Sequence analysis of C2-V4 of HIV-1 env genes in Fuxin / 预防医学

Objective@#To learn the sequence characteristics of C2-V4 of HIV-1 env genes and the epitope variation of representative broadly monoclonal neutralizing antibody in Fuxin, so as to provide evidence for the HIV-1 variation trend and the biological characteristics of V3 loop.@*Methods @#The whole blood samples of 112 HIV-1 cases in Fuxin Health Service Center from 2018 to 2019 were collected and the DNA was extracted. The C2-V4 of env genes were amplified by nested-PCR and the PCR products were subjected to sequencing. The bioinformatics analysis was carried out using MEGA software, and the V3-tip motifs, co-receptors, net charge and characteristic amino acids were analyzed using HIV Database. @*Results@#Totally 101 effective gene sequences were obtained, and 5 types of V3-tip motifs were found. Among them, 77 pieces of GPGQ ( 76.24% ) were found in CRF01_AE, CRF07_BC, CRF65_cpx and G subtypes; 19 pieces of GPGR ( 18.81% ) were found in CRF01_AE, CRF07_BC and B subtypes; 3 pieces of GPGH, 1 piece of GPGK and 1 piece of GPGA were only found in CRF01_ AE subtype. The co-receptor was mainly CCR5 ( 84, 83.17% ) . The net charge numbers of V3 loops in CRF01_ AE, CRF07_ BC, B, CRF65_cpx and G were 3.28±1.17, 3.22±0.92, 4.25±0.83, 2.50±0.50 and 3, respectively. The mutation rates of neutralizing antibodies binding b12 and VRC01 were 0-9.90%. The deletion rates of N-glycosylation sites of 295 and 332 were 18.81% and 14.85%, without the loss of both sites.@*Conclusions @#The HIV-1 strains in Fuxin from 2018 to 2019 are macrophage-tropic and non-syncytium-inducing, with GPGQ as the main type of V3-tip motif, CCR5 as the main co-receptor, slow replication and low ability to escape neutralizing antibodies.

Progress in HIV-1 Env trimer design / 生物工程学报

Currently, HIV-1 vaccine development has still been a hot pot in the AIDS research. HIV-1 glycoprotein Env is the sole target in the virion surface that mediates the membrane fusion between the virion and cell in the HIV-1 infection process. Env protein is the significant immunogen for HIV-1 vaccine development. In recent years, there have been breakthroughs in the Env trimer research. For example, the strategies including SOSIP, NFL2P, and UFO had been applied to design and generate HIV-1 Env trimer. The improvement of quantity and stability is beneficial to achieve the HIV-1 native-like Env trimer for elicitation of strong neutralizing antibody responsing in animal immunization. This review focuses on the different strategies for Env trimer design and compares their advantages and disadvantages, combining with our work to give some advice, which might provide relevant information for the future HIV-1 immunogen design.

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.

Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil / Determinação da heterogeneidade do genoma do vírus da leucose enzoótica bovina no estado de Santa Catarina, Brasil

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.(AU)

O bovine leukemia virus (BLV) é um membro da família Retroviridae, gênero Deltaretrovirus, e o principal agente viral causador de perdas econômicas em rebanhos leiteiros. Diversos estudos têm sido feitos sobre os genótipos de BLV, e foram encontrados pelo menos sete em amostras de diferentes partes do mundo. O objetivo deste estudo foi realizar a caracterização molecular de amostras de BLV de bovinos leiteiros soropositivos no estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos de 31 propriedades, e fez-se inicialmente a sorologia por meio do teste de imunodifusão em gel de ágar. Após a sorologia, 191 amostras soropositivas foram então submetidas à extração de DNA, e em 77 amostras se realizou a reação da polimerase em cadeia (PCR), para a amplificação de um fragmento de 440 pb do gene env. Dezenove amostras foram submetidas à análise do polimorfismo dos fragmentos de restrição por digestão do fragmento da PCR por cinco enzimas de restrição: BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia apontaram 42% de animais soropositivos (191/454) e 68% de propriedades positivas (21/31). Na PCR, 80,52% (62/77) dos animais apresentaram-se positivos. A análise do polimorfismo dos fragmentos de restrição identificou cinco genótipos circulantes no estado, e a maior prevalência foi observada no genótipo X (47,4%). Este estudo permite-nos conhecer alguns dos genótipos virais presentes em bovinos leiteiros do estado de Santa Catarina, bem como identificar a existência de novas variantes e sua prevalência atual, e os resultados são úteis para futuros estudos epidemiológicos.(AU)

