HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil. .

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.

Target-directed catalytic metallodrugs

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vs metal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements.

Analysis of Xenotropic Murine Leukemia Virus-Related Virus (XMRV) in Korean Blood Donors in a Medical Center / 대한수혈학회지

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) has been detected in peripheral blood mononuclear cells (PBMNs), therefore, it has been regarded as being infectious and transmittable by transfusion. Thus, we attempted to detect XMRV in blood samples in order to confirm the absence of XMRV from blood donors. METHODS: We achieved 165 blood donors and four chronic fatigue syndrome (CFS) patients. We performed real-time polymerase chain reaction using the LightCycler 480 (Roche, Penzberg, Germany) for the gag and env genes of the XMRV genome. DNA was extracted from peripheral blood samples. We used Uracil-N-Glycosylase in order to prevent contamination and DNA extracted from mouse embryonic fibroblasts (MEF) for amplification control. RESULTS: No XMRV was detected in any of the blood donors in both the gag and env genes. In four CFS patients, amplification was not detected in the gag gene. In two of four CFS patients, amplifications were detected and the melting temperature was in agreement with that of MEF control in the env gene. CONCLUSION: Although XMRV was not present in blood samples from blood donors, this is the first report on XMRV in Korean blood donors. We confirmed the absence of XMRV in Korean blood donors, the same as studies reported in other countries.

Distribution of human immunodeficiency virus type 1 subtypes in the state of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Variability in HIV-1 partial genomic sequences in Costa Rican patients: analysis with different bioinformatics tools / Variabilidad de secuencias genómicas parciales del VIH-1 en pacientes costarricenses: análisis con diferentes herramientas bioinformáticas

OBJECTIVE: To estimate subtype and genomic variability in the HIV pol gene of Costa Rican patients by using different bioinformatics tools and to use this information to establish new policies to better manage these patients. METHODS: A total of 113 pol sequences available from Costa Rican patients under highly active antiretroviral therapy were analyzed by using the Genotyping, REGA, Stanford, and MEGA programs. The pol sequences came from 77 virologic failures (VF) and 36 basal samples (BS). Of the 77 VF, 22 also were sequenced in the env region. RESULTS: No major differences were found among the variables studied. However, there was a tendency for more variability in VF patients with a high baseline viral load. In the pol gene, 75 percent-83 percent of BS and 66 percent-75 percent of VF samples were pure B subtype by Genotyping and REGA, respectively. The other samples presented variations related mainly to circulating recombinant form CRF12 by genotyping or to CRF17 or -29 by phylogenetic analysis or a new possible BD recombinant with all programs. In the Stanford program, all variable samples showed a subtype B with high polymorphism. The variability in the env sequences was lower than that in the pol region. CONCLUSION: The B subtype is predominant in Costa Rican HIV-positive patients. There is high variability within sequences with potential recombination between B and F or D subtypes. The BD recombinant has not been previously reported. This high variability is likely the result of possible recombinant events, nonadherence to antiretroviral therapy, sexual intercourse without protection, and many sexual partners. Similar studies should be done in other countries in the Region, in particular in those places with extensive immigration, in order to decrease the possibility of virus variability as well as the cost of antiretroviral therapy.

OBJETIVOS: Determinar el subtipo y la variabilidad genómica del gen pol del VIH de pacientes costarricenses mediante diferentes herramientas bioinformáticas y el uso de esta información para establecer nuevas políticas para mejorar el diagnóstico y el tratamiento de estos pacientes. MÉTODOS: Se analizaron 113 secuencias del gen pol de pacientes costarricenses bajo tratamiento antirretrovírico de gran actividad mediante cuatro programas: Genotyping, REGA, Stanford y MEGA. Las secuencias pol analizadas provenían de 77 casos considerados fracasos virológicos (FV) y 36 muestras iniciales (MI). También se secuenció la región env de 22 de los 77 FV. RESULTADOS: No se encontraron diferencias importantes entre las variables estudiadas. No obstante, se observó una tendencia a una mayor variabilidad en los pacientes FV que tenían una elevada carga viral inicial. Con respecto al gen pol, 77-83 por ciento de las MI y 66-75 por ciento de las muestras de los FV eran del subtipo B puro según Genotyping y REGA, respectivamente. Las otras muestras presentaron variaciones relacionadas principalmente con la forma recombinante en circulación CRF-12 según Genotyping, con la CRF-17 o la CRF-29 según el análisis filogenético, o una nueva posible forma recombinante BD según todos los programas. Con el programa Stanford, todas las muestras variables reflejaron un subtipo B con elevado polimorfismo. La variabilidad de la secuencia env fue menor que la de la región pol. CONCLUSIONES: El subtipo B fue el predominante en los pacientes positivos al VIH en Costa Rica. Existe una alta variabilidad en las secuencias con una posible recombinación entre los subtipos B, y F o D. La forma recombinante BD no se había notificado antes. Esta elevada variabilidad parece ser el resultado de posibles eventos de recombinación, la falta de adhesión al tratamiento antirretrovírico, las relaciones sexuales sin protección y numerosas parejas sexuales. Se deben emprender estudios similares en otros países de la Región, en particular en los lugares con mucha inmigración, para reducir tanto la posibilidad de que el virus varíe como el costo del tratamiento antirretrovírico.

