Dissertation > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)

Phenotypic Analysis, Sequences Analysis, Envelope Glycoprotein Expression and Construction of Infectious Molecular Clones of Human Immunodeficiency Virus Type 1 CRF01_AE from Fujian Province, China

Author HuangHaiLong
Tutor YanYanSheng;YanPingPing
School Fujian Medical
Course Pathogen Biology
Keywords Human immunodeficiency virus 1 Viral tropism Phenotype CRF01_AE Full-length glycoprotein 120 gene Full-length genome Sequence analysis Infectious molecular clones
CLC R373
Type PhD thesis
Year 2007
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Human Immunodeficiency Virus (HIV) was the pathogen of Acquired Immunodeficiency Syndrome (AIDS), which is a member of class Retroviridae and genus Lentiviridae. Basing on twice surveys of HIV-1 molecular epidemiology, HIV CRF01_AE strain has been introduced into inland neighboring provinces from southeastern coast areas and southwester border area, the prevalence of HIV CRF01_AE strains are increasing. In this study, we first reported that HIV CRF01_AE pathogen, full-length glycoprotein 120 gene genetic characterization and expression, HIV-1 CRF01_AE full-length sequences genetic characterization and construction of infectious molecular clones of HIV-1 CRF01_AE, which may be the basis on pathogen properties, genetic diversity, vaccines, diagnosis methods and infection mechanism. This study is divided into five parts.1. Primary viral strains were isolated from 4 Fujian HIV-1 positive blood samples and the study of their biological characteristics1) 4 HIV-1 CRF01_AE strains were isolated, Fj200604 isolate could infect MT-2 cell successively and induce syncytium, but others could not infect MT-2 cell; Fj200601, Fj200602 and Fj200603 used CCR5 as coreceptor, but Fj200604 used CCR5 as coreceptor.2) The biological characteristics of 4 HIV-1 isolates were linked to their env V3 loop sequence variability; the biological phenotype of HIV-1 strains can be predicted with its V3 loop sequence.2. Sequences analysis of the full-length glycoprotein 120 gene from HIV-1 CRF01_AE in Fujian, China1) Phylogenetic trees showed that the 21 HIV-1 subtype CRF01_AE strains from Fujian clustered with the AE reference strain, overall genetic distance is 9.5±2.5 %2) All sequences had four types of V3 loop central motif: GPGQ (72.24 %), GPGR (19.04 %), GPGH (4.76 %), and GQGQ (4.76 %). Predictions for the potential use of co-receptors disclosed that 76.19 % of 21 sequences probably use CCR5 coreceptor, 4.76 % sequences probably use CCR4/CCR5, and 19.04 % could not be predicted.3) Amino acid sequences show that V3 region is relatively conserved, whereas V1, V2, V4, V5 loop are more variable.4) The N-linked glycosylation sites of 21 sequences were relatively conserved.3. Cloning and expression of HIV-1 CRF01_AE full-length gp120 gene in Drosophila S2 cell1) Construct the recombinant expression plasmid of full-length gp120 gene and△V1/V2 gp120 gene, transient expression confirmed them can secret from S2 cell .3) Establish stably S2 cells line of secretion expression gp120 and△V1/V2.4. Genetic characterization of CRF01_AE full-length Human Immunodeficiency Virus Type 1 sequences from Fujian, China1) The cloning and characterization of 13 CRF01_AE full-length isolates were first reported in China, the method of HIV-1 full-length genome amplification was set up.2) The sequences of 13 CRF01_AE full-length isolates have been submitted to GenBank database.3) The phylogeny analysis of the 13 sequences showed that they clustered with HIV-1 CRF01_AE isolates; these isolates were separated into a few distinct subgroups and were dispersed among the Thailand isolates. 4) The intersample diversity among Fujian isolates (excluding hypermutated sequence Fj061) was significantly higher than that among the Thailand isolates on the level of the full-length genome, gag, pol and env genes analysis; Hypermut program confirmed only Fj061 was a hypermutated sequence5) Bootscan analysis using reference isolates from each subtype from A to J and recombination subtype HIV-1 CRF01_AE showed no new intersubtype recombination in all 13 sequences.6) In all 13 isolates did not carry any putative drug resistance position in the protease and RT sequences analyzed.5. Construction and biological characterization analysis of full-length HIV CRF01_AE Infectious Molecular Clones1) A full-length clone and A-B chimeric full-length clone can infect donor PBMCs and replicate in PBMCs, the active of A-B chimeric full-length clone is better than A full-length clone and PNL4-3.2) A-B chimeric full-length clone used CCR5 as coreceptor, but A full-length clone is not determined.3) The V3 sequence was found to match the corresponding sequence of the plasmid clone, indicating that the virus cultivated in PBMCs was derived from the molecular clone.

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