Bovine Leukemia Virus Genotypes Research Articles

Genetic Variability of Bovine Leukemia Virus: Evidence of Dual Infection, Recombination and Quasi-Species.

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.

Phylogenetic analysis of the partial sequences of the env and tax BLV genes reveals the presence of genotypes 1 and 3 in dairy herds of Antioquia, Colombia

Bovine leukemia virus (BLV) is a retrovirus that primarily infects dairy cows. Although few studies have also used the tax gene, phylogenetic studies of BLV use mostly the env gene. The aim of this work was to establish the circulating genotypes of BLV in specialized dairy cattle from Antioquia, Colombia. Twenty blood samples from Holstein Friesian cows were collected, and their DNA was isolated. A PCR was performed for a partial region of the env and tax genes. A phylogenetic analysis was carried out using the maximum likelihood and Bayesian methods for both genes. Nineteen sequences were identified as genotype 1 by env and tax genes. Only one sequence was clustered with genotype 3 and had the highest proportion of different nucleotide sites compared to other strains. Four amino acid substitutions in the 134 amino acid residue fragment of the Env protein were identified in the Colombian sequences, and three new amino acid substitutions were reported in the 296 amino acid residue fragment of the Tax protein. R43K (Z finger), A185T (Activation domain), and L105F changes were identified in the genotype 3 sample. This genotype has been reported in the United States, Japan, Korea, and Mexico, but so far, not in Colombia. The country has a high rate of imported live animals, semen, and embryos, especially from the United States. Although it is necessary to evaluate samples from other regions of the country, the current results indicate the presence of two BLV genotypes in specialized dairy herds.

Изучение генетической вариабельности выделенных изолятов вируса лейкоза крупного рогатого скота в Белгородской области

Abstract. The purpose of the research Was to study genetic diversity of bovine leukemia virus isolates obtained in the Belgorod Region using restriction fragment length polymorphism method (RFLP). Scientific novelty. Bovine leukosis is one of the most common chronic infectious diseases of cattle in many countries of the world, which causes significant economic losses in the livestock industry. The typing of bovine leukemia virus (BLV), the study of its genetic structure, the evaluation of the mutation vector and a more detailed disclosure of the biological properties of the pathogen represent fundamental and applied value. Methods. The object of the research was 3-4-year-old cows infected with leukemia virus (n = 10), identified by serological methods in disadvantaged dairy farms. The immunodiffusion (ID) test, hematologic studies, PCR, genotyping, statistical processing of obtained data were conducted. Results. Conducted hematological studies determined the stage of the leukemic process in each animal. The target env fragment of the BLV gene (444 bp) was amplificated by 2-stage nested PCR, and this region was genotyped for all studied leukemia virus isolates using the restriction fragment length polymorphism (RFLP) method. In the course of the work, specific regions of the BLV env (gp51) gene, 970 bp long, were also obtained. We have given a primary assessment of the genetic diversity of BLV with the establishment of a genetic group (Belgian genotype according to RFLP). In the course of the work, specific regions of the env gp51 BLV gene, 444 bp long, were obtained. These fragments will be used for further DNA sequencing followed by phylogenetic analysis and determination of amino acid changes in the structure of the surface glycoprotein (gp51) of the bovine leukemia virus. Monitoring studies of BLV genotypes and the study of antigenic changes in the pathogen will allow timely development of the latest means of controlling and restricting the spread of bovine leukosis and improvement of diagnostic serological and PCR test systems.

