Int. J. Cancer: 36,651-655 (1985) 0 1985 Alan R. Liss, Inc.
PREVALENCE OF HTLV-I IN ARCTIC REGIONS Marjorie ROBERT-GUROFF~, Jeffrey CLARK3, Anne P. LANIER~, Gunhild BECK MAN^, Mads MELBYE~, Peter EBBESEN~, William A. BLATTNER3 and Robert c. GALLO Laboratory of Tumor Cell Biology and 3Environmental Epidemiology Branch, National Cancer Institute, Bethesda, MD 20205; 4Arctic Investigations Laboratory, Centers for Disease Control, Anchorage, AK 99501, Department of Medical Genetics, University of Umea, Umea, Sweden; 61nstitute of Cancer Research, Radiumstationen, DK-8OOO Aarhus C, Denmark.
Sera of native inhabitants of Arctic regions were In 1982 we reported the isolation of a new HTLV assayed for antibodies to HTLV-I by the ELlSA tech- subtype, termed HTLV-11, from cells of a patient with firm antibody specificity. Residents of 7 widely separated Alaskan villages exhibited prevalence rates anaraman et al. t 1982). HTLV-11 is readily distin- of 0 to I ~ o / ~ for HTLV-I antibodies. L~~~ than I 01~ of guishable from HTLV-I by immunologic and molecular Greenland Eskimos were HTLV-I antibody-positive. biologic Criteria, but iS ak0 Clearly related to the pro- Residents of 3 northern Swedish regions ranged in totype virus by these same criteria. An additional HTLV-I antibody prevalence from 0 to 5%. Sera of HTLV-I1 isolate has been obtained from cells of an healthy native inhabitants of Alaska and northern intravenous drug user, who died of AIDS (Hahn et al., Sweden were similarly assayed for antibodies to 1984). However, except in sera of i.v. drug abusers HTLV-II. No additional sera were shown to be positive (Tedder et al., 1984; Robert-Guroff, unpublished), no for HTLV-II antibodies. While some of the HTLV-I evidence of widespread infection by HTLV-II has been antibody-positive sera exhibited cross-reactivity with HTLV-II antigens, competition experiments using dis- found in any seroepidemiologic studies. Moreover, rupted ~ ~ ~ v - 1 1 or purified HTLV-I p24 as test anti- HTLV-11 has not been associated with a m particular gens indicated that the primary antibody response in human malignancy, including hairy-cell leukemia or all cases tested was elicited by HTLV-I. Our results AIDS. In the course of our studies with HTLV-11, we show that HTLV-I distribution is not restricted to en- learned that the patient from whose cells HTLV-I1 was demic areas in warm, humid climates, but extends to originally isolated had lived for Some time in an Es- Arctic regions. Within these regions, HTLV-I exhibits kimo village in Alaska @layney 1983). This the same restricted distribution seen in other areas howledge provided a second stimulus to investigate where virus infection is prevalent. The Arctic does not HTLV in Arctic regions. we therefore studied Sera of seem to be a reservoir for HTLV-II infection. The origin of HTLV-I in Arctic is not known. one Alaskan Eskimos, Greenland Eskimos, and Swedish may speculate that foreign visitors introduced the vi- Lapps to determine the Prevalence of HTLV-1 and -11 rus into Aleut and Lapp populations, and that it has in these populations. been maintained there and restricted in i t s distribu- tion as a result of close familial relationships.
nique followed by Ompetition experiments to On- rarely encountered T-cell hairy-cell leukemia (Kaly-
MATERIAL AND METHODS
The human T-cell leukemia (lymphotropic) virus sera type I (HTLV-I) has been causally associated with an Sera of 333 Alaskan Eskimos, originally collected aggressive malignancy of mature T-cells in adults typ- in 1975 and 1981 and stored at -60C until testing, ified by adult T-cell leukemia (ATL) (Gallo et al., were selected from 7 Eskimo communities widely dis- 1983; Robert-Guroff and Gallo, 1983) first described tributed throughout the state. Persons from all age in Japan (Uchiyama et al., 1977). Both HTLV-I and groups were tested in villages 1 and 2, while only ATL have been shown to be endemic in certain regions those between the ages of 40 and 59 were tested in the of the world, especially the southwestern islands of other 5 communities. In this latter group, random Japan (Hinuma et al., 1982; Robert-Guroff et al., samples were chosen from available sera in order to 1983), the Caribbean islands (Blattner et al., 1982, provide an equal distribution by sex and 10-year age 1983; Gessain et al., 1984), the countries surrounding group. Sixteen additional sera from close relatives of the Caribbean basin (Merino et al., 1984; Robert- an antibody-positive Eskimo and 52 sera from healthy Guroff et al., 1984), and parts of subsaharan Africa Alaskan natives or from Alaskan natives with various (Hunsmann et al., 1983; Biggar et al., 1984; Saxinger malignancies were also included in the study. Sera of et al., 1984~) . All these endemic areas have in com- 504 Greenland Eskimos (Ebbesen et al., 1981; Melbye mon warm, humid climates. While HTLV-I is ineffi- et al., 1984) were obtained in 1978 and stored frozen ciently transmitted, with the primary route thought to until use. The sera represented all age groups between involve intimate contact and infected cells rather than 11 and 80, but the majority represented persons be- cell-free virus, it has been speculated that parasitic tween ll and 40 years of age. Males and females were infestations involving insect vectors common in tropi- equally represented in each age group. Two hundred cal and semitropical endemic areas might play a role sera were obtained from Swedish Lapps between the in the life cycle of the virus and in its persistence in ages of 11 and 70, although the majority ranged in age populations inhabiting these areas. In spite of climato- logic differences, parasitic infestations also occur with high frequency in some Arctic populations. Therefore, as part of a geographic seroepidemiologic survey, we undertook an investigation of the prevalence of HTLV- I in Arctic regions.
