Evolutionary Spread and Recombination of Porcine E
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病菌学杂志 2005年第1期
Paul-Ehrlich-Institut, Langen, Germany
ABSTRACT
Different Suiformes with increasing phylogenetic distance to the common pig (Sus scrofa) were assayed for the presence of porcine endogenous retroviruses (PERV) in general (pol gene), while the distribution of long terminal repeat (LTR) types (with or without repeats in U3) and env genes (classes A, B, and C) were determined in detail. PERV was not detectable in the most distantly related species, while classes PERV-A and PERV-B are present in Suiformes originating in the Pliocene epoch, and class PERV-C was detectable only in S. scrofa and in closely related species originating in the Holocene epoch. This distribution pattern of PERV classes is in line with our previous study on the age of PERV (45) and suggests an African origin of about 7.5 million years ago (MYA) and a gradual spread of PERV through the Suiformes. It seems likely that PERV-C originated more recently (1.5 to 3.5 MYA) by recombination with a homologue of unknown descent, while the origin of the repeatless LTR was a separate event approximately 3.5 MYA.
TEXT
Porcine endogenous retroviruses (PERV) pose a serious challenge in xenotransplantation, i.e., the transfer of cells, tissues, or organs from one species to another for therapeutic reasons, since they are present in all cells and in almost every individual pig (27) and are transmitted vertically.
Endogenous retroviruses have been detected in the genomes of all vertebrate species (4, 14), and their general organization corresponds to that of exogenous retroviruses (4, 40, 46); however, most are replication-incompetent, while only a minority are functional, as reported, e.g., for mice (5, 10, 41) and pigs (1, 2, 21, 26, 28, 29).
For gamma-type PERV, three different classes exist, designated PERV-A, PERV-B, and PERV-C (1, 23, 42). The first two classes (7, 42) productively infect human cells in vitro, thus posing a serious risk in xenotransplantation, while the latter (1) does not replicate on human cells. There are only minor genetic differences between the classes, being most prominent in the receptor-binding domain of the Env protein. In addition, there are two different types of long terminal repeats (LTRs) that significantly affect the replication properties of single viruses (37, 47) through binding of transcription factor NF-Y (36). PERV-A and PERV-B proviruses demonstrate LTRs that harbor distinct 39-bp repeats in U3 which enable high transcription levels and adaptation to new host cells by multimerization of these repeats, with the transcriptional activity being generally stronger if more repeats are present. On the other hand, PERV-A and PERV-C were found to display repeatless LTRs (37, 47). Although sequences homologous to the 39-bp repeat are present in these LTRs, they are not organized in a repetitive manner and do not show multimerization as a response to replication cycles in their hosts. This LTR type confers a very low transcriptional activity, and we propose that it originated as an adaptation to endogenous replication of PERV. This leaves PERV-A as the only PERV harboring both types of LTR.
We recently determined the age of PERV as having an upper limit of approximately 7.6 x 106 years, while the repeatless LTR type evolved approximately 3.4 x 106 years ago and is therefore the phylogenetically younger structure (45). The age determined for PERV correlates with the time of separation of pig species (Suidae, Sus scrofa) from their closest relatives, American-born peccaries (Tayassuidae, Pecari tajacu), 7.4 x 106 years ago. While the time of the phylogenetic split was calculated by using mitochondrial DNA sequences (19), which tend to underestimate the time, archaeological data suggest a split of Suidae and Tayassuidae in the Eocene epoch, about 15 MYA (43, 44).
To extend the above-mentioned study on PERV's relative age, we analyzed a wide array of Suidae of Eurasian and African origin, as well as samples of the Tayassuidae as the closest evolutionary relatives of pigs, for the presence or absence of PERV. In anthropological studies, the presence or absence of human endogenous retroviruses in various hominoids has been used successfully to designate the age of the proviruses (17, 18, 39). Therefore, we aimed for a similar approach to define the age and spread of the different PERV genotypes more accurately.
Sample acquisition. Suiformes of various species were assayed. They are listed below in decreasing order of relationship to Sus scrofa. The epoch when the phylogenetic split occurred, as determined by archaeological evidence (43, 44) and genetic reconstruction (8, 11, 15, 24), is given in parentheses: Sus barbatus, Sus celebensis (Holocene), Potamochorus porcus, Potamochorus larvatus (Pleistocene), Phacochorus africanus, Babyrousa babyrussa (Pliocene), and Tayassuidae pecari (Eocene). A detailed classification of the families of Suidae and Tayassuidae, including subfamilies and genera, is given in Fig. 1.
Phylogenetic analysis. Recombination is an important process that has impact on biological evolution at different levels. Recombination reshuffles existing variation and even creates new variants at the amino acid level. It breaks down linkage disequilibrium and, especially, has a significant impact on the evolution of viral pathogens (12, 13). Common phylogenetic analysis depicts the history of a number of sequences as a tree where two sequences share a common ancestor. This model fails when recombination is present, because a sequence now is no longer limited to a single ancestor. But this poses a problem only if one tries to reconstruct a phylogenetic tree from a sample for which it is unknown if recombination has occurred. Here, we try to determine whether recombination played a role in PERV evolution. A number of methods have been developed to detect recombination, and their relative performance has been analyzed (9, 30, 48). In accordance with these comparisons, we have chosen SimPlot because it performed well for sequences as divergent as different PERV envelope classes. It produces very few false-positive results, and uses a phylogenetic method (bootstrapping) for detection, which is compatible with other analyses, and has a graphical interface, making comparison of analyses easy.
