_Archives Arch Virol (1992) 123:243-253

Vi rology © Springer-Verlag 1992 Printed in Austria

Human

s p u m a v i r u s a n t i b o d i e s in sera f r o m A f r i c a n p a t i e n t s

C. Mahnke 1, P. Kashaiya 2, J. Riissler 1, H. Bannert 1, A. Levin3, W. A. Blattner4, M. Dietrich 5, J. Luande2, M. LOchelt 1, A. E. Friedman-Kien6, A. L. Komaroff7, P. C. Loh 8, M.- E. Westarp 9, and R. M. Fliigel1

1projektgruppe Humane Retroviren, Angewandte Tumorvirologie, DKFZ, Heidelberg, Federal Republic of Germany aTanzania Tumourcenter, Ocean Road Hospital, Dar-Es-Salaam, Tanzania 3RTI Unit, Clinical Research Centre, Medical Research Council, Harrow, U.K. 4National Cancer Institute, Bethesda, Maryland, U.S.A. 5Bernhard-Nocht-Institut fiir Tropenmedizin, Hamburg, Federal Republic of Germany 6New York Medical Center, New York, New York, U.S.A. 7Brigham and Women's Hospital, Havard Medical School, Boston, Massachusetts, U.S.A. s University of Hawaii at Manoa, Honolulu, Hawaii, U.S.A. 9Neurologie der Universit/it Ulm im RKU, Ulm, Federal Republic of Germany Accepted September 13, 1991 Summary. Serum samples collected from patients with a wide variety of diseases from African and other countries were tested for antibodies to the human spumaretrovirus (HSRV). A spumaviral env-specific ELISA was employed as screening test. Out of 3020 human sera screened, 106 were found to be positive (3.2%). While the majority of patients' sera from Europe (1581) were negative, 26 were positive (1.6%). Sera from healthy adult blood donors (609), from patients with multiple sclerosis (48), Graves' disease (45), and chronic fatigue syndrome (41) were negative or showed a very low prevalence for spumaviral env antibodies. A higher percentage of seropositives (6.3%) were found among 1338 African patients from Tanzania, Kenya, and Gabon. Out of 1180 patients from Tanzania, 708 suffered from tumors, 75 from AIDS, and 128 had gynecological problems; 51 of the Tanzanian patients were HSRV seropositive (4.3%). A particularly high percentage of 16.6% seropositives were identified among nasopharyngeal carcinoma patients (NPC) from Kenya and Tanzania consistent with results reported 10 years ago. However, 20 nasopharyngeal carcinoma patients from Malaysia were HSRV-seronegative. In selected cases, sera from seropositive individuals were reacted with proteins from HSRVinfected cells in vitro. HSRV env-and gag-specific antibodies were specifically detected by these sera in Western blots. The results indicate spumavirus infections in human patients with various

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diseases at a relatively low prevalence worldwide; in African patients, however, the prevalence of spumavirus infections is markedly higher. Introduction

A human spumavirus was isolated from the tymphoblastoid cells of a nasopharyngeal carcinoma patient (NPC) by Achong etal. [1]. This virus isolate (HSRV) was characterized by nucleotide sequencing, transcription mapping, and molecularbiological studies [16, 18, 13]. These studies revealed that the HSRV genome encodes the gag, pol, and env genes typical for all known retroviruses [4] and, in addition, regulatory genes, the bel genes [6, 7, 18, 23]. This genomic arrangement indicates that spumaviruses belong to the complex retroviruses [5, 7]. The b e l l gene was characterized as a transcriptional transactivator that activates the HSRV long terminal repeat (LTR) region, and to a lesser extent also that of the HIV-1 LTR [11]. Characterization of the HSRV splicing pattern revealed that part of the b e l l gene is combined via splicing with the bel2 gene resulting in still another HSRV gene product, termed Bet, a protein of 56kDa [13]. Besides the bel and bel2 proteins, Bet was indeed identified in HSRV-infected cells by indirect immunofluorescence and protein blotting as an abundantly expressed viral antigen [13]. For other interesting features of HSRV, see recent reviews [5, 7]. Spumaviruses were isolated from many different species and, frequently from monkeys and apes [20]. Previous seroepidemiological studies showed the presence of spumavirus antibodies in NPC patients and controls from Kenya [2, 17] and in inhabitants of Pacific Islands [2, 17, 14]. To date, spumaviruses also called foamy viruses, have not been unambiguously associated with a defined human disease, and have been described "as viruses in search of a disease" [26]. Spumaviruses were repeatedly isolated from patients with subacute granulomatous thyroiditis de Quervain [-27]. It is of interest in this context that experimentally infected rabbits underwent an immunosuppression [9]. In an effort to approach this problem, HSRV sequence-specific test systems are currently developed in our laboratory by employing defined molecular HSRV clones and expression of recombinant viral proteins. The central part of the HSRV env gene was used for the synthesis of a recombinant viral env antigen [ 15]. This envpx antigen was used for the development and establishment of an HSRV-specific ELISA system. The env-specific ELISA detects HSRV antibodies in human sera and has been employed as a first screening test to study the prevalence of HSRV infections in patients. The results of the envspecific ELISA reported here show that HSRV infections occurred in individuals with various diseases. Materials and methods Cells and virus