Molecular analysis of a fragment of bovine leukemia virus env gene by Nested-PCR in dairy cows from Pasto, Nariño / Análisis molecular de un fragmento del gen env del virus de leucosis bovina, por PCR anidada en vacas lecheras de Pasto, Nariño / Análise molecular pela reação em cadeia da polimerase de um fragmento do gene env do vírus de leucose bovina aninhada em vacas leiteiras de Pasto, Nariño

Abstract: Introduction: Routine diagnosis for bovine leukemia is performed using indirect serologic tests, although it is recommended to use polymerase chain reaction (PCR), which allows a reliable diagnosis in early stages and in young animals. Moreover, by amplifying the DNA, it is possible to sequence fragments of the virus, which allows to identify genotypes and to construct phylogenetic trees. Objective: To analyze a fragment of the env gene of bovine leukemia virus (BLV) isolated from indirect ELISA-positive animals in dairy farms in the municipality of Pasto (Nariño). Materials and methods: Once the presence of BLV was established in 48 animals over two years old in seven dairy farms in the municipality of Pasto by indirect ELISA test, a nested PCR test was performed to confirm diagnosis and to sequence a fragment of the env gene in positive animals and their daughters. The sequences obtained were compared with the seven genotypes described worldwide by MEGA 6 program. Results: The sequences of the compared fragments do not differ from those described; but they allow their grouping into different clusters according to their similarity. The genotypes found in these farms correspond to genotype 1 and 2 described in the literature. Conclusions: Only one genotype was found on farms with proper recovery and adequate biosecurity measures, and on farms with less control in management and recovery practices, both genotypes described for the region were evident.

Resumen: Introducción: El diagnóstico de rutina para la leucosis bovina se realiza con pruebas serológicas indirectas, aunque se recomienda usar la reacción en cadena de la polimerasa (PCR), que permite un diagnóstico confiable en fases iniciales y en animales jóvenes. Además, mediante la amplificación del ADN se pueden secuenciar fragmentos del virus, que permite identificar los genotipos presentes y construir árboles filogenéticos. Objetivo: Analizar un fragmento del gen env del virus de leucosis bovina (VLB) aislado de animales positivos a ELISA indirecta en fincas lecheras del municipio de Pasto, Nariño. Materiales y métodos: Una vez establecida la presencia del VLB en 48 animales mayores de dos años en siete fincas lecheras del municipio de Pasto mediante la prueba de ELISA indirecta, se realizó una prueba de PCR anidada para confirmar el diagnóstico y secuenciar un fragmento del gen env en los animales positivos y sus hijas. Las secuencias obtenidas se compararon con los siete genotipos descritos en el mundo mediante el programa MEGA 6. Resultados: Las secuencias de los fragmentos comparados no difieren de las descritas; pero permiten su agrupación en diferentes clústeres de acuerdo con su similitud. Los genotipos encontrados en estas fincas corresponden al genotipo 1 y 2 descritos en la literatura. Conclusiones: En las fincas con reposición propia y adecuadas medidas de bioseguridad solo se encontró un genotipo y en las fincas con menor control en las prácticas de manejo y de reposición se encontraron los dos genotipos descritos para la región.

