No genetic evidence for involvement of Deltaretroviruses in adult patients with precursor and mature T-cell neoplasms
© Burmeister et al; licensee BioMed Central Ltd. 2007
Received: 20 September 2006
Accepted: 07 February 2007
Published: 07 February 2007
The Deltaretrovirus genus comprises viruses that infect humans (HTLV), various simian species (STLV) and cattle (BLV). HTLV-I is the main causative agent in adult T-cell leukemia in endemic areas and some of the simian T-cell lymphotropic viruses have been implicated in the induction of malignant lymphomas in their hosts. BLV causes enzootic bovine leukosis in infected cattle or sheep. During the past few years several new Deltaretrovirus isolates have been described in various primate species. Two new HTLV-like viruses in humans have recently been identified and provisionally termed HTLV-III and HTLV-IV. In order to identify a broad spectrum of Deltaretroviruses by a single PCR approach we have established a novel consensus PCR based on nucleotide sequence data obtained from 42 complete virus isolates (HTLV-I/-II, STLV-I/-II/-III, BLV). The primer sequences were based on highly interspecies-conserved virus genome regions. We used this PCR to detect Deltaretroviruses in samples from adult patients with a variety of rare T-cell neoplasms in Germany.
The sensitivity of the consensus PCR was at least between 10-2 and 10-3 with 100% specificity as demonstrated by serial dilutions of cell lines infected with either HTLV-I, HTLV-II or BLV. Fifty acute T-cell lymphoblastic leukemia (T-ALL) samples and 33 samples from patients with various rare mature T-cell neoplasms (T-PLL, Sézary syndrome and other T-NHL) were subsequently investigated. There were no cases with HTLV-I, HTLV-II or any other Deltaretroviruses.
The results rule out a significant involvement of HTLV-I or HTLV-II in these disease entities and show that other related Deltaretroviruses are not likely to be involved. The newly established Deltaretrovirus PCR may be a useful tool for identifying new Deltaretroviruses.
Retroviruses are the main etiologic agents in a variety of malignant diseases in animals . Bovine leukemia virus (BLV) was the first Deltaretrovirus to be discovered in 1969 by electron microscopy , but it was not until 1985 that the first complete nucleotide sequence of an isolate was reported . Since the discovery of HTLV-I  and HTLV-II  and their closely related simian counterparts STLV-I  and STLV-II  several Deltaretrovirus isolates have been described in various non-human primate species. In 1994, a third simian Deltaretrovirus, later designated as STLV-III, was identified in a Hamadryas Baboon (Papio Hamadryas) [8–10]. Until recently no human counterparts of STLV-III were known, but in 2005 two independent research groups described human isolates that showed high homology to STLV-III and were considered to be HTLV-III isolates [11, 12]. Moreover, a fourth Deltaretrovirus was identified in a single human individual from the Rain Forest in Cameroon. It did not show sufficient homology to be classified as primate T-cell lymphotropic virus (PTLV) type I, II or III and was thus considered to be a species representative of a hitherto unknown putative PTLV-IV virus group .
HTLV-I and STLV-I are etiologically linked to the induction of certain T-cell lymphomas/leukemias in their hosts [13, 14]. The oncogenic action of the virus is mediated by the viral tax and rex genes that act as transcription factors, thereby promoting cell growth and malignant transformation However, the etiology of many human malignant T-cell and T-/NK-cell disorders is still not well understood. On the other hand a great deal of knowledge has been gained in the last years on the molecular biology of Deltaretroviruses, since a large number of new isolates have been described. To investigate the possible involvement of Deltaretroviruses in various human T-cell neoplasms, we have constructed a novel Deltaretrovirus consensus PCR based on nucleotide sequence alignments of all 42 complete Deltaretrovirus isolates published to date. Highly conserved virus genome regions were identified that allowed the construction of a generic PCR, capable of detecting all known Deltaretroviruses.
Accession number of the 42 virus isolates used in the nucleotide sequence alignments.