The detection of PERV in different Suiformes was based on five samples per species on average. The reader should bear in mind that, as no PERV-C locus is known that can be followed through time, the determined sequences represent independent snapshots of different specimens of different species. Analyses of modern pigs suggest a number of (6 to 10) replication-competent proviruses (26), 30 to 50 full-length PERV (1, 2, 28, 29), and 100 to 200 loci encompassing partial proviruses (35). Even under the assumption of less frequent PERV integration in older Suiformes, there should be enough sequences for detection by PCR. Thus, we believe that the limited number of animals should not pose a problem when interpreting samples testing negative. A cytochrome b phylogenetic tree was generated to calibrate the relative age of PERV in different Suiformes samples (Fig. 2A), including the archaeological data on Suiformes fossils. This tree is in line with previous studies using cytochrome b sequences (31) or archaeological data (43, 44), but includes three additional families not analyzed hitherto (S. celebensis, P. porcus, and P. larvatus). PERV sequences are completely undetectable in T. pecari and in B. babyrussa (Eocene and Miocene epochs, respectively), while the earliest presence of PERV happens in P. africanus associated with the late Miocene or early Pliocene epoch (3.5 to 7.5 MYA) (Fig. 2A and B), which confirms our recent study of the age of PERV (45). Only PERV-A can be detected in samples of P. africanus, while the earliest appearance of PERV-B is in the slightly younger P. porcus associated with the early Pleistocene epoch (3.5 to 7.5 MYA) (Fig. 2A and B). In contrast, PERV-C is detected for the first time in the much younger S. barbatus of the early Holocene epoch (0.1 to 1.5 MYA) (Fig. 2A and B). The repeatless LTR, on the other hand, has been detectable since appearing in P. larvatus and P. porcus of the late Pleistocene epoch (1.5 to 3.5 MYA) (Fig. 2B) (45). While we therefore assume a separate origin for PERV-C and the repeatless LTR, both could have emerged together as discussed below.
Patience et al. (28) also analyzed a series of Suiformes for the presence of beta- and gamma-retroviruses, and while our results match at large, we disagree on some minor points. As it is difficult to acquire exotic DNA samples in the first place, we were not able to repeat the study with the exact set of Suiformes used by Patience et al.; some species may differ in subspecies, and only a limited number of specimens were available for analysis. In addition, the primer sets used by Patience et al. and by us differ in sequence and therefore may lead to different results, particularly when only minor sequence variations have an impact on the detection limit. Hence, these differences may account for all minor disagreements. In contrast to our own findings, Patience at al. detected PERV-C in the sample of P. larvatus of the Pleistocene epoch, therefore shifting the emergence of PERV-C by one epoch to 1.5 to 3.5 MYA, though coinciding with the appearance of the repeatless LTR.
Analysis of interclass mosaicism. SimPlot, an interactive 32-bit program for Windows, was created to plot sequence similarity versus position (32). Briefly, SimPlot calculates and plots the percent identity of the query sequence to a panel of reference sequences in a sliding window, which is moved stepwise across the alignment. The window and step sizes are adjustable. Alignments were analyzed for recombination breakpoints by maximization of 2 as previously described (34, 38).
In general, the homologies between PERV-A and PERV-C are approximately 85%, while the similarities between PERV-B and either PERV-A or PERV-C barely exceed 70%. In general, gamma-type retroviruses, including PERV, share a common homology of approximately 60%. This fact and the occurrence of repeatless LTRs in both PERV-A and PERV-C but not in PERV-B (see above), leads to the assumption of a common evolutionary origin for these two classes. A possible recombination of PERV classes is most likely in the LTR or env sequences, and analysis revealed negligible sequence variations in gag and pol (data not shown).
Detection of recombination was carried out by comparing PERV env sequences obtained from various Suiformes to reference sequences for PERV-A (AJ133817) (7), PERV-B (AJ133818) (7), and PERV-C (AF038600) (1). Some PERV env sequences obtained from "old" Suiformes showed the PERV-A or PERV-C sequence and an analysis was therefore not necessary, while A/C recombinant sequences obtained from these samples showed a high degree of homology. Because of this finding and to keep the representation in Fig. 3 as simple as possible, the analyses shown in Fig. 3 were displayed with only one representative sequence per species.
The first recombination event between PERV envelope genes is detectable in S. celebensis, affecting the 3' end of a PERV-A env, which acquires some sequence homology of PERV-C (Fig. 3 B). This change affects mostly the C-terminal region of the env gene, but does not change the class of the env gene (Fig. 3 A). Therefore, S. celebensis is still considered negative for PERV-C as revealed for the other evolutionary older Suiformes (Fig. 2 B). This observation has two important implications. The change leads to a slightly truncated R peptide, with this R peptide structure being associated with repeatless LTRs in PERV-A proviruses. In addition, a truncated cytoplasmic tail has been shown to increase the fusogenic potential of several gamma-type retroviruses (6, 16, 33) and of PERV-A and PERV-B (3). As PERV-C seems to harbor only repeatless LTRs, it is conceivable that recombination happened between only one repeatless PERV-A provirus and the unknown PERV-C progenitor. The changed R peptide may have played an essential role in the origin of PERV-C by enabling the incorporation of the modified envelope into the capsid in the first place or by offsetting some of the limitations posed by the less active LTR through larger fusogenicity. However, the latter would also apply to repeatless PERV-A proviruses (3). A second recombination event is detectable with S. barbatus, affecting the receptor binding domain between nucleotide positions 300 and 900 of the env gene (Fig. 3 C), thus creating a new class of PERV (Fig. 3 A) and enabling the detection of PERV-C for the first time (Fig. 2 B). In samples from S. scrofa, PERV-C was detectable with reasonable abundance, as the homology with evolutionary older forms is already as high as 98%. Therefore, mostly point mutations are sufficient to change the query sequences to match the env reference sequence almost perfectly (Fig. 3 D).
There was no detectable recombination event between PERV-B and either PERV-A or PERV-C in the samples (data not shown), but recent publications of PERV sequences suggest that the different PERV classes still recombine (20, 49).
Recombination has been well documented for RNA viruses (50) and most likely involves similarity-assisted template switching (22, 25). The frequency of successful intertypic genetic exchanges is determined by, among other factors, (i) properties inherent to the process of viral replication, that is, the error susceptibility of viral reverse transcriptase and cellular RNA polymerase II; (ii) the frequency of cross-species transmission; (iii) the viability of the recombinant progeny; and (iv) the evolutionary gain.
Conclusion. PERV originated in African members of the Suidae family about 7.5 MYA (Fig. 2A and B), with repeat-harboring U3 sequences representing the exclusive type of LTR. The repeatless LTR developed during the early Pliocene epoch (3.5 MYA) in African Suidae, with its weak transcriptional activity most likely being an adaptation to an endogenous replication cycle. It is difficult to conclude whether PERV-A and PERV-B developed independently or whether both virus classes originated from a similar event like the A and C recombination described here. If so, the event took place too early to be clarified in this study. PERV-C, being much more closely related to PERV-A than to PERV-B, did not arise in the same epoch as PERV-A and PERV-B, but originated nearly 3.5 million years later due to a recombination event between PERV-A and an unknown ancestor. While the A and C recombination coincides with the appearance of the repeatless LTR, we assume that these events are independent from each other. Furthermore, we suggest that the recombination process leading to PERV-C involved an unknown ancestor and a PERV-A variant with a repeatless LTR.
