ISSN 10683674, Russian Agricultural Sciences, 2014, Vol. 40, No. 2, pp. 149–152. © Allerton Press, Inc., 2014. Original Russian Text © B.V. Syomin, I.M. Donnik, A.Ya. Samyilenko, 2014, published in Doklady Rossiiskoi Akademii Sel’skokhozyaistvennykh Nauk, 2014, No. 1, pp. 62–65.
VETERINARY
Capability of Bovine and Human Leukemia Viruses for Interspecies Transfer B. V. Syomina, I. M. Donnikb, and A. Ya. Samyilenkoa a
AllRussia Research and Technology Institute for Biological Industry, Moscow oblast, 141142 Russia email:
[email protected] b Ural State Agrarian University, Yekaterinburg, 620075 Russia Received October 21, 2013
Abstract—For the first time, the analysis for the possibility of leukemia virus transmissions from cattle to humans and from humans to cattle has been performed. A set of algorithms to identify in silico capability for interspecific transfers of leukemia viruses was developed. Keywords: Bovine Leukemia Virus (BLV), Human Tcell Leukemia Virus (HTLV), leukemia viruses, inter species virus transfer, in silico analysis DOI: 10.3103/S1068367414020207
Leukemia virus infection is one of the main causes of leukemia induction in humans and animals. Leuke mia viruses of different organisms are similar in struc tures and code genes of the same type. They represent RNA viruses or retroviruses (a family of retroviruses) that are included in the delta specific group according to the international classification [1]. The peculiarity of retroviruses included in that group, in contrast to the representatives of Retroviridae, is that they do not have their own homologs in the population of endoge nous viruses that largely inhabit the genome of practi cally all eukaryotes [2]. This indicates that delta viruses derived from a common exogenous ancestor relatively not long ago, and, maybe, that they are potentially capable of interspecies dissemination. Par ticularly, there is an assumption indicating the possi bility of interspecific transmission of bovine leukemia virus (BLV). It was experimentally determined that BLV is able to infect sheep, rabbits, swine, capybaras, water buffaloes, and chimpanzees; it was proved with sheep that the infection leads to malignization [4]. However, it is not ascertained until now whether BLV can infect a human and Human Tcell Leukemia Virus (HTLV types) can infect cattle. The objectives of the study are the analysis for a possibility of leukemia virus transmissions from cattle to humans and vice versa, and development of algorithms to reveal in silico capacity for interspecific transmission of leukemia viruses.
tional Nucleotide Sequence Database (GenBank) (Table 1). Aminoacid and nucleotide sequences were analyzed using server standard programs at the NIH (United States) and the EMBO (United Kingdom and France). Particularly, the BLAST program was used to find similar sequences from the database for nucle otide and aminoacid sequences, the ClustalW pro gram was used for their multiple alignments, and the FASTA program was used to search sequence similar ity and identity. RESULTS AND DISCUSSION At the initial stage, phylogenetic relationships of leukemia viruses were compared. It is known that the aminoacid sequence of the Gag locus is the most degenerate among all genes of retroviruses (i.e., Gag sequences of different retrovirus species are more diverse); in spite of this, Gag proteins form the parti cles in a similar way [5]. Consequently, aminoacid sequences of the Gag were compared to estimate phy logenetic similarity of viruses. The CNRS program (France) was used for this purpose. The analysis revealed, as expected, a considerable relationship of Table 1. Nucleotide sequences
METHODS Fulllength nucleotide and aminoacid sequences of BVL and HTLV were selected from the Interna 149
Virus BLV HTLV1 HTLV2 HTLV3
Identification number in the GenBank K02120 J02029.1 AF139382 DQ093792
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Table 2. Comparative analysis of aminoacid sequences of BLV and HTLV genes Gag
Env
Pol
Tax
Rex
Viruses similarity/identity of sequences, % BLV/HTLV3 47/34.5
37/26
58/44
33/23
36/27
viral HTLV types with each other. Relationship between BLV and HTLV, which diverged from a com mon ancestor approximately 0.5 million years ago, i.e., at the time when modern humans and artiodac tyla existed, was revealed. Thus, from an evolutionary genetics viewpoint, viruses diverged into separate spe cies not so long ago. Therefore, the further analysis for sequences involved was of interest. It is revealed through the analysis of identity/similarity in amino acid sequences of similar proteins that HTLV3 (one of the human leukemia viruses) has a significant (approximately 30%), i.e., not random, similarity with BLV (Table 2). In principle, approximately 30% of aminoacid sequence identity indicates their func tional identity [6]; however, proof becomes a convinc ing argument by similarity in functional domains of aminoacid sequences [6, 7]. At the next stage of the work, the most identi cal/similar regions in the BLV and HTLV3 gene sequences, which appeared to be in the functional domains responsible for the formation of participles, reverse transcription, virus intracellular transport of immature viral participle, and budding of viruses, were