The propagation of HSRV in human embryoniclung cells(HEL) was as recently described [6].

Spumaretrovirus antibodies in human sera

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Human sera Most African serum samples were from tumor patients of the Ocean Road Hospital, Tanzania Tumour Centre, Dar-Es-Salaam and collected from 1989 to 1991. The sera from Gabon were taken from individuals who received blood transfusions. Sera from Kenya were from nasopharyngeal carcinoma patients [ 17] and were taken between 1975 and 1979; 14 serum samples were from healthy individuals. All sera from NPC patients from Kenya were EBV-positive and negative for HTLV-I antibodies. The diagnosis for all Kenyan NPC patients was histopathologically confirmed. Dr. S. Kook, University of Malaysia, Kuala Lumpur, provided 40 sera; 20 were from Malaysian NPC patients and 20 control sera from healthy individuals. Sera from 609 healthy adult blood donors were kindly provided by Dr. Rother and Dr. Roelcke, Institut fiir Immunologie der Universit/it Heidelberg. Dr. Mathias, Klinisches Labor der Chirurgischen Universit~itsklinik, Heidelberg, kindly provided 920 sera from patients with various diseases. All sera sent under code to the Heidelberg laboratory. Protein extractions and Western blot analysis Cells in 25 cm 2 flask were washed, collected by scraping with PBS and low speed sedimentation, suspended in 150 gl distilled water and subjected to three cycles of freezing and thawing. Protein sample buffer was added and the lysate was incubated for 5 rain at 100 °C prior to electrophoresis on 14% polyacrylamide gels. Pre-stained protein markers (GibcoBRL, Karlsruhe) were run in parallel for molecular weight determination. The gels were directly stained with Coomassie Brilliant Blue or, alternatively, used for Western blotting. The transfer of proteins onto nitrocellulose membranes BA 85 (Schleicher & Schiill, Dassel) was performed with a semi-dry system according to the manufacturers' instructions (cti GmbH; Idstein). Free binding sites were saturated with 3% fat-depleted dry milk powder in PBS containing 0.1% Tween 20. Patients sera were diluted 1 : 250. Rabbit anti envpx sera were used at a dilution of 1 : 500 in 3% fat-depleted milk powder in 0.1% Tween 20/PBS. Protein A-coupled peroxidase (Sigma, M/inchen) was used at a dilution of 1 : 10,000 and reacted with a dye solution of 3 ml 4-chloro-l-naphthol (Sigma Co., Mfinchen) 0.03% freshly prepared, 47 ml water, and 10 gl H202 (30%) to detect specifically bound antibodies [81. After 5rain the reaction was stopped by adding water. E L I S A test for spumaviral env antibodies The tests were essentially performed as described by Mahnke et al. [14] with minor modifications. Recombinant envpxantigen (0.05-0.1 ~g/well) was pipetted into 96-well microtiter plates (Flow Labs., Meckesheim). After 1 h incubation at room temperature, the wells were washed three times with 0.15 ml of 0.1% Tween 20 in PBS (S 8/12 microtiterplate washer, Flow Labs.). After decanting, 1001~1 of 3% bovine serum albumin (BSA) in 0.1% Tween 20 in PBS were added to each well followed by an incubation of 1 h at room temperature. Subsequently, the plates were decanted and washed as before and test serum samples or rabbit antibodies against envpx were added in 50 ~1 0.1% Tween 20 in PBS. The plates were tightly sealed and incubated for 1 h at37 °C. The plates were washed, dried and subsequently 50 gl of a protein A-peroxidase solution (1 mg/ml) were added at a dilution of 1:250 in 0.1% Tween 20 in PBS. After sealing in plastic bags, the plates were incubated at 37 °C for 60 rain. After washing and drying 100 ~tl of a substrate solution were added to each well. To prepare the substrate solution, 28 mg of o-phenylendiamine dihydrochloride (Sigma Co., Deisenhofen) was dissolved in 100ml of 100raM citric acid (pH 5.0) and to 15ml of this solution, 5 gl 30% hydrogen peroxide were added shortly before use. After incubating for 15 rain at room temperature in a dark room, the color reaction was stopped

