htlv-i infection associated with disease in aboriginal indians from british columbia: a serological...

Clinical and Diagnostic

Clinical and Diagnostic Virology Virology E L S E V I E R 2 (1994) 67-78

HTLV-I infection associated with disease in aboriginal Indians from British Columbia: a serological and PCR

analysis

G.A. Dekaban a.b.,, J.J.F. Oger c, D. Foti c, E.E. King a, D.J. Waters d, F.J. Picard a'f J. Arp a,b, D. Werker e, G.P.A. Rice a,f

a Immunology Group, The John P. Robarts Research Institute, London, Ontario, Canada Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

c Neurology, University of British Columbia, Vancouver, British Columbia, Canada d Human Retrovirus Section~AIDS Vaccine Program, NCI-FCRDC, P.R.l./DynCorp, Frederick, MD, USA

e Department of Health Care andEpidemiology, U.B.C., Vancouver, British Columbia, Canada f CNS Department, University Hospital, London, Ontario, Canada

(Received 13 August 1993; revised 4 November 1993; accepted 9 November 1993)

Abstract

Background: Recent epidemiological studies have demonstrated the presence of HTLV-I and its close relative, HTLV-II in several aboriginal populations in North, Central and South America but not in Canadian Indian populations. HTLV-II appears to be more prevalent than HTLV-I in aboriginal populations of the Americas. Recently several clinical cases of HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a case of adult T-cell leukemia (ATL) have been identified in British Columbian Indians. This data suggests that a new endemic area of HTLV-I infection may be present within British Columbian Indian population. However, it has recently been shown that HTLV-II may also be associated with a neurological disease similar to HAM/TSP.

Objectives: The purpose of the work reported here was to demonstrate whether HTLV-I, HTLV-II or both were responsible for the diseases seen in the British Columbian Indians.

Study design: In this study serological and gene amplification techniques were used to determine whether HTLV-I or HTLV-II was present in four families and three unrelated individuals all from different bands of aboriginal Native Indians in British Columbia. In each family, one member had an HTLV-associated disease, three cases of HAM/TSP and one case of ATL. Of the three individual aboriginal natives unrelated to the four families, two had HAM/TSP while the third was asymptomatic for HTLV-associated diseases.

*Corresponding author. Fax: + 1 (519) 663-3789. Abbreviations: ATL, adult T-cell leukemia; BC, British Columbia; BCI, British Columbian Indian; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; HTLV-I or II, Human T-lymphotropic virus type I or II; PCR, polymerase chain reaction; RIPA, radioimmunoprecipitation assay.

0928o0197/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0928-0197(93)E0045-H

68 G. A. Dekaban et al./Clinical and Diagnostic Virology 2 (1994) 67-78

Results: This study demonstrated the presence of HTLV-I in the aboriginal Indians with disease and in some of their family members. HTLV-II was not detected in any of the British Columbian Indians tested in this study.

Conclusions: These British Columbian Indians represent the first Canadian aboriginal Indians with HTLV-I infection and associated diseases. Furthermore, the British Columbia Indian population may represent a previously unrecognized endemic population of HTLV-I infection.

Key words." HTLV-I; Aboriginal Indian; Canada; Infection; HAM/TSP

1. Introduction

HTLV-I was the first human retrovirus to be associated with a human disease known as adult T-cell leukemia (ATL) (Mann et al., 1991). HTLV-I has also been firmly associated with a neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAMfrSP) (Mann et al., 1991). More recently HTLV-I infection has been associated with several other diseases (Ijichi et al., 1991; LaGrande et al., 1991) and immunosuppression (Nakada et al., 1984; Katsuki et al., 1987; Miyazaki et al., 1991). In addition to HTLV-I, a close relative, HTLV-II, has also been isolated. However, the linkage o f the latter virus with specific disease etiology remains uncertain (Mann et al., 1991; Murphy, 1993).