Oligonucleotide sequences. Porcine cytochrome b sequences were amplified by nested PCR with outer forward primer 5'-GCT TAC CCT TTC CAA CTA GGC TTC-3' and outer reverse primer 5'-TTC GAA GTA CTT TAA TGG GAC AAG-3' and inner forward primer 5'-CAC ACA CTA GCA CAA TGG ATG CC-3' and inner reverse primer 5'-GAG GAT ACT AAT ATT CGG ATT GTT AT-3' by using a regimen of 35 cycles of denaturation (94°C for 30 s), annealing (58°C for 30 s), and elongation (72°C for 90 s). PERV polymerase sequences were detected by PCR with the specific primer pair PERV-pol-forward (5'-TTG ACT TGG GAG TGG GAC GGG TAA C-3') and PERV-pol-reverse (5'-GAG GGT CAC CTG AGG GTG TTG GAT-3') by using a regimen of 35 cycles of denaturation (94°C for 1 min), annealing (58°C for 1 min), and elongation (72°C for 3 min) as described in reference 7. For enhanced sensitivity, a nested PCR was performed with the same regimen with the following primer pair: PERV-pol-forward-nested (5'-GGT AAC CCA CTC GTT TTC TGG TCA-3') and PERV-pol-reverse-nested (5'-GAG CTG TGT AGG GCT TCG TCA AAG ATG-3'). The chances of detecting pol genes of non-PERV gamma-retroviruses or even from those of other related virus classes are negligible. PERV envelope gene class-specific detection was done with the same PCR regimen used for cytochrome b detection with the following primer pairs: PERV-A forward (5'-ATC CTA CCA GTT ATA ATC AAT-3') and PERV-A reverse (5'-GAT TAA AGG CTT CAG TGT GG-3'), PERV-B forward (5'-GGA TAA ATG GTA TGA GCT GG-3') and PERV-B reverse (5'-GCT CAT AAA CCA CAG TAC TAT-3'), and PERV-C forward (5'-CAC CTA TAC CAG CTC TGG ACA ATT-3') and PERV-C reverse (5'-TAA ACA ACC AGG CTC CAT TCT AAA-3'). Envelope gene reference sequences used in SimPlot (32) analysis were taken from previously published, full-length viruses PERV-A (AJ133817) (7), PERV-B (AJ133818) (7), and PERV-C (AF038600) (1).
Nucleotide sequence accession numbers. Sequences analyzed from Suiformes samples in this study were submitted to GenBank. Cytochrome b alignment: AY534296 through AY534303. PERV env sequences: S. scrofa (AY534304), S. barbatus (AY534305), and S. celebensis (AY534306). Additional env sequences were obtained from all species but showed significant homologies with already-published sequences. These sequences can be obtained from the authors.
ACKNOWLEDGMENTS
This study was supported by a grant from the German Ministry of Health (Bundesministerium für Gesundheit und Soziale Sicherung), Bonn, Germany.
We thank Stewart Lowden, Royal (Dick) School of Veterinary Studies, University of Edinburgh (United Kingdom) for supplying genomic DNA samples from various Suiformes used in this study. We thank the reviewers for critical reading and helpful suggestions on improving the manuscript.
Present address: Centre for Nanostructural Bioengineering, University of Queensland, 4072 Brisbane, Australia.
REFERENCES
Akiyoshi, D. E., M. Denaro, H. Zhu, J. L. Greenstein, P. Banerjee, and J. A. Fishman. 1998. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J. Virol. 72:4503-4507.
Armstrong, J. A., J. S. Potterfield, and A. T. DeMadrid. 1971. C-type virus particles in pig kidney cell lines. J. Gen. Virol. 10:195-198.
Bobkova, M., J. Stitz, M. Engelstadter, K. Cichutek, and C. J. Buchholz. 2002. Identification of R-peptides in envelope proteins of C-type retroviruses. J. Gen. Virol. 83:2241-2246.
Boeke, J. D., and J. P. Stoye. 1997. Retrotransposons, endogenous retroviruses and the evolution of retroelements, p. 343-436. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses, vol. 16. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Boone, L. R., P. L. Glover, C. L. Innes, L. A. Niver, M. C. Bondurant, and W. K. Yang. 1988. Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes. J. Virol. 62:2644-2650.
Christodoulopoulos, I., and P. M. Cannon. 2001. Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J. Virol. 75:4129-4138.
Czauderna, F., N. Fischer, K. Boller, R. Kurth, and R. R. T?njes. 2000. Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells. J. Virol. 74:4028-4038.
Douzery, E., and F. M. Catzeflis. 1995. Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia). J. Mol. Evol. 41:622-636.
Drouin, G., F. Prat, M. Ell, and G. D. Clarke. 1999. Detecting and characterizing gene conversions between multigene family members. Mol. Biol. Evol. 16:1369-1390.
Frankel, W. N., J. P. Stoye, B. A. Taylor, and J. M. Coffin. 1989. Genetic identification of endogenous polytropic proviruses by using recombinant inbred mice. J. Virol. 63:3810-3821.
Geisler, J. H. 2001. New morphological evidence for the phylogeny of Artiodactyla, Cetacea, and Mesonychidae. Am. Mus. Novit. 344:1-53.
Gibbs, A., C. H. Calisher, and F. Garcia-Arenal. 1995. Molecular basis of virus evolution. Cambridge University Press, Cambridge, United Kingdom.
Gibbs, M. J., and G. F. Weiller. 1999. Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus. Proc. Natl. Acad. Sci. USA 96:8022-8027.
Herniou, E., J. Martin, K. Miller, J. Cook, M. Wilkinson, and M. Tristem. 1998. Retroviral diversity and distribution in vertebrates. J. Virol. 72:5955-5966.
Irwin, D. M., T. D. Kocher, and A. C. Wilson. 1991. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32:128-144.
Januszeski, M. M., P. M. Cannon, D. Chen, Y. Rozenberg, and W. F. Anderson. 1997. Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J. Virol. 71:3613-3619.
Johnson, W. E., and J. M. Coffin. 1999. Constructing primate phylogenies from ancient retrovirus sequences. Proc. Natl. Acad. Sci. USA 96:10254-10260.
