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Objective@#To analyze the variation characteristics of HIV-1 Gp120 sequences in men who have sex with men (MSM) in Guangzhou.@*Methods@#Plasma samples were collected from HIV-1 infected MSM before antiretroviral treatment. Viral RNA was extracted from plasma. Gp120 gene sequences were amplified by reverse transcription and nested-PCR using specific primers. Phylogenetic tree, length polymorphism, amino acid characteristics of V3 loop, co-receptors and signature amino acids were analyzed.@*Results@#The phylogenetic tree were divided into 4 clusters, and the most prevalent subtypes were CRF07_BC (34/61, 55.74%) and CRF01_AE (24/61, 39.34%). Majority of HIV-1 Gp120 sequences had 496-515 amino acids. Among five hypervariable regions, the V1 region had the highest levels of length polymorphism and V3 region had the lowest. The top four peptide of V3 loop were GPGQ (56/58, 96.55%). Most of the co-receptors HIV-1 strains used was CCR5(50/58, 86.21%)according to four methods of comprehensive prediction. There are four signature amino acids in CRF01_AE subtype strains, and the frequency of occurrence was 0.75-0.83; there are eight signature amino acids in CRF07_BC subtype strains, and the frequency was 0.74-0.94.@*Conclusions@#The length of Gp120 sequences in MSM in Guangzhou has a high polymorphism. The top four peptide of V3 loop, co-receptor and signature amino acid of V3 ring have formed unique patterns.
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Objective To investigate the prevalence and characteristics of non-nucleoside reverse transcriptase inhibitors (NNRTIs)resistance-related gene mutations among the AIDS patients with virological suppression failure in Guangdong Province 2015.Methods Plasma samples from AIDS patients receiving highly active antiretroviral therapy for more than one year with viral loads > 1000 copies/mL from Guangdong province (except Shenzhen)were collected from January to December 2015.Total 612 HIV-1 gene fragments were amplified from plasma samples using self-developed lab method.Sub-genotypes were determined by phylogenetic tree according to the sequences,NNRTIs resistance-related mutations were determined in Stanford University HIV-1 Drug Resistance Database. The NNRTIs-resistance, the relationships of NNRTIs resistance-related mutations with baseline CD4 +T lymphocyte counts,transmission routes,antiviral regimens and HIV-1 genotypes were analyzed.SPSS 17.0 software was used to analyze the data.Results In 612 patients with virological suppression failure,the main NNRTIs resistance-related mutations were K103 (26.80%),Y181 (14.71 %),V179 (13.73%),G190 (11 .44%) and V106 (10.62%).The susceptibility rate of 310 patients (50.65%)to NNRTIs had changed,the highly resistant rate to nevirapine was 49.51 %,which was higher than that of efavirenz (43.14%),etravirine (5.56%) and rilpivirine (12.25%),respectively,and the differences were statistically significant (χ2 =5.00,296.3 and 198.0,all P 200 cells/μL was lower than that in those with baseline CD4 +T lymphocyte counts <200 cells/μL (χ2 =17.93,P <0.01 );the incidence rate of drug resistance was lower in intravenous drug abusers than that of sexually transmitted patients (χ2 =44.21 ,P <0.01 );while the incidence of drug resistance in patients receiving NVP-containing regimens was higher than that in those receiving EFV-containing regimens (χ2 =8.93,P <0.01 ),and the incidence rate was higher in patients with CRF01 _AE than that in those with CRF07_BC and CRF08 _BC (χ2 =8.46 and 8.47,P <0.01 ).Conclusions The results suggest that compliance education and follow-up should be strengthened in patients with high baseline CD4 +T lymphocyte counts and intravenous drug users,and patients with liver diseases should avoid using drugs containing NVP regimens.
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Objective To investigate the relationship between HBV mutations in the precore (PC)/core promoter region and the liver histological changes in HBeAg negative CHB patients. Method A total of 71 HBeAg negative CHB patients with liver biopsy from April 2012 to Dec 2013 were enrolled. DNA was extracted from blood serum, then the HBV S gene and PC/core promoter region were amplified by semi-nested PCR and sequenced. The relationship between significant liver histological changes and viral factors were analyzed by Logistic regression analysis. Results The incidence of significant necroinflammation (15.8% vs. 27.3%, χ2 =1.398, P = 0.237) and significant fibrosis (71.1% vs. 84.4%, χ2= 1.926, P = 0.165) were found to be similar between patients infected with HBV genotype B and genotype C . By Logistic regression analysis including risk factors of age, sex, HBV genotype and mutations (T1753V,A1762T/G1764A,A1846T and G1896A), the A1762T/G1764A mutation in HBV associated with significant necroinflammation (OR = 4.296, P = 0.037), while factors of age, sex, genotype and other mutation were not associated with significant liver histological changes. (all P > 0.05). Conclusion Mutation in PC/core promoter region of HBV may act as a marker to evaluate the liver histological changes.
