tion of MOMJ and MOMS, of which passages 25 and 14 were ..... Although MOMS had received transfusion six times, she .... Int J Cancer 1982;30:257-264. 6.
AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 10, Number 9, 1994 Mary Ann Liebert, Inc., Publishers
Familial Transmission and Minimal Sequence Variability of Human T-Lymphotropic Virus Type I (HTLV-I) in Zaire HSIN-FU
LIU,1
ANNE-MIEKE VANDAMME,1 KAYEMBE KAZADI,2 HERWIG JAN DESMYTER,1 and PATRICK GOUBAU1
CARTON,3
ABSTRACT
previously reported a strong familial clustering of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Zaire, suggesting a familial transmission of the virus together with the presence of cofactors. In the present study among 84 relatives of 16 HTLV-I-positive or HAM/TSP index cases, we found that all 15 seropositive children had a seropositive mother and that all 15 children with a seropositive father but a seronegative mother were seronegative. Lymphocytes of 17 relatives from 2 families with a familial HTLV-I-associated neuropathy were tested in 2 polymerase chain reaction (PCR) assays amplifying pol and tax/rex gene fragments. The 10 seropositive individuals were PCR positive for HTLV-I and the 7 seronegatives were negative in both PCR assays. The PCR results showed no evidence for a long lag period between infection with HTLV-I and seroconversion. The HTLV-I long terminal repeat (LTR) of these 10 individuals, related in the first to the fourth degree, was amplified and sequenced. Identical sequences were found within the families except for one woman infected with two variants, one being the familial strain and the other a mutated one with a single nucleotide substitution in the 755 sequenced nucleotides of the LTR region. The family strain and the mutant were both present in two samples taken 1 year apart. Together, the HTLV-I serology, PCR, and sequencing results point toward mother-to-child transmission as the main mode of HTLV-I infection in this population. Comparison of the LTR sequences of the two families with other HTLV-I strains from different geographical regions shows that the Zairean HTLV-I strains form a separate cluster. In contrast to other retroviruses, which have high mutation rates, HTLV-I is genetically very stable as illustrated by the identical LTR sequences within families and by the small differences between the Zairean strains. Our group
INTRODUCTION
Human virus,
T is
type I (HTLV-I), a retroof adult T cell leukemia/ chronic neurological disease,
LYMPHOTROPic virus an
etiological agent
lymphoma (ATL)1,2 and of a HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP).3,4 HTLV-I is endemic in southern Japan,2 the Caribbean basin,5 and sub-Saharan Africa.6,7 There is now also considerable evidence of its prevalence in South America8,9 and in Melanesia.10 HTLV-I is transmitted through blood transfusion, sexual contacts, or from mother to child, usually by breast feeding. Kazadi-Kayembe et al1 previously described a cluster of familial HAM/TSP in Equateur (Zaire). The familial clustering of
HAM/TSP suggests that in Zaire mother-to-child transmission of HTLV-I within families may be the main mode of infection. In the present study, we investigated the familial transmission of HTLV-I and the intra- and interfamilial genomic variability of the virus in the Equateur region. We focused our genomic sequence study on the long terminal repeat (LTR) of the virus. Sequence variation is higher in the LTR than, for example, in the envelope (env) gene, which is well conserved in the case of HTLV-I." The LTR is therefore more informative for the evaluation of genome variability and for phylogenetic analysis. The LTR is also interesting because studies on the murine leukemia virus have shown that it contains determinants of cell tropism and pathogenicity.12 This remains to be demonstrated in the human retroviruses. Studies
'Rega Institute for Medical Research and University Hospitals, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium. 2Centre Neuropsychopathologique, University of Kinshasa, Kinshasa, Zaire. department of Neurology, University Hospitals, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium. 1135
1136
LIU ET AL.