Immunogenicity of plasmid DNA and adenoviral vectors encoding HIV-1 subtype B env gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct DNA and recombinant adenovirus vector vaccines containing an env gene from the prevalent subtype B strain in China and try to use them for therapeutic and prophylactic vaccines.</p><p><b>METHODS</b>The candidate plasmid DNA vaccine pVR-gp160 and recombinant adenovirus vaccine rAdV-gp160 were constructed separately. BALB/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gp120-specific cellular responses and antibody levels were detected by ELISPOT and ELISA respectively.</p><p><b>RESULTS</b>DNA vaccine alone and combined vaccines in a DNA prime/rAdV-gp160 boost vaccination regimen induced high level of Gp120-specific cellular responses. While low level of Gp120-specific antibodies were elicited in all groups.</p><p><b>CONCLUSION</b>DNA and rAdV vaccines could efficiently express Gp160 protein and activate specific cellular responses.</p>

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.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07_BC. A highly replication-competent simian/human immunodeficiency viruses (SHIV) construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell (PBMC), establish infection in Chinese rhesus macaque.</p><p><b>METHODS</b>A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques (Macaca mulatta) was infected with one SHIV via intravenous infusion.</p><p><b>RESULTS</b>One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection.</p><p><b>CONCLUSIONS</b>We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF_07BC recombinant strain.</p>

Surveillance of subtype and genetic variation of the circulating strains of HIV-1 in Thailand.

Two HIV-1 strains, CRF01_AE and subtype B', were reported in Thailand during the early years of the epidemic. Recently, an intersubtype recombination of HIV-1 strain was found in Thailand. Eight-hundred and twenty-eight samples collected during years 1995-2004 from high-risk groups in Bangkok, northern, northeastern, and southern region of Thailand were studied. HIV-1 env nucleotide sequences were used for phylogenetic analysis of the circulating HIV-1 strain. By single HIV-1 region (env) genotyping, CRFO1_AE was found in 97.3% and HIV-1 subtype B was found in 2.7%. A predominance of CRF01_AE was found in all geographic regions. Parallel analysis of the HIV-1 gag and env genes demonstrated that 2.1% and 4.0% of recombinant HIV-1 strains were found using p17 and p24 region sequences, respectively. The recombinant gag gene was also found in one southern isolate. Phylogenetic analysis of HIV-1 isolated from 20 provinces in 2002 suggested the northern and northeastern isolates were more related than the southern isolates which had the lowest genetic diversity of 0.13. The GPGQ V3 loop tip was also present in isolates from all regions. The molecular epidemiological data from this study may be useful for surveillance design as well as targeting prevention efforts. It also provides information regarding new antigenic regions of circulating strains responsible for the HIV-1 epidemic in Thailand.

Difficulties in the diagnosis of HTLV-2 infection in HIV/AIDS patients from Brazil: comparative performances of serologic and molecular assays, and detection of HTLV-2b subtype