Kim, H. S., R. V. Wadekar, O. Takenaka, B.-H. Hyun, and T. J. Crow. 1999. Phylogenetic analysis of HERV-K LTR-like elements in primates: presence in some new world monkeys and evidence of recent parallel evolution in these species and in Homo sapiens. Arch. Virol. 144:2035-2040.
Kim, K. I., J. H. Lee, K. Li, Y. P. Zhang, S. S. Lee, J. Gongora, and C. Moran. 2002. Phylogenetic relationships of Asian and European pig breeds determined by mitochondrial DNA D-loop sequence polymorphism. Anim. Genet. 33:19-25.
Klymiuk, N., M. Muller, G. Brem, and B. Aigner. 2003. Recombination analysis of human-tropic porcine endogenous retroviruses. J. Gen. Virol. 84:2729-2734.
Krach, U., N. Fischer, F. Czauderna, and R. R. Tonjes. 2001. Comparison of replication-competent molecular clones of porcine endogenous retrovirus class A and class B derived from pig and human cells. J. Virol. 75:5465-5472.
Lai, M. M. 1992. RNA recombination in animal and plant viruses. Microbiol. Rev. 56:61-79.
Le Tissier, P., J. P. Stoye, Y. Takeuchi, C. Patience, and R. A. Weiss. 1997. Two sets of human-tropic pig retrovirus. Nature 389:681-682.
Montgelard, C., F. M. Catzeflis, and E. Douzery. 1997. Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences. Mol. Biol. Evol. 14:550-559.
Nagy, P. D., and A. E. Simon. 1997. New insights into the mechanisms of RNA recombination. Virology 235:1-9.
Niebert, M., C. Rogel-Gaillard, P. Chardon, and R. R. T?njes. 2002. Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells. J. Virol. 76:2714-2720.
Niebert, M., and R. R. T?njes. 2003. Analyses of prevalence and polymorphisms of six replication-competent and chromosomally assigned porcine endogenous retroviruses in individual pigs and pig subspecies. Virology 313:427-434.
Patience, C., M. Switzer, Y. Takeuchi, D. J. Griffiths, M. E. Goward, W. Heneine, J. P. Stoye, and R. A. Weiss. 2001. Multiple groups of novel retroviral genomes in pigs and related species. J. Virol. 75:2771-2775.
Patience, C., Y. Takeuchi, and R. Weiss. 1997. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3:282-286.
Posada, D., and K. A. Crandall. 2001. Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proc. Natl. Acad. Sci. USA 98:13757-13762.
Randi, E., V. Lucchini, and C. H. Diong. 1996. Evolutionary genetics of the suiformes as reconstructed using mtDNA sequencing. J. Mamm. Evol. 3:163-195.
Ray, S. C. 1998. SimPlot for Windows (version 1.6). S. C. Ray, Baltimore, Md.
Rein, A., J. Mirro, J. G. Haynes, S. M. Ernst, and K. Nagashima. 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68:1773-1781.
Robertson, D. L., B. H. Hahn, and P. M. Sharp. 1995. Recombination in AIDS viruses. J. Mol. Evol. 40:249-259.
Rogel-Gaillard, C., N. Bourgeaux, A. Billault, M. Vaiman, and P. Chardon. 1999. Construction of a swine BAC library: application to the characterization and mapping of porcine type C endoviral elements. Cytogenet. Cell Genet. 85:205-211.
Scheef, G., N. Fischer, E. Flory, I. Schmitt, and R. R. T?njes. 2002. Transcriptional regulation of porcine endogenous retroviruses released from porcine and infected human cells by heterotrimeric protein complex NF-Y and impact of immunosuppressive drugs. J. Virol. 76:12553-12563.
Scheef, G., N. Fischer, U. Krach, and R. R. T?njes. 2001. The number of a U3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells. J. Virol. 75:6933-6940.
Smith, J. M. 1992. Analyzing the mosaic structure of genes. J. Mol. Evol. 34:126-129.
Steinhuber, S., M. Brack, G. Hunsmann, H. Schwelberger, M. P. Dierich, and W. Vogetseder. 1995. Distribution of human endogenous retrovirus HERV-K genomes in humans and different primates. Hum. Genet. 96:188-192.
Stoye, J. P. 2001. Endogenous retroviruses: still active after all these years? Curr. Biol. 11:914-916.
Stoye, J. P., and J. M. Coffin. 1987. The four classes of endogenous murine leukemia virus: structural relationships and potential for recombination. J. Virol. 61:2659-2669.
Takeuchi, Y., C. Patience, S. Magre, R. Weiss, P. Banerjee, P. LeTissier, and J. Stoye. 1998. Host range and interference studies of three classes of pig endogenous retrovirus. J. Virol. 72:9986-9991.
Thenius, E. 1990. Even-toed ungulates, p. 1-15. In S. P. Parker (ed.), Grzimek's encyclopedia of mammals, vol. 5. McGraw-Hill, New York, N.Y.
Thenius, E. 1970. Zur Evolution und Verbreitungsgeschichte der Suidae (Artiodactyla, Mammalia). S. S?ugetierk. 35:321-342.
T?njes, R. R., and M. Niebert. 2003. Relative age of proviral porcine endogenous retrovirus sequences in Sus scrofa based on the molecular clock hypothesis. J. Virol. 77:12363-12368.
Wilkinson, D. A., D. L. Mager, and J. A. Leong. 1994. Endogenous human retroviruses, p. 465-535. In J. A. Levy (ed.), The Retroviridae, vol. 1. Plenum Press, New York, N.Y.
Wilson, C. A., S. Laeeq, A. Ritzhaupt, W. Colon-Moran, and F. K. Yoshimura. 2003. Sequence analysis of porcine endogenous retrovirus long terminal repeats and identification of transcriptional regulatory regions. J. Virol. 77:142-149.
Wiuf, C., T. Christensen, and J. Hein. 2001. A simulation study of the reliability of recombination detection methods. Mol. Biol. Evol. 18:1929-1939.
Wood, J. C., G. Quinn, K. M. Suling, B. A. Oldmixon, B. A. Van Tine, R. Cina, S. Arn, C. A. Huang, L. Scobie, D. E. Onions, D. H. Sachs, H. J. Schuurman, J. A. Fishman, and C. Patience. 2004. Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature swine. J. Virol. 78:2494-2501.