Resumo: Introdução: O diagnóstico de rotina para a leucose bovina se realiza com provas serológicas indiretas, mesmo que se recomenda usar a reação em cadeia da polimerase (PCR), que permite um diagnóstico confiável em fases iniciais e em animais jovens. Além do mais, mediante a amplificação do ADN se podem sequenciar fragmentos do vírus, que permite identificar os genótipos presentes e construir árvores filogenéticas. Objetivo: Analisar um fragmento do gene env do vírus de leucose bovina (VLB) isolado de animais positivos a ELISA indireta em fazendas leiteiras do município de Pasto, Nariño. Materiais e métodos: Uma vez estabelecida a presença do VLB em 48 animais maiores de dois anos em sete fazendas leiteiras do município de Pasto mediante a prova de ELISA indireta, se realizou uma prova de PCR aninhada para confirmar o diagnóstico e sequenciar um fragmento do gene env nos animais positivos e suas filhas. As sequências obtidas foram compararam com os sete genótipos descritos no mundo mediante o programa MEGA 6. Resultados: As sequências dos fragmentos comparados não diferem das descritas; mas permitem a sua agrupação em diferentes clusters de acordo com sua similitude. Os genótipos encontrados nestas fazendas correspondem ao genótipo 1 e 2 descritos na literatura. Conclusões: Nas fazendas com reposição própria e adequadas medidas de biossegurança somente se encontrou um genótipo e nas fazendas com menos controle nas práticas de manejo e de reposição se encontraram os dois genótipos descritos para a região.

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.

Quantitative analysis of human endogeneous retrovirus family W env1 in placentas of patients with preeclampsia in iodine deficiency areas / 中华地方病学杂志

Objective To detect the expression of human endogeneous retrovirus family W env1 (HERVWE1) in placentas of normal pregnant women and patients with preeclampsia and explore its role in the pathogenesis of preeclampsia.Methods Fifty-two cases of placental tissues provided by Shandong Maternity Hospital were from iodine deficiency areas (county or city:.Zhangqu, Changqing, Pingyin) of Shandong Province, including 30 cases of preeclampsia placentas as case group, 22 cases of normal term pregnancy placentas as control group.The mRNA expression of HERVWE1, human chorionic somator mammotropin (HCS), chorionic specific transcription factor (GCMa) and amino acyl carrier 2 (ASCT-2) in placentas of the two groups were determined by real-time PCR, and the protein expression of HERVWE1 was determined by Western blotting.Results The mRNA expression levels of HERVWE1 (0.149 ± 0.045 vs.0.409 ± 0.028) and HCS (336.600 ± 50.100 vs.815.600 ± 101.300) in placentas of patients with preeclampsia were significantly lower than those in the control group (t =25.60, 20.40,all P ＜ 0.05).There were no significant difference in GCMa (0.022 ± 0.007 vs.0.024 ± 0.009) and ASCT-2 (0.423 ± 0.050 vs.0.438 ± 0.060) between the two groups (t =0.87, 0.95, all P ＞ 0.05);Western blotting analysis showed that expression of HERVWE1 protein (0.340 ± 0.010 vs.0.580 ± 0.010) in placentas of the case group was lower than that in normal pregnant women (t =85.50, P ＜ 0.05).Conclusion The expression of HERVWE1 is significantly decreased in placentas of the case group and is associated with pathogenesis of preeclampsia.

Preparation and identification of Env-pseudotyped subtype B HIV-1 / 军事医学

Objective To prepare the Env-pseudotyped subtype B HIV-1 with enhanced green fluorescent protein ( EG-FP) gene,explore HIV-1 infection mechanisms and develop feasible methods of identification .Methods The Env-pseudo-typed viruses were packaged in HEK293T cells by cotransfection, and the reporter gene and P24 protein were detected by PCR, Western blot and ELISA .Reporter gene amplification , viral titration assay and a single round of infection assay were performed after the env-pseudotyped viruses infected HIV-1 permissive cell .Results and Conclusion A generation and identification method of the pseudotyped HIV-1 was established . The Env-pseudotyped subtype B HIV-1 has been prepared, which is able to infect SupT1 and TZM-bl cells through infection assay .