The current diagnosis of human T-lymphotropic virus type-2 (HTLV-2) infection is based on the search of specific antibodies; nevertheless, several studies conducted in Brazil pointed deficiencies of the commercially available kits in detecting HTLV-2, mostly in HIV/AIDS patients. This study searched for the presence of HTLV-1 and -2 in 758 HIV/AIDS patients from Londrina, Paraná, Brazil. Serum samples were screened for HTLV-1/2 antibodies using two EIA kits (Vironostika and Murex), and confirmed by WB (HTLV Blot 2.4, Genelabs). The results obtained by EIA disclosed 49 (6.5 percent) reactive sera: 43 positive by both EIA kits, and six with discordant results. WB confirmed HTLV-1 infection in seven samples (0.9 percent) and HTLV-2 in 21 sera (2.8 percent). Negative and indeterminate results were detected in four (0.5 percent) and 16 (2.1 percent) sera, respectively. Blood from 47 out of 49 HTLV seroreactive patients were collected and analyzed for the presence of env, LTR and tax genomic segments of HTLVs by PCR. PCR confirmed six cases of HTLV-1 and 37 cases of HTLV-2 infection (14 out of 16 that were found to be WB indeterminate). Restriction analysis of the env PCR products of HTLV-2 disclosed 36 isolates of HTLV-2a/c subtype, and one of HTLV-2b subtype. These results emphasize the need of improving serologic tests for detecting truly HTLV-2 infected patients from Brazil, and confirm the presence of HTLV-2b subtype in the South of this country.

O diagnóstico de infecção por HTLV-2 se baseia na pesquisa de anticorpos específicos, entretanto, vários estudos conduzidos no Brasil têm apontado falhas nos kits sorológicos disponíveis no mercado em detectar HTLV-2, principalmente nos pacientes com HIV/aids. Este trabalho avaliou a presença de infecção por HTLV-1 e -2 em 758 pacientes HIV/aids de Londrina, Paraná, Brasil. Amostras de soro foram analisadas quanto à presença de anticorpos anti-HTLV-1/2 por dois kits de EIA (Vironostika e Murex) e confirmados por WB (HTLV Blot 2.4, Genelabs). Os resultados obtidos pelos testes sorológicos mostraram 49 (6,5 por cento) soros reagentes: 43 positivos para ambos os kits e seis com resultados discordantes. O WB confirmou infecção por HTLV-1 em sete soros (0,9 por cento) e HTLV-2 em 21 soros (2,8 por cento). Resultados negativos e indeterminados foram detectados, respectivamente, em quatro (0,5 por cento) e 16 (2,1 por cento) soros. Amostras de sangue de 47 dos 49 pacientes com sorologia reagente foram avaliadas quanto à presença de segmentos do genoma dos HTLVs (env, LTR e tax), usando a técnica de PCR. As PCRs confirmaram seis casos de infecção por HTLV-1 e 37 casos por HTLV-2 (14 dos 16 cuja sorologia resultou WB indeterminada). A subtipagem de HTLV-2 por análise de restrição enzimática de produtos da PCR env mostrou 36 isolados de subtipo HTLV-2a/c e um HTLV-2b. Esses resultados reforçam a necessidade de melhorar o diagnóstico de infecção por HTLV-2 no Brasil e confirmam a presença do subtipo HTLV-2b na região sul do país.

Development of Neutralization Assay using Murine Leukemia Virus (MuLV) Pseudotyped with Japanese encephalitis Virus (JEV) env Gene

The envelope (E) glycoprotein of JEV is the major antigen to elicit neutralizing antibody (NAb) against JEV infection. In order to develop a rapid and safe neutralization assay system for evaluation of the JEV vaccine strains, we constructed JEV-pseudotyped viruses with JEV env genes (Nakayama-NIH, Beijing-1). The titers of JEV-pseudotyped viruses with NK and BJ strains were 4.0x10(4) IFU/ml and 1.3x10(5) IFU/ml in Vero cell cultures, respectively. We have analyzed the neutralization activity of immunized mouse sera with JEV-NK and JEV-BJ pseudotyped viruses. The neutralizing antibody titers of NK and BJ (50% reduction of virus) were about 1:10,000 at each immunized sera. Compared with conventional plaque reduction neutralization test (PRNT), the method using JEV-pseudotyped virus has desirable advantages such as more rapid, easier, and non-biohazardous. This neutralization assay system might be useful to evaluate NAb activity against JEV vaccine strains or vaccine candidates.