Genetic diversity of BLV, common in the territory of the Kochenevsky district of the Novosibirsk region

This study evaluated bovine leukemia virus (BLV) prevalence in Kochenevsky district, Novosibirsk region,Russia. BLV prevalence data in farmsteads of private farmers and large livestock farms were obtained; phylogenetic analysis was conducted to define BLV genotypes in the study region. PCR analysis was used to detect BLV proviral cDNA; focal virus spread was identified. The BLV prevalence among the cattle of the private farmsteads ranged from 0% to 79% across the study areas. Among the livestocks of the Kochenevsky district, 72% of samples were positive for BLV. The BVL 1000-bp genome fragment coding the env gene region was obtained for 191 samples. The following phylogenetic analysis was performed and 162 sequences were classified as genotype 4 and the remaining 29 sequences were classified as genotype 7. BLV 7 genotype was identified among Proskudnoe private farmsteads only, while BLV genotype 4 was widespread across the region. Both BLV genotype 7 (n=17) and 4 (n=126) were detected in the dairy farm. Phylogenetic analysis revealed distinct clusters (with a bootstrap support value і75) of BLV sequences most likely indicating to the several transmission routes of the infection in the Kochenevsky district. The study results can be used to replenish fundamental knowledge about the BLV evolution and development of novel BLV prevention and elimination approaches.

Molecular Characterization of Bovine Leukemia Virus with the Evidence of a New Genotype Circulating in Cattle from Kazakhstan.

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.

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens.

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.

Genetic analysis of the pX region of bovine leukemia virus genotype 1 in Holstein Friesian cattle with different stages of infection.

The pX genetic region of bovine leukemia virus (BLV) includes four genes with overlapping reading frames that code for the Tax, Rex, R3, and G4 proteins. These proteins are involved in the regulation of transcriptional and post-transcriptional viral expression, as well as having oncogenic potential. Our goal was to investigate the pathogenicity of the pX region of BLV genotype 1 in terms of lymphocytosis, lymphomas, and proviral DNA load. We screened 724 serological samples from mixed-age Holstein Friesian cattle from six states in Mexico. Peripheral blood leukocytes (PBLs) were isolated from whole blood with anticoagulant, and genomic DNA was extracted from the PBLs using a commercial kit. Then, a set of primers that hybridize in conserved regions of the BLV pX region were used, which allowed for PCR standardization to detect proviral DNA in infected cells. Positive amplicons were sequenced using the Sanger method, resulting in 1156-nucleotide-long final sequences that included the four pX region genes. The experimental group consisted of 30 animals. Twelve of these had lymphocytosis, six had lymphoma, and 12 were apparently healthy cattle without any signs of lymphocytosis or lymphoma. The presence of lymphoma was detected in six bovine tumor tissues using histopathology, and the presence of BLV was detected by in situ hybridization. Phylogenetic analysis demonstrated that the 30 sequences were associated with genotype 1, and the genetic distance between the sequences ranged from 0.2% to 2.09%. We identified two sequences in the G4 gene: one with a three-nucleotide deletion resulting in the loss of a leucine (AGU_7488L, in a cow with lymphocytosis), and one with a nine-nucleotide deletion resulting in the loss of leucine, proline, and leucine (AGU_18A, in a cow without lymphocytosis). Analysis of the PX region indicated that positive selection had occurred in the G4, rex, and R3 genes, and we found no difference in proviral DNA load between the studied groups. We were unable to establish an association between variations in the pX region and the development of lymphocytosis, lymphoma, asymptomatic status, or proviral DNA load in BLV-infected cattle.

Molecular Characterization of the env Gene of Bovine Leukemia Virus in Cattle from Pakistan with NGS-Based Evidence of Virus Heterogeneity.

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.

ТЕХНОЛОГИЯ ГЕНОДИАГНОСТИКИ BOVINE LEUKEMIA VIRUS У КРУПНОГО РОГАТОГО СКОТА, В ВЫРАБАТЫВАЕМОМ СЫРЬЕ И ПРОИЗВОДИМОЙ ПРОДУКЦИИ

