A novel real time PCR assay for bovine leukemia virus detection using mixed probes and degenerate primers targeting novel BLV strains.

The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease in cattle. We previously developed the quantitative real-time PCR (qPCR) assay to measure the proviral loads of BLV using coordination of common motif (CoCoMo) degenerate primers. We here found four single mutations within the probe region of the original BLV-CoCoMo-qPCR assay, three of which have negative impact on its sensitivity in the probe sequences of the long terminal regions of the BLV-CoCoMo-qPCR-2 assay, using genomic DNA from 887 cows from 27 BLV-positive farms via a nationwide survey conducted in 2011 and 2017 in Japan. Therefore, the modified probes were designed to completely match the three BLV mutant strains identified here. Moreover, we examined the optimum ratio of the concentration to be mixed with the wild type and three new BLV TaqMan probes were designed here using genomic DNAs extracted from cattle naturally infected with the wild type BLV strain and three mutant strains. Finally, we successfully established an improved assay maintained the original sensitivity and reproducibility and can detect novel BLV strains.

Detection and Molecular Characterization of Bovine Leukemia Virus in Egyptian Dairy Cattle.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), the most common neoplastic disease in cattle worldwide. The first EBL outbreak in Egypt was reported in 1997. To date, there are few studies regarding BLV diagnosis using only serological detection and no studies investigating the distribution of BLV provirus, which is the retroviral genome integrated into the host genome, in Egypt. The genetic characteristics of Egyptian BLV strains are also unknown. Therefore, we aimed to detect BLV provirus and determine BLV genetic variability among dairy cattle in Egypt. We collected 270 blood samples of dairy cattle from 24 farms located in five provinces in Egypt. Out of the 270 samples, 58 (21.5%) were positive for BLV provirus. Phylogenetic analysis based on 18 420-bp selected sequences out of 50 isolates of the BLV env-gp51 gene demonstrated that Egyptian BLV isolates were clustered into genotype-1 and-4, among 11 genotypes detected worldwide. Furthermore, phylogenetic analysis and alignment of the 501-bp sequence of the env-gp51 gene revealed that at least six genetically different strains are present in Egypt. Genotype-1 isolates comprised four different strains (G1-a, G1-b, G1-c, and G1-d) and genotype-4 isolates included two different strains (G4-x and G4-y). Moreover, in one farm with 100% infection rate, we identified three isolates of G1-a strain, 35 isolates of G4-x strain, and two isolates of G4-y strain. Overall, this study provides the new report on molecular prevalence of BLV in Egypt and records the coexistence of BLV genotype-1 and-4 in Egyptian cattle.

BoLA-DRB3 genetic diversity in Highland Creole cattle from Bolivia.

The genetic diversity of the BoLA-DRB3 gene has been reported in different cattle breeds owing to its central role in the immune response. However, it is still unknown in hundreds of cattle breeds, especially native populations. Here, we studied BoLA-DRB3 genetic diversity in Highland Creole cattle (CrAl) from Western Bolivia, raised at altitudes between 3800 and 4200 m. DNAs from 48 CrAl cattle were genotyped for BoLA-DRB3 exon 2 alleles using polymerase chain reaction-sequence-based typing (PCR-SBT). The results were compared with 1341 previously reported data from Tropical Creole cattle and other breeds raised in the region. Twenty-three BoLA-DRB3 alleles were identified in CrAl, including the BoLA-DRB3*029:02 variant previously detected in other Creole cattle. Observed and expected heterozygosity were 0.87 and 0.93, respectively. Nucleotide diversity and the number of pairwise difference values were 0.078 and 19.46, respectively. The average number of nonsynonymous and synonymous substitutions were 0.037 and 0.097 for the entire BoLA-DRB3 exon 2, and 0.129 and 0.388 for the antigen-binding site, respectively. Venn analysis and the review of the IPD-MHC database and the literature showed that 2 of 64 alleles were only detected in CrAl, including BoLA-DRB3*029:01 previously reported in African cattle and *048:01 detected in Philippine cattle. Two additional alleles, BoLA-DRB3*007:02 and *029:02, were only present in CrAl and Lowland Creole cattle. Principal Component Analysis (PCA) showed that Bolivian Creole cattle breeds were closely located but they were distant from the Colombian Hartón del Valle Creole. FST analysis showed a low degree of genetic differentiation between Highland and Lowland Bolivian Creole cattle (FST = 0.015). The present results contribute to increasing our knowledge of BoLA-DRB3 genetic diversity in cattle breeds.