Human immunodeficiency virus type- 1 subtypes of infected patients in Espírito Santo, Brazil

Genetic variability of human immunodeficiency virus type - 1(HIV-1) is a potential threat for both diagnosis and treatment of HIV/AIDS, as well as the development of effective vaccines. Up to now, HIV subtypes circulating among HIV-positive patients in the state of Espírito Santo were not known. In the present study, blood samples from 100 therapy-naïve HIV-1 infected patients were collected and the HIV subtype was determined through the Heteroduplex Mobility Assay (HMA). Ninety-seven out of 100 studied samples were subtyped by HMA, 73 samples (75.2 percent) were from subtype B, 9 (9.3 percent) from subtype F, 3 (3.1 percent) from subtype C, 6 (6.2 percent) Benv/Fgag, and another 6 (6.2 percent) Fenv/Bgag, what suggests that recombinant viruses were present in the studied samples. Twenty-eight percent of the subtype B samples were represented by the Brazilian B" subtype, which were identified by RFLP with Fok I. Data presented here demonstrate that the epidemiological characteristics of the HIV epidemic in the state of Espírito Santo are similar to those from the other Southeastern states and helped to better understand the genetic polymorphism of HIV in Brazil.

Human immunodeficiency virus type 1 strains epidemic in Shenzhen / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the epidemic status of human immunodeficiency virus type 1 (HIV-1) subtypes in Shenzhen and to study their transmission source and routes.</p><p><b>METHODS</b>HIV-1 env and gag genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 122 HIV-1 carriers confirmed in Shenzhen. The C2-V3 region (about 450 bp) of HIV-1 env and P17/ P24 region were sequenced.</p><p><b>RESULTS</b>Among 122 samples, 6 HIV-1 strains including 3 circulating recombinant forms (CRFs) of CRF01_AE, CRF08_BC, CRF07_BC and 3 subtypes of B', B, C were found in Shenzhen, and the percentages were 45.1% (55/122) for CRF01_AE, 31.1% (38/122) for CRF08_BC, 6.6% (8/122) for CRF07_BC, 14.8% (18/122) for B' subtype, 1.6% (2/122) for B subtype, and 0.8% (1/122) for C subtype. The intragroup genetic distances were (4.455 +/- 1.478)%, (2.997 +/- 1.345)%, (4.380 +/- 2.024)%, (5.186 +/- 2.487)%, and (4.869 +/- 2.638)%, respectively. In comparison with the sequence of respective international strains 01AE. TH. 90. CM240, 97CNGX-9F, CN. 97. C54A, B. US. 83. JRFL, and RLA2, the genetic distances were (5. 228 +/- 0.823)%, (3.634 +/- 1.073)%, (4.233 +/- 1.119)%, (4.950 +/- 2.564)%, and (5.795 +/- 2.198)%, respectively. The major subtypes found in injection drug users (IDUs) were CRF07_BC, CRF08_BC, and CRF01_AE strains. CRF01_AE and B' strains were epidemic mainly in sexual workers.</p><p><b>CONCLUSION</b>There are 3 HIV-1 subtypes (B', B, C) and 3 CRFs (CRF01_AE, CRF08_BC, CRF07_BC) epidemics in Shenzhen. The predominant subtypes varies among different transmission routes. While CRF01_AE is predominant among sexual workers, CRF08_BC and CRF01_AE are major subtypes among IDU population.</p>

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

Human immunodeficiency virus type-1 (HIV-1) subtypes in Jamaica / Subtipos del virus tipo-1 de la inmunodeficiencia humano (VIH-1) en Jamaica

The subtypes of 141 isolates of human immunodeficiency virus type-1 (HIV-1) from Jamaica were determined by a combination of env and gag heteroduplex mobility analysis (HMA) genotyping. The majority of HIV-1 isolates were subtype B (131/141, 93.0); one (0.8) isolate each of subtypes C, D and E was found and 7 (4.9) were indeterminate. These results and the failure of the sets of primers used to amplify some of the HIV-1 isolates provide strong evidence of genetic diversity of the HIV/AIDS epidemic in Jamaica. Surveillance of the circulating HIV-1 genetic subtypes is a pre-requisite for developing regional vaccine strategies and understanding the transmission patterns of the virus. This is the first study of its kind in Jamaica and the findings complement data from other Caribbean countries. This work supports the view of colleagues from the French and Spanish-speaking Caribbean that an epidemiological network supported by regional laboratories will help track this epidemic accurately with positive outcomes for the public.

