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Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the Pestivirus genus. Infection with BVDV causes a disease with a wide spectrum of clinical symptoms, most often mild, although infections with this virus constitute a serious economic problem all over the world. The virus is characterized by a high genetic variability, while the accumulation of single mutations leads to the formation of its new variants. The aim of this study was to better understand the complicated pathogenesis of this disease at the molecular level via the analysis of the transcriptome of cells infected with this virus. The bovine kidney cell line (MDBK), the cytopathic (cp) reference strain, and two non-cytopathic (ncp) BVD virus field strains were used in transcriptomic studies. The cell transcriptome was tested 24 and 72 h after infection. The results of the microarray analysis revealed changes in the expression levels of numerous genes. Genes with changed expression as a result of infection with the cp strain caused changes in the expression levels of a large number of genes and enriched a number of pathways. Genes with increased expression levels were enriched among other pathways involved in the cell cycle, while genes with reduced expression levels enriched pathways mostly related to metabolism. Genes with increased expression levels as a result of infection with ncp strains enriched a much smaller number of pathways, among them, pathways related to signaling activity 24 h post-infection and serine biosynthetic pathways both 24 and 72 h post-infection. Pathways enriched by genes with reduced expression levels were related to the innate immune response (72 h post-infection) or metabolism (24 and 72 h post-infection). The results of microarray studies can help us to better understand the host’s response to BVDV infection.

Recent studies have shown that bovine leukemia virus (BLV) sequences can be classified into seven distinct genotypes based on full gp51 sequence. This classification was based on available sequence data that mainly represented the BLV population that is circulating in cattle from the US and South America. In order to aid with a global perspective inclusion of data from Eastern Europe is required. In this study we examined 44 BLV isolates from different geographical regions of Poland, Belarus, Ukraine, and Russia. Phylogenetic analysis based on a 444bp fragment of env gene revealed that most of isolates belonged to genotypes 4 and 7. Furthermore, we confirmed the existence of a new genotype, genotype 8, which was highly supported by phylogenetic computations. A significant number of amino acid substitutions were found in the sequences of the studied Eastern European isolates, of which 71% have not been described previously. The substitutions encompassed mainly the C-part of the CD4+ epitope, zinc binding peptide region, CD8+ T cell epitope, and overlapping linear epitope E. These observations highlight the use of sequence data to both elucidate phylogenetic relationships and the potential effect on serological detection of geographically diverse isolates.

Characterization of the global genetic diversity of the bovine leukemia virus (BLV) is an ongoing international research effort. Up to now BLV sequences have been classified into eleven distinct genotypes. Although BLV genotyping and molecular analysis of field isolates were reported in many countries, there is no report describing BLV genotypes present in cattle from Pakistan. In this study we examined 27 env gene sequences from BLV-infected cattle coming from four farms located in Khyber Pakhtunkwa, Gilgit Baltisan and Punjab provinces. Phylogenetic analyses revealed the classification of Pakistani sequences into genotypes G1 and G6. The alignment with the FLK-BLV sequence revealed the presence of 45 mutations, namely, seven in genotype G1 and 33 in genotype G6. Five mutations were found in both, G1 and G6 genotypes. Twelve amino acid substitutions were found in the analyzed sequences, of which only one P264S was specific for sequences from Pakistan. Furthermore, a certain degree of nucleotide heterogeneity was identified by NGS. These results highlight the need for further study on the importance of genetic variability of BLV, especially in the context of its pathogenicity and potential effect on serological detection.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease that has worldwide distribution. Whilst it has been eradicated in most of Western Europe and Scandinavia, it remains a problem in other regions, particularly Eastern Europe and South America. For this study, in 2013, 24 cattle from three farms in three regions of Moldova were screened by ELISA and nested PCR. Of these cattle, 14 which were PCR positive, and these were molecularly characterized based on the nucleotide sequence of the env gene and the deduced amino acid sequence of the encoded gp51 protein. Our results demonstrated a low level of genetic variability (0-2.9%) among BLV field strains from Moldova, in contrast to that observed for other retroviruses, including human immunodeficiency virus (HIV) (20-38%) Mason IL (Trudy vologod moloch Inst 146–164, 1970 ) and equine infectious anemia virus (EIAV) (~40%) Willems L et al (AIDS Res Hum Retroviruses 16(16):1787–1795, 2000 ), where the envelope gene exhibits high levels of variation Polat M et al (Retrovirology 13(1):4, 2016 ). Sequence comparisons and phylogenetic analysis revealed that BLV genotype 7 (G7) is predominant in Moldova and that the BLV population in Moldovan cattle is a mixture of at least three new sub - genotypes: G7D, G7E and G4C. Neutrality tests revealed that negative selection was the major force operating upon the 51-kDa BLV envelope surface glycoprotein subunit gp51, although one positively selected site within conformational epitope G was detected in the N-terminal part of gp51. Furthermore, two functional domains, linear epitope B and the zinc-binding domain, were found to have an elevated ratio of nonsynonymous to synonymous codon differences. Together, these data suggest that the evolutionary constraints on epitopes G and B and the zinc-binding domains of gp51 differ from those on the other domains, with a tendency towards formation of homogenous genetic groups, which is a common concept of global BLV diversification during virus transmission that may be associated with genetic drift.