Worobey, M., and E. C. Holmes. 1999. Evolutionary aspects of recombination in RNA viruses. J. Gen. Virol. 80:2535-2543.(Marcus Niebert and Ralf R)
ABSTRACT
Different Suiformes with increasing phylogenetic distance to the common pig (Sus scrofa) were assayed for the presence of porcine endogenous retroviruses (PERV) in general (pol gene), while the distribution of long terminal repeat (LTR) types (with or without repeats in U3) and env genes (classes A, B, and C) were determined in detail. PERV was not detectable in the most distantly related species, while classes PERV-A and PERV-B are present in Suiformes originating in the Pliocene epoch, and class PERV-C was detectable only in S. scrofa and in closely related species originating in the Holocene epoch. This distribution pattern of PERV classes is in line with our previous study on the age of PERV (45) and suggests an African origin of about 7.5 million years ago (MYA) and a gradual spread of PERV through the Suiformes. It seems likely that PERV-C originated more recently (1.5 to 3.5 MYA) by recombination with a homologue of unknown descent, while the origin of the repeatless LTR was a separate event approximately 3.5 MYA.
TEXT
Porcine endogenous retroviruses (PERV) pose a serious challenge in xenotransplantation, i.e., the transfer of cells, tissues, or organs from one species to another for therapeutic reasons, since they are present in all cells and in almost every individual pig (27) and are transmitted vertically.
Endogenous retroviruses have been detected in the genomes of all vertebrate species (4, 14), and their general organization corresponds to that of exogenous retroviruses (4, 40, 46); however, most are replication-incompetent, while only a minority are functional, as reported, e.g., for mice (5, 10, 41) and pigs (1, 2, 21, 26, 28, 29).
For gamma-type PERV, three different classes exist, designated PERV-A, PERV-B, and PERV-C (1, 23, 42). The first two classes (7, 42) productively infect human cells in vitro, thus posing a serious risk in xenotransplantation, while the latter (1) does not replicate on human cells. There are only minor genetic differences between the classes, being most prominent in the receptor-binding domain of the Env protein. In addition, there are two different types of long terminal repeats (LTRs) that significantly affect the replication properties of single viruses (37, 47) through binding of transcription factor NF-Y (36). PERV-A and PERV-B proviruses demonstrate LTRs that harbor distinct 39-bp repeats in U3 which enable high transcription levels and adaptation to new host cells by multimerization of these repeats, with the transcriptional activity being generally stronger if more repeats are present. On the other hand, PERV-A and PERV-C were found to display repeatless LTRs (37, 47). Although sequences homologous to the 39-bp repeat are present in these LTRs, they are not organized in a repetitive manner and do not show multimerization as a response to replication cycles in their hosts. This LTR type confers a very low transcriptional activity, and we propose that it originated as an adaptation to endogenous replication of PERV. This leaves PERV-A as the only PERV harboring both types of LTR.
We recently determined the age of PERV as having an upper limit of approximately 7.6 x 106 years, while the repeatless LTR type evolved approximately 3.4 x 106 years ago and is therefore the phylogenetically younger structure (45). The age determined for PERV correlates with the time of separation of pig species (Suidae, Sus scrofa) from their closest relatives, American-born peccaries (Tayassuidae, Pecari tajacu), 7.4 x 106 years ago. While the time of the phylogenetic split was calculated by using mitochondrial DNA sequences (19), which tend to underestimate the time, archaeological data suggest a split of Suidae and Tayassuidae in the Eocene epoch, about 15 MYA (43, 44).
To extend the above-mentioned study on PERV's relative age, we analyzed a wide array of Suidae of Eurasian and African origin, as well as samples of the Tayassuidae as the closest evolutionary relatives of pigs, for the presence or absence of PERV. In anthropological studies, the presence or absence of human endogenous retroviruses in various hominoids has been used successfully to designate the age of the proviruses (17, 18, 39). Therefore, we aimed for a similar approach to define the age and spread of the different PERV genotypes more accurately.
Sample acquisition. Suiformes of various species were assayed. They are listed below in decreasing order of relationship to Sus scrofa. The epoch when the phylogenetic split occurred, as determined by archaeological evidence (43, 44) and genetic reconstruction (8, 11, 15, 24), is given in parentheses: Sus barbatus, Sus celebensis (Holocene), Potamochorus porcus, Potamochorus larvatus (Pleistocene), Phacochorus africanus, Babyrousa babyrussa (Pliocene), and Tayassuidae pecari (Eocene). A detailed classification of the families of Suidae and Tayassuidae, including subfamilies and genera, is given in Fig. 1.
Phylogenetic analysis. Recombination is an important process that has impact on biological evolution at different levels. Recombination reshuffles existing variation and even creates new variants at the amino acid level. It breaks down linkage disequilibrium and, especially, has a significant impact on the evolution of viral pathogens (12, 13). Common phylogenetic analysis depicts the history of a number of sequences as a tree where two sequences share a common ancestor. This model fails when recombination is present, because a sequence now is no longer limited to a single ancestor. But this poses a problem only if one tries to reconstruct a phylogenetic tree from a sample for which it is unknown if recombination has occurred. Here, we try to determine whether recombination played a role in PERV evolution. A number of methods have been developed to detect recombination, and their relative performance has been analyzed (9, 30, 48). In accordance with these comparisons, we have chosen SimPlot because it performed well for sequences as divergent as different PERV envelope classes. It produces very few false-positive results, and uses a phylogenetic method (bootstrapping) for detection, which is compatible with other analyses, and has a graphical interface, making comparison of analyses easy.
