1 Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands
2 Laboratoire Retrovirus, UR36 IRD, 911 Avenue Agropolis, BP 5045, 34032 Montpellier Cedex 1, France
Correspondence
Jonathan Heeney
heeney{at}bprc.nl
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ABSTRACT |
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EMBL database accession numbers of the STLVcpz sequences described in this study are Y18902, Y18903 and AJ493584AJ493594.
Published ahead of print on 11 December 2002 as DOI 10.1099/vir.0.18778-0.
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INTRODUCTION |
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In 1982, the existence of naturally occurring HTLV-related viruses in non-human primates was first demonstrated by the presence of anti-HTLV antibodies in Japanese macaques (Macaca fuscata) (Miyoshi et al., 1982). Since then, many simian T-cell lymphotropic viruses (STLV) from different lineages (STLV-I, STLV-II, STLV-L) have been characterized from a wide range of non-human primate species (Digilio et al., 1997
; Giri et al., 1994
; Goubau et al.; 1994
; Ishikawa et al., 1987
; Mahieux et al., 1997a
; Meertens et al., 2001
; errienet et al., 2001
; NSaksena et al., 1994
; Takemura et al., 2002
; Vandamme et al., 1996
). With regard to the presence of STLV-I infections in great apes, serologically related, but clearly distinct, viruses have been found in orangutans (Ibuki et al., 1997
; Verschoor et al., 1998
), common chimpanzees, pygmy chimpanzees and gorillas (Giri et al., 1994
; Ishikawa et al., 1987
; Koralnik et al., 1994
; Saksena et al., 1994
; Vandamme et al., 1996
; Voevodin et al., 1997
). STLV-I primarily causes asymptomatic infections in their natural hosts but rare cases of STLV-associated leukaemia and/or lymphoma have been reported in African green monkeys, baboons, macaques and gorillas (Franchini & Reitz, 1994
). HTLV-I and STLV-I cannot be separated into different phylogenetic clusters or clades (Saksena et al., 1993
), suggestive of multiple inter-species virus transmissions in the past and present (Koralnik et al., 1994
).
Little is known about the modes of transmission of STLV in non-human primates. In this study, we examined the incidence and possible modes of transmission of HTLV-I-like infections in a closed breeding colony of Western common chimpanzees (Pan troglodytes verus), all descending from a group of founder animals originating from Sierra Leone. A reduced rate of transmission was monitored of a virus characterized as STLVcpz, a virus that naturally infects chimpanzees.
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METHODS |
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Blood was drawn from all colony animals on a regular yearly basis as part of the routine health surveillance programme. Serum and peripheral blood mononuclear cells (PBMCs) were separated and stored at -20 and -80 °C, respectively. Few serum samples were available prior to 1980.
Detection of antibodies cross-reactive with HTLV-I antigens.
Serum samples from the chimpanzees were assayed for antibodies to HTLV-I antigens by an ELISA developed in-house (Warren et al., 1998). The specificity of the ELISA responses was confirmed by Western blot (WB) analysis using HTLV blot 2.4 (Genelabs Diagnostics), which detects both anti-HTLV-I and anti-HTLV-II antibodies.
PCR and sequence analysis.
Genomic DNA was isolated from PBMCs using standard DNA isolation procedures. Primers env-1, env-2 and env-22 were used in a semi-nested PCR for the amplification of a 522 bp fragment from the envelope gene of HTLV-I/STLV-I (Ibrahim et al., 1995; Koralnik et al., 1994
). Genomic DNA (1 µg) was used as template for each PCR assay. Amplification reactions were performed in the presence of 200 µM of each dNTP, 50 pmol of each primer, 10 mM Tris/HCl (pH 8·3), 50 mM KCl and 1·25 U TaqGold (Applied Biosystems). The reaction mixtures for the env PCR contained 2·3 and 2·1 mM MgCl2 in combination with the outer and semi-nested primer sets, respectively. Both amplification reactions were performed for 35 cycles consisting of a 30 s denaturation step at 94 °C, a 30 s annealing step at 56 °C and an elongation step of 1 min at 72 °C.
PCR fragments were analysed on a 1·5 % agarose gel, bands were cut out and PCR fragments isolated using the QIAquick Gel Extraction kit (Qiagen). Fragments were cloned in the pGEM-T cloning vector (Promega) and sequenced using the Sequenase DNA Sequencing kit, version 2.0 (USB). Sequences were analysed using GENEWORKS, version 2.3 (IntelliGenetics).
Phylogenetic analysis.
Analysis of the HTLV/STLV sequences was performed using the MACVECTOR, version 6.0, and ASSEMBLYLIGN software packages (Oxford Molecular). Phylogenetic analysis of nucleotide sequences was performed using PAUP, version 4.0b10 (Swofford, 2002). Pairwise distances were calculated using the HKY85 method and the neighbour-joining method was used to create a phylogram.
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RESULTS |
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Of all 197 chimpanzees tested, 22 showed antibodies to HTLV-I antigens in their blood (Table 1). The majority of cases (n=20) were found in the group of founder animals, while only two HTLV-positive animals were detected in the offspring born in the colony. All positive ELISA results were confirmed by HTLV WB analysis.