Characterization of bovine MHC DRB3 diversity in global cattle breeds, with a focus on cattle in Myanmar.

BACKGROUND: Myanmar cattle populations predominantly consist of native cattle breeds (Pyer Sein and Shwe), characterized by their geographical location and coat color, and the Holstein-Friesian crossbreed, which is highly adapted to the harsh tropical climates of this region. Here, we analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus that has been linked to the immune response, in Myanmar cattle populations. METHODS: Blood samples (n = 294) were taken from two native breeds (Pyer Sein, n = 163 and Shwe Ni, n = 69) and a cattle crossbreed (Holstein-Friesian, n = 62) distributed across six regions of Myanmar (Bago, n = 38; Sagaing, n = 77; Mandalay, n = 46; Magway, n = 46; Kayin, n = 43; Yangon, n = 44). In addition, a database that included 2428 BoLA-DRB3 genotypes from European (Angus, Hereford, Holstein, Shorthorn, Overo Negro, Overo Colorado, and Jersey), Zebuine (Nellore, Brahman and Gir), Asian Native from Japan and Philippine and Latin-American Creole breeds was also included. Furthermore, the information from the IPD-MHC database was also used in the present analysis. DNA was genotyped using the sequence-based typing method. DNA electropherograms were analyzed using the Assign 400ATF software. RESULTS: We detected 71 distinct alleles, including three new variants for the BoLA-DRB3 gene. Venn analysis showed that 11 of these alleles were only detected in Myanmar native breeds and 26 were only shared with Asian native and/or Zebu groups. The number of alleles ranged from 33 in Holstein-Friesians to 58 in Pyer Seins, and the observed versus unbiased expected heterozygosity were higher than 0.84 in all the three the populations analyzed. The FST analysis showed a low level of genetic differentiation between the two Myanmar native breeds (FST = 0.003), and between these native breeds and the Holstein-Friesians (FST <  0.021). The average FST value for all the Myanmar Holstein-Friesian crossbred and Myanmar native populations was 0.0136 and 0.0121, respectively. Principal component analysis (PCA) and tree analysis showed that Myanmar native populations grouped in a narrow cluster that diverged clearly from the Holstein-Friesian populations. Furthermore, the BoLA-DRB3 allele frequencies suggested that while some Myanmar native populations from Bago, Mandalay and Yangon regions were more closely related to Zebu breeds (Gir and Brahman), populations from Kayin, Magway and Sagaing regions were more related to the Philippines native breeds. On the contrary, PCA showed that the Holstein-Friesian populations demonstrated a high degree of dispersion, which is likely the result of the different degrees of native admixture in these populations. CONCLUSION: This study is the first to report the genetic diversity of the BoLA-DRB3 gene in two native breeds and one exotic cattle crossbreed from Myanmar. The results obtained contribute to our understanding of the genetic diversity and distribution of BoLA-DRB3 gene alleles in Myanmar, and increases our knowledge of the worldwide variability of cattle BoLA-DRB3 genes, an important locus for immune response and protection against pathogens.

New evidence of bovine leukemia virus circulating in Myanmar cattle through epidemiological and molecular characterization.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and causes serious problems for the cattle industry. In this study, we examined the prevalence of BLV infection and the distribution of BLV genotypes in cattle in the northern, central, and southern parts of Myanmar. The prevalence of BLV infection among Myanmar cattle (37.04%) in this study was markedly higher than the prevalence (9.1%) observed in our earlier study in which BLV was detected from the limited number of cattle only from a small area of Myanmar. Phylogenetic analysis of partial env-gp51 sequence of the isolated BLV strains revealed that there are at least three BLV genotypes (genotype-1, genotype-6, and genotype-10) in Myanmar, which have also been detected in the neighboring countries. We performed this study to estimate the BLV proviral load, which is a major diagnosis index for determining the virus transmission risk. The cattle of the three test regions with warm, wet, and humid climatic conditions (upper Sagaing, Yangon, and Kayin) exhibited a high mean proviral load, while cattle of three other regions with low annual rainfall and very high temperature (Mandalay, Magway, and upper Bago) exhibited a low mean proviral load. Further, the level of proviral load and the prevalence of BLV infection in Myanmar native cattle (N = 235) were lower than that in the hybrid cattle (Holstein Friesian × Myanmar native) (N = 62). We also observed that the cattle with high risk for BLV transmission, which have high proviral load, may enhance the BLV infection rate. Hence, to control BLV transmission, it is necessary to eliminate these cattle with high-risk for BLV transmission and to diagnose BLV provirus in cattle in the remaining regions/states of Myanmar sharing a boundary with neighboring countries.