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by adding 100 pl 2 M sulphuric acid. Optical densities were determined by an automated ELISA reader (Biotec EL 310) at 490 nm. As negative controls for the ELISA, routinely, human sera from healthy blood donors and from symptome-free individuals, and, separately, 50 ~tl of a 2% BSA solution were used. Human sera that reacted with the envpx antigen under the assay conditions used, gave negative results when the recombinant fusion protein was omitted from the assay. Diluted rabbit hyperimmune sera (1:2000) against the HSRV envpx proteins served as positive controls. Routinely, test sera from patients were diluted 1:250. Results

The spumaviral e n v - s p e c i f i c E L I S A that was used in this study detects antibodies against HSRV [-15]. The results of a first screening of 3020 patients sera from different geographical areas given in Tables 1 and 2 as a survey indicate that the prevalence of HSRV infection varies from country to country. A relatively high percentage of HSRV seropositive subjects was found when hospital patients from Dar-Es-Salaam (4.3%) were compared to those from a hospital in Heidelberg (2.7%). Since the clinical status of the majority of the patients in both hospitals was quite different, this might have contributed to the unequal distribution observed. A closer inspection of the diseases of the HSRV env-seropositive patients revealed that an obvious association of HSRV with a given type of clinical status was not readily apparent. The data also suggest that HSRV is not distinctively associated with certain diseases. They include multiple sclerosis, Graves' disease, and chronic fatigue syndrome (Table 1). Most of these diseases have been suggested to be of viral etiology and were consequently included in this first screening [3, 12]. There was a significantly higher prevalence of HSRV infections in certain African tumor patients, particularly in individuals with nasopharyngeal carcinomas, Kaposi's sarcomas and some other tumors (Table 2). However, groups of patients with similar diseases from other countries were seronegative. Since the HSRV envpx does not cross-react with the HIV-1 env antibodies under the conditions used, it is not surprising that sera from AIDS patients in Europe and the majority of sera from Tanzanian AIDS patients were HSRVseronegative. The fact that we found 17% HSRV-seropositives among Tanzanian AIDS patients indicates that the sera contained antibodies against both HIV-1 and HSRV. To confirm that those human sera that had reacted positively in the HSRV env ELISA screening indeed contained HSRV-specific antibodies, Western blots were performed using cell culture-derived HSRV antigen. Lysates from HSRV-infected and uninfected HEL fibroblasts were separately run o n a 14% polyacrylamide gel under denaturing conditions, the proteins were subsequently blotted on nitrocellulose membranes and reacted with those human sera that had scored strongly positive in the env ELISA screening. Figure I shows the Western blots with HSRV positive patients' sera from various countries at a 1 : 250 dilution of human sera. High molecular weight bands that

247

Spumaretrovirus antibodies in human sera Table 1. Survey of results of HSRV env-specific ELISA screening Clinical status

Origin (country)

Blood donors Hospital patients MS

Germany Germany Japan, U.S.A., Germany U.S.A. Germany Japan Kenya Tanzania Gaboon Tanzania Tanzania

609 920 48

0 25 0

41 (19) 45 12 (4) 26 (14) 708 100 39 128

0 0 1 (1) 4 (1) 37 6 7 7

Tanzania Tanzania

52 255

0 15

0 5.9

3020

102

3.4

Chron. fatigue syn. Graves' disease Kawasaki disease NPC Tumor patients Blood transfusion AIDS, ARC Gynecol. patients Healthy, pregnant, mild diseases Unknown diagnosis Sum

