Генетическое разнообразие вируса лейкоза крупного рогатого скота и его распространение в районах Республики Дагестан

ABSTRACTBackground and Aim:Bovine leukemia virus (BLV) is a globally distributed retrovirus that causes enzootic bovine leu-kosis, a chronic infection associated with significant economic losses in cattle. Conventional serological diagnostic tools such as agar gel immunodiffusion and enzyme-linked immunosorbent assay detect anti-BLV antibodies but cannot identify proviral DNA, especially in early infections or in calves with maternal antibodies. This study aimed to develop a sensitive and specific duplex quantitative polymerase chain reaction (qPCR) assay targeting the env gene of BLV with β-actin as an internal control and apply it for molecular surveillance and genotyping of BLV in cattle from six regions of Kazakhstan.Materials and Methods:A total of 1,680 bovine DNA samples from cattle aged over 3 years were collected from six administrative regions of Kazakhstan. A duplex qPCR assay was developed using primers targeting a conserved region of the BLV env gene and bovine β-actin. Sensitivity was assessed using plasmid and genomic DNA dilutions, and specificity was tested against existing WOAH-recommended and commercial polymerase chain reaction (PCR) protocols. Positive samples with cycle threshold <28 were subjected to nested PCR and Sanger sequencing for genotyping. Phylogenetic analysis was conducted using the maximum likelihood method.Results:The developed qPCR assay demonstrated a sensitivity of 20 plasmid copies for the env gene and 6 genomic equivalents for β-actin per reaction, with high specificity comparable to international standards. The overall BLV infection rate was 38.9%, ranging from 13% in Pavlodar to 60.5% in East Kazakhstan. Among 149 sequenced positive samples, four genotypes (G1, G4, G7, and G8) were identified. Genotype G4 was predominant, comprising 79.2% of sequences and present in all six regions.Conclusion:The duplex qPCR assay is a robust, sensitive, and cost-effective diagnostic tool for detecting BLV provirus, including in animals with maternal antibodies or early-stage infections. The regional genotypic distribution underscores the need for tailored control strategies. This molecular surveillance provides essential baseline data for national BLV eradication programs and contributes to global BLV epidemiological mapping.

Bovine leukemia virus (BLV) belongs to the genus, Deltaretrovirus of the family, Retroviridae and it is the causative agent of enzootic bovine leukosis. The prevalence of BLV in three provinces in the Red River Delta Region in the North of Vietnam, Hanoi, Vinhphuc and Bacninh was studied from April 2017 to June 2018. A total of 275 blood samples collected from cattle were used for serum isolation and DNA extraction. Of these samples, 266 sera were subjected to ELISA test for detecting antibody against BLV gp51 protein and 152 DNA samples were used to detect the 444 bp fragment corresponding to a part of the gp51 region of the env by nested PCR. The results showed that 16.5% (n=44) and 21.1% (n=32) of samples were positive for BLV gp51 antibody and BLV proviral DNA, respectively. Phylogenetic analysis of the partial (423 bp) and complete (913 bp) BLV env-gp51 gene indicated that Vietnamese strains were clustered into genotypes 1, 6 and 10 (G1, G6 and G10). Of those genotypes, G1 genotype was dominant; G6 strains were designated as G6e and G6f subgenotypes; the existence of genotype 10 was confirmed for the first time in Vietnam. The present study provides important information regarding the prevalence of BLV infection and genetic characteristics of BLV strains identified in Vietnam, contributing to promote the establishment of disease control and eradication strategies in Vietnam.

ABSTRACT: This study aimed to evaluate the effect of bovine leukemia virus (BLV) on the systemic profile of naturally infected dairy heifers during the transition period. Pregnant Holstein and Jersey heifers (n=24) were distributed in pairs into two experimental groups: (BLV+) and (BLV-). Animals in the BLV+ group were divided into two subgroups based on the median BLV proviral load (high and low). The animals were then assessed at weeks -3, -2, -1, calving time (0), +1, +2, and +3. Blood samples were obtained for hematological and biochemical analyses, as well as haptoglobin measurements. Farm BLV screening revealed a herd BLV prevalence of 57.25% and heifer BLV prevalence of 38.7%. Mean corpuscular hemoglobin concentration was the only hematological variable for which group interaction was observed, with BLV+ cattle having higher values (33.29 ± 3.39%) than BLV- cattle (31.08 ± 2.31%). Aspartate aminotransferase activity was higher in the BLV+ heifers. The BLV+ group had greater incidence of inflammation (haptoglobin ≥ 2.0 mg/dL). Fibrinogen concentrations were also higher at weeks 0 and +1 in BLV+ heifers than in BLV- heifers. A high proviral load affected total leukocyte and lymphocyte count; however, this profile was not observed in the low proviral load and paired BLV- heifers. To our knowledge, this is the first study to report the impact of BLV infection on the health of dairy heifers during the transition period, demonstrating the effect of proviral load on white blood cell changes and early inflammation in infected animals.

