ++ +q- NT ++ -}-+ ++ q-q ~-+ NT -- NT + NT NT bit t t NT -- NT 4. NT NT NT NT NT NT NT NT NT bit q-q * IFA titre: + + +, i> 1 : 1024; + +, 1 : 128 to 1:512; +, 1 : 16 to 1:64; -, < 1 : 16. t A.p., A. peninsulae; MJ~, M. fbrtis; M.p., M. pennsylvanicus. :~ NT, Not tested.

5 t~ Table 3. Cross IAHA tests with rat immune sera Serum A Chen SR-11 TR-352 TP GP R22 CG38-83 CLS 1/452 Sotkamo PH-I Reciprocal IAHA titre* against t" )" A. agrarius Human R. norvegicus kor A A A KOR C.I' JPN JPN USA USA KOR A Chen SR-11 TR-352 TP GP * IAHA titre: +++, 1>1:1024; ++, 1:128 to 1:512; +, 1:16 to 1:64; -, <1:16. C. glareolus M. r A ~ pennsylvanicus CH USSR USSR FIN USA R22 CG38-83 CLS 1/452 Sotkamo PH-I t _ _ _ _ _ _

6 Oo Table 4. Cross IFA tests with monoclonal antibodies against SR-11 Reciprocal IFA titre* against A R. norvegicus C. glareolus A.a.~f A.a.'~ Hum.t A.a.'[' Hum.t A.a.t Hum.t A.p.t A r.x ~ MJ~'f M.p.~f Ig KOR KOR KOR CHI CHI USSR USSR USSR JPN JPN USA USA KOR CI~I USSR USSR USSR FIN USSR USA Antibody isotype A9 Chen SR-II TR-352 TP GP R22 CG38-83 CLS 1/452 CG-1820 Sotkamo 5302 PH-I D9-5-8 G 1 DI-5-8 G1 C2-4-5 G2a E G2a A7-4-4 GI BI-4q G1 D2-4-8 G 1 C G _ ~ _ *IFAtitre:++,)l:1024;+,l:16tol:128;-,<l:16. ~A.a.,A. agrarius;hum.,human;a.p.,a.~ninsulae;m~,m.~rt~;m.p.,m, penn~lvani~s. Table 5. Cross IFA tests with monoclonal antibodies against Reciprocal IFAAtitre* against R. norvegicus C. glareolus A.a.~f A.a.~ Hum.~ A.a.~( Hum.t A.a.~ Hum.t A.p.$ A x ~ M.f.~ M.p.t Ig KOR KOR KOR CHI CHI USSR USSR USSR JPN JPN USA USA KOR CIJI CUSSR USSR USSR FIN USSR USA Antibody isotype A9 Chen SR-II TR-352 TP GP R22 CG38-83 CLS 1/452 CG-1820 Sotkamo 5302 PH-I D3-4-6 G NT~; G5-8-4 G2a C2-9-3 G2a D4-3-5 M B G3 D G2a C G D4-8-8 G B G2a D G2a * IFA titre: + +, ~ 1 : 1024; +, 1 : 16 ~ 1 : 128; -, < 1 : 16. A.a., A. agrarms; Hum., human; A~., A. ~ni~u~e; M~, M. ~rt~; M~., M. pen~ylvan~us. ~, Not tested.