Epidemiological studies have demonstrated HTLV-I to be distributed around the world but in geographically clustered areas (Mann et al., 1991). More interestingly HTLV-I and II have been identified in several aboriginal Indian populations in the Americas and in aboriginals from several Pacific Rim countries (Mann et al., 1991; Sherman et al., 1992; Bastian et al., 1993). There have been no previously published reports of HTLV-I infection and disease association in Canadian aboriginal Indians. One Canadian Indian tribe has been examined for the presence of a retroviral infection, including HTLV-I, with negative results (Black et al., 1988). One report found some evidence of HTLV-I infection in Alaskan aboriginals (Robert-Guroff et al., 1985) and this observation has now been corroborated (Davidson et al., 1990; Kaplan et al., 1993). Recently, evidence of HTLV-II infection in association with a neurological disease very similar to HAM/TSP has been described in aboriginal Indians from New Mexico (Hjelle et al., 1992). We report here serological and gene amplification results which demonstrate the presence of HTLV-I infection in four families from different Canadian aboriginal Indian bands located on Vancouver Island and coastal regions of British Columbia. The HTLV-I-associated disease, HAM/TSP, was found in a member of three different families and ATL was found in a member of a fourth family. In addition, three unrelated aboriginal Indians were identified who are HTLV-I-positive and two of them have HAM/TSP.

2. Materials and methods

Subjects In the first family (BCI 1), a husband (BCI 1-1) was diagnosed with a case of

ATL in British Columbia in 1987 and he died in 1988. He and his wife (BCI 1-2) were confirmed HTLV-I-seropositive by the Laboratory Centre for Disease Control

G. A. Dekaban et al./Clinical and Diagnostic Virology 2 (1994) 67- 78 69

in Ottawa, Ontario (Dr. J.M. Connors, BC Cancer Agency, personal communication). We obtained serum and heparinized whole blood samples from the wife of the deceased ATL patient, one of his sons and this son's wife and their two children (BCI 1-2 to 1-6, respectively). In the three other families (BCI 2, BCI 3, BCI 4), the mother/wife (BCI 2-1, 3-1 and 4-1 ) were diagnosed with a myelopathy clinically consistent with HAM/TSP. Serum and heparinized blood were obtained from each HAM/TSP patient and, where possible, from their husbands (BCI 2-2, 3-2 and 4-2) and their children (BCI 2-3, 2-4 and 2-5; BCI 4-3 and 4-4). Three additional aboriginal Indians were also tested because they were referred to us with neurological disorders. Two of three Indians (BCI 5-1 and BCI 6-1 ) turned out to have myelopa- thies clinically consistent with HAM/TSP and the third (BCI 7-1) has a dementing illness. All subjects and guardians of minors gave informed consent for blood collection and guidelines outlined by the Local Human Ethics Committee were followed. Complete clinical case histories of the HAM/TSP patients BCI 2-1, 3-1, 4-1 and 5-1 will be published elsewhere (Oger et al., 1993) and are referred to in that publication as HAM-l, HAM-2, HAM-3 and HAM-4, respectively.

Serological analysis The serological assays performed on the serum samples for the detection of

HTLV-I antibody were the HTLV-I whole virus ELISA (Genetic Systems) and the recombinant HTLV-I p21E envelope ELISA (Cambridge Biotech). The synthetic peptide ELISA kits capable of distinguishing HTLV-I and II were obtained from Olympia and Biochem Immunosystems Inc. All ELISA assays were performed according to the manufacturer's specifications. Western blot assays performed either with the HTLV-I whole virus p21e enhanced Western Blot Kit (Cambridge Biotech) or the HTLV Blot 2.3 Kit (Cellular Products) were performed with a 1:100 dilution of the patient serum sample according to the manufacturer's specifications. The Western blots performed with recombinant HTLV-I gp63 (rgp63) produced by a baculovirus expression system and radioimmunoprecipitation assays (RIPA) of metabolically-labeled HTLV-I infected M J-2 cells were performed as described previously (Ford et al., 1992; Arp et al., 1993).