Absence of bovine leukemia virus proviral DNA in Japanese human blood cell lines and human cancer cell lines.

Bovine leukemia virus (BLV) infects cattle worldwide and causes B-cell lymphoma in cattle. BLV has been identified in human breast and lung cancer and in blood, but the association of BLV and human cancer is controversial. In this study, we investigated the existence of BLV in 145 Japanese human blood cell lines and 54 human cancer cell lines, using a new highly sensitive PCR assay that can amplify even one copy of BLV using LTR primers different from those in previous studies on BLV provirus in breast cancer. All samples were found negative for BLV provirus, suggesting that BLV is unlikely to infect humans.

Broadly applicable PCR restriction fragment length polymorphism method for genotyping bovine leukemia virus.

Bovine leukemia virus (BLV) is a causative agent of enzootic bovine lymphoma (EBL). BLV is prevalent worldwide, and ten genotypes have been classified based on the sequence of the envelope glycoprotein (gp51) gene. In this study, we present a simple and generally applicable PCR restriction fragment length polymorphism (PCR-RFLP) method to identify BLV genotypes. While the genotyping results obtained by previously described PCR-RFLP methods matched only 78.96% to the results of phylogenetic analysis, we demonstrated that our PCR-RFLP method can identify 90.4% of the sequences available in the database in silico. The method was validated with 20 BLV sequences from EBL tumor tissues and 3 BLV sequences from blood of BLV infected cattle, and was found to show high specificity. We utilized this method to determine genotypes of blood samples from 18 BLV seropositive cattle in Kanagawa and Niigata, as well as 12 EBL cattle in Chiba, Japan. Our analysis with the modified PCR-RFLP detected two genotypes, Genotypes 1 and 3. Genotype 1 was detected as the main genotype, while Genotype 3 was sporadically observed. This technique can be used as a reliable system for screening a large number of epidemiological samples.

Epidemiology and genetic diversity of bovine leukemia virus.

Bovine leukemia virus (BLV), an oncogenic member of the Deltaretrovirus genus, is closely related to human T-cell leukemia virus (HTLV-I and II). BLV infects cattle worldwide and causes important economic losses. In this review, we provide a summary of available information about commonly used diagnostic approaches for the detection of BLV infection, including both serological and viral genome-based methods. We also outline genotyping methods used for the phylogenetic analysis of BLV, including PCR restriction length polymorphism and modern DNA sequencing-based methods. In addition, detailed epidemiological information on the prevalence of BLV in cattle worldwide is presented. Finally, we summarize the various BLV genotypes identified by the phylogenetic analyses of the whole genome and env gp51 sequences of BLV strains in different countries and discuss the distribution of BLV genotypes worldwide.

The molecular epidemiological study of bovine leukemia virus infection in Myanmar cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and affects both health status and productivity. However, no studies have examined the distribution of BLV in Myanmar, and the genetic characteristics of Myanmar BLV strains are unknown. Therefore, the aim of this study was to detect BLV infection in Myanmar and examine genetic variability. Blood samples were obtained from 66 cattle from different farms in four townships of the Nay Pyi Taw Union Territory of central Myanmar. BLV provirus was detected by nested PCR and real-time PCR targeting BLV long terminal repeats. Results were confirmed by nested PCR targeting the BLV env-gp51 gene and real-time PCR targeting the BLV tax gene. Out of 66 samples, six (9.1 %) were positive for BLV provirus. A phylogenetic tree, constructed using five distinct partial and complete env-gp51 sequences from BLV strains isolated from three different townships, indicated that Myanmar strains were genotype-10. A phylogenetic tree constructed from whole genome sequences obtained by sequencing cloned, overlapping PCR products from two Myanmar strains confirmed the existence of genotype-10 in Myanmar. Comparative analysis of complete genome sequences identified genotype-10-specific amino acid substitutions in both structural and non-structural genes, thereby distinguishing genotype-10 strains from other known genotypes. This study provides information regarding BLV infection levels in Myanmar and confirms that genotype-10 is circulating in Myanmar.