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.

Distribution of enteric bacteria in the sediments of Parangipettai and Cuddalore coast of India.

Distribution of major groups of enteric bacteria viz., Escherichia coli, Shigella spp., Salmonella spp., Vibrio parahaemolyticus and Vibrio cholerae was studied in the sediment samples collected from different depths of 5, 15, 25 and 35 m in Parangipettai and Cuddalore coast during March, 2009 to February, 2010. Among the bacterial population in Parangipettai, V. parahaemolyticus was found to be maximum with an occurrence of 55% followed by Shigella spp. (15%), V. cholerae (12%), Salmonella spp. (12%) and E. coli (6%). In Cuddalore, as an oddity, Vibrio cholerae emerged as dominant species with an incidence of 60% of the total. V. parahaemolyticus came next in the order with an incidence of 14%, followed by E.coli (11%), Shigella spp. (8%) and Salmonella spp. (7%). Similarly, monsoon and premonsoon seasons registered higher bacterial populations in both the regions. When the depth wise results were viewed, 5 and 15 m depths showed maximum (V. parahaemolyticus 3.7x 10 CFU g-1 in Parangipettai; V. cholera - 8.6x10 CFU g-1 in Cuddalore) bacterial population compared to subsequent depths. Cluster and MDS also showed distinct spatial and seasonal variations of bacterial populations in both the regions. Biota-Environment (BIO - ENV) method revealed the combinations of temperature, salinity and TOC as the best match influencing bacterial population.

The Comparison of Genetic Variation in the Envelope Protein Between Various Immunodeficiency Viruses and Equine Infectious Anemia Virus / 中国病毒学·英文版

The envelope protein(Env) of lentiviruses such as HIV,SIV,FIV and EIAV is larger than that of other retroviruses.The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus,demonstrating that envelope is crucial for vaccine.We compared Env variation of the four kinds of lentiviruses.Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor,and the relationship of HIV and EIAV was the furthest.EIAV had the shortest Env length and the least number of potential N-linked glycosylation sites(PNGS) as well as glycosylation density compared to various immunodeficiency viruses.However,HIV had the longest Env length and the most PNGS.Moreover,the alignment of HIV and SIV showed that PNGS were primarily distributed within extracellular membrane protein gp120 rather than transmembrane gp41.It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env.There are low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.

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.

Sequence variation in the env region of SHIV-XJ02170 during in vivo passaging / 中华微生物学和免疫学杂志

Objective To identify the variation in the Env region of SHIV-XJ02170 during passaging in Chinese origin Rhesus Macaques.Methods Fragments of the SHIV-XJ02170 gp160 and gp120 gene were amplified by PCR and RT-PCR separately from the blood samples of SHIV-XJ02170 infected animals at the peak viral load time point.Purified RT-PCR product was ligated into T easy vector and transformed into JM109 competent cells,18 clones were selected by PCR method and sequenced by ABI 3730DNA sequencers.The gene distances(divergence,diversity)were calculated using DISTANCE.Results In all,the SHTV-XJ02170 gp120 gene evolved forward along the virus passaging.It could be found that viral divergence from the founder strain serially enhanced during in vivo passaging,but in the early phase of each passage,SHIV-XJ02170 gp120 gene evolved toward ancestral state upon transmission to a new host.All of the SHIV-XJ02170 strains had V3 loop central motif(GPGQ)and were predicted to be using CCR5 on the basis of the critical amino acids within V3 loop.Conclusion There was significant increase in the genetic distance during serial passaging,and SHIV-XJ02170 gp120 gene evolved forward along passaging.This could partly explain why the virus infectivity was enhanced during in vivo passaging.

The impact of the modification of 2G12 and 2F5 neutralizing epitopes of the HIY-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity / 中华微生物学和免疫学杂志

Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn I . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus (BC02, BC03, BC04, BC05 and BC12) were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can't be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BG03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.