The detection of PERV in different Suiformes was based on five samples per species on average. The reader should bear in mind that, as no PERV-C locus is known that can be followed through time, the determined sequences represent independent snapshots of different specimens of different species. Analyses of modern pigs suggest a number of (6 to 10) replication-competent proviruses (26), 30 to 50 full-length PERV (1, 2, 28, 29), and 100 to 200 loci encompassing partial proviruses (35). Even under the assumption of less frequent PERV integration in older Suiformes, there should be enough sequences for detection by PCR. Thus, we believe that the limited number of animals should not pose a problem when interpreting samples testing negative. A cytochrome b phylogenetic tree was generated to calibrate the relative age of PERV in different Suiformes samples (Fig. 2A), including the archaeological data on Suiformes fossils. This tree is in line with previous studies using cytochrome b sequences (31) or archaeological data (43, 44), but includes three additional families not analyzed hitherto (S. celebensis, P. porcus, and P. larvatus). PERV sequences are completely undetectable in T. pecari and in B. babyrussa (Eocene and Miocene epochs, respectively), while the earliest presence of PERV happens in P. africanus associated with the late Miocene or early Pliocene epoch (3.5 to 7.5 MYA) (Fig. 2A and B), which confirms our recent study of the age of PERV (45). Only PERV-A can be detected in samples of P. africanus, while the earliest appearance of PERV-B is in the slightly younger P. porcus associated with the early Pleistocene epoch (3.5 to 7.5 MYA) (Fig. 2A and B). In contrast, PERV-C is detected for the first time in the much younger S. barbatus of the early Holocene epoch (0.1 to 1.5 MYA) (Fig. 2A and B). The repeatless LTR, on the other hand, has been detectable since appearing in P. larvatus and P. porcus of the late Pleistocene epoch (1.5 to 3.5 MYA) (Fig. 2B) (45). While we therefore assume a separate origin for PERV-C and the repeatless LTR, both could have emerged together as discussed below.
Patience et al. (28) also analyzed a series of Suiformes for the presence of beta- and gamma-retroviruses, and while our results match at large, we disagree on some minor points. As it is difficult to acquire exotic DNA samples in the first place, we were not able to repeat the study with the exact set of Suiformes used by Patience et al.; some species may differ in subspecies, and only a limited number of specimens were available for analysis. In addition, the primer sets used by Patience et al. and by us differ in sequence and therefore may lead to different results, particularly when only minor sequence variations have an impact on the detection limit. Hence, these differences may account for all minor disagreements. In contrast to our own findings, Patience at al. detected PERV-C in the sample of P. larvatus of the Pleistocene epoch, therefore shifting the emergence of PERV-C by one epoch to 1.5 to 3.5 MYA, though coinciding with the appearance of the repeatless LTR.
Analysis of interclass mosaicism. SimPlot, an interactive 32-bit program for Windows, was created to plot sequence similarity versus position (32). Briefly, SimPlot calculates and plots the percent identity of the query sequence to a panel of reference sequences in a sliding window, which is moved stepwise across the alignment. The window and step sizes are adjustable. Alignments were analyzed for recombination breakpoints by maximization of 2 as previously described (34, 38).
In general, the homologies between PERV-A and PERV-C are approximately 85%, while the similarities between PERV-B and either PERV-A or PERV-C barely exceed 70%. In general, gamma-type retroviruses, including PERV, share a common homology of approximately 60%. This fact and the occurrence of repeatless LTRs in both PERV-A and PERV-C but not in PERV-B (see above), leads to the assumption of a common evolutionary origin for these two classes. A possible recombination of PERV classes is most likely in the LTR or env sequences, and analysis revealed negligible sequence variations in gag and pol (data not shown).
Detection of recombination was carried out by comparing PERV env sequences obtained from various Suiformes to reference sequences for PERV-A (AJ133817) (7), PERV-B (AJ133818) (7), and PERV-C (AF038600) (1). Some PERV env sequences obtained from "old" Suiformes showed the PERV-A or PERV-C sequence and an analysis was therefore not necessary, while A/C recombinant sequences obtained from these samples showed a high degree of homology. Because of this finding and to keep the representation in Fig. 3 as simple as possible, the analyses shown in Fig. 3 were displayed with only one representative sequence per species.
The first recombination event between PERV envelope genes is detectable in S. celebensis, affecting the 3' end of a PERV-A env, which acquires some sequence homology of PERV-C (Fig. 3 B). This change affects mostly the C-terminal region of the env gene, but does not change the class of the env gene (Fig. 3 A). Therefore, S. celebensis is still considered negative for PERV-C as revealed for the other evolutionary older Suiformes (Fig. 2 B). This observation has two important implications. The change leads to a slightly truncated R peptide, with this R peptide structure being associated with repeatless LTRs in PERV-A proviruses. In addition, a truncated cytoplasmic tail has been shown to increase the fusogenic potential of several gamma-type retroviruses (6, 16, 33) and of PERV-A and PERV-B (3). As PERV-C seems to harbor only repeatless LTRs, it is conceivable that recombination happened between only one repeatless PERV-A provirus and the unknown PERV-C progenitor. The changed R peptide may have played an essential role in the origin of PERV-C by enabling the incorporation of the modified envelope into the capsid in the first place or by offsetting some of the limitations posed by the less active LTR through larger fusogenicity. However, the latter would also apply to repeatless PERV-A proviruses (3). A second recombination event is detectable with S. barbatus, affecting the receptor binding domain between nucleotide positions 300 and 900 of the env gene (Fig. 3 C), thus creating a new class of PERV (Fig. 3 A) and enabling the detection of PERV-C for the first time (Fig. 2 B). In samples from S. scrofa, PERV-C was detectable with reasonable abundance, as the homology with evolutionary older forms is already as high as 98%. Therefore, mostly point mutations are sufficient to change the query sequences to match the env reference sequence almost perfectly (Fig. 3 D).
There was no detectable recombination event between PERV-B and either PERV-A or PERV-C in the samples (data not shown), but recent publications of PERV sequences suggest that the different PERV classes still recombine (20, 49).
Recombination has been well documented for RNA viruses (50) and most likely involves similarity-assisted template switching (22, 25). The frequency of successful intertypic genetic exchanges is determined by, among other factors, (i) properties inherent to the process of viral replication, that is, the error susceptibility of viral reverse transcriptase and cellular RNA polymerase II; (ii) the frequency of cross-species transmission; (iii) the viability of the recombinant progeny; and (iv) the evolutionary gain.
Conclusion. PERV originated in African members of the Suidae family about 7.5 MYA (Fig. 2A and B), with repeat-harboring U3 sequences representing the exclusive type of LTR. The repeatless LTR developed during the early Pliocene epoch (3.5 MYA) in African Suidae, with its weak transcriptional activity most likely being an adaptation to an endogenous replication cycle. It is difficult to conclude whether PERV-A and PERV-B developed independently or whether both virus classes originated from a similar event like the A and C recombination described here. If so, the event took place too early to be clarified in this study. PERV-C, being much more closely related to PERV-A than to PERV-B, did not arise in the same epoch as PERV-A and PERV-B, but originated nearly 3.5 million years later due to a recombination event between PERV-A and an unknown ancestor. While the A and C recombination coincides with the appearance of the repeatless LTR, we assume that these events are independent from each other. Furthermore, we suggest that the recombination process leading to PERV-C involved an unknown ancestor and a PERV-A variant with a repeatless LTR.