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From the compiled data of the entire chimpanzee colony over two decades, it is clear that transmission of this virus was a relatively rare event. Prior to 1986, one seroconversion event was documented, while between 1986 and 2001, only two seroconversions were observed. Ch-So is an adult female belonging to the founder population. Serum samples from Ch-So, which were collected prior to 1973, were negative in ELISA and WB analyses (Fig. 2, lane 3) but this animal seroconverted in 1973 (Fig. 2
, lane 4). The two other seroconversions occurred more recently in adult chimpanzees that were born in captivity in the colony. These infections were linked to two separate events. One transmission of STLVcpz occurred during a fight between two males, Ch-Co and Ch-Bi. During this event, the aggressor, Ch-Bi, who drew blood, later became STLVcpz-seropositive (Fig. 2
, lanes 56, Ch-Bi and lane 7, Ch-Co). The other case involved a seronegative female, Ch-Ev, who was in a breeding programme with a male, Ch-Fr, who was unknowingly infected with STLV. The actual event associated with transmission could not be documented specifically but seroconversion occurred shortly after conception (Fig. 2
, lanes 89, Ch-Ev and lane 10, Ch-Fr).
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DISCUSSION |
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The discrepancy between incidence rates of founder animals and offspring in our colony (54 versus 1 %) can, in part, be explained by the history of the older animals. In the 1970s, five female founder animals received blood cell transfusions and/or skin grafts from donors now recognized as STLVcpz carriers. The likelihood of iatrogenic transmission is made plausible by the documented seroconversion of Ch-So. This animal first tested positive shortly after having received a blood transfusion and skin transplantation from Ch-Lo who was STLVcpz-infected, as tested in retrospect on sera from 1975 and 1986. Analysis of plasma from Ch-So sampled 23 years after the transfusion event revealed infection with a variant that differed in only 3 aa with the sequence of the donor, Ch-Lo. Comparable sequence differences were described by others who also reported minor sequence differences 8 years after transmission of STLV from macaques to baboons (Voevodin et al., 1996). The 15 other STLV-positive founders (41 %) have not had comparable treatment and most likely acquired the infection via a natural route of transmission. Since that time, serological monitoring of virus infections in our colony has minimized the chance of iatrogenic transfer of STLVcpz infection. Indeed, the infection rate of animals born in this colony has dropped to only two cases of transmission in 160 animals (1 %).
In humans, HTLV-I is transmitted commonly from mother-to-child through breast-feeding (Chiodo et al., 1986; Miyoshi et al., 1992
; Nyambi et al., 1996
). In contrast to the human situation, this appears to be an uncommon mode of transmission in this chimpanzee breeding colony. A considerable proportion of the 160 animal offspring, 79animals, were natural births from STLV-positive females. Depending on the individual situation, all the animals were breast-fed for a minimum of 3 months before weaning but the majority continued to nurse for periods of a year or more. After weaning, none of the babies were found to be infected with STLVcpz. Anti-STLV antibodies in milk and transient maternal antibody titres in sera from newborn animals were detectable by ELISA, but PCR analysis from milk samples from infected nursing females did not reveal the presence of virus (data not shown).
When compared to the data of the wild-derived founder chimpanzees, the transmission rate in the colony after 1986 was very low. The nature of social management of the colony may have attributed to the low number of new infections. Over the past 20 years, these animals have been socially grouped into stable family units and age-related peer groups. We propose that such social configurations reduce transmission, which may occur in the wild following blood-to-blood contact resulting from territorial fighting of competitive family groups or raids by troops of mature males. The latter may be an explanation for the 70 % infection rate among the wild-caught male founders when related to the figures of naturally infected female founders (eight animals; 30 %).
Clearly, the virus found in our colony was introduced by a number of naturally infected founder animals and is not an HTLV-I strain introduced by iatrogenic transfer. We have characterized the virus as STLVcpz belonging to a larger group of chimpanzee viruses, comprising STLV PTR-114.1, -3570 and -X43, and members of the large HTLV-I/STLV-I central African subtype B clade. Several authors (Koralnik et al., 1994; Voevodin et al., 1997
) have speculated that chimpanzees may be infected with distinct STLV strains depending on the subspecies and geographical distribution. The STLVcpz from previous studies were derived from chimpanzees that had either an indeterminate geographical origin or may have acquired the infection while in captivity with animals from other subspecies or origin. Since the specific origin and subspecies of the colony of animals is known, our data imply that the origin of this group of STLVcpz is Pan troglodytes verus of the Sierra Leone region.
From our study, it is apparent that mother-to child transmission in chimpanzees is very uncommon. Based on only a few examples, we suspect that the majority of transmission events in the wild occurs during social conflicts among rival troops or families. Furthermore, to sustain this STLV infection in chimpanzee populations, such events must occur relatively frequently in wild, free-ranging chimpanzees.
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ACKNOWLEDGEMENTS |
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Received 15 August 2002;
accepted 27 November 2002.