Bovine leukemia virus long terminal repeat variability: identification of single nucleotide polymorphisms in regulatory sequences

Abstract

BackgroundLimited data are available on the incidence of variations in nucleotide sequences of long terminal repeat (LTR) regions of Bovine Leukemia Virus (BLV). Consequently, the possible impact of SNPs on BLV LTR function are poorly elucidated. Thus, a detailed and representative study of full-length LTR sequences obtained from sixty-four BLV isolates from different geographical regions of Poland, Moldova, Croatia, Ukraine and Russia were analyzed for their genetic variability.MethodsOverlap extension PCR, sequencing and Bayesian phylogenetic reconstruction of LTR sequences were performed. These analyses were followed by detailed sequence comparison, estimation of genetic heterogeneity and identification of transcription factor binding site (TFBS) modifications.ResultsPhylogenetic analysis of curated LTR sequences and those available in the GenBank database reflected the acknowledged env gene classification of BLV into 10 genotypes, and further clustered analysed sequences into three genotypes - G4, G7 and G8. Additional molecular studies revealed the presence of 97 point mutations distributed at 89 positions throughout all 64 LTR sequences. The highest rate of variability was noted in U3 and U5 subregions. However, the variability in regulatory sequences (VR) was assessed as lower than the variability within non-regulatory sequences (VNR) for both, U3 and U5 subregions. In contrast, VR value for R subregion, as well as for the total LTR, was higher than the VNR suggesting the existence of positive selection. Twelve unique SNPs for these LTR sequences localized in regulatory and non-regulatory elements were identified. The presence of different types of substitutions lead to the abrogation of present or to the creation of additional TFBS.ConclusionThis study represents the largest study of LTR genetic variability of BLV field isolates from Eastern part of Europe. Phylogenetic analysis of LTRs supports the clustering BLV variants based on their geographic origin. The SNP screening showed variations modifying LTR regulatory sequences, as well as altering TFBS. These features warrant further exploration as they could be related to proviral load and distinctive regulation of BLV transcription and replication.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1062-z) contains supplementary material, which is available to authorized users.

Polymorphism of Haplotypes of Bovine Leukemia Virus

“…Data from the previous study indicate an increase in viral load and white blood cell count when C> G is replaced [38], which clearly refutes the assumption that the BLV masking vector is determined by the LRT region. A comparison of our own studies and published data [2,19,36] more likely confirms the hypothesis about the function of the LRT region a regulator of the viral particles synthesis. Taking into account the principles of genetic control, it is difficult to predict [14] that the regulator itself could be a factor in recognition of the virion by the host's immune system on the basis of the "friend or foe" principle.…”

“…It is important to note that it is the haplotype I that carries the SNP determined by the endonuclease BseI, which makes it possible to verify the assumption of a number of scientists [30,38] about the need for these substitutions so that the virus can "escape" from the reaction of the host's immune system. On the other hand, the LRT region contains genetic elements for regulating the rate of synthesis of viral particles of BLV [19], and as our recent studies [38] show, the intensity of replication of viral particles may well be a consequence of mutations in the LRT region. But it is worth remembering that an increase in the rate of reproduction, all other things being equal, will certainly provoke a more intense response of the immune system, which can lead to the death of both the virus carrier [38] and the virus if it fails to penetrate the body of the next host.…”

“…Although BLV is a virus with a rather conservative nucleotide sequence [15,16], the RNA molecule is still a rather unstable and mutable structure [17]. Over the past ten years, many new BLV strains have been reported in the world [18], while there is still no guarantee that the researchers have described all existing varieties [19]. In turn, it is the nucleotide sequence of the virus itself which is the target for the most modern and accurate screening method -PCR analysis [20][21][22].…”

“…BLV genes such as pol, env, and gag are quite well studied and are used in screening test systems [23][24][25]. At the same time, the BLV LTR region is considered to be a rather poorly studied structure [19], which makes it important not only to study its molecular structure, but also creates the prospect of developing test systems based on primers specific to the LTR region.…”

Biological Diversity of Haplotypes of the Bovine Leukemia Virus (BLV)

“…BLV хоть и является вирусом с достаточно консервативной нуклеотидной последовательностью [11; 12], молекула РНК всё же представляет собой довольно неустойчивую и мутабильную структуру [13]. За последние десять лет в мире было зарегистрировано немало новых штаммов BLV [14], при этом гарантии того, что исследователями описаны все существующие разновидности по-прежнему нет [15]. В свою очередь, именно нуклеотидная последовательность вируса сама по себе является мишенью для наиболее современного и точного метода скрининга -ПЦРанализа [16][17][18].…”