Oligonucleotide sequences. Porcine cytochrome b sequences were amplified by nested PCR with outer forward primer 5'-GCT TAC CCT TTC CAA CTA GGC TTC-3' and outer reverse primer 5'-TTC GAA GTA CTT TAA TGG GAC AAG-3' and inner forward primer 5'-CAC ACA CTA GCA CAA TGG ATG CC-3' and inner reverse primer 5'-GAG GAT ACT AAT ATT CGG ATT GTT AT-3' by using a regimen of 35 cycles of denaturation (94°C for 30 s), annealing (58°C for 30 s), and elongation (72°C for 90 s). PERV polymerase sequences were detected by PCR with the specific primer pair PERV-pol-forward (5'-TTG ACT TGG GAG TGG GAC GGG TAA C-3') and PERV-pol-reverse (5'-GAG GGT CAC CTG AGG GTG TTG GAT-3') by using a regimen of 35 cycles of denaturation (94°C for 1 min), annealing (58°C for 1 min), and elongation (72°C for 3 min) as described in reference 7. For enhanced sensitivity, a nested PCR was performed with the same regimen with the following primer pair: PERV-pol-forward-nested (5'-GGT AAC CCA CTC GTT TTC TGG TCA-3') and PERV-pol-reverse-nested (5'-GAG CTG TGT AGG GCT TCG TCA AAG ATG-3'). The chances of detecting pol genes of non-PERV gamma-retroviruses or even from those of other related virus classes are negligible. PERV envelope gene class-specific detection was done with the same PCR regimen used for cytochrome b detection with the following primer pairs: PERV-A forward (5'-ATC CTA CCA GTT ATA ATC AAT-3') and PERV-A reverse (5'-GAT TAA AGG CTT CAG TGT GG-3'), PERV-B forward (5'-GGA TAA ATG GTA TGA GCT GG-3') and PERV-B reverse (5'-GCT CAT AAA CCA CAG TAC TAT-3'), and PERV-C forward (5'-CAC CTA TAC CAG CTC TGG ACA ATT-3') and PERV-C reverse (5'-TAA ACA ACC AGG CTC CAT TCT AAA-3'). Envelope gene reference sequences used in SimPlot (32) analysis were taken from previously published, full-length viruses PERV-A (AJ133817) (7), PERV-B (AJ133818) (7), and PERV-C (AF038600) (1).
Nucleotide sequence accession numbers. Sequences analyzed from Suiformes samples in this study were submitted to GenBank. Cytochrome b alignment: AY534296 through AY534303. PERV env sequences: S. scrofa (AY534304), S. barbatus (AY534305), and S. celebensis (AY534306). Additional env sequences were obtained from all species but showed significant homologies with already-published sequences. These sequences can be obtained from the authors.
ACKNOWLEDGMENTS
This study was supported by a grant from the German Ministry of Health (Bundesministerium für Gesundheit und Soziale Sicherung), Bonn, Germany.
We thank Stewart Lowden, Royal (Dick) School of Veterinary Studies, University of Edinburgh (United Kingdom) for supplying genomic DNA samples from various Suiformes used in this study. We thank the reviewers for critical reading and helpful suggestions on improving the manuscript.
Present address: Centre for Nanostructural Bioengineering, University of Queensland, 4072 Brisbane, Australia.
REFERENCES
Akiyoshi, D. E., M. Denaro, H. Zhu, J. L. Greenstein, P. Banerjee, and J. A. Fishman. 1998. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J. Virol. 72:4503-4507.
Armstrong, J. A., J. S. Potterfield, and A. T. DeMadrid. 1971. C-type virus particles in pig kidney cell lines. J. Gen. Virol. 10:195-198.
Bobkova, M., J. Stitz, M. Engelstadter, K. Cichutek, and C. J. Buchholz. 2002. Identification of R-peptides in envelope proteins of C-type retroviruses. J. Gen. Virol. 83:2241-2246.
Boeke, J. D., and J. P. Stoye. 1997. Retrotransposons, endogenous retroviruses and the evolution of retroelements, p. 343-436. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses, vol. 16. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Boone, L. R., P. L. Glover, C. L. Innes, L. A. Niver, M. C. Bondurant, and W. K. Yang. 1988. Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes. J. Virol. 62:2644-2650.
Christodoulopoulos, I., and P. M. Cannon. 2001. Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J. Virol. 75:4129-4138.
Czauderna, F., N. Fischer, K. Boller, R. Kurth, and R. R. T?njes. 2000. Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells. J. Virol. 74:4028-4038.
Douzery, E., and F. M. Catzeflis. 1995. Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia). J. Mol. Evol. 41:622-636.
Drouin, G., F. Prat, M. Ell, and G. D. Clarke. 1999. Detecting and characterizing gene conversions between multigene family members. Mol. Biol. Evol. 16:1369-1390.
Frankel, W. N., J. P. Stoye, B. A. Taylor, and J. M. Coffin. 1989. Genetic identification of endogenous polytropic proviruses by using recombinant inbred mice. J. Virol. 63:3810-3821.
Geisler, J. H. 2001. New morphological evidence for the phylogeny of Artiodactyla, Cetacea, and Mesonychidae. Am. Mus. Novit. 344:1-53.
Gibbs, A., C. H. Calisher, and F. Garcia-Arenal. 1995. Molecular basis of virus evolution. Cambridge University Press, Cambridge, United Kingdom.
Gibbs, M. J., and G. F. Weiller. 1999. Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus. Proc. Natl. Acad. Sci. USA 96:8022-8027.
Herniou, E., J. Martin, K. Miller, J. Cook, M. Wilkinson, and M. Tristem. 1998. Retroviral diversity and distribution in vertebrates. J. Virol. 72:5955-5966.
Irwin, D. M., T. D. Kocher, and A. C. Wilson. 1991. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32:128-144.
Januszeski, M. M., P. M. Cannon, D. Chen, Y. Rozenberg, and W. F. Anderson. 1997. Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J. Virol. 71:3613-3619.
Johnson, W. E., and J. M. Coffin. 1999. Constructing primate phylogenies from ancient retrovirus sequences. Proc. Natl. Acad. Sci. USA 96:10254-10260.