EMBL/Genbank/DDBJ accession number
[EMBL:AY563954] (Brazilian isolate), [EMBL:AY563953] (Brazilian isolate), [GenBank:NC_001436], [EMBL:AF259264] (isolate WHP from China), [EMBL:AF139170] (from an HTLV-I/II seroindeterminate patient), [EMBL:J02029] (Japanese ATL isolate), [EMBL:AF033817], [EMBL:L03561], [EMBL:D13784] (Caribbean isolate), [EMBL:L02534] (Melanesian isolate), [EMBL:U19949] (isolate from an ATL patient), [EMBL:AF042071] (isolate from Germany), [EMBL:L36905] (from a patient with post-transfusion spastic paraparesis)
[GenBank:NC_001488], [EMBL:AF326584] (Brazilian isolate), [EMBL:AF326583] (Brazilian isolate, strain RP329), [EMBL:AF412314] (with HIV coinfection), [EMBL:AF139382] (Brazilian isolate), [EMBL:AF074965] (isolate from a Guahibo Indian from Venezuela), [EMBL:M10060], [EMBL:L11456] (Guyami Indian isolate), [EMBL:Y14365] (Congolese Bambute Efe Pygmy isolate), [EMBL:X89270] (Italian isolate), [EMBL:L20734], [EMBL:Y13051] (African isolate, subtype b)
[GenBank:NC_000858] (from a naturally infected tantalus monkey from Central Africa), [EMBL:AY590142] (in Macaca arctoides), [EMBL:Z46900] (from Celebes macaques), [EMBL:AF074966] (isolate Tan90 from Central African Republic)
[GenBank:NC_001815], [EMBL:Y14570] (STLV-PP from Pan paniscus), [EMBL:U90557] (from Pan paniscus)
[EMBL:AF517775] (from Papio hamadryas papio from Senegal), [GenBank:NC_003323] (from red-capped mangabeys (Cercocebus torquatus) from Cameroon), [EMBL:AY217650] (from Theropithecus gelada), [EMBL:AY222339] (from a red-capped mangabey – Cercocebus torquatus torquatus – from Nigeria), [EMBL:Y07616] (STLV-PH969 from a Hamadryas baboon)
[GenBank:NC_001414], [EMBL:AF257515] (from a Holstein cow from Argentina), [EMBL:K02120] (Japanese isolate), [EMBL:AF033818], [EMBL:D00647] (Australian isolate)
It should be noted that the PCR sensitivity could be further increased to 10-3 – 10-4 (while retaining specificity) by lowering the annealing temperature to 60°C and increasing the number of PCR cycles but at the expense of a stronger 657 bp sideband.
Patient and disease characteristics.
Disease entity (N)
Median age (range) [years]
Precursor T-cell (50)
Early T-cell lymphoblastic
Cortical (thymic) T-cell lymphoblastic
Mature T-cell lymphoblastic
Mature T-cell (31)
Sézary syndrome and Mycosis fungoides
Ki-1 large T-cell lymphoma
Intestinal T-cell lymphoma
Other (unspecified) peripheral T-NHL
NK cell disorders (2)
29 and 78
None of the investigated samples yielded a PCR product indicative of the presence of a Deltaretrovirus. The parallel investigation of positive controls led to the expected results.
While the etiological involvement of HTLV-I in endemic adult T-cell leukemia/lymphoma is beyond dispute there have been repeated controversies whether this virus might also play a role in other T-cell neoplasms such as T-prolymphocytic leukemia , Sézary syndrome or Mycosis fungoides [20–22]. The situation is further complicated by the fact that the classification of T-NHLs has been evolving and changing over the years as new disease entities are recognized and refined diagnostic criteria are established [23, 24]. Thus the results of older studies may not always be fully transferable to today's situation. Some investigators have also suggested that a truncated HTLV-I may play a role in certain T-NHLs [19, 25]. On the other hand HTLV-II has not been convincingly linked to any specific malignant T-cell disorder. The simian Deltaretroviruses are implicated in lymphomatous diseases in various simian hosts (reviewed in ). The newly discovered HTLV-III and HTLV-IV isolates have not yet been fully characterized, and their distribution or possible involvement in human disease is unknown.
A few previous investigations performed on precursor T-cell neoplasms in Germany were mainly based on HTLV-I serology  which may not be as reliable as nucleic acid-based techniques . Germany has a low HTLV-I seroprevalence, but virus isolates without linkage to endemic areas have occasionally been reported .
