Presence of bovine leukemia virus genotypes 1 and 3 in antioquia, colombia / Presencia de los genotipos 1 y 3 del virus de la leucosis bovina en antioquia, colombia

SUMMARY Bovine leukemia virus (BLV) is an immunosuppressant retrovirus that primarily affects dairy livestock, its target cells are B lymphocytes in which it integrates its genome infecting cattle for life. It is important to identify the distribution of the BLV in the region and to reconstruct its evolutionary history through phylogenetic trees, for the province of Antioquia this is the first report of the BLV genotypes. The aim of this study was to identify the genotype of BLV circulating in dairy cattle of different regions of the province of Antioquia, Colombia. DNA was extracted from 8 Holstein cows. Nested PCR was performed to amplify a fragment of 444 pb of the env gene. The env viral gene codes for surface protein gp51, gene is highly conserved and it is used for phylogenetic analysis. Obtained amplicons were sequenced, manually aligned in MEGA V7 program, and compared to 53 viral env gene sequences registered in GenBank. Phylogenetic analysis was performed by Maximum Likelihood and Bayesian methods. Two circulating genotypes were found: the most common genotype was 1, found in seven samples; they grouped with sequences from EE. UU, Argentina and Japan; only one sample was classified as genotype 3 and was grouped with samples from EE. UU and Japan. At least two genotypes (1 and 3) of BLV are circulating in Antioquia; however, more cattle and herds should be evaluated to elucidate the diversity and distribution of BLV in Colombia.

RESUMEN 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.

Genetic analysis of env and gag gene fragments of bovine leukemia virus identified in cattle from Korea / 대한수의학회지

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. This study was conducted to clarify the molecular characteristics of BLVs obtained from a specific region in Korea. Proviral BLVs were detected in anti-BLV antibody-positive blood samples by PCR. Env and gag fragments were sequenced and compared to previously published reference sequences. Analysis of the env gene sequence revealed that the YI strain was highly similar to genotype 1, including United States and Japanese strains. The gag gene sequence had the highest degree of similarity with a Japanese strain.

Construction of JSRV-NM Pseudovirions by High Efficiency Packaging System of the Lentivirus / 天津医药

Objective To overcome the fact that SRV-NM virus can only multiple and amplify through partially pu-rified jaagsiekte retrovirus inoculated intratracheally in sheep but it cannot be augmented using in vitro cell culture, we con-structed JSRV-NM pseudovirions based on high efficiency packing system of lentivirus. Methods Lentivirus of three high efficiency packing plasmids system pMD.G, pCMV-HIV 8.2 and pHIV-eGFP was developed, and JSRV-NM-env coated plasmid pCMVJSRV-NM was used to substitute VSV-G virus coated plasmid pMD.G then co-transfected into 293T cells to replicate, package and produce restructured JSRV-NM pseudovirions. Gene expression of pseudovirion was determined through WPRE using real time PCR; Virus infectivity was detected through inoculating JSRV-NM pseudovirions into 24 pore plates. Results We construct JSRV-NM pseudovirions successfully based on the lentivirus system. JSRV-NM pseudo-virions can also be concentrated to higher titer (108 TU/mL detected by real time PCR by ultracentrifugation without signifi-cant loss of activity. JSRV-NM and VSV-G pseudovirions infected on Hela cells (both MOI= 3) respectively and no obvi-ous difference were shown on their infection efficiency detected by real time PCR. Conclusion Based on lentivirus system, JSRV-NM pseudovirions can be multipled and amplified in 293T cell culture in vitro. JSRV-NM pseudovirions is stable without loss its infection activity and the requirements of biological laboratory safety II was also met. JSRV-NM pseudoviri-ons will provide a useful tool for further study of JSRV-NM-env infection across species or its induction of lung adenocarci-noma.

Fixations of the HIV-1 env gene refute neutralism: New evidence for pan-selective evolution

We examined 103 nucleotide sequences of the HIV-1 env gene, sampled from 35 countries and tested: I) the random (neutral) distribution of the number of nucleotide changes; II) the proportion of bases at molecular equilibrium; III) the neutral expected homogeneity of the distribution of new fxated bases; IV) the hypothesis of the neighbor infuence on the mutation rates in a site. The expected random number of fxations per site was estimated by Bose-Einstein statistics, and the expected frequencies of bases by matrices of mutation-fxation rates. The homogeneity of new fxations was analyzed using χ2 and trinomial tests for homogeneity. Fixations of the central base in trinucleotides were used to test the neighbor infuence on base substitutions. Neither the number of fxations nor the frequencies of bases ftted the expected neutral distribution. There was a highly signifcant heterogeneity in the distribution of new fxations, and several sites showed more transversions than transitions, showing that each nucleotide site has its own pattern of change. These three independent results make the neutral theory, the nearly neutral and the neighbor infuence hypotheses untenable and indicate that evolution of env is rather highly selective.

Analysis on Quasispecies of HIV-1 env Gene by Single Clone per PCR

The human immunodeficiency viruses (HIV) exhibit tremendous genetic variability in their hosts. It is mainly due to two factors: the error-prone nature of the viral reverse transcriptase enzyme and the effects of environmental constraints such as antiviral therapy, cellular tropism, or HIV-specific immune responses. These quasispecies show fluctuation both in the overall divergence and diversity between individual sequences with different duration after primary infection. For better understand the viral quasispecies, we have performed the longitudinal genetic analysis of HIV-1 env gene V1-C5 region (1.2 kb) by two molecular cloning methods. Diversity indicated that 'single clone per PCR' value was higher than that of 'multiple clones per PCR' in subjects: 0.58-3.15 in subject 1 (P<0.05) and 0.28-2.25 in subject 2 (P<0.05). But divergence was similar in both molecular cloning methods. Phylogenetic analysis of longitudinal sequences at different sampling stages revealed the existence of different topologies individually. These data suggested that 'single clone per PCR' is more efficient than 'multiple clones per PCR' in genetic diversity analysis.