Gene amplification Gene amplifications were performed on DNA obtained from peripheral blood

lymphocytes (PBL) obtained from whole heparinized blood and purified on Hypaque gradients as previously described (Dekaban et al., 1992a) or directly on 50-100 #1 of heparinized whole blood according to the method of Kawaski (1990). All DNA preparations were tested for their ability to support an amplification using Beta- actin primers as previously described (Dekaban et al., 1992a). The polymerase chain reaction (PCR) conditions with primers for the HTLV-I p24, pol, env and pXH regions and the HTLV-II pol and env regions were as previously described (Dekaban et al., 1992b). Analysis of the amplification products by 1% agarose gel electrophore- sis and Southern blot analysis were conducted as described previously (Dekaban et al., 1992a). The PCR reactions utilized Taq polymerase supplied by Promega Corp. (Madison, WI).


3. Results

Serological analysis A serological analysis of the serum collected from the wife of the ATL patient

(BCI 1-1 ) confirmed that she was seropositive for HTLV-I (BCI 1-2 of Table 1 ) and seronegative for HTLV-II as determined by the two peptide ELISA's capable of distinguishing HTLV-I from HTLV-II. A positive confirmatory Western blot was obtained (Fig. 1A, lane 7). Subject BCI 1-2 was also seropositive by Western blot to HTLV-I rgp63 and in a RIPA. All the other family members examined were seronegative in all of the serological assays (BCI 1-3, BCI 1-4, BCI 1-5 and BCI 1-6 of Table 1 ).

In a second family (BCI 2), the wife with a clinical diagnosis consistent with HAM/TSP and her husband were both seropositive for HTLV-I (BCI 2-1 and BCI 2-2 of Table 1). The two differential HTLV-I/II peptide ELISA results confirmed that they were positive for HTLV-I but not HTLV-II. The confirmatory Western blots and RIPA performed with BCI 2-1's serum were positive for HTLV-I (Table 1 and Fig. 1A, lane 12). The husband, BCI 2-2, was only weakly positive in RIPA

T A B L E 1

Results of the serological profile of the B.C. aboriginal indians

Sample Diagnosis Whole virus rp21e Differential rpg63 Western R I P A

E L I S A E L I S A H T L V - I / I I blot peptide-specific E L I S A a

BCI 1-1 A T L b + N .D . c N .D . N .D . N .D .

1-2 A S Y M P a + + H T L V - I + + +

1-3 U N A F F E C T E D . . . . N .D .

1-4 U N A F F E C T E D . . . . N .D .

1-5 U N A F F E C T E D . . . . N .D .

1-6 U N A F F E C T E D . . . . N .D .

BCI 2-1 H A M / T S P + + H T L V - I + + +

2-2 A S Y M P + - H T L V - I + + W + e

2-3 U N A F F E C T E D . . . . N .D .

2-4 U N A F F E C T E D . . . . N .D .

2-5 U N A F F E C T E D . . . . N .D .

BCI 3-1 H A M / T S P + + H T L V - I + + N .D .

3-2 A S Y M P + + H T L V - I + + N .D .


4-2 A S Y M P + + H T L V - I + + N .D .

4-3 A S Y M P + - H T L V - I + + N .D .

4-4 U N A F F E C T E D - - - N .D . N .D .

BCI 5-1 H A M / T S P + + H T L V - I + + N . D .


BCI 7-1 A S Y M P + + H T L V - I + + N .D .

aBoth HTLV-I/II Peptide Specific ELISAs gave the same result; bELISA test performed by Centers Disease Control, Ottawa; No other sample was available for further testing; ~not determined; dasymptomatic; °weak positive.

G. A. Dekaban et al./Clinical and Diagnostic Virology 2 (1994) 67-78 71

A

D53 -

° , ,

o,~o- i

1

B

ii!!i !iii!4i iilii!