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Objective To detect the change of hepatitis C virus (HCV)RNA in the peripheral blood mononuclear cells (PBMC)and serum of patients with chronic hepatitis C (CHC)during treatment with peg-interferon α-2a (Peg IFNα-2a)plus ribavirin (RBV),and to analyze the clinical significance of HCV RNA detection in PBMC.Methods The peripheral blood samples of 20 CHC patients who visited Department of Infectious Diseases in Guangzhou No.8 People′s Hospital from June 2013 to December 2014,were collected during treatment with Peg IFNα-2a+RBV at different time points (week 0,2,4, 12,24,36 and 48).Serum and PBMC were separated.Accurate fluorescence quantification assay (Cobas TaqMan real time polymerase chain reaction[PCR])was used to detect HCV RNA level in serum,while real-time PCR and nest-PCR were applied to detect HCV RNA in PBMC.Categorical data were analyzed byχ2 test.Results Accurate fluorescence quantification of serum HCV RNA showed that HCV RNA level decline rapidly after treatment (F = 148.06,P < 0.01 ),and 18 patients achieved HCV RNA undetectable at week 12 of treatment.The positive rate of nest-PCR was higher than real-time PCR (all P <0.01).Comparison of HCV RNA levels in serum and PBMC from 20 cases found that,the clearance rate of HCV RNA in PBMC was postponed.Two patients whose HCV RNA in PBMC kept detectable relapsed at week 24 after end of treatment.Conclusions HCV RNA can be detected in PBMC of CHC patients and the positive rate of nest-PCR is higher than real-time PCR.Antiviral therapy is effective on HCV both inside and outside PBMC,but the clearance rate of HCV RNA in PBMC is postponed compared with that in serum.Slow clearance of HCV in PBMC may be a risk factor for relapse after end of treatment.
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Objective To investigarte the immune status in the patients with dengue fever .Methods The flow cytometry was used to detect the T lymphocytes in the patients with dengue fever for analyzing their immune status .Results Compared with the reference range in the healthy individuals ,it was found that the T lymphocyte proportion in the patients with dengue fever was sig‐nificantly reduced compared with the healthy individuals .The percentages of CD3+CD4+ and CD3+CD8+ lymphocytes were (36 .54 ± 9 .78)% and (17 .7 ± 10 .01)% respectively ,which had statistical difference compared with the control group(P<0 .05) ,CD3+CD4+ lymphocyte count was (49 .98 ± 240 .2)cells/μL ,the difference was statistically significant (P<0 .05) ,CD3+ CD8+ lympho‐cytes count was (380 .9 ± 364 .6)cells/μL ,the difference was statistically significant(P<0 .05) .Conclusion The immune status in the patients with dengue fever is abnormal ,T lymphocyte percentage is significantly reduced compared with the healthy individuals .
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Objective To investigate the characteristics of V3 loop amino acid sequences of human immunodeficiency virus type 1 (HIV-1) quasi-species in long-term non-progressors (LTNP)infected with HIV. Methods End-point limiting dilution polymerase chain reaction (EPLD PCR) was used to amplify the env gene c2-v3-c3 region of single HIV-1 provirus from five LTNPs at sequential time points. The PCR products were then sequenced and the amino acid sequences of V3 loop were analyzed by sequence confirm analysis technology. Results The results showed that there were one to ten kinds of polymorphisms in the V3 region of HIV-1 quasi-species which were found from the serial samples of the five LTNP. However, the sequences of the predominant strains were either completely consistent or at most changed at one or two residues in the serial samples of individual patient. The tetramer compositions of the tip of V3 loop were consistent in each patient. It was GPGR in four patients and GPGK in one patient. It was speculated the co-receptor of HIV-1 was CC chemokine receptor (CCR)-5 based on the amino acids at the residue 11 and residue 25 of V3 loop and the net charge of V3 loop. Conclusions There are various polymorphisms at the HIV V3 loop in LTNP. However, the tetramer composition of the tip part of V3 loop is stable. The LTNP are very likely infected with non-syncytium inducing (NSI) strain.
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Objective To detect and analyze the haemagglutinin (HA) gene of the first influenza A-H1N1 viral strain isolated in Guangdong Province during an influenza A pandemic in 2009.Methods A-H1N1 virus strain was isolated from the throat swab of the first patient diagnosed with A-H1N1 virus infection in Guangdong Province in 2009. Viral nucleonic acid was extracted from supernatant of cell culture and amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) with HA gene-specific primers. The product was cloned, sequenced, and the homology was analyzed. Results A 1710 bp HA gene of the first influenza A-H1N1 viral strain in Guangdong Province in 2009 was acquired, which was named as A/GuangzhouSB/01/2009 (H1N1) HA with GenBank access No. GQ268003. The homology of the studied HA gene and the 277 influenza A (H1N1) isolates reported in the epidemic areas was 99.0%-99.8%, and as high as 99.8% when compared with the isolates reported in the United States where the patient had traveled. When the studied HA gene was compared with 25 isolates of Chinese seasonal A-H1N1 virus, the homology was 72.3%-85.6%. Conclusions The homology of the first isolated A-H1N1 viral strain in Guangdong Province in 2009 and epidemic influenza A-H1N1 virus is high, while it is low compared with Chinese seasonal A-H1N1 virus.
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detection of HIV-1 quasispecies in HIV-1 infected populations with low level viral load.
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Objective To identify the pathogens that cause hand, foot and mouth disease (HFMD) in adults and analyze the nucleotide sequences characteristics of enterovirus 71 (EV71). Methods The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the enterovirus from the samples of four adult HFMD patients. The 227 bp amplified segments of EVT1 were then sequenced and compared with the sequences of previously isolated EVT1 strains available from GenBank by homogeneity and phylogenetic tree analyses. Results All the results of RT-PCR with enterovirus universal primers and EVT1 specific primers were positive. The EV71 sequences analysis showed that the four new sequences (named as GZ19610, GZ99310, GZ99355 and GZ46477) shared 96.0% to 99.1% nucleotide identify themselves and shared 96.9% to 100.0% homology with the strain Fuyang/17.08/3 isolated in 2008 from Fuyang, Anhui Province. Phylogenetic tree analysis showed that the genotype of the four new sequences was all subtype C4, they were the same sub-genotype as those strains isolated from Chinese mainland and Chinese Taiwan in 2004, and the genetic distance between them was most closely. Conclusions EV71 can cause adult HFMD. Compared with the nucleotide sequences of EV71 strains that isolated now and formerly in China