The causes of most malignant T-cell disorders are only partially understood. Numerous recurrent genetic aberrations have been described  but a clear and detailed model of disease development still does not exist. Oncogenic viruses such as Epstein-Barr virus or HTLV-I are well established causative factors in various human T-cell lymphomas and leukemias . It appears possible that yet undetected Deltaretroviruses may play a role in human T-cell malignancies.
We developed a consensus PCR for detecting Deltaretroviruses based on highly conserved genomic sequences of all published complete Deltaretrovirus isolates. Since this PCR is based on highly interspecies conserved sequence motifs it may also be capable of detecting related but hitherto unknown Deltaretroviruses. However, despite high sensitivity and specificity of our PCR approach, no Deltaretrovirus-positive cases were found in our series of samples.
The results rule out a role of known Deltaretroviruses in the disease entities under investigation here. The involvement of a hitherto undetected Deltaretrovirus is not completely excluded but rendered more unlikely. Truncated proviruses that have lost their 5'-region with the tRNA binding site may also escape detection by our PCR system. Despite these negative results, our newly established consensus PCR may be a useful tool in the search for and characterization of new Deltaretroviruses in primates and other mammals.
The following cell lines were used: BL3.1 (infected with BLV, a bovine lymphoma cell line, ), MJ [G11] (infected with HTLV-I, derived from a human cutaneous T-cell lymphoma, ) and Mo T (infected with HTLV-II, derived from a patient with hairy cell leukemia, ). All cell lines were obtained from the ATCC (Acc No CRL-2306, TIB-8294, and TIB-8066, respectively). Cell culture was done according to the recommendations of the supplier. DNA isolated from the cell lines was used to prepare serial dilution rows.
DNA was isolated from sample material or cell lines using the PureGene kit (Biozym Diagnostik, Hessisch Oldendorf/Germany) and dissolved in Tris/EDTA buffer at a concentration of 60 ng/μl.
Preparation of cell line dilution series
Serial dilutions of cell line DNA in DNA from buffy coats of blood donors were prepared as recently described .
All samples were obtained for diagnostic purposes, and we retrospectively investigated archived material. The patients had given their consent for scientific investigations. The T-precursor samples were obtained within the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL). Our study complied with the Helsinki Declaration.
The HotStarTaq kit (QIAGEN, Hilden/Germany) was used with 200 ng sample DNA, 400 nM of each PCR primer delta-F 5'-CARKTGGGGGCTCGTCCGGGAT-3' and delta-R 5'-GGCCTGGAGGCGYTCHRGTTTAA-3', buffer conditions and polymerase mix as recommended by the supplier. The primers were optimized derivatives of those previously published  and HPLC-purified. The following cycler program was used on a GeneAmp 2400 Cycler (PerkinElmer): 94°C for 15 minutes, 40 cycles (94°C for 20 seconds, 62°C for 20 seconds, 72°C for 90 seconds), 4°C.
Nucleotide sequence alignments
All available complete nucleotide sequences from Deltaretrovirus isolates were collected from the EMBL/Genbank/DDBJ database (Table 1), converted into FASTA file format, and aligned using the ClustalX software .
The PHYLIP program package , version 3.65 for MacOS X, with the program modules dnacomp and drawgram was used with the default parameters to construct a phylogenetic tree from the aligned sequences.
Immunophenotyping by FACS analysis was done essentially by standard methods described elsewhere .
human T-cell lymphotropic virus
simian T-cell lymphotropic virus
bovine leukemia virus
primate T-cell lymphotropic virus
polymerase chain reaction
Non Hodgkin lymphoma
acute lymphoblastic leukemia
The authors are grateful to Mara Molkentin, Barbara Komischke and Rita Lippoldt for their skillful technical assistance. They are indebted to Prof. Harald Stein (Head of the Dept. of Pathology, Charité Campus Benjamin Franklin) for giving them access to lymphoma DNA samples. TB and SS were supported by the Deutsche Krebshilfe (German Cancer Aid, grant 10-1988-Bu1). TB is also supported by the Berliner Krebsgesellschaft (Berlin Cancer Society). Dr. J. Weirowski critically read the manuscript.
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