BackgroundBovine 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.ResultsIn 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0239-z) contains supplementary material, which is available to authorized users.

Bovine leukemia virus (BLV) is a retrovirus responsible for lymphoproliferative disorders in cattle. Although infections of BLV in animals are well known, little is known about its capacity to infect humans. This study investigated the presence of anti-BLV antibodies and BLV proviruses in human and cattle samples. An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect anti-BLV antibodies while nested PCR was employed to identify BLV provirus sequences. The overall prevalence of anti-BLV antibodies in human and cattle samples were 12.50% and 16.73%, respectively. When using ELISA as a reference test, sensitivity and specificity for nested PCR were 0.625 and 0.970, respectively. The predictive value of a positive test was 0.862 and the predictive value of a negative test was 0.897. The percentage of cattle correctly classified by nested PCR assay was 89.1%. Nested PCR and Southern blot analysis, using primers specific for BLV gag sequences, revealed that BLV proviruses were detectable in cattle and human samples. Our results highlight the risk of human exposure to BLV and the need for further investigations to determine whether BLV infection poses a health hazard for humans.

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.

Vertical transmission of bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds

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.

Disease caused by bovine leukemia virus (BLV) results in significant economic losses to the livestock industry. To date, there is only one report describing the strains found in Italy. BLV strains (n = 24), collected between 2012 and 2016 from four different Italian regions, were genetically analyzed by direct sequencing of a portion of the BLV env gene, and the sequences were compared with those in the GenBank database. The Italian BLVs clustered into genotypes G2, G4, G6, G7, and G8, revealing a high level of BLV genetic heterogeneity in Italy. This study provides a basis for further investigations into the evolutionary relationship between BLV strains.

Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?

Bovine leukemia virus (BLV) is distributed worldwide. BLV has many effects on the health status and productivity of infected animals and is a potential risk for humans. In this study, we aimed to investigate the presence of and genotype bovine leukemia viruses on Jordanian dairy farms. Nested PCR coupled with RFLP and direct sequencing of a partial fragment of the env gene were carried out. Two BLV genotypes were found, genotypes 1 and 6. These genotypes were identified by nested PCR-RFLP of 444 bp of the env gene by restriction digestion with HaeIII, Bcl I and Pvu II. However, BLV-Jordan-10 seems to represent an entirely new genotype in our phylogenetic analysis. The nucleotide sequence identity between these two Jordanian BLV genotypes (1 and 6) was 96.2 %. The nucleotide sequence identity between Jordanian BLV genotype 1 and other reference BLV genotype 1 strains ranged from 99 % to 99.5 %. The nucleotide sequence similarity of the Jordanian BLV genotype 6 to other BLV genotypes ranged from 90 % to 96.7 %. A neutralizing motif and CD8(+) T-cell epitope were found in the env protein of both Jordanian isolates. In this study, we documented the presence of two BLV genotypes (1 and 6) on Jordanian dairy farms.

Bovine leukemia virus (BLV) infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. BLV is the etiological agent of enzootic bovine leukosis. Recently we developed a new quantitative real-time PCR method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals [1]. In this study, we analyzed a kinetic of the provirus and relevance of the BLV antibody titer. First, we experimentally compared the sensitivity of our methods with previously reported for BLV provirus detection real-time PCR system, and determined the high sensitivity of our developed BLV-CoCoMo-qPCR. Next, we estimated the sensitivities of the antibody detection methods such as ELISA, PHA and AGID and the provirus load estimated by BLV-CoCoMo-qPCR, using a total of 391 cattle. In three methods, high false-negative rate were observed at the range (100 copies/105cells) of low provirus copy number cattle. Meanwhile, a number of cattle with high antibody titer cannot be detected provirus. To investigate the reasons for the results, two cattle were experimentally infected with BLV and followed-up the titer of serum antibody and proviral load. We detected that proviral load were suppressed at the high antibody titer stage and it increased at the low antibody titer stage. In this study, we clearly detected the inverse proportion between antibody titer and proviral load for the first time in natural host. It suggested that quantification of proviral load is very important to halt the spread of BLV.