A new genotype of bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis.

BACKGROUND: Bovine leukemia virus (BLV) is a member of retroviridae family, together with human T cell leukemia virus types 1 and 2 (HTLV-1 and -2) belonging to the genes deltaretrovirus, and infects cattle worldwide. Previous studies have classified the env sequences of BLV provirus from different geographic locations into eight genetic groups. To investigate the genetic variability of BLV in South America, we performed phylogenetic analyses of whole genome and partial env gp51 sequences of BLV strains isolated from Peru, Paraguay and Bolivia, for which no the molecular characteristics of BLV have previously been published, and discovered a novel BLV genotype, genotype-9, in Bolivia. RESULTS: In Peru and Paraguay, 42.3 % (139/328) and over 50 % (76/139) of samples, respectively, were BLV positive. In Bolivia, the BLV infection rate was up to 30 % (156/507) at the individual level. In Argentina, 325/420 samples were BLV positive, with a BLV prevalence of 77.4 % at the individual level and up to 90.9 % at herd level. By contrast, relatively few BLV positive samples were detected in Chile, with a maximum of 29.1 % BLV infection at the individual level. We performed phylogenetic analyses using two different approaches, maximum likelihood (ML) tree and Bayesian inference, using 35 distinct partial env gp51 sequences from BLV strains isolated from Peru, Paraguay, and Bolivia, and 74 known BLV strains, representing eight different BLV genotypes from various geographical locations worldwide. The results indicated that Peruvian and Paraguayan BLV strains were grouped into genotypes-1, -2, and -6, while those from Bolivia were clustered into genotypes-1, -2, and -6, and a new genotype, genotype-9. Interestingly, these results were confirmed using ML phylogenetic analysis of whole genome sequences obtained by next generation sequencing of 25 BLV strains, assigned to four different genotypes (genotypes-1, -2, -6, and -9) from Peru, Paraguay, and Bolivia. Comparative analyses of complete genome sequences clearly showed some specific substitutions, in both structural and non-structural BLV genes, distinguishing the novel genotype-9 from known genotypes. CONCLUSIONS: Our results demonstrate widespread BLV infection in South American cattle and the existence of a new BLV genotype-9 in Bolivia. We conclude that at least seven BLV genotypes (genotypes-1, -2, -4, -5, -6, -7, and -9) are circulating in South America.

BLV-CoCoMo-qPCR-2: improvements to the BLV-CoCoMo-qPCR assay for bovine leukemia virus by reducing primer degeneracy and constructing an optimal standard curve.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. Because BLV infection can remain clinically silent, the proviral load is an important index for estimating disease progression. CoCoMo-qPCR, an assay developed to estimate BLV proviral load, allows the highly sensitive detection of BLV originating in different countries. Here, we developed a modified version of the CoCoMo-qPCR assay, the "BLV-CoCoMo-qPCR-2" assay, which uses optimized degenerate primers. We also constructed a new plasmid standard. Finally, we used both assays to examine DNA samples from BLV-infected cattle and compared the results.

Detection and molecular characterization of bovine leukemia virus in Philippine cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. However, there are no comprehensive studies on the distribution of BLV in the Philippines, and the genetic characteristics of Philippine BLV strains are unknown. Therefore, the aim of this study was to detect BLV infections in the Philippines and determined their genetic variability. Blood samples were obtained from 1116 cattle from different farms on five Philippine islands, and BLV provirus was detected by BLV-CoCoMo-qPCR-2 and nested PCR targeting BLV long terminal repeats. Out of 1116 samples, 108 (9.7 %) and 54 (4.8 %) were positive for BLV provirus, as determined by BLV-CoCoMo-qPCR-2 and nested PCR, respectively. Of the five islands, Luzon Island showed the highest prevalence of BLV infection (23.1 %). Partial env gp51 genes from 43 samples, which were positive for BLV provirus by both methods, were sequenced for phylogenetic analysis. Phylogenetic analysis based on a 423-bp fragment of the env gene revealed that Philippine BLV strains clustered into either genotype 1 or genotype 6. Substitutions were mainly found in antigenic determinants, such as the CD4(+) T-cell epitope, the CD8(+) T-cell epitope, the second neutralizing domain, B and E epitopes, and these substitutions varied according to genotype. This study provides comprehensive information regarding BLV infection levels in the Philippines and documents the presence of two BLV genotypes, genotypes 1 and 6, in this population.