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Received: June 18, 1985.
652 ROBERT-GUROFF ET AL
from 50 to 70. Males and females were equally repre- sented. One hundred sera were obtained from healthy individuals residing near the Swedish-Finnish border. Most of the people represented were between 11 and 40 years of age, although a few sera were from adults of 40 to 60 years. Again, males and females were equally represented. Sera from 100 healthy donors in Vasterbotten County, Sweden, represented primarily males between the ages of 21 and 50. immunologic Assays
Sera were analyzed for antibodies to HTLV-I and -11 by the previously described ELISA technique (Sax- inger and Gallo, 1983) with either disrupted HTLV-I or HTLV-I1 as test antigen. Confirmation that ob- served antibody reactivities were specific for either virus was obtained by competition assays using ex- tracts of the appropriate virus-producing cells as com- petitor as well as extracts of non-virus-producing cells and fetal calf serum (Robert-Guroff et al . , 1982). In some cases, sera reacted specifically with both HTLV- I and HTLV-I1 antigens. In order to determine which virus elicited the primary antibody response, competi- tion experiments were carried out in the ELISA system using HTLV-I p24-coated Bio-EnzaBeads (Litton Bio- netics, Kensington, MD) as the test antigen and HTLV- I-producing HUT102 cell extract or HTLV-II-produc- ing C3-44 cell extract as competitor. In cases where serum reactivity against HTLV-I p24 was minimal, similar competition experiments were carried out us- ing disrupted HTLV-I1 as test antigen. The bead assay was carried out as follows. Limiting dilutions of sera were incubated with the HTLV-I-coated Bio-Enza- Beads in 250 pl PBS containing 20% normal goat serum, 0.05% Tween-20, 2% Trasylol (Mobay, New York, NY), and serial dilutions of competing proteins at the indicated concentrations. The beads were incu- bated with gentle agitation for 1 hr at room tempera- ture and then washed as described in the Litton Bio- EnzaBead manual in PBS-Tween. The beads were then incubated for 1 hr at room temperature with an appro- priate dilution of peroxidase-conjugated goat anti-hu- man IgG in PBS-Tween containing 1% normal goat serum. Following washing, the last 3 washes being in PBS without detergent, the beads were incubated in peroxidase substrate solution consisting of 0.005 % H202 and 0.05 % orthophenylenediamine in citrate buffer, PH 5.0, for 20 min at room temperature in the dark. The reaction was stopped by the addition of 50 p1 4N H2S04 per well. Results were expressed as a percentage of the control absorbance reading obtained in the absence of any competitor. When disrupted HTLV-I1 was used as the test antigen, the competition experiments were carried out similarly on limiting dilutions of sera in the presence of competitors, and the standard ELISA technique was used throughout the procedure. Sera were judged to possess antibody elic- ited primarily by HTLV-I if, when tested against HTLV-I p24, extracts of C3-44 cells did not fully compete for the reactivity, or the competition curve had a lesser slope than that obtained following compe- tition with HUT102 cell extract. Against HTLV-I1 as test antigen, type-I sera gave competition curves of similar slope when either HUT102 or C3-44 cell ex- tract was used as competitor. Sera were judged to possess antibody elicited by HTLV-I1 if, when tested against HTLV-I1 as test antigen, C3-44 cell extract
was a more effective competitor than HUT102 cell extract. Against HTLV-I p24 as test antigen, type I1 sera were more efficiently competed by C3-44 extract or gave similar cornpetition curves with C3-44 and HUT102 cell extracts as competitors.
Antibody titers were determined on serial dilutions of test sera in the ELISA system against disrupted HTLV-I and/or -11 as test antigen. Titer is expressed as the reciprocal of the serum dilution at which the absorbance of the test serum eq