Aim. Assessment of the incidence of leukemia virus in cattle using PCR diagnostics in herds of the Republic of Dagestan and study of the molecular genetic characteristics of circulating viruses. Materials and Methods. 150 cattle blood samples were examined. PCR diagnostics of samples for the presence of bovine leukosis virus (BLV) were carried out using the RealBest‐Vet DNA BLV test system and a laboratory set of primers. Some of the samples were sequenced using the Sanger method and their phylogenetic analysis was performed. Results. Out of 150 samples, 24 samples were positive for the presence of BLV. In the Untsukulskiy district, no BLV DNA was detected in any of the 16 samples. In the Karabudakhkentskiy district out of 40 – in 2 (5 %), in Buynakskiy district – in 1 out of 30 (3.3 %) and in Babayurtovskiy district–in 21 out of 60 samples BLV was detected (35 %). For 13 BLV‐positive samples, fragments of the env gene measuring 1000 bp were obtained and deciphered. According to phylogenetic analysis, 7 samples of BLV belong to the 7th, and 6 – to the 4th genotype of BLV. The BLV genotype 4 isolated in the Babayurtovskiy district clusters with viruses from Kazakhstan, while viruses of genotype 4 from other farms cluster with Russian BLV. The studied samples of genotype 4 did not form common clusters. For the BLV genotype 7 isolated in farms of the Babayurtovskiy district, on the contrary, a combination of sequences into one cluster of genetically similar viruses was observed. Conclusion. Significant differences in the incidence of leukemia virus in livestock on farms in Dagestan were revealed. No patterns were found in the registration of cattle cases with a specific breed of cattle or with the age of the animal. It has been shown that viruses of both genotypes 7 and 4 circulate in the Republic. For BLV 4, it is assumed that there are different ways of its introduction into farms but no associated chains of virus spread have been found. For BLV genotype 7, transmission of BLV has been registered, which indicates the need to strengthen leukemia prevention measures on farms.

Bovine leukemia virus (BLV) single-stranded cDNA was used to study the distribution of DNA sequences in tissues (normal or malignant) from bovine, ovine, porcine and human origin. After recycling against normal bovine DNA, BLV (3H), cDNA hybridized with bovine enzootic tumor DNA but did not hybridize with normal bovine DNA. These results indicate that BLV is an exogenous RNA oncogenic virus and confirm that enzootic bovine leukosis (EBL) is an infectious disease. Proviral BLV sequences were also detected in buffy coat cells of animals in persistent lymphocytosis (PL) and carrying antibodies to BLV but no tumors. In animals at the tumor stage of EBL, the proviral sequences were found in buffy coat cells, in solid tumors (lymphosarcomas) and in organs infiltrated with tumoral lymphoid cells but not in apparently normal organs. No hybridization above background was observed between BLV (3H) cDNA and DNAs extracted from buffy coat cells and tumors corresponding to sporadic forms of bovine leukosis, pig leukemia and some human leukemias and sarcomas.

We investigated the infection of Bovine Leukemia Virus (BLV) related to other pathogens [Neospora caninum, Bovine Herpesvirus-1 (BoHV-1), Bovine Viral Diarrhea Virus (BVDV), and pathogenic bacteria] in 80 bovine aborted fetuses. The materials comprised whole fetuses, fetal organs, and placenta. The BLV was diagnosed by nested-PCR (env gp51 BLV gene), the identification of viral genotypes by sequencing, and the phylogenetic analysis by neighborjoining and maximum composite likelihood methods. The other pathogens and diagnoses were, respectively: Neospora caninum (nested-PCR), BoHV-1 (nested-PCR), BVDV (PCR), Brucella spp. (isolation and identification), Leptospira spp. (PCR), aerobic bacteria [Enterobacteriaceae, Gram positive cocci, Trueperella (Arcanobacterium) pyogenes] and micro-aerophilic (Campylobacter spp., Histophilus somni, and Listeria monocytogenes) by isolation and identification. BLV fetal antibodies were identified by ELISA kit. Thirteen (16.25%) fetuses were positive by BLV nested-PCR. Phylogenetic analysis revealed BLV genotypes 1, 5, and 6, which are frequently found in cattle in Brazil, Argentina, and Uruguay. No fetuses were positive for BLV antibodies by ELISA. A single case of coinfection with BLV was found for each of the pathogens Trueperella (Arcanobacterium) pyogenes, Klebsiella spp., and Streptococcus spp. were isolated as a pure or representing the preponderance of bacteria in a pooled culture. In the 67 BLV-negative fetuses, pathogens identified were single cases of Trueperella (Arcanobacterium) pyogenes, Staphylococcus aureus, and Brucella abortus; 2 of Escherichia coli; 3 of bovine viral diarrhea virus; and 4 of Neospora caninum. No pathogens were found in 55 fetuses. The low number of BLV positive samples infected or no by other pathogens didn´t allow performing statistical analysis for understanding if there were significative differences among not infected and infected BLV fetuses. Because BLV is an immunosuppressive agent and predisposes the cow to other pathogens, its connection with Leukemia or abortions need additional studies with bigger sampling, for elucidating pathogenesis in the pregnant cow and in the fetus. The rates of BLV transplacental transmission show the necessity of prophylactic measures in Brazilian cattle herds, in order to avoid infection in utero.