The most important task of the dairy cattle industry is to obtain high quality raw milk. To achieve it, a set of measures is required, including aimed at increasing the biological safety of produced raw materials. The aim of the study was to create a scientific and methodological basis for the Bovine leukemia virus (BLV) gene diagnostics in a combined format of pathogen indication and identification. This required updating the strategy of BLV PCR-RFLP genotyping, consistent with its phylogenetic classification, taking into account the growing knowledge about the genetic diversity of 11 genotypes of the studied viral pathogen. When staging nested PCR, oligonucleotide primers were used, which initiate at the final stage of the reaction the production of a 444 bp env-gene fragment of the pathogen. Five restriction endonucleases were used in PCR-RFLP BLV genotyping of: PvuII, SspI, AsuHPI, HaeIII, and BstX2I. As a result of verification of the developed Bovine leukemia virus method for gene identification with an updated genotyping strategy, a technical result was obtained, expressed in the ability to identify all 11 BLV genotypes discovered to date by interpreting the generated 58 genotype-associated combinations of PCR-RFLP profiles.

Bovine leukemia virus genotype surveillance in cattle at a slaughterhouse in Aichi Prefecture, Japan, in 2019 using polymerase chain reaction combined with restriction fragment length polymorphism.

Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) is commonly used for genotyping bovine leukemia virus (BLV) in slaughterhouses. However, unclassified BLV genotypes have been sporadically reported. To assess the current status of BLV genetic characterization in cattle, PCR-RFLP was performed on blood samples of 170 cattle (84 Japanese Black, 60 Japanese Black x Holstein, and 26 Holstein) from 17 farms (5 prefectures) at a slaughterhouse in Aichi Prefecture in 2019. A total of 65 samples (38.2%) were BLV positive, and genotype 1 was the most predominant (56/65 samples), followed by genotypes 3 (6 samples) and 5 (1 sample), and two unclassified samples. No relationship between the genotypes and breeds was observed. Sequence and phylogenetic analyses demonstrated that unclassified BLV genotypes clustered with genotype 1 sequences were, therefore, not new genotypes.

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.

Prevalence and molecular epidemiology of bovine leukemia virus in Colombian cattle

Bovine leukemia virus (BLV) is one of the five agents considered most significant for cattle. It is important to determine the prevalence and molecular epidemiology of BLV throughout the country in order to gain a more thorough understanding of the current situation of BLV and to reveal the possibility of masked genotypes that the primers used by OIE are unable to identify. Blood samples were collected at random from 289 cows distributed in 75 farms across the country. PCR amplification of env, gag and tax gene segments was performed. The obtained amplicons were sequenced and then subjected to phylogenetic analyses. A total of 62% of the cows present at 92% of the farms were BLV-positive for gag fragment. Genotype 1 was exclusively detected by env gene segment when analyzed using previously reported primers. However, tax gene analysis revealed circulation of genotype 6 variants, which were also detected based on env gene analysis with newly designed primers. These results indicate that current genotyping approaches based on partial env sequencing may bias BLV genetic variability approaches and underestimate the diversity of the detected BLV genotypes. This report is one of the first molecular and epidemiological studies of BLV conducted in Colombia, which contributes to the global epidemiology of the virus; it also highlights the substantial impact of BLV on the country's livestock and thus is a useful resource for farmers and government entities.

Molecular characterization of bovine leukemia virus reveals existence of genotype 4 in Chinese dairy cattle

Bovine leukemia virus (BLV) causes enzootic bovine leucosis and is widely spread worldwide, except several European countries, Australia and New Zealand. Although BLV is highly prevalent in China, information about the genetic diversity and evolutionary dynamics of BLV among Chinese dairy herds is still lacking. To determine the genetic variability of BLV, 219 cows from four cities of Ningxia province of China were screened for BLV infection by fluorescence resonance energy transfer (FRET)-PCR and sequencing, 16 selected positive samples were subjected to molecular characterization. Phylogenetic analysis using the neighbor-joining (NJ) method on complete sequences of envelope (env) gene of BLV obtained from China and those available in GenBank (representing BLV genotypes 1–10) revealed that those Chinese strains belonged to genotypes 4 and 6. Totally, 23 mutations were identified and 16 of them were determined to be unique mutations among Chinese strains. Alignment of the deduced amino acid sequences demonstrated six mutations in glycoprotein 51 (gp51) and three mutations in glycoprotein 30 (gp30) located in the identified neutralizing domain (ND), CD8+ T cell epitope, E-epitope, B-epitope, gp51N12 and cytoplasmic domain of transmembrane protein. This study reported for the first time the BLV genotype 4 in China, and further studies are warranted to compare its immunogenicity and pathogenicity with other BLV genotypes.