revealed. In addition to the data on similarity (Table 3), identical ultrashort motifs responsible for binding to cellular signaling proteins required for intracellular transport of immature participles, such as signals of sumoylation [7], the PPPY classic proline motif required for transport to a plasma membrane and for budding [8], signals of phosphorylation, etc. [7, 9], are in the similar positions in the Gag aminoacid sequences of both involved retroviruses. Thus, the pre sented analysis proves that Gag HTLV3 and BLV pos sess similar functional domains, which means they can form not only morphologically similar participles [10, 11] but also can function in a cross functional manner in the cells. Thus, Gag BLV is capable to form a viral participle in the human cell and provide its transport to a plasma membrane; it must be the same as that for HTLV3 in bovine cells. As is known, Pol codes the reverse transcriptase (revertase), RNAcase H, and integrase. Revertase and RNAcase H possess similar functional domains and even in evolutionary distant species of viruses; that is why the data on their similarity in HTLV3 and BLV are not presented. A comparison of the functional domains of integrases of both viruses has revealed that integration of DNA copy of HTLV3 into the host cell genome takes place through the identical mechanism (Table 4). Similar motifs were found in the Env transmem brane and surface peptides (cubunits) (Table 5). Intro duction of Env glycoproteins of both retroviruses into the plasma membrane of the cell takes place through
Table 3. Comparison of functional domains in Gag HTLV3 and BLV aminoacid sequences Gag HTLV3/BLV
Similarity/Identity, %
22 HWLNFLQAAYRLQPGPSEFDFHQLRKFL 50 . | | | .| | : | . | | . | .| | . .| | . .| : .: : 16 DWLNLLQSAQRLNPRPSPSDFTDLKNYI 44
Functional means
62.1/48.3
Matrix
134 HRPWQMKDLQAIKQEVSSSAPGSPQFMQTIRLAVQQFDPT 184 | | .| . : : : | | . | | : | : . : .| || | . .: : | | : || | : . | . || | 119 HRAWALRELQDIKKEIENKAPGSQVWIQTLRLAILQADPT 169
77.5/52.5
Capsid (CA)
234 EYQNLWLSA 243 :| | | | | | .| 220 QYQNLWLQA 230
88.9/77.6
Capsid (CA)
248 ILQGPEEPFGSFVERLNVAL 278 |: || |.| . . ..| |.| |.: :| 244 IVQGPAESSVEFVNRLQISL 264
65/50
Region of the histocompatibility
349 PCFRCGQVGHWSRDC 362 || :| | .:. | | |: | | | 347 PCYRCLKEGHWARDC 360
87/67
Nucleocapsid (NC)
364 KQPRPPPGPCPVCQDPTHWKRDCPQLKT 392 | . . .| | | | | | | : | :| | : | | | | | | | .| | : 362 KATGPPPGPCPICKDPSHWKRDCPTLKS 390
96/83
Nucleocapsid (NC)
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Table 4. Comparison of functional domains in BLV and HTLV3 integrase aminoacid sequences Similar BLV and HTLV3 integrase sequences
Similarity/Identity, %
591 VFVDTYSGA 599 | :|| | | ||| 1223 VWVDTYSGA 1231
100/88.9
657 HVPYNPTSSGLDERTNGLLKLLLSKYHLD 685 | : | | | | | | | | | . | | | | | : | | . | : | | | .| | 1289 HIPYNPTSSGLVERTNGILKTLISKYLLD 1317
89.7/79.3
780 WVPWRLLK 787 | :|| | | || 1413 WIPWRLLK 1420
100/87.5
Table 5. Comparative analysis of Env aminoacid sequences Env, BLV, and HTLV3 Conserved sequences
Similarity/Identity, %
Functional means
275 NALLLPP 282 | :::| | | 290 NSVVLPP 297
100/88.9
Gp 51 Surface peptide
365 AQNRRGLDWLY 375 | | || | | || .|: 377 AQNRRGLDLLF 387
89.7/79.3
Gp 32 Transmembrane pep tide
423 NWDLGLT 429 || || | |: 431 NWDLGLS 437
100/87.5
Gp 32 Transmembrane pep tide
an identical mechanism. However, any significant similarity/identity in the Env surface subunit was not revealed. It is the peptide that ensures specific interac tions between a virus and a cell plasma membrane dur ing infection. In other words, specificity of both HTLV to human cells and BLV to bovine cells is ensured by the Env surface subunit. Thus, the performed analysis shows that both BLV and HTLV3 function identically; BLV and HTLV3 are capable to reproduce in human cells and bovine cells, respectively. Simultaneously, both viruses are specialized for their species hosts. This may be indi cated by the absence of identical extensive sequences in their Tax and Rex proteins. However, on the other hand, Tax and Rex BLV and HTLV3 have similarities with more than 30% identity (Table 2). The main argument for a specialization of viruses is the absence of identity in extensive regions within aminoacid sequence of the Env surface peptide (Table 5). That is, specific Env availability ensures rapid absorption and introduction of a virus into the cell of its new species host. This fact does not mean BLV is incapable of introduction into the human cells and that HTLV3 is incapable of introduction into bovine cells. Interspe cific spread of viruses can occur in the exosomal path way [12, 13]. Within such a mechanism, retroviral par ticles acquire exosomallike lipid and protein compo RUSSIAN AGRICULTURAL SCIENCES