7 Serotyping of HFRS viruses 985 Table 6. Serotypes of hantaviruses defined by IFA and IAHA tests using polyclonal and monoclonal antibodies Serotype 1 (Apodemus type) Subtype 1 Serotype 2 (Rattus type) Serotype 3 (Clethrionomys type) Serotype 4 (Microtus type) Subtype 2 Subtype 3 Subtype 1 Subtype , A9, (A. agrarius), Chen, 4605 (human) (A. agrarius), (human) 4590 (A. peninsulae) SR-I 1, TR-352, Girard Point, Tchoupitoulas, R22, ( R. norvegicus) CG-1820, Sotkamo (C. glareolus) CG38-83, CLS 1/452 (C. glareolus) PH-I (M. pennsylvanicus), 4590 (M. fortis) 4590 isolated from A. peninsulae showed a quite different pattern and reacted with all eight clones. Thus 4590 was clearly discriminated from other Apodemus strains, suggesting that the strain from A. peninsulae might form a third subtype among Apodemus strains. Strain 4605 isolated from a patient seemed to be antigenically quite similar to strain isolated from A. agrarius, but not to the strain isolated from A. peninsulae. The reaction patterns of the Clethrionomys strains were different from that of Clone D1-5-8 did not react with four Clethrionomys strains. Although there were some differences between strain CG-1820 and Sotkamo, and between CG38-83 and CLS 1/452, clear discrimination of the patterns of the CG-1820 and Sotkamo group from that of the CG38-83 and CLS 1/452 group was possible. The reaction pattern of 5302 isolated from M. fortis in far-eastern U.S.S.R. was exactly similar to that of PH-I isolated from M. pennsylvanicus in the U.S.A., and almost similar to that of strain CG Pattern analysis indicated that the Apodemus strains were of three subtypes, the Clethrionomys strains of two subtypes and the Microtus strains of a single subtype. Cross IFA using monoclonal antibodies against Ten monoclonal antibodies against were also employed (Table 5). The patterns for the Apodemus strains, the human strain isolated in far-eastern U.S.S.R. and were identical to that of , whereas those of , A9 and Chen were different from that of The patterns of A9 and Chen, both isolated in China, were identical. The reaction patterns of CG and Sotkamo and of CG38-83 and CLS 1/452 were relatively common, and those of the former group were clearly different from those of the latter, whereas those of 5302 and PH-I were identical. However, these all could be clearly discriminated from DISCUSSION Serological analysis with polyclonal sera can be used for antigenic classification, and monoclonal antibodies can provide further detailed antigenic analyses. So far, several investigators have proposed that hantavirus isolates can be classified into three (Schmaljohn et al., 1985; Goldgaber et al., 1985) or four (Lee et al., 1985b) serotypes. In the present study, 20 HFRS strains were analysed; the conclusions and proposed antigenic classification are shown in Table 6. These 20 strains were divided into at least four groups, according to their rodent hosts, i.e. Type 1 (Apodemus type), Type 2 (Rattus type), Type 3 (Clethrionomys type) and Type 4 (Microtus type). IFA tests demonstrated that Type 3 can be further divided into two subtypes: subtype 1 (strains CG-1820, Sotkamo) and subtype 2 (strains CG38-83, CLS 1/452). Further analysis using IAHA tests and panels of monoclonal antibodies against SR-11 and demonstrated that Type 1 can be subdivided into three subtypes: subtype 1 (strains , A9, Chert, and 4605), subtype 2 (strains 83-61, ) and subtype 3 (strain 4590). Our conclusion confirms partially the serotypic classification of hantaviruses by Lee et al. (1985b) and the analyses using monoclonal antibodies have provided a more detailed antigenic classification of hantaviruses, especially the U.S.S.R. strains. The analyses indicated that the Clethrionomys and Microtus strains were antigenically distinct from the Apodemus and rat strains. Strain 5302 isolated from M.fortis in the far-eastern U.S.S.R. was quite different to Apodemus strains from the same areas and closer to PH-I isolated from M.