Detection and molecular characterization of bovine leukemia virus in various regions of Iran.

Purpose. Bovine leukemia virus (BLV) infects cattle worldwide, imposing an economic impact on the dairy cattle industry. The purpose of this study was to evaluate the molecular epidemiology of BLV in Iran.Methodology. Blood samples taken from 280 cows aged over 2 years old from 13 provinces of Iran were used for leukocyte count and blocking ELISA. Genomic DNA was extracted from the peripheral blood leukocytes of BLV-infected samples and fetal lamb kidney cells to perform PCR of partial env, rex and tax genes and long-terminal-repeat region. The PCR products were sequenced, the phylogenetic tree of each gene was constructed, and nucleotide and amino acid sequence pair distances were calculated.Results. The frequency of BLV infection was 32.8 % among animals and was 80 % among provinces. In BLV seropositive animals, the rate of persistent lymphocytosis was 36.9 %. The constructed phylogenetic trees showed the presence of two BLV genotypes (1 and 4) in Iranian strains. As previous studies, our results showed that the env gene was more variable than previously thought, the Rex protein could withstand more amino acid changes compared to the Tax protein, and no significant differences were observed in average changes of the nucleotide of these genes between clinical stages.Conclusions. Our data indicates an increase in the frequency of this infection in Iran. This is the first study report of the presence of BLV genotype 4 in Iranian farms. These findings may have an important role in the control and prevention of BLV infection in Iran and other countries.

Short communication: Genotyping and single nucleotide polymorphism analysis of bovine leukemia virus in Chinese dairy cattle

Bovine leukemia virus (BLV) causes enzootic leucosis in cattle and is classified into 10 genotypes with a worldwide distribution, except for several European countries, Australia, and New Zealand. Although BLV is widespread in Chinese cows with the positive rate of 49.1% at the individual level, very little is known about the BLV genotype in dairy cattle in China. To determine BLV genetic variability in cows in China, 112 BLV-positive samples from 5 cities in China were used for BLV molecular characterization in this study. Phylogenetic analysis using the neighbor-joining method on partial env sequence encoding gp51 obtained from 5 Chinese cities and those available in GenBank (n = 53, representing BLV genotype 1-10) revealed the Chinese strains belonged to genotype 6. Seven unique SNP were identified among Yancheng, Shanghai, and Bengbu strains out of the total 12 SNP identified in Chinese strains. The genotyping coupled with SNP analysis of BLV can serve as a useful molecular epidemiological tool for tracing the source of pathogens. This study highlights the importance of genetic analysis of geographically diverse BLV strains to understand BLV global genetic diversity.

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.

Bovine Leukemia Virus in Bovine Aborted Fetuses

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.

