| Part One: Sequence Variation of p24 and gp41 Coding Region and Subtype Analysis of Human Immunodeficiency Virus Type 1 in Henan Province and Shanghai of ChinaObjective To compare sequence variations of the p24 coding region in gag gene and subtype phylogenetic between Henan and Shaghai, two regions in China with different HTV-1 transmission modes. Methods Plasma specimens were collected from 38 HIV-1 infected patients, of which 23 from Henan province and 15 from Shanghai. Viral RNAs were extracted and regions of gag p24 and env gp41 were amplified using the method of RT-PCR and Nested-PCR. The 693 nucleotides of p24 region and 460 nucleotides of gp41 immunodominant region (IDR) were determined, then phylogenetic analyses were performed. Results Subtyping showed that 81.6% (31/38) were B subtype. All the 23 Henan specimens were B subtype. Of 15 Shanghai specimens, 8 were B subtype ,1 was A subtype , 5 were CRF01_AE and 1 was CRF02_AG. Comparing to the consensus sequence (cons_B, from HIV database), nucleotide substitution in B subtype of Henan was 1.6%~4.2%, with an average of 3.2%, while that of Shanghai was 2.0%~3.8%, with an average of 3.4%. No G-to-A hypermutation was observed in the all variations. Intrapatient distances were calculated by pairwise comparison of each sequence. As for B subtype, the mean value of intrapatient distance of p24 region was 3.6% and that of gp41 IDR was 4.4%. And for env gp41 IDR of B subtype, the mean variation value of Henan was 3.7%,while that of Shanghai was 4.6%(P<0.05). The intersubtype distance between B subtype and other subtypes was 14.5%~16.8%.The two consensus sequences of Henan and Shanghai B subtype obtained by CLUSTAL-X were aligned to cons_B, both the substitution for predicted amino acid were 2.2%(5/231). Of all the substitutions they shared the same three: A14P, I91V and E180D, but the disparity between the two consensus sequences was not significant (0.9%).Phylogenetic analyses implied that most specimens were B subtype, and all the B subtype specimens from Henan and many of B subtype Shanghai specimens were actually B'subtype. Conclusion HTV-1 B subtype was dominant in Henan and Shanghai of China and seqeunces in the p24 binding site of gag gene remain highly conserved. Part Two: Prevalence of Drug-Resistant Human Immunodeficiency Virus Type 1 in Therapy-Naive and antiretroviral-treated Patients of China Objective To comprehend the emergence and prevalence of drug-resistant mutations in reverse transcriptase and protease coding regions in human immunodeficiency virus type 1 (HlV-l)-infected patients treated with antiretroviral drugs (ARV), and the primary(or transmitted) drug resistance in treatment-naive patients. Methods Plasma specimens were collected from 181 HIV-1 patients of Henan, Anhui province and Shaghai. The entire protease gene and the first 1-251 amino acids of the reverse transcriptase gene were amplified by RT-PCR from viral RNA and sequenced. The sequences were analyzed with HIV drug resistance algorithm by Stanford HFV-db program (http://hivdb.stanford.edu/)and genotypic resistance mutations were determined to particular anti-HIV drugs, then phylogenetic analysis was performed on the pol gene sequences for subtype determination. Results One hundred pol gene sequences were obtained and were divided into two groups according to exposure to ARV or not: ARV treatment group(group A, n=44) and treatment-naive group(group B, n=56). In group A, 70% (31/44) presented with virus mutations associated with resistance to at least one antiretroviral drug of the three common antiretroviral drug: nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). The distribution of the amino acid resistance mutations was as follows: to NRTI in 34% (15/44) include K65R, M184V, M41L, V75I/T, A62V, K70E/N, L74V, D67G, E44D, T69D, V118I, F116Y, Q151M, L210W and T215Y; to NNRTI in 68% (30/44) include K103N, Y181C, G190A/S, V106A, F227L, K101E, Y188L, V108I, V179E; and 2% (1/44) showed PI associated major resistance (M46I> V82F> L90M). In group B, the overall prevalence of viruses with drug-resistant mutations was 7% (4/56), and 2% (1/56) showed mutations indicating NRTI resistance(T69N); 5% (3/56) showed NNRTI resistance(K101Q, K103Q, Y181C), no major resistance associated with PI was found. All the 100 specimens presented with minor mutations(polymorphism) in the protease gene such as L63P, V77I, I93L, A71V and M36I. All the B subtype specimens showed...
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