Background Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis. The SU or surface subunit, gp51, of its envelope glycoprotein is involved in receptor recognition and virion attachment. It contains the major neutralizing and CD4+ and CD8+ T cell epitopes found in naturally infected animals. In this study, we aimed to determine global variation and conservation within gp51 in the context of developing an effective global BLV vaccine. Results A total of 256 sequences extracted from the NCBI database and collected in different parts of the world, were studied to identify conserved segments along the env gene sequences that encode the gp51 protein. Using the MEME server and the conserved DNA Region module for analysis within DnaSP, we identified six conserved segments, referred to as A–F, and five semi-conserved segments, referred to as G–K. The amino acid conservation ranged from 98.8 to 99.8% in conserved segments A to F, while segments G to K had 89.6–95.2% conserved amino acid sequence. Selection analysis of individual segments revealed that residues of conserved segments had undergone purifying selection, whereas, particular residues in the semi-conserved segments are currently undergoing positive selection, specifically at amino acid positions 48 in segment K, 74 in segment G, 82 in segment I, 133 and 142 in segment J, and residue 291 in segment H. Each of the codons for these six residues contain the most highly variable nucleotides within their respective semi-conserved segments. Conclusions The data described here show that the consensus amino acid sequence constitutes a strong candidate from which a global vaccine can be derived for use in countries where eradication by culling is not economically feasible. The most conserved segments overlap with amino acids in known immunodeterminants, specifically in epitopes D–D′, E-E′, CD8+ T-cell epitopes, neutralizing domain 1 and CD4+ T-cell epitopes. Two of the segments reported here represent unique segments that do not overlap with previously identified antigenic determinants. We propose that evidence of positive selection in some residues of the semi-conserved segments suggests that their variation is involved in viral strategy to escape immune surveillance of the host.

We have characterized the intrahost genetic variation in the bovine leukemia virus (BLV) by examining 16 BLV isolates originating from the Western Siberia–Tyumen and South Ural–Chelyabinsk regions of Russia. Our research focused on determining the genetic composition of an 804 bp fragment of the BLV env gene, encoding for the entire gp51 protein. The results provide the first indication of the quasi-species genetic nature of BLV infection and its relevance for genome-level variation. Furthermore, this is the first phylogenetic evidence for the existence of a dual infection with BLV strains belonging to different genotypes within the same host: G4 and G7. We identified eight cases of recombination between these two BLV genotypes. The detection of quasi-species with cases of dual infection and recombination indicated a higher potential of BLV for genetic variability at the intra-host level than was previously considered.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) and has worldwide distribution. Infections with BLV have been reported in cattle from Kazakhstan but the virus has not yet been thoroughly characterized. In this study, we detect and estimate the level of BLV proviral DNA by qPCR in DNA samples from 119 cattle naturally infected with BLV, from 18 farms located in four different geographical regions of Kazakhstan. Furthermore, we conducted the phylogenetic and molecular analysis of 41 BLV env-gp51 gene sequences from BLV infected cattle. Phylogenetic analysis showed the affiliation of sequences to two already known genotypes G4 and G7 and also to a new genotype, classified as genotype G12. In addition, a multivariate method was employed for analysis of the association between proviral load and different variables such as the geographical location of the herd, cattle breeds, age of animals, and the presence of particular BLV genotypes. In summary, the results of this study provide the first evidence on molecular characterization of BLV circulating in cattle from Kazakhstan.

The objective of this study was to determine the true seroprevalence of bovine leukemia virus (BLV) infection in dairy cattle from Pakistan at the animal and herd-level. We tested 1380 dairy cattle from 451 herds and 92 water buffalo. The sera were tested by ELISA and the results were analyzed using Bayesian inference. The median posterior estimate of the herd level true BLV prevalence was 1.4%, with a 95% credible interval (CI) 0.7–3.1, whereas the median posterior estimate of the within-farm true seroprevalence was 3.8% with a 95% CI 2.8–4.8. All 92 sera collected from water buffalo were negative. Several risk factors potentially associated with seropositivity to BLV infections in Pakistan were analyzed using logistic regression model based on calculation of an odds ratio (OR). The study showed an association between seropositivity and medium herd (≥50) size (OR = 23.57, 95% CI: 3.01–103.48). Common housing of indigenous cattle with exotic-breed cattle (OR = 0.67, 95% CI: 06–2.35) or housing indigenous or their crossbred cattle with exotic-breed cattle (OR = 0.95, 95% CI: 0.14–3.01) had no effect on the BLV seroprevalence. Similarly, common housing of cattle and water buffalo was not risk factor for increased BLV seropositivity (OR = 27.10, 95% CI: 0.63–119.34).

Background Limited 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. Methods Overlap 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. Results Phylogenetic 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 (V R ) was assessed as lower than the variability within non-regulatory sequences (V NR ) for both, U3 and U5 subregions. In contrast, V R value for R subregion, as well as for the total LTR, was higher than the V NR 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. Conclusion This 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.

Bovine leukaemia virus (BLV) is the aetiological agent of enzootic bovine leukosis. The aim of the study was to ascertain the ability of qPCR to detect proviral BLV DNA in various tissues from slaughtered cattle when BLV was suspected but serological testing was not possible. Three types of tissues were collected during sanitary slaughtering of 22 cattle naturally infected with BLV: spleen, lymph node and muscle. The proviral load (PVL) was estimated in this tissue by a real-time quantitative PCR (qPCR) for BLV based on the gene. To measure provirus copy number per 10 cells, the bovine histone H3 family 3A gene was also amplified by qPCR. The PVL was the highest in the spleen and ranged there from 1 to 59,188 copies/10 cells, with four cases in which no proviral DNA was detected. In the lymph nodes the PVL ranged from 2 to 6,888 copies/10 cells, with seven cases in which no copies were detected. The lowest PVL was recorded in DNA from muscle samples and ranged from 1 to 119 copies/10 cells; no BLV was detected in 6 out of 22 samples. The BLV qPCR is a suitable tool for the detection of proviral BLV DNA in various tissues when infection is suspected and no blood or other fluids are available for serological examination.