No. of patients' sera No. of tested (healthy seroindividuals) positives

Percentage seropositives

0 2.7 0 0 0 8.3 15.3 5.2 6.0 17.9 5.4

Remarks

healthy adults surgical diseases fulminant (6)

infants 1975-79

HIV-1 + (39) no tumors healthy (19)

Table 2. Diagnosis of tumor patients and results of HSRV-env ELISA screening Clinical status

Origin (country)

No. of patients' sera No. of tested (healthy seroindividuals) positives

Percentage seropositives

Cervical carcinoma Squameous cell ca NPC

Tanzania Tanzania Tanzania, Kenya Malaysia Tanzania Tanzania Tanzania Tanzania Tanzania Tanzania Tanzania Germany

381 63 42a (14)

16 1 7 (1)

4.2 1.6 16.6

20 (20) 58 56 40 37 18 12 27 6

0 (0) 7 3 3 1 2 2 0 0

0 12.1 5.4 7.5 2.7 11.1 16.6 0 0

NPC Kaposi's sarcoma Esophageal ca Lymphomas Breast carcinoma Larynx carcinoma Ovarian carcinoma Other tumors AIDS

a Includes 26 sera from Kenyan NPC patients collected from 1975-1979

Remarks

m

EBV + , HTLV-I

HIV-1 + (8)

HIV-1 + (6)

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Fig. 1. Detection of HSRV-specific proteins by Western blotting. Lysates from HSRVinfected (1, 3, 5, 7, 9) and uninfected (2, 4, 6, 8, and 10) human embryonic lung fibroblasts were electrophoresed, blotted, and reacted with sera from a patient with a Burkitt lymphoma (1 and 2), cervical carcinoma (3 and 4), neurological disease (5 and 6), and Kaposi's sarcoma (7 and 8). Arrows mark HSRV env glycoprotein bands of 170 and 130kDa. As positive control for Western blotting, rabbit anti envpx (9) was reacted with lysates from HSRVinfected cells. The HSRV glycoproteins gp 130, gp 64 (SU) and gp 47 (TM) are marked by arrows (9); M pre-stained marker proteins. All electrophoretic separations were done at 14% polyacrylamide concentration correspond to the HSRV e n v gene products were identified under the conditions used (marked by arrows). Viral antigens of 160-170, 130, and 47kDa, respectively, that were previously identified [19, 24, 15] and shown to correspond to the e n v precursor glycoproteins with and without the leader signal peptide, and to the TM protein [24]. Lane 9 (Fig. 1) shows a positive control in which rabbit hyperimmune serum against e n v p x was used to identify the HSRV e n v glycoproteins from infected HEL cells. Three bands of 130, 64, and 47 kDa consistent with the previously reported values for the HSRV e n v precursor, the surface (SU), and the transmembrane protein (TM) were found [24, 19]. Alternatively, the gp 130 might be a heterodimer of gp 64 (SU) and gp 47 (TM) that comigrates with the e n v precursor of 130 kDa. Independent of the origin of this band, the HSRV glycoprotein of 130 kDa comigrated with bands detected by HSRV-specific human sera. It was, furthermore, significant that human control sera from seronegative patients did not react with proteins from HSRV-infected cells (not shown). To ascertain that the protein bands detected by the human serum sample of an American AIDS patient with a Kaposi's sarcoma were HSRV-specific, the Western blots were repeated under different conditions of gel electrophoresis (10%) that allowed for a higher resolution of the high molecular weight HSRV antigens. Figure 2 shows the e n v gp 130 is clearly detectable by the serum sample