Comparison of the Immunogenicities of HIV-1 Mutants Based on Structural Modification of env / 中国病毒学·英文版

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17 percent for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75 percent for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

Epidemiological comparisons of codon usage patterns among HIV-1 isolates from Asia, Europe, Africa and the Americas

To investigate the genomic properties of HIV-1, we collected 3,081 sequences from the HIV Sequence Database. The sequences were categorized according to sampling region, country, year, subtype, gene name, and sequence and were saved in a database constructed for this study. The relative synonymous codon usage (RSCU) values of matrix, capsid, and gp120 and gp41 genes were calculated using correspondence analysis. The synonymous codon usage patterns based on the geographical regions of African countries showed broad distributions; when all the other regions, including Asia, Europe, and the Americas, were taken into account, the Asian countries tended to be divided into two groups. The sequences were clustered into nine non-CRF subtypes. Among these, subtype C showed the most distinct codon usage pattern. To determine why the codon usage patterns in Asian countries were divided into two groups for four target genes, the sequences of the isolates from the Asian countries were analyzed. As a result, the synonymous codon usage patterns among Asian countries were divided into two groups, the southern Asian countries and the other Asian countries, with subtype 01_AE being the most dominant subtype in southern Asia. In summary, the synonymous codon usage patterns among the individual HIV-1 subtypes reflect genetic variations, and this bioinformatics technique may be useful in conjunction with phylogenetic methods for predicting the evolutionary patterns of pandemic viruses.

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.

Phylogenetic Analysis of env Gene V3-V5 Region of HIV-1 Subtype A Isolates from Korean

Phylogenetic analysis was conducted to monitor transmission of HIV and to investigate the genetic structure of primary isolates from 12 HIV-1 subtype A infected Koreans. The individuals infected with subtype A viruses had been diagnosed as HIV-1 seropositives during the period 1987 to 1995 and blood samples have been collected from 1991 to 1997. DNA of each individual was isolated from uncultured or cultured peripheral blood mononuclear cells. V3-V5 (0.7 kb) fragment of HIV-1 rev gene was amplified by nested polymerase chain reaction and the PCR products were sequenced. The mean value of the divergence of nucleotide of HIV-1 euv V3-V5 fragment was 17.0+/-4.06% (8.6~25.8%) within HIV-1 subtype A isolates from Koreans. This diversity was higher than those of African isolates (13.7+/-2.66%). In the phylogenetic tree, Korean subtype A isolates were not grouped together, but intermingled into African isolates. The results of this study suggested that HIV-1 subtype A variants be introduced from multiple sites of Africa into Korea and the big genetic diversity of Korea HIV-1 subtype A isolates may be further influenced by the range of geographic locations in which the infection occurred rather than the elapsed time between infection and collection of samples and the disease progression.

Phylogenetic Analysis of env Gene V3-V5 Region of HIV-1 Subtype A Isolates from Korean

Phylogenetic analysis was conducted to monitor transmission of HIV and to investigate the genetic structure of primary isolates from 12 HIV-1 subtype A infected Koreans. The individuals infected with subtype A viruses had been diagnosed as HIV-1 seropositives during the period 1987 to 1995 and blood samples have been collected from 1991 to 1997. DNA of each individual was isolated from uncultured or cultured peripheral blood mononuclear cells. V3-V5 (0.7 kb) fragment of HIV-1 rev gene was amplified by nested polymerase chain reaction and the PCR products were sequenced. The mean value of the divergence of nucleotide of HIV-1 euv V3-V5 fragment was 17.0+/-4.06% (8.6~25.8%) within HIV-1 subtype A isolates from Koreans. This diversity was higher than those of African isolates (13.7+/-2.66%). In the phylogenetic tree, Korean subtype A isolates were not grouped together, but intermingled into African isolates. The results of this study suggested that HIV-1 subtype A variants be introduced from multiple sites of Africa into Korea and the big genetic diversity of Korea HIV-1 subtype A isolates may be further influenced by the range of geographic locations in which the infection occurred rather than the elapsed time between infection and collection of samples and the disease progression.