~ii!!,i~ii:i;i~iii .~ii!:iii'i)ii:i~ii

ii~;i? i!iiiii

,ii:i!i ii iii'!:

i!il

iii~i! ii!iiiii !ii!i i~!iii

iiiii~ ii~ili iii!~:ii iilii

iii!!i iiii~!i! i!ili! iii:i

2 2

)

) )

IN:

C

1 2 3 4 5 6 7 8 9 i0

se~Control-

rPg]p46-II-

p53 ~

p36-

p3~-

p~8-

~24-

p19-

r ~ 2 1 e

Fig. 1. Results of the Western blot assays with sera from the aboriginal native Indians. The Western blots for.panels A and B were generated with the Cambridge Biotech kits, while the Panel C Western blots were obtained with the Cellular Products Kit. A: Results for families BCI 1 and BCI 2 from the two coastal bands: lane 1, is the HTLV-I kit positive serum control; lane 2, kit negative serum control; lane 3, HTLV-II kit positive serum controls; lane 4, BCI 2-2, lane 5, BCI 2-3; lane 6, BCI 2-4; lane 7, BCI 1-2; lane 8, BCI 1-6; lane 9, BCI 1-3; lane 10, BCI 1-4; lane 11, BCI 1-5; lane 12, BCI 2-1; lanes 13, 15 and 16 are negative controls from Canadians with neurological diseases other than HAM/TSP. Lane 17 is a Canadian asymptomatic HTLV-I-positive serum sample and lane 14 is a Canadian HAM/TSP patient serum sample of Caribbean origin. B: Family BCI 3 and a repeat of BCI 2-2 serum samples. Lanes 1-3 are the same as in Panel A; lane 4, BCI 2-5, BCI 2-2 first serum sample (same one tested in the Western blot shown in Panel A); lane 6, BCI 2-2 second serum sample; lane 7, an HTLV- I-positive asymptomatic Brazilian; lanes 8, 11 and 12, negative control sera; lane 9, BCI 3-2 and lane 10, BCI 3-1. C: Family BCI 4 and three unrelated Indians. Lane 1 kit HTLV-I-positive control serum; lane 2, kit negative control serum; lane 3, kit HTLV-II-positive control serum; lane 4, our own negative control serum; lane 5, BCI 4-1; lane 6, BCI 4-3; lane 7, BCI 5-1; lane 8, BCI 4-2; lane 9, BCI 6-1; lane 10, BCI 7-1.


and for HTLV-I in the differential HTLV-I/II peptide ELISAs. The confirmatory Western blot using the p21e enhanced Western blot kit for BCI 2-2 (Fig. 1A, lane 4) was suggestive of HTLV-II infection based on previously published criteria (Lipka et al., 1991). The presence of HTLV-II was suggested because of the greater reactivity to p24 than to p19 in the presence of a p21e positive band. This pattern of reactivity was also seen in the HTLV-II-positive control lanes (Fig. 1A, lane 3). However, when the Western blot was repeated with a 2nd serum sample (taken a few months later) and with a different lot of the HTLV-I p21e enhanced Western blot strips, BCI 2-2 was dearly positive for p19 (Fig. 1B, lanes 5 and 6). The intensity of BCI 2-2's p19 band, however, was still weaker than that observed for p21e or p24. The rp21e ELISA was also negative for BCI 2-2 under conditions specified by the assay. The reason for these discrepancies is unclear but may be related to a low titre of HTLV-I antibody in BCI 2-2 or sequence variability in the region of the B-cell epitopes of p19. The other family members of the second family (BCI 2-3 to BCI 2-5) were negative for HTLV in all of the assays performed.

In the third family (BCI 3), the wife (BCI 3-1) with HAM/TSP was seropositive in all the assays for HTLV-I including the confirmatory Western blot (Table 1 and Fig. 1B, lane 10). The husband (BCI 3-2) was also seropositive in all the assays for HTLV-I (Table 1 and Fig. 1B, lane 9). The differential HTLV-I/II peptide ELISA was positive for HTLV-I but not HTLV-II.