Los subtipos de 141 aislados del virus tipo 1 de la inmunodeficiencia humno (VIH-1) en Jamaica, fueron determinados combinando la genotipificación por análisis de heterodúplex (HMA) en los genes env y gag. La mayor parte de los aislados HIV-1 fueron del subtipo B (131/141, 93.0%), se halló uno (0.8%) aislado para cada uno de los subtipos C, D y E, en tanto que 7 (4.9%) fueron indeterminados. Estos resultados y el fallo de los conjuntos de primers usados para amplificar algunos de los aislados de VIH-1, ofrecen fuerte evidencia de la diversidad epidémica del VIH/SIDA en Jamaica. La vigilancia de los subtipos genéticos de VIH-1 en circulación, constituye un pre-requisito, tanto para desarrollar estrategias de vacunas a nivel regional, como para entender los patrones de transmisión del virus. Este es el primer estudio de este tipo en Jamaica, y nuestros hallazgos complementan los datos obtenidos en otros países del Caribe. Coincidimos con nuestros colegas del Caribe francófono e hispano-parlante en cuanto a que una red epidemiológica apoyada por los laboratorios regionales, nos ayudaría a continuar rastreando esta epidemia con exactitud, y con resultados positivos para el público.

Early detection of HIV-1 in infants by PCR.

Infants of HIV-infected mothers are at great risk of becoming infected with HIV during childbirth. Many infants acquire HIV during labor and delivery. Others can acquire HIV through the mixing of fetal and maternal blood as the placenta separates. The duration of membrane rupture, acute chorioamnionitis and invasive delivery techniques that increase the baby's contact with the mother's blood have been associated with higher risks of MTCT during labor and delivery. HIV is present in breast milk and risk of its transmission during breastfeeding depends on several factors, including: infant age, pattern of breastfeeding, breastfeeding duration, breast health, maternal viral load and maternal immune status. Infants born to HIV infected mothers carry their mother's antibodies in their blood into the second year of life, even if the infants themselves are not infected. For this reason, standard HIV antibody tests cannot reliably confirm HIV infection in infants until after the maternal antibodies have disappeared. Tests that can diagnose pediatric HIV infection accurately during the first year of life include HIV-PCR, CD4/CD8 counts, P24 antigen tests, and viral cultures. PCR offers an effective tool to reliably diagnose HIV in a pediatric age group. Nineteen infants born by normal delivery to HIV-1 seropositive mothers were studied by PCR for the HIV1 env gene. Thirteen babies (68.5%) were negative whereas 6 babies were found to be positive (31.5%). Although PCR is one of the most useful tests for this clinical situation, it is not definitive. Therefore, PCR should be interpreted with caution and repeated at regular defined intervals, preferably lasting until the HIV antibody status of the infant is resolved.

Construction and expression of a Rev-dependent TNF-R1 expressing HIV-infected-cell injurious vectors / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Rev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE).</p><p><b>METHODS</b>Molecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope.</p><p><b>RESULTS</b>The new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours.</p><p><b>CONCLUSIONS</b>Rev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.</p>

Genomic Features of Retroelements and Implications for Human Disease

Most of the endogenous retroviral genes integrated into the primate genome after the split of New World monkeys in the Oligocene era, approximately 33 million years ago. Because they can change the structure of adjacent genes and move between and within chromosomes they may play important roles in evolutionas well as in many kinds of disease and the creation of genetic polymorphism. Comparative analysis of HERVs (human endogenous retroviruses) and their LTR (long terminal repeat) elements in the primate genomes will help us to understand the possible impact of HERV elements in the evolution and phylogeny of primates. For example, HERV-K LTR and SINE-R elements have been identified that have been subject to recent change in the course of primate evolution. They are specific elements to the human genome and could be related to biological function. The HERV-M element is related to the superfamily of HERV-K and is integrated into the periphilin gene as the truncated form, 5'LTR-gag-pol-3'LTR. PCR and RT-PCR approaches indicated that the insertion of various retrotransposable elements in a common ancestor genome may make different transcript variants in different primate species. Examination of the HERV-W elementrevealed that env fragments were detected on human chromosomes 1, 3-7, 12, 14, 17, 20, and X, whilst the pol fragments were detected on human chromosomes 2-8, 10-15, 20, 21, X, and Y. Bioinformatic blast search showed that almost full-length of the HERV-W family was identified on human chromosomes 1-8, 11-15, 17, 18, 21, and X. Expression analysis of HERV-W genes (gag, pol, and env) in human tissues by RT-PCR indicated that gag and pol were expressed in specific tissues, whilst env was constituitively expressed in all tissues examined. DNA sequence based phylogenetic analysis indicated that the gag, pol and env genes have evolved independently during primate evolution. It will thus be of considerable interest to expand the current HERV gene information of various primates and disease tissues.

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.