Spumaretrovirus antibodies in human sera

249

from the Kaposi's sarcoma patient (lane 1), since it comigrates with the protein band that reacted with rabbit hyperimmune serum against HSRV e n v p x (lane 4). In cell extracts from uninfected HEL, however, a corresponding band was not detectable with either the Kaposi's sarcoma patient serum or with the anti e n v p x antiserum (lanes 2 and 5). This result indicates that the immunodetection of the HSRV e n v antibodies was specific and independent of the polyacrylamide concentration. Figure 2 also revealed that an additional HSRV-specific protein band of an apparent molecular weight of approximately 160-170 kDa (lane 1) had reacted with the patient's serum. This polypeptide species probably corresponds to HSRV SU and/or TM oligomers. The p 160-170 protein species is remarkable, since it was detectable by the Kaposi's sarcoma patient serum (Fig. 2) and could be an HSRV e n v multimer as described for HIV-1 e n v glycoproteins that have been reported to be resistant to treatment with SDS [21]. The relatively high level of p 170 might explain the apparent absence of antigens to the monomeric and less stable form of the HSRV gp 47 TM protein in the human sera used. To confirm the results obtained with ELISA screening tests, Western blots were carried out using antiserum against the capsid antigen of the HSRV g a g gene. The capsid antigen gene had been expressed in E. coli and purified (A. Bartholomfi etal., in press). Figure 3 illustrates that some of the e n v p x seropositive human sera showed a characteristic double band at 78 and 74 kDa that is also readily detectable in HSRV-infected cells along with another doublet of

Fig. 2. Detection of HSRV-specific env proteins by Western blotting. Extracts of HSRVinfected (1 and 4) and uninfected HEL cells (2 and 5) were electrophoresed in a 10% polyacrylamide gel, blotted, and reacted with the serum of Kaposi's sarcoma patient (1 and 2) or with rabbit antiserum against e n v p x (4 and 5). 3 and 6 Pre-stained marker proteins of the sizes indicated. The thick arrow marks the HSRV gp 130, the thin arrows mark the gp 170 and another HSRV env protein of approximately 190 kDa

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Fig. 3. Detection of HSRV gag-specific antigens by Western blotting. Lysates from HSRVinfected HEL cells were electrophoresed in a 14% polyacrylamide gel, blotted and reacted with either rabbit antiserum against HSRV capsid antigen (1, 2, and 6) or with sera from human patients. Apparently healthy patient from the Cook Islands (4, 8, and 16); patient with neurological disease (5 and 9); tumor patients from Tanzania (7 and 11); patients with neurological diseases from Germany (12, 13, and 14); healthy individuals as controls from Germany (10) and Kenia (•5); 3 was reacted with rabbit antiserum against envpx for comparison and as molecular weight markers (see Fig. 1, lane 9)

60 and 58 kDa. The double bands that specifically reacted with the antiserum against the H S R V capsid antigen correspond to H S R V gag precursors and processing intermediates (A. Bartholom/i et al., in press). In some h u m a n sera, both the 78 and 74 and the 60 and 58 k D a double bands were detectable (Fig. 3). Taken together, the results suggest that the h u m a n sera tested contained HSRV-specific antibodies.

Discussion The results of the HSRV-specific ELISA screening tests indicate that H S R V infections in man occur in a relatively wide spectrum of diseases. The identification of H S R V env-specific proteins by immunoblot analysis with four sera from patients with completely different clinical status, Burkitt lymphoma, cervical carcinoma, Kaposi's sarcoma, and patients with neurological diseases supports this notion. It cannot be ruled out, however, that H S R V is directly or indirectly associated with one or the other of these clinical diseases. The association of H S R V with a given clinical status might be obscured by various factors, such as false positive ELISA results, incorrect diagnoses, or by sec-

Spumaretrovirus antibodies in human sera

251

ondary and even multiple infections of the same patient by different diseasecausing agents. It should be noted that is seems possible that HSRV acts in concert with various other viruses, i.e., HIV-1 or herpesviruses. Keller etal. recently reported that the HSRV bel 1 gene products transactivates the HIV-1 LTR [11]. In general, infections by any one virus that results in immunosuppresion might contribute to the broad spectrum of diseases observed. The relatively high seroprevalence of HSRV in certain African tumor patients, such as NPC, is consistent with studies carried out late in the 1970s [2, 17]. It is worth mentioning that atiquots of the same serum samples collected in the 1970s from Kenyan NPC patients and controls gave similar results, although different methods (neutralization tests and indirect immunofluorescence assays) were used at that time. Recently, HSRV gag-specific antigen and antiserum became available by expression cloning of part of the HSRV gag gene (A. Bartholom/i et al., in press). Immunoblots were carried out with sera from patients that had been seropositive for env in ELISA and in Western blots. It was found that the human sera showed immunoreactivity with HSRV gag protein from virally infected cells but no reactivity with lysates from uninfected cells. A characteristic and gag-specific double band of 78 and 74 kDa was detectable in HSRV env-positive sera. These results corroborate the data obtained with the envpx tests and strongly indicate that antibodies against HSRV (or a closely related retrovirus) from two different genomic regions, gag and env, occur in human patients. Nevertheless, the data reported here have to be verified by HSRV-specific polymerase chain reactions. As noted above, the consistently negative results for sera from patients with multiple sclerosis, Graves' disease, and chronic fatigue syndrome for which a retrovirus association was suspected [3, 12] makes it rather unlikely that HSRV plays a major role in any one of these diseases. Only a very limited number of sera from patients with autoimmune diseases, Sj6gren's syndrome and SLE were assayed for the presence HSRV antibodies. The results were difficult to interpret due to unspecific cross-reactions in the controls. It remains to be clarified whether the immunoreactivity is due to autoantibodies or to HSRV antibodies. The geographical distributions of seroprevalence of HSRV reported here and previously [17, 14, 10] shows that HSRV infections have occurred in many different countries from which sera were obtained. When the sera from Indians of an isolated village in Venezuela were tested, HSRV seropositives were not found [10]. It is, nevertheless, obvious from the limited data that a higher incidence of HSRV infections seem to have occurred in African countries when compared to other regions. This may be due to that there are many endemic viral infections in subtropical and tropical Africa.