Kim, H. S., R. V. Wadekar, O. Takenaka, B.-H. Hyun, and T. J. Crow. 1999. Phylogenetic analysis of HERV-K LTR-like elements in primates: presence in some new world monkeys and evidence of recent parallel evolution in these species and in Homo sapiens. Arch. Virol. 144:2035-2040.
Kim, K. I., J. H. Lee, K. Li, Y. P. Zhang, S. S. Lee, J. Gongora, and C. Moran. 2002. Phylogenetic relationships of Asian and European pig breeds determined by mitochondrial DNA D-loop sequence polymorphism. Anim. Genet. 33:19-25.
Klymiuk, N., M. Muller, G. Brem, and B. Aigner. 2003. Recombination analysis of human-tropic porcine endogenous retroviruses. J. Gen. Virol. 84:2729-2734.
Krach, U., N. Fischer, F. Czauderna, and R. R. Tonjes. 2001. Comparison of replication-competent molecular clones of porcine endogenous retrovirus class A and class B derived from pig and human cells. J. Virol. 75:5465-5472.
Lai, M. M. 1992. RNA recombination in animal and plant viruses. Microbiol. Rev. 56:61-79.
Le Tissier, P., J. P. Stoye, Y. Takeuchi, C. Patience, and R. A. Weiss. 1997. Two sets of human-tropic pig retrovirus. Nature 389:681-682.
Montgelard, C., F. M. Catzeflis, and E. Douzery. 1997. Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences. Mol. Biol. Evol. 14:550-559.
Nagy, P. D., and A. E. Simon. 1997. New insights into the mechanisms of RNA recombination. Virology 235:1-9.
Niebert, M., C. Rogel-Gaillard, P. Chardon, and R. R. T?njes. 2002. Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells. J. Virol. 76:2714-2720.
Niebert, M., and R. R. T?njes. 2003. Analyses of prevalence and polymorphisms of six replication-competent and chromosomally assigned porcine endogenous retroviruses in individual pigs and pig subspecies. Virology 313:427-434.
Patience, C., M. Switzer, Y. Takeuchi, D. J. Griffiths, M. E. Goward, W. Heneine, J. P. Stoye, and R. A. Weiss. 2001. Multiple groups of novel retroviral genomes in pigs and related species. J. Virol. 75:2771-2775.
Patience, C., Y. Takeuchi, and R. Weiss. 1997. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3:282-286.
Posada, D., and K. A. Crandall. 2001. Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proc. Natl. Acad. Sci. USA 98:13757-13762.
Randi, E., V. Lucchini, and C. H. Diong. 1996. Evolutionary genetics of the suiformes as reconstructed using mtDNA sequencing. J. Mamm. Evol. 3:163-195.
Ray, S. C. 1998. SimPlot for Windows (version 1.6). S. C. Ray, Baltimore, Md.
Rein, A., J. Mirro, J. G. Haynes, S. M. Ernst, and K. Nagashima. 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68:1773-1781.
Robertson, D. L., B. H. Hahn, and P. M. Sharp. 1995. Recombination in AIDS viruses. J. Mol. Evol. 40:249-259.
Rogel-Gaillard, C., N. Bourgeaux, A. Billault, M. Vaiman, and P. Chardon. 1999. Construction of a swine BAC library: application to the characterization and mapping of porcine type C endoviral elements. Cytogenet. Cell Genet. 85:205-211.
Scheef, G., N. Fischer, E. Flory, I. Schmitt, and R. R. T?njes. 2002. Transcriptional regulation of porcine endogenous retroviruses released from porcine and infected human cells by heterotrimeric protein complex NF-Y and impact of immunosuppressive drugs. J. Virol. 76:12553-12563.
Scheef, G., N. Fischer, U. Krach, and R. R. T?njes. 2001. The number of a U3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells. J. Virol. 75:6933-6940.
Smith, J. M. 1992. Analyzing the mosaic structure of genes. J. Mol. Evol. 34:126-129.
Steinhuber, S., M. Brack, G. Hunsmann, H. Schwelberger, M. P. Dierich, and W. Vogetseder. 1995. Distribution of human endogenous retrovirus HERV-K genomes in humans and different primates. Hum. Genet. 96:188-192.
Stoye, J. P. 2001. Endogenous retroviruses: still active after all these years? Curr. Biol. 11:914-916.
Stoye, J. P., and J. M. Coffin. 1987. The four classes of endogenous murine leukemia virus: structural relationships and potential for recombination. J. Virol. 61:2659-2669.
Takeuchi, Y., C. Patience, S. Magre, R. Weiss, P. Banerjee, P. LeTissier, and J. Stoye. 1998. Host range and interference studies of three classes of pig endogenous retrovirus. J. Virol. 72:9986-9991.
Thenius, E. 1990. Even-toed ungulates, p. 1-15. In S. P. Parker (ed.), Grzimek's encyclopedia of mammals, vol. 5. McGraw-Hill, New York, N.Y.
Thenius, E. 1970. Zur Evolution und Verbreitungsgeschichte der Suidae (Artiodactyla, Mammalia). S. S?ugetierk. 35:321-342.
T?njes, R. R., and M. Niebert. 2003. Relative age of proviral porcine endogenous retrovirus sequences in Sus scrofa based on the molecular clock hypothesis. J. Virol. 77:12363-12368.
Wilkinson, D. A., D. L. Mager, and J. A. Leong. 1994. Endogenous human retroviruses, p. 465-535. In J. A. Levy (ed.), The Retroviridae, vol. 1. Plenum Press, New York, N.Y.
Wilson, C. A., S. Laeeq, A. Ritzhaupt, W. Colon-Moran, and F. K. Yoshimura. 2003. Sequence analysis of porcine endogenous retrovirus long terminal repeats and identification of transcriptional regulatory regions. J. Virol. 77:142-149.
Wiuf, C., T. Christensen, and J. Hein. 2001. A simulation study of the reliability of recombination detection methods. Mol. Biol. Evol. 18:1929-1939.
Wood, J. C., G. Quinn, K. M. Suling, B. A. Oldmixon, B. A. Van Tine, R. Cina, S. Arn, C. A. Huang, L. Scobie, D. E. Onions, D. H. Sachs, H. J. Schuurman, J. A. Fishman, and C. Patience. 2004. Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature swine. J. Virol. 78:2494-2501.
Worobey, M., and E. C. Holmes. 1999. Evolutionary aspects of recombination in RNA viruses. J. Gen. Virol. 80:2535-2543.(Marcus Niebert and Ralf R)