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

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

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

OBJECTIVETo study the epidemic status of human immunodeficiency virus type 1 (HIV-1) subtypes in Shenzhen and to study their transmission source and routes.

METHODSHIV-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.

RESULTSAmong 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.

CONCLUSIONThere 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.

Adolescent ; Adult ; China ; epidemiology ; Female ; Genes, env ; genetics ; Genes, gag ; genetics ; Genes, pol ; genetics ; HIV Infections ; epidemiology ; HIV-1 ; genetics ; Humans ; Male ; Middle Aged ; Molecular Epidemiology ; Polymerase Chain Reaction

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

BACKGROUNDRev 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).

METHODSMolecular-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.

RESULTSThe 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.

CONCLUSIONSRev 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.

AIDS Vaccines ; immunology ; Apoptosis ; Biolistics ; Cell Line, Tumor ; Gene Products, rev ; physiology ; Genes, env ; physiology ; Genetic Vectors ; Humans ; Keratinocytes ; metabolism ; Plasmids ; Receptors, Tumor Necrosis Factor, Type I ; genetics

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

OBJECTIVETo study the effect of NP9 on the growth of transplanted nasopharyngeal carcinoma (NPC) in nude mice and explore the mechanisms involved.

METHODSRecombinant pRc/CMV2-NP9 plasmid was constructed and transfected into the NPC cell lines by lipofectamine 2000. Cell clones stably expressing NP9 were obtained by detecting the mRNA expression of NP9 in G418-resistant clones with RT-PCR. The tumorigenicity and size of transplanted tumors were assessed after inoculation of NPC cells and their transgene clones into Balb/C mice. The expression of PCNA and cyclin D1 in transplanted tumors was detected by immunohistochemistry.

RESULTSThe expression of NP9 was detected in some of NP9 gene-transfected G418-resistant clones of CNE1 and SUNE1. In vivo experiments showed that the tumorigenicity of CNE19 clone was decreased significantly compared to that of CNE1 and its vector control, and the transplanted tumors grew more slowly from SUNE1/NP9 than from SUNE1 and SUNE1/vector. Compared with the vector control, the expression of cyclin D1 and PCNA in CNE1/NP9 transplants was decreased.

CONCLUSIONNP9 inhibits tumorigenicity and growth of NPC transplanted tumor by down-regulating the expression of cyclin D1 and PCNA.

Animals ; Cyclin D1 ; biosynthesis ; genetics ; Endogenous Retroviruses ; genetics ; Female ; Gene Products, env ; biosynthesis ; genetics ; Genes, Tumor Suppressor ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Neoplasm Transplantation ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Tumor Cells, Cultured

OBJECTIVETo characterize CRF01_AE strains of recombinant human immunodeficiency virus type-1 (HIV-1) found in the Second National Molecular Epidemiology Study on HIV in China and to analyze its sequence variation in the env V3-C3 region during the First National Molecular Epidemiology Study (NMES1, 1996 - 1998) to the Second National Molecular Epidemiology Study (NMES2, 2001 - 2002).

METHODSDNA was extracted from peripheal blood mononuclear cells of the subjects with HIV infection. The env C2-V4 region of HIV-1 was amplified with nested polymerase chain reaction (n-PCR). PCR products were directly sequenced using ABI 377 DNA sequencer, then the gene-based phylogenetic tree was constructed and its variation of amino acids was analyzed with GCG software.

RESULTSTotally, 169 strains of recombinant HIV-1 CRF01_AE were identified from blood samples collected from different high risk groups in 17 of 31 provinces, municipalities and autonomous regions all over China by the end of 2002. Although sexual transmission still dominated during NMES1 (62.2%, 23/37) and NMES2 (55.3%, 73/132), prevalence of HIV-1 CRF01_AE in intravenous drug users (IDUs) increased to 41.6% (57/137) during NMES2 from 27% (10/37) during NMES1. Phylogenetic tree analysis showed that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 did not cluster with those prevalent in the subjects infected by sexual transmission during NMES2 and those in IDUs during NMES1. The amino acid residues of V3 region of HIV-1 CRF01_AE in IDUs were relatively conservative, but the sixth, eighth, ninth, tenth, twelfth, fifteenth, sixteenth amino acid residues of C3 region displayed regular changes.

CONCLUSIONSHIV-1 CRF01_AE strain has been introduced into inland provinces from southeastern coast areas and southwestern border areas, with an increasing prevalence in IDUs. The sequence of env V3-C3 region of recombinant HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 was obviously different from that during NMES1, suggesting that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 might come from a new source and have a potential to spread.

Amino Acid Sequence ; China ; epidemiology ; Genes, env ; genetics ; Genetic Variation ; Genome, Viral ; HIV Infections ; epidemiology ; genetics ; virology ; HIV-1 ; classification ; genetics ; Humans ; Molecular Epidemiology ; Molecular Sequence Data ; Phylogeny ; RNA, Viral ; genetics ; Recombination, Genetic ; Sequence Homology, Amino Acid ; Substance Abuse, Intravenous ; virology