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

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

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

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

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.
Base Sequence ; Chromosomes, Human ; Endogenous Retroviruses ; Genes, env ; Genome ; Genome, Human ; Humans* ; Phylogeny ; Platyrrhini ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Primates ; Retroelements* ; 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

BACKGROUND: Previous data have been reported that subtype B is prevalent in South Korea, but neither the extent nor the proportion of subtypes could be evaluated. This study was designed to analyze the distribution of HIV-1 subtypes, temporal instructions and transmission dynamics between epidemiological groups. METHODS: 1,280 Koreans had been diagnosed as HIV seropositive during the period 1985 to 2000. Among them, 134 individuals were selected for this molecular epidemiological study. 134 DNAs were isolated from uncultured or cultured peripheral blood mononuclear cells. V3-V5 (0.7 kb) fragment of HIV-1 env gene was amplified by nested polymerase chain reaction and was sequenced. RESULTS: HIV-1 isolates from thirty-seven homosexuals were all subtype B (100%). On the other hand, 66 isolates from 94 heterosexuals were subtype B (70%) and 28 were non B subtypes (30%:13 A, 4 C, 2 D, 8 E, 1 G). Only subtype B strains were isolated from 73 males who were infected with HIV inside Korea while 16 B and 20 non B subtype strains were isolated from 36 males who were HIV infected outside of Korea. However, B and non B strains were isolated half and half from females who were infected inside Korea except one. CONCLUSION: The HIV-1 subtype B strains are prevalent in Korea from the early HIV infection until present in both homo and heterosexuals. Non B strains have been transmitted from men who were infected outside Korea to their spouses and casual partners. So, we need further study to monitor subtype B and non B HIV transmission in epidemiological groups of Korea.
DNA ; Female ; Genes, env ; Hand ; Heterosexuality ; HIV ; HIV Infections ; HIV-1* ; Hominidae ; Homosexuality ; Humans ; Korea* ; Male ; Molecular Epidemiology ; Polymerase Chain Reaction ; Spouses

Xenotransplantation of porcine organs has the potential to overcome the acute shortage of human tissues and organs for human transplantation. Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply. However, this procedure is also associated with a number of safety issues related to zoonotic infections. Among such zoonotically important pathogens, porcine endogenous viruses (PERVs) represent the most concerned virus as they persist asymptomatically and show germline transmission in pigs. They belong to gamma retroviruses and are of three types viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from miniature pigs to analyze the envelope gene of PERVs. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.1-TOPO vectors and sequenced. A total of 51 env clones were obtained from two miniature pigs, types M149 and T1111. Phylogenetic analysis of these genes revealed the presence of only PERV type A and B in the proportion of 45% and 55%, respectively. Among these, 9 clones had the correct open reading frame: eight were PERV type A and one PERV type B. Since both these PERV types are polytropic and have the capacity to infect human cells, our data raise a concern that proviral PERVs might have the potential to generate infectious viruses during or after xenotransplantation in humans.
Clone Cells ; Cloning, Molecular* ; Cloning, Organism ; DNA ; Genes, env ; Humans ; Methods ; Open Reading Frames ; Polymerase Chain Reaction ; Retroviridae ; Swine* ; Transplantation, Heterologous ; Zoonoses