Genetic identification of bovine leukaemia virus

Molecular genetic research methods make it possible to evaluate the genetic diversity of bovine leukemia virus (BLV) and are the most informative approaches to its genetic identification. Molecular genetic research methods work well for the phylogenetic analysis of sequenced nucleotide DNA sequences of the provirus, as well as for the polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) according to the phylogenetic classification of the pathogen. The purpose of the research was to study the scientific and methodological approaches to the genetic identification of bovine leukemia virus, integrated into the molecular monitoring of infection of cattle with BLV genotypes. The authors used PCR-RFLP-genotyping and comparative phylogenetic analysis of aligned nucleotide sequences of the env gene fragment of the BLV provirus isolates to detect the genotypic affiliation of the cattle from twenty-one livestock farms of the Republic of Tatarstan. As a result, isolates of four out of ten BLV genotypes were found in the Tatarstani cattle, namely genotypes 1, 4, 7, and 8. The research involved a comparative analysis of 505 nucleotide sequences of a fragment of the BLV env gene, including those deposited in GenBank NCBI. The analysis confirms the inconsistency of several earlier PCR-RFLP typing strategies with the current approach in assessing the genotypic diversity by phylogenetic analysis. The improved strategy of PCR-RFLP genotyping of BLV corresponds with its modern phylogenetic classification. The strategy makes it possible to identify all the known genotypes of the viral pathogen. Its validity has been proved by in silico modelling of restrictogrammes and a phylogenetic analysis of the env gene fragment of 57 reference isolates of ten BLV genotypes that generate 57 genotype-associated combinations of diagnostically significant PCR-RFLP profiles.

Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil

ABSTRACT: Bovine leukemia virus (BLV) is a member of Retroviridae family, genus Deltaretrovirus, and the main viral agent responsible for economic loses in dairy herds. Some studies have been carried out about BLV genotypes, and at least seven genotypes were found out in samples of different regions of the world. The objective of this study was to identify BLV samples from seropositive dairy cattle in Santa Catarina state, Brazil, using molecular techniques. Blood samples were collected (454) from dairy cattle from 31 different farms, and serology using agar gel immunodiffusion test (AGID) was performed. After that, 191 seropositive samples were submitted to DNA extraction, and in 77 samples the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene was performed. Nineteen DNA samples were subjected to restriction fragment length polymorphism (RFLP) analysis by digestion of the PCR fragment by five restriction endonucleases - BamHI, HaeIII, Tru9I, TaqI, and MwoI. It was found 42% seropositive animals (191/454) and 68% positives of the farms (21/31). The PCR showed 80.5% (62/77) of animals positive. The RFLP analysis identified five different genotypes dispersed by Santa Catarina state, with the highest prevalence for genotype X (47.4%). Overall, our results identified the viral genotypes present in dairy cattle and the prevalence of new variants in representative farms from Santa Catarina state.

Presencia de los genotipos 1 y 3 del virus de la leucosis bovina en Antioquia, Colombia

El virus de la leucosis bovina (BLV) es un retrovirus inmuno- supresor que afecta, principalmente, al ganado lechero; sus células diana son los linfocitos B, en los cuales, integra su genoma, infectando al ganado de por vida. Es importante identificar la distribución del BLV en la región y reconstruir su historia evolutiva, a través de árboles filogenéticos; para el departamento de Antioquia, este es el primer reporte de los genotipos del BLV . El objetivo de este estudio fue identificar el genotipo de BLV que circula en ganado lechero de diferentes regiones del departamento de Antioquia, Colombia . Se extrajo ADN de 8 vacas Holstein . Se realizó una PCR anidada, para amplificar un fragmento del gen env de 444 pb . El gen env viral codifica la proteína de superficie gp51, altamente conservado y es usado en análisis filogenéticos . Los amplicones obtenidos se secuenciaron, se alinearon manualmente en el programa MEGA V7 y se compararon con 53 secuencias del gen env viral, registradas en GenBank . El análisis filogenético, se realizó por métodos de Máxima Verosimilitud y Bayesianos . Se encontraron dos genotipos circulantes: el genotipo más común fue 1, hallado en siete muestras, agrupadas con secuencias de EE . UU, Argentina y Japón; solo una muestra se clasificó como genotipo 3 y se agrupó con muestras de EE . UU y Japón . Al menos dos genotipos (1 y 3) de BLV están circulando en Antioquia; sin embargo, se deben evaluar más bovinos y hatos para elucidar la diversidad y la distribución de BLV en Colombia.