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.
Clone Cells* ; Cloning, Molecular ; Genes, env* ; Genetic Variation ; HIV ; HIV-1* ; Polymerase Chain Reaction* ; RNA-Directed DNA Polymerase ; Tropism

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.
Animals ; Asian Continental Ancestry Group ; Cattle* ; Enzootic Bovine Leukosis ; Genes, env ; Genes, gag* ; Genotype ; Humans ; Korea ; Leukemia Virus, Bovine* ; Polymerase Chain Reaction ; United States

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.
Animals ; Blood Donors ; DNA ; Fatigue Syndrome, Chronic ; Fibroblasts ; Freezing ; Genes, env ; Genes, gag ; Genome ; Humans ; Mice ; Real-Time Polymerase Chain Reaction ; Xenotropic murine leukemia virus-related virus

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.
Africa ; Disease Progression ; DNA ; Genes, env* ; Genes, rev ; Genetic Structures ; Genetic Variation ; Geographic Locations ; HIV ; HIV-1* ; Korea ; Polymerase Chain Reaction

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.
Africa ; Disease Progression ; DNA ; Genes, env* ; Genes, rev ; Genetic Structures ; Genetic Variation ; Geographic Locations ; HIV ; HIV-1* ; Korea ; Polymerase Chain Reaction

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.
Animals ; Antibodies, Neutralizing ; Asian Continental Ancestry Group* ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Genes, env* ; Glycoproteins ; Humans ; Leukemia Virus, Murine* ; Mice ; Neutralization Tests ; Vero Cells

Human immunodeficiency virus type 1 (HIV-1) phenotype plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected persons is associated with a rapid decline of CD4+T cells (CD4+), rapid disease progression, and reduced survival time after AIDS diagnosis. We have reported the effects of Korean red ginseng (KRG) on HIV-1 infected patients. To investigate whether KRG affects HIV-1 at gene level and there is a correlation between genotype and decline of CD4+, the C2-V3 region of env gene from 65 HIV-1 isolates were cloned and sequenced. Distributions of subtype were subtype B 57 (88%), subtype A 4 (6%), subtype C 2 (3%), subtype G 1 (2%), and subtype H 1 (2%). The prevalences of SI according to the number of CD4+ are as follows; 40% (6/15) in CD4+ <100/ ul, 14% (1/7) in 100-200/ul, and 2% (1/43) in >200/ul. Seventy-five percent (6/8) of SI were detected in rapid progressor with the decline of CD4+ over 60/ul per year. The correlation between SI genotype and the detection of immune complex dissociated (ICD) p24 antigen was significant (p<0.001). In the 40 patients followed-up over 60 months by CD4+, there was significant correlation between annual decrease of CD4+ and duration of KRG intake (R=-0.380, p<0.01), whereas no correlation between CD4+ and zidovudine (ZDV) was observed. The intrapatient variation of amino acid level showed significant inverse correlation with the months of KRG intake (R=-0.47, p<0.01). These results suggest that the determination of genotype by C2- V3 sequencing may be used for the evaluation of prognosis of AIDS patient, and long-term intake of KRG may prevent or delay the progression from NSI to SI.
Antigen-Antibody Complex ; Clone Cells ; Diagnosis ; Disease Progression ; Genes, env* ; Genotype ; HIV* ; HIV-1* ; Humans* ; Panax* ; Phenotype ; Prevalence ; Prognosis ; Sequence Analysis* ; Zidovudine

BACKGROUND: Limited data are available on genetic subtypes of HIV- 1 in Korean AIDS patients. To determine subtypes of HIV-1 in Korean patients, we analyzed nucleotide sequences of the env gene of HIV-1 and constructed a phylogenetic tree. METHODS: Nineteen patients infected with HIV-1 were enrolled. The median CD4 + count was 85/mm 3. Peripheral blood mononuclear cells (PBMC) were collected and co-cultivated with pre-stimulated PBMC from HIV-seronegative donors for 7 ~14 days. DNA was extracted from cultured lymphocytes and proviral V3 region of the env gene was amplified by nested polymerase chain reaction (PCR) for direct sequencing without cloning. Sequence analysis was performed by cycle- sequencing and dye terminator methods with an automated DNA Sequenator. The sequences were aligned with nines ets of reference sequences for each subtype by Clustal method. Phylogenetic tree was constructed by the neighbor j oining method. RESULTS: Eighteen of the nineteen sequences fell into subtype B (95%) and one was subtype A (5%). The patient nfected with subtype A was an ex-prostitute and had engaged in sexual contact with sailors who are generally regarded as one of the highest risk groups of HIV infection in Korea. The tetrameric motifs at the tip of the V3 loop were comprised of GPGR (six cases, 32%), GPGS (three cases, 16%), GPGQ, GPGG, GPGK, APGS (one case each, 5%) CONCLUSION: Subtype B is predominant clade of HIV-1 isolated from Korean patients and only one case showed subtype A.
Base Sequence ; Clone Cells ; Cloning, Organism ; DNA ; Genes, env ; HIV Infections ; HIV-1 ; Humans ; Korea ; Lymphocytes ; Military Personnel ; Phylogeny ; Polymerase Chain Reaction ; Sequence Analysis ; Tissue Donors

OBJECTIVETo identify subtypes of human immunodeficiency virus 1 (HIV-1) strains and their distribution, infection sources, and the trends of HIV infection in Jiangsu province.

METHODSAnticongulated bloods from 46 infected persons were collected to separate previrus DNA. HIV-1 env genes were then amplified by nested-PCR and sequenced for their C2-V3 region so as to identify subtypes. The analysis of consensus sequence, genetic distance and phylogenetic tree were conducted with GCG software.

RESULTSBy the end of 2001, there had been six subtypes of HIV-1 strains identified in Jiangsu province: A, B, B', C, D and E. The predominant subtypes were C (accounting for 40.48%) and B' (accounting for 38.10%). Subtype C accounted for 86.67% among injecting DUs while subtype B' accounted for 91.67% among commercial blood donors and receivers.

CONCLUSIONSubtype B'among commercial blood donors was brought to Jiangsu from neighboring provinces. The outbreak of HIV-1 infection among local DUs was caused by subtype C from Xinjiang province. Findings from HIV/AIDS molecular epidemiologic study suggest that it is challenging for Jiangsu to treat patients, apply vaccine, prevent and control AIDS in the future.

China ; epidemiology ; Genes, env ; genetics ; HIV Infections ; blood ; epidemiology ; HIV Seroprevalence ; HIV-1 ; classification ; genetics ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA

BACKGROUNDThe 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.

METHODSA 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.

RESULTSOne 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.

CONCLUSIONSWe 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.

Animals ; Chimera ; Genes, env ; HIV-1 ; genetics ; physiology ; Humans ; Macaca mulatta ; Proviruses ; genetics ; Receptors, CCR5 ; physiology ; Simian Immunodeficiency Virus ; genetics ; physiology