8 986 K. SUGIYAMA AND OTHERS pennsylvanicus in the U.S.A. Rat virus strains isolated in other parts of the world have been found to be antigenically similar. Thus antigenic relationships depend more on the rodent host of origin than the site of isolation. On the other hand, Microtus strains were found to be similar to one of the Clethrionomys subtypes, that including the CG-1820 and Sotkamo strains. Furthermore, virus strains isolated from A. agrarius and A. peninsulae in the far-eastern U.S.S.R. were antigenically different. It is interesting that there are two subtypes of Clethrionomys-borne virus in the European part of the U.S.S.R.; strains CG38-83 and CLS 1/452 were more crossreactive with the Apodemus strains, whereas the CG-1820 strain was clearly less cross-reactive with the Apodemus strains. It is not known whether a mutation occurred in a CG-1820-type virus to extend the host range from Clethrionomys to Apodemus or vice versa. The analysis also indicated that the panels of monoclonal antibodies are useful for determination of the antigenic types of viruses recovered from human patients in endemic areas. Antigenic typing may be important for an understanding of the ecology and evolution of HFRS viruses and for the development of HFRS vaccines and control measures. The authors wish to thank Dr M. Tatsumi, Department of Veterinary Science, NIH, Tokyo, Japan, for his advice in hybridoma procedures. We thank Ms T. Kohara and M. Ogata for technical assistance. This work was supported in part by a scientific research grant from the Ministry of Health and Welfare, Government of Japan (1984) and a Research Grant, WP/CDS/ICP/011, from the WHO. REFERENCES ARIKAWA, J., TAKASHIMA, I., HASHIMOTO, N., MORITA, C., SUGIYAMA, K., MATSUURA, Y., SHIGA, S. & KITAMURA, T. (1985). Epidemiological study of haemorrhagic fever with renal syndrome related virus infection among urban rats in two islands in Tokyo Bay, Japan. Acta virologica 29, BRUMMER-KORVENKONTIO, M., VAHERI, A., VON BONSDORFF, C.-H., VUORIMIES, J., MANNI, T., PENTTINEN, K., OKER- BLOM, N. & L~DEVJRTA, J. (1980). Nephropathia epidemica: detection of antigen in bank voles and serologic diagnosis of human infection. Journal of Infectious Diseases 141, GOLDGABER, D., GIBBS, C. J., JR, GAJDUSEK, D. C. & SVEDMYR, A. (1985). Definition of three serotypes of hantavirnses by a double sandwich ELISA with biotin-avidin amplification system. Journal of General Virology 66, INOUYE, S., MATSUNO, S. & KONO, R. (1981). Difference in antibody reactivity between complement fixation and immune adherence haemagglutination tests with virus antigens. Journal of Clinical Microbiology 14, KITAMURA, T., MORITA, C., KOMATSU, T., SUGIYAMA, K., ARIKAWA, J., SHIGA, S., AKAO, Y., IMAIZUMI, K., OYA, A., URASAWA, S., TAKEDA, H. & HASHIMOTO, N. (1983). Isolation of a virus of haemorrhagic fever with renal syndrome (HFRS) through a cell culture system. Japanese Journal of Medical Science and Biology 37, K(3HLER, G. & MILSTEIN, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, London 256, LEDUC, J. W., SMITH, G. A. & JOHNSON, K. M. (1984). Hantaan-like viruses from domestic rats captured in the United States. American Journal of Tropical Medicine and Hygiene 33, LEE, H. w., LEE, P. W. & JOHNSON, K. i. (1978). Isolation of the etiologic agent of Korean haemorrhagic fever. Journal of lnfectious Diseases 137, LEE, H. W., LEE, P. W., L.~-IDEVIRTA, J. & BRUMMER-KORVENKONTIO, M. (1979a). Aetiological relation between Korean haemorrhagic fever and nephropathia epidemica. Lancet i, LEE, H. w., LEE, P. W., TAMURA, i., TAMURA, T. & OKUNO, Y. (1979b). Etiological relation between Korean haemorrhagic fever and epidemic haemorrhagic fever in Japan. Biken Journal 22, LEE, P. W., GAJDUSEK, O. C., GIBBS, C. J., JR & XU, Z. Y. (1980). Aetiological relation between Korean haemorrhagic fever with renal syndrome in People's Republic of China. Lancet i, LEE, P. W., AMYX, H. L., YANAGIHARA, R., GAJDUSEK, D. C., GOLDGABER, D. & GIBBS, C. J., JR (1985a). Partial characterization of Prospect Hill virus isolated from meadow voles in the United States. Journal of Infectious Diseases 152, LEE, P. W., GIBBS, C. J. JR, GMDUSEK, D. C. & YANAGIHARA, R. (19853). Serotypic classification of hantaviruses by indirect immunofluorescent antibody and plaque reduction neutralization tests. Journal of Clinical Microbiology 22, oi, v. T. & HERZENBERG, L. A. (1980). I~nmunoglobulin-producing hybrid cell lines. In Selected Methods in Cellular Immunology, vol. 1, pp Edited by B. B. Mishetl & S. M. Shiigi. San Francisco: W. H. Freeman. SCHMALJOHN, C. S. & DALRYMPLE, J. M. 0983). Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae. Virology 131, SCHMALJOHN, (3. S., HASTY, S. E., DALRYMPLE, J. M., LEDUC, J. W., LEE, H. W., VON BONSDORFF, C. H., BRUMMER- KORVENKONTIO, M., VAHERI, A., TSAI, T. F., REGNERY, H. L., GOLDGABER, D. & LEE, P. W. (1985). Antigenic and genetic properties of viruses linked to haemorrhagic fever with renal syndrome. Science 227,

9 Serotyping of HFRS viruses 987 SONG, G., HANG, C. S., LIAO, H. X., NI, D. S., QUI, X. Z., SHEN, H. K., GAO, G. Z., ZHOU, N., DU, Y. L., ZHAO, J. N., XU, J. K, KONG, B. X., WANG, Z. S., Ztth~O, Z. Q. & WU, Y. S. (1982a). Adaptation of EHF virus isolated in Apodemus agrarius to cell cultures. Chinese Journal of Microbiology and Immunology 2, (In Chinese.) SONG, G., HANG, C. S., LIAO, H. X., QU1, X. Z., GAO, G. Z., DU, Y. L., ZHAO, J. N., XU, J. K. & KONG, B. X. (1982b). Isolation of EHF-related agent from Rattus norvegicus captured from patients' home in endemic area of the mild type of haemorrhagic fever. Acta microbiologica sinica 22, (In Chinese.) SUGIYAMA, K., MATSUURA, Y., MORITA, C., MORIKAWA, S., KOMATSU, T., SHIGA, S., AKAO, Y. & KITAMURA, K. (1984a). Determination by immune adherence HA of the antigenic relationship between Rattus- and Apodemus-borne viruses causing haemorrhagic fever with renal syndrome. Journal of Infectious Diseases 149, 472. SUGIYAMA, K., MATSUURA, Y., MORITA, C., SHIGA, $., AKAO, Y., KOMATSU, T. & KITAMURA, T. (1984b). An immune adherence assay for discrimination between etiologic agents of haemorrhagic fever with renal syndrome. Journal of lnfectious Diseases 149, SUGIYA~.A, K., r~orita, C., ~ a, TSUURA, 't., SmOA, S., KOMATSU, T., MORmAWA, S. & KITA~tURA, T. (1984C). Isolation of a virus related to haemorrhagic fever with renal syndrome from urban rats in a nonendemic area. Journal of Infectious Diseases 149, 473. SVEDMYR, A., LEE, H. W., BERGLUND, A., HOORN, B., NYSTROM, K. & GAJDUSEK, D. C. (1979). Epidemic nephropathy in Scandinavia is related to Korean haemorrhagic fever. Lancet i, 100. SVEDMYR, A., LEE, P. W., 6AJDUSEK, D. C., GIBBS, C. J., JR & NYST~6M, K. (1980). Antigenic differentiation of the viruses causing Korean haemorrhagic fever and epidemic (endemic) nephropathy of Scandinavia. Lancet ii, TKACrIENKO, E. A, DONETS, M. A. & REZAPKIN, G. V. (1982). Serotypes of HFRS (haemorrhagic fever with renal syndrome) virus in East European and Far Eastern USSR. Lancet i, 863. TKACHENKO, E. A., BASHKIRTSEV, V. N., VAN DER GROEN, G., DZAGUROVA, T. K., IVANOV, A. P. & RYLTSEVA, E. V. (1984). Isolation in Vero-E6 ceils of hanta virus from Clethrionomys glareolus captured in the Bashkiria area of the USSR. Annales de la Soci~t~ belge de m~decine tropicale 64, TSAI, T. F., BAUER, S. P., SASSO, D. R., WHITFIELD, S. G., McCORMICK, J. B., CARAWAY, T. C., McFARLAND, L., BRADFORD, n. & ~U~TA, T. (1985). Serological and virological evidence ofa hantaan virus-related enzootic in the United States. Journal of Infectious Diseases 152, YAMANISHI, K., DANTAS, J. R., JR, TAKAHASHI, M., YAMANOUCHI, T., DOMAE, K., KAWAMATA, J. & KURATA, T. (1983). Isolation of haemorrhagic fever with renal syndrome virus from tumor specimen of a rat. Biken Journal 26, YANAGIHARA, R., SVEDMYR, A., AMYX, H. L., LEE, P. W., GOLDGABER, D., GAJDUSEK, D. C., GIBBS, C. J., JR & NYSTROM, K. (1984). Isolation and propagation of nephropathia epidemica virus in bank voles. Scandinavian Journal of Infectious Diseases 16, (Received 22 July 1986)