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sitions to the extent of their transport into the cells. As a result of this, they are involved in biogenesis of intraluminal vesicles (ILVs) and, thus, can be spread between cells without Env glycoprotein of their mem brane. For example, such a mechanism was demon strated in detail for a relative of the analyzed viruses, HIV [14]. Unfortunately, any direct tests for revealing leuke mia viruses in species different from their species hosts have not been performed up to now. Moreover, a spe cific primer set in the case of PCR and a very careful serological analysis are required to arrange such tests, since it is known that test blood sera react equally with HTLV and BLV proteins; that is, proteins of these two types of viruses expose equal antigenic determinants, and they are often immunologically indistinguishable [15]. Direct observations for possibility of interspecies transfer of leukemia viruses were performed in Sverd lovsk oblast once. There were more than 90% of ani mals with a positive reaction of immunodiffusion (RID) in the herds on dairy farms of the region from 1991 to 1993; simultaneously, from 15 to 24% of cows with lymphocytosis specific for disease development were indicated among them. In connection with the fact that culling such animals was not practically car ried out, they were in the common herds for a long time. In addition, no prevention measures were 2014
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applied for the service staff. Representatives of worker families that worked on the dairy commodity farms for 15 to 20 years were retrospectively interviewed in Brod pos’olok in 1994–1996. It was ascertained that 11 per sons (seven milkers, three cattlemen, and one veteri narian) left their employment up to that time. Malig nant neoplasms (MN) were found in 10 persons of them during 5 to 10 years after leaving the employ ment. Data on one man were not available because of his leaving for another region. The morbidity structure was not similar: two cases of lungs and trachea MN, two cases of stomach MN, one case of hide MN, one case of mammary gland MN, and one case of dysemia. Dysemia (chronic lymphocytic leukemia) was found in a woman who worked as a veterinary assistant more than 20 years. In addition, malignant neoplasms were found in men aged from 50 to 60 years, in four women aged from 45 to 50, and in three women aged from 40 to 45. In 1994, blood samples were drawn from 10 work ers (three cattlemen, six milkers, and one veterinary assistant) on a dairy commodity farm which was leu kemia unsafe from 1968; further, serological testing with BLV antigens were performed using RID. Each of them showed a negative result. A collective blood serum sample was formed of 10 samples, and experi mental contamination was introduced into three 6 monthold rams. They were tested in RID every two weeks during 8 months. Then, they were slaughtered. The results of serological testing did not reveal any positive reaction in all animals during the experiment. Clinical testing also did not reveal any specific to leu kemia changes. However, a neoplasm of a round shape with rough surface and 8 × 7 cm in size was found in mesentery of one animal during pathoanatomical studying. Considerable hyperplasia of mesenterial lymphatic nodes and a lack of symptoms of inflamma tion was found in the other animal. The diagnoses such as lymphosarcoma in ram 1 and hyperplasia of mesenterial lymphatic nodes in ram 3 were made using histological analyses. Therefore, the presented data on existence of leu kemia virus transport between an animal and a human require a thorough experimental check since new recombinant viruses combining genes of animal and human viruses that might be potentially more virulent and pathogenic than the present certain viruses are very likely to emerge. This topic is especially urgent in the Russian Federation, where a leukemia virus has been circulating in cow herds of many regions for decades. ACKNOWLEDGEMENTS We thank Anna Bakanova, a master of the Moscow State Academy of Veterinary Medicine and Biotech nology for her assistance in analyzing the array of aminoacisequencebased data on viruses with com puter software.
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Translated by O. Zhiryakova
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