In the fourth family, the mother/wife (BCI 4-1 ) was also afflicted with HAM/TSP and was seropositive in all the serological assays (Table 1 and Fig. 1C, lane 5). We were unable to test her husband. However, her daughter (BCI 4-3) and the daughter's husband (BCI 4-2) are HTLV-I seropositive (Fig. 1C, lanes 6 and 8). Like BCI 2-2, BCI 4-3 was also negative in the rgp21e, but also had a low level of antibody as determined by her Western blot results. BCI 4-3's Western blot to whole virus protein indicated an indeterminate result with only p19 and the rgp21e bends being present. Using our recombinant gp63 envelope protein, she had a low titer as compared to other BCI samples we have tested (data not shown). The HTLV-IflI differential peptide ELISA indicated HTLV-I, but not HTLV-II, was present in all three individuals of this family. This was further confirmed by the fact that BCI 4-1 and 4-2 were also positive to the recombinant HTLV-I specific peptide but not to the HTLV-II specific peptide at the top of the Cellular Products Western blot strips. Serum from BCI 4-3 did not react with either of the Western blot HTLV-I or II peptides presumably due to low titres of antibody. The only other child in this family (BCi 4-4) was negative for HTLV-I antibody.

We have also obtained samples from three other unrelated aboriginal British Columbia Indians who were referred to us because they had neurological disorders. Two of them have HAM/TSP (BCI 5-1 and BCI 6-1) and one has a dementia (BCI 7-1). All were seropositive for HTLV (Table 1 and Fig. 1). The presence of only HTLV-I but not HTLV-II was indicated by the differential HTLV-IflI peptide specific ELISA results (Table 1 ) and by the reactivity to the HTLV-I specific recombinant peptide at the top of the Cellular Products Western blot strip (Fig. 1C, lanes 7, 9 and 10).

G. A. Dekaban et al. ~Clinical and Diagnostic Virology 2 (1994) 67- 78 73

Gene amplification In order to confirm the serological results and unambiguously determine the

presence of HTLV-I and not HTLV-II, gene amplifications were performed using PCR. HTLV-I specific PCR primers for the gag p24 region and an N-terminal region ofpol amplified HTLV-I sequences in all seropositive aboriginal Indians (Table 2). Additional primer pairs for the HTLV-I envelope gene and the pXn region amplified HTLV-I specific sequences from BCI 1-2, BCI 2-1, BCI 2-2, BCI 3-1 and BCI 3-2. Representative examples of the PCR experiments are shown in Figs. 2 and 3. Proviral sequences were detected by PCR in DNA prepared from whole blood (Fig. 2, lanes 14-16) and in DNA obtained from purified PBLs (Fig. 2, lanes 10-12). The weaker result obtained from BCI 2-2 (Fig. 2, lane 16) using DNA directly from whole blood was probably due to the presence of PCR inhibitors as a result of partial hemolysis. Purified PBL DNA from BCI 2-2 yielded an unambiguous positive result (Fig. 2, lane 12). Similar results were obtained for BCI 3-1 and BCI 3-2, as shown in Fig. 3 which shows representative results for the pol, env and p X n primers. BCI 2-2 and BCI 4-3 that had exhibited aberrant Western blot patterns were clearly HTLV-I- positive by PCR.

The absence of HTLV-II was supported by the lack of amplification with pol and

T A B L E 2

S a m p l e L o c a t i o n o f P C R p r i m e r p a i r s

H T L V - P H T L V - I I b

p 2 4 gag pol env tax pol env

B C I 1-1 c N . D . a N . D . N . D . N . D . N . D . N . D .

1-2 + + + + - -

1-3 - - N . D . N . D . - -

1 - 4 - - N . D . N . D . - -

1 - 5 - - N . D . N . D . - -

1 - 6 - - - - N . D . N . D . - -

B C I 2-1 + + + + - -

2 -2 + + + + - -

2 -3 - - N . D . N . D . - -

2 - 4 - - - - N . D . N . D . - - -

2 -5 - - N . D . N . D . - -

B C I 3-1 + + + + - -

3 -2 + + + + - -

B C ! 4-1 + + + + - -

4 - 2 + + N . D . N . D . - -

4 -3 + + N . D . N . D . - -

4 - 4 - - N . D . N . D . - -

B C I 5-1 + + N . D . N . D . - -

a H T L V - I p r i m e r p a i r s a r e spec i f i c f o r H T L V - I o n l y ; b H T L V - I I p r i m e r p a i r s a r e spec i f i c f o r H T L V - I I

on ly ; Cno s a m p l e w a s a v a i l a b l e f o r t h i s p a t i e n t w i t h A T L ; d n o t d e t e r m i n e d .

74 G. A. Dekaban et aL /Clinical and Diagnostic Virology 2 (1994)67-78

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20PB 10 "710"610510-410-310-210 "1

296 bp

Fig. 2. Southern blot hybridization of the gene amplification products obtained by PCR from the two families from the two coastal bands (BCI 1 and BCI 2) using HTLV-I pol primers which amplify a 296 bp fragment. Lanes 1, 9, 13, 17 and 19 are sham DNA extract blanks; lane 8 is a blank lane; lane 2, BCI 1-4; lane 3, BCI 1-2; lane 4, BCI 1-3; lane 5, BCI 2-4; lane 6, BCI 1-5, lane 7, BCI2-3; lane 10, BCI 2-1 (HAM/TSP), lane 11, BCI 1-1 (ASYMP); lane 12, BCI 2-2 (ASYMP); lanes 14 to 16 same as lanes 10 to 12 except DNA was obtained directly from 100 ul of whole blood; lane 18, BCI 2-5; lane 20, BCI 2-5, but DNA was obtained from 100 ul of blood; lane PB, PCR water blank control. 10 -7 to 10-1 represents a serial dilution of HTLV-I infected M J-2 cells in uninfected H9 cells. Unless stated otherwise, the above DNA samples were obtained from purified PBLs.

e n v primers specific for HTLV-II in DNA obtained from all of the screened aboriginal Indians (Table 2 and Fig. 3B).

4. Discussion

HTLV-I/II infection has been reported in a number of aboriginal Indians from various geographic regions of the Americas except in Canada (Lairmore et al., 1990; Zamora et al., 1990; Hjelle et al., 1991; Mann et el., 1991). With the possible exception of the reported HTLV-I infection in Alaska natives (Kaplan et al., 1993), there are no other confirmed reports of HTLV-I infection in aboriginal Indians from North America. These are the first Canadian aboriginal Indians reported to have HTLV-I infection and associated diseases. The serological and gene amplification results presented here clearly demonstrate that HTLV-I, but not HTLV-II, was present in all twelve of the aboriginal Indians who tested positive for HTLV in bands from Vancouver Island and coastal regions of British Columbia.

The risk factors for transmission of HTLV-I in these BC aboriginal Indians follow the usual patterns. Blood transfusions were risk factors in some cases. It should be noted that, at the time these transfusions would have taken place (20-30 years ago), the only source of blood was from other aboriginal band members except in cases where patients were transferred to a major city center (to our knowledge, this did not occur). Only in the last three years has the Canadian Red Cross been supplying


A HTLV- I

p24 env PXII

M 12 3 4 5 12 3 4 5 12 3 4 5

B HTLV- I I

pol env

M 12345 12345

Fig. 3. Gene amplification products obtained by PCR of DNA purified from the Family BCI 3. A: Amplification results obtained with HTLV-I specific primer pairs for the p24 region of gag (p24), the gp46 region of envelope (env) and the pXll open reading frame of the X region (pX.). Lane 1, BCI 3-1; lane 2, BCI 3-2; lane 3, HTLV-I-positive control DNA (the 10 -z dilution used in Fig. 2); lane 4, normal human DNA control; lane 5, PCR water blank. M, marker DNA, PM-2 digested with HaelII. B: Amplification results obtained with HTLV-II specific primers for the reverse transcriptase region (pol) and the gp46 region of envelope (env). The lane designations are the same as described above for A.


screened blood to aboriginal Indian clinics. Multiple sexual partners and intravenous drug abuse were also risk factors in two cases.