Acknowledgements We are indebted to many of our colleagues from different countries who generouslyprovided human sera. We acknowledge in particular Dr. Matthias, General Surgery Hospital, Hei-

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delberg for his cooperation. The authors thank Kunitada Shimotohno, National Cancer Research Institute, Tokyo for his critical comments and support and Dr. Eberhardt, Ocean Road Hospital for his active support. We thank Harald zur Hausen for his personal interest in this research and for his support. This research was financed by a grant from the Commission of the European Community (TS*CT87-0186-D). This study was supported in part by contract #NOI-CP-85603 between the National Cancer Institute, U.S.A. and Research Triangle Institute, U.S.A. to W. A. Blattner and A. Levine; and by a grant from the National Institutes of Health to A. L. Komaroff.

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of naturally occurring antibodies to human syncytial virus in East African populations. J Gen Virol 47:399-406 Muranyi W, Fliigel RM (1991) Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures. J Virol 65:727-735 Netzer KO, Rethwilm A, Maurer B, ter Meulen V (1990) Identification of the major immunogenic structural proteins of human foamy virus. J Gen Virol 71:1237-1241 Neumann-Haefelin D, R~thwilm A, Bauer G, Gudat F, zur Hausen H (1983) Characterization of a foamy virus isolated from Cercopithecusaethiops lymphoblastoid cells. Med Microbiol Immunol 172:75-86 Pinter A, Honnen WJ, Tilley SA, Bona C, Zaghouani H, Corny M, Zolla-Pazner S (1989) Oligomeiic structure of gp 41, the transmembrane protein of human immunodeficiency virus type 1. J Virol 63:2674-2679 Rethwilm A, Darai G, RSsen A, Maurer B, Ftiigel RM (1979) Molecular cloning of the genome of human spumaretrovirus. Gene 59:19-28 Rethwilm A, Erlwein O, Baunach G, Maurer B, ter Meulen V (1991) The transcriptional transactivator of human foamy virus maps to the bel 1 genomic region. Proc Natl Acad Sci USA 88:941-945 Tobaly-Tapiero J, Santillana-Hayat M, Giron ML, Guillemin MC, Rozain F, Peries J, Emanoil-Ravier R (1990) Molecular differences between two immunologically related spumaretroviruses: the human prototype HSRV and the chimpanzee isolate SFV 6. AIDS Res Hum Retrovir 6:951-957 Weiss RA, Teich N, Varmus HE, Coffin JM (eds) (1985) RNA tumor viruses, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 25-207 Weiss RA (1988) A virus in search of a disease. Nature 333:497-498 Werner J, Gelderblom H (1979) Isolation of foamy virus from patients with de Quervain thyroiditis. Lancet ii: 258-259

Authors' address: Dr. R. M. Fliigel, Projektgruppe Humane Retroviren, Angewandte Tumorvirologie, DKFZ, Im Neuenheimer Feld 280, D-W-6900 Heidelberg, Federal Republic of Germany. Received August 1, 1991

Human spumavirus antibodies in sera from African patients.

Serum samples collected from patients with a wide variety of diseases from African and other countries were tested for antibodies to the human spumare...
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