suggest that the genomic variability of HTLV-I depends on geography more than on pathologies.13,14
MATERIALS AND METHODS
Study groups One hundred members from 16 families with HTLV-I and associated neurological diseases were tested serologically. Among these, the MO family from Lisala, which was described by Kazadi-Kayembe et al.1 as the M family previously, and the MW family from Mbandaka were further studied in detail. Among the infected members, some of the MO family have HAM/TSP, and two of the MW family have a flaccid quadriparesis with an as yet unproven causative relation with HTLVI. Blood was drawn in 1988 and 1990 for HTLV serology from relatives of HAM/TSP cases and of HTLV-I-positive index cases, detected in previous studies.7,15
Serological assay Screening was done with an HTLV-I whole virus lysate enzyme-linked immunosorbent assay (ELISA; Abbott, Abbott Park, IL) and screening reactives were further tested with an indirect immunofluorescence assay on MT-2 cells (IFA/MT-2) and with an HTLV-I virus lysate Western blot (Diagnostic Biotechnology, Singapore) and for some samples with a matrix dot-blot assay.16 The matrix assay is an automated HTLV-I dot-blot immunoassay in which serum is tested against purified viral pl9sa« antigen and the recombinant antigens p21f"v and P24«"«, spotted on nitrocellulose. Only sera reacting with both piqgag ancj p24s"s antigens together with one env antigen (p21env or gp46) were considered as HTLV antibody positive. HTLVpositive sera were further typed with specific ELISAs containing synthetic peptides derived from the outer membrane glycoprotein of HTLV-I and HTLV-II.16
Cell culture blood mononuclear cells (PBMCs) were sepaFicoll with sodium metrizoate (Lymphoprep; Nycomed Pharma, Oslo, Norway) and washed twice in RPMI1640. The cells were resuspended to a concentration of 106 cells/ml in RPMI-1640 Dutch modification (Life Technologies, Paisley, U.K.) with 15% inactivated fetal calf serum (Life Technologies), phytohemagglutinin (PHA; 2 pg/ml) (Wellcome, Dartford, U.K.), Polybrene (2 pg/ml), 2 mM L-glutamine (Life Technologies), and gentamycin (20 pg/ml) (Life Technologies). After 3 days, cells from seronegative individuals were frozen with 8% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) in liquid nitrogen; these cells are further refered to as passage 2. Cells from the HTLV seropositives were washed and resuspended in a medium similar to the above and with human recombinant interleukin 2 (IL-2; 10 U/ml) (Boehringer GmbH, Mannheim, Germany) but without PHA. Between 2.5 and 7.5 ml of this suspension was added to an equal volume of a 106 cells/ml suspension of cord blood mononuclear cells that had been similarly stimulated for 2 to 4 days with PHA. The cord cells were also washed and resuspended in the IL-2 medium before being added to the donor
Peripheral
rated
on
lymphocytes. Medium was changed twice a week and the cells were followed for immortalization, for antigen expression by immunofluorescence with a polyclonal anti-HTLV-I-positive human serum, and for the production of p24 core antigen (common epitopes of HTLV-I and -II detected) by ELISA on the cul-
fluid (Coulter, Hialeah, FL). In the first weeks fresh stimulated cord cells were added when the cell numbers were falling. The cell lines that were obtained grow spontaneously widiout IL-2 but growth is enhanced by IL-2. ture
Detection
ofproviral DNA
The cultured lymphocytes (passage 2 or 3, with the exception of MOMJ and MOMS, of which passages 25 and 14 were analyzed, respectively) of 14 MO family members and 3 MW family members were treated by proteinase K digestion. DNA was extracted by phenol-chlorofonp extraction and ethanol precipitation and was then resuspended in Milli-Q water (Millipore, Bedford, CT). MT-4, HTLV-II Mo cells, and HUT78 cells were used as HTLV-I positive, HTLV-II positive, and negative controls, respectively, in the subsequent polymerase chain reaction (PCR). A globin PCR17 was done to control the quality of the extracted DNA. The PCR was performed with the primer pair SKI 10-SK111 amplifying a 186-base pair (bp) fragment of the pol region of HTLV-I and HTLV-II, which was subsequently detected by liquid hybridization using SKI 12 or SK188 as HTLV-I- or HTLV-II-specific probe, respectively.18 DNA from 105 cells was used under standard reagent conditions, being a 50-pl PCR of 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, a 200 pM concentration of each dNTP, a 1 pM concentration of each primer, and 1.25 units of Taq polymerase (AmpliTaq; PerkinElmer, Norwalk, CT). Amplification was carried out in a TCI DNA thermal cycler (Perkin-Elmer) for 40 cycles: 94°C for 1 min 30 sec, 55°C for 1 min, and 72°C for 1 min. Five microliters of the amplified pol fragment together with 32P end-labeled probe was denatured in a final concentration of 100 mM NaCl at 95°C for 10 min and hybridized at 55°C for 60 min. This liquid hybridization mixture was separated on a 6% polyacrylamide gel, and subsequently the gel was dried and autora-
diographed.
Nested PCR using the tax/rex primer sets TRIO 1-104" designed to amplify both HTLV-I and -II was performed on the same samples under standard reagent conditions but with 3 mM MgCl2 instead of 2 mM MgCl2 Amplification with the outer TR101-102 primer set was carried out in a Gene Amp PCR system 9600 (Perkin-Elmer) with 35 cycles of 95°C for 30 sec, 50°C for 30 sec, and 72°C for 45
sec.
Two microliters of the
amplification product was used for the inner PCR (TR103-104) with 25 cycles of 95°C for 1 min, 50°C for 1 min
outer
30 sec, and 72°C for 1 min 30 sec in a Biometra TrioThermoblock (Biometra, Göttingen, Germany). This 159-bp PCR product was visualized in a 6% polyacrylamide gel by ethidium bromide staining and ultraviolet (UV) transillumination.
Sequencing of HTLV-I long terminal repeat The HTLV-I LTR of 10
PCR-positive family
members
(MOMS [passages 14, 25, and 27], MODI [passages 2, 25, and 27], MOGA [passage 2], MOMA [passage 3], MOBE [passage
1137
FAMILIAL HTLV-I IN ZAIRE
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