In three of the four families (BCI 1, 2 and 3), the spouse without HTLV-I disease was also infected with HTLV-I. The asymptomatic HTLV-I carrier was the husband in two families (BCI 2 and 3), while the wife was in the other family (BCI 1). In all three couples, sexual transmission of HTLV-I could have occurred. We have not been able to test the spouse of BCI 4-1. The fact that both partners were infected is not uncommon since similar clustering of HTLV-I infection in married couples has been reported in Japan (Stuver et al., 1993). In only one case was there suggestive evidence of transmission at birth or by breast milk transmission (BCI 4-1 to BCI 4-3); however, BCI 4-3's husband is also HTLV-I-positive. Thus, at present, the source of BCI 4-3's infection is unclear. It is also interesting to note that the mother of BCI 4-1 was breastfeeding BCI 4-1 while being treated for a T-cell leukemia (in the 1950's) of which she died shortly thereafter.

The origin of the virus, that led to the HTLV-I infections in these Indians of distinctly different Indian band heritage and located some distance apart from each other in isolated coastal and interior areas of British Columbia, is unknown. No connections were established between the families or the three unrelated individuals. We cannot assess the true origin of the viruses since we have not as yet been able to determine how endemic HTLV-I is in the aboriginal Indian bands, of which these aboriginal Indians are members. Studies in Japan suggest that there exists 1000 seropositive individuals for each case of HAM/TSP (Kaplan et al., 1990). If the Japanese epidemiological data are extrapolated to this ethnic group, which comprises approximately 75,000 individuals (Government of Canada), seropositivity could be expected to be found in 5.3% of the Aboriginal population (data based on the number of full-blooded Natives answering to single ancestry in British Columbia from Canada 1991 Census). At the moment, we cannot rigorously exclude the possibility that the HTLV-I infections that we are currently observing originate from a source outside the Indian community; although we feel this unlikely. Two cases of HTLV-I-associated ATL in Caribbean immigrants have been observed in British Columbia (Dr R.D. Gascoyne, BC Cancer Agency, personal communication); thus, HTLV-I originating from the Caribbean is present within the province of British Columbia. In addition, Japanese fishermen have been known to stop at various locations along the BC coast. A nucleotide sequence analysis of the British Columbian aboriginal HTLV-I is required to determine its relatedness to the currently-defined five or six major geographic groups (USA, Japanese, Caribbean, West and Central African and Pacific Rim). The finding of HTLV-I in British Columbian Indians is consistent and supportive of the recently suggested idea that HTLV-I may have come from the Old World when early migrants crossed the Bering Strait during the last ice age (Kaplan et al., 1993).

It will also be important to sequence the p19 region of BCI 2-2 in order to resolve the abnormal Western blot pattern that was observed. This is important from a diagnostic point of view since relying solely on Cambridge Biotech Western blotting system may lead to an HTLV-II misdiagnosis if a similar Western blot pattern is observed. In such a case, it may be necessary to use the differential peptide ELISA,


PCR or a Cellular Product-type of Western blot as a further test to unambiguously determine whether HTLV-I or II is present.

By further expansion of the epidemiological and molecular analysis of the HTLV-I isolates from coastal and interior aboriginal Indian bands in British Columbia, we will be able to obtain a clearer picture as to the origin and extent of HTLV-I infection in this province. Until this report, HTLV-I infection and the diseases of ATL and HAM/TSP were confined to recent immigrants and descendants from the HTLV-I endemic areas of the Caribbean basin and countries of South America. In addition, further analysis of the British Columbian aboriginal isolates may determine whether infection is a relatively recent event following pathways similar to those that have led to HIV infections in Canadian aboriginal Indians, or whether these HTLV-I isolates are unique to British Columbian aboriginal Indians.

Acknowledgements

Supported by the Medical Research Council of Canada and in part by the National Cancer Institute, Division of Health and Human Services, under Contract number NO1-CO-74102 with Program Resources, Inc. DynCorp. G.A.D. and G.P.A.R. are supported by Ontario Ministry of Health Career Scientist Awards. J.A. is supported by an MRC of Canada Studentship. We thank Barbara Carroll for her contribution to the preparation of this manuscript. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services or the Ontario Ministry of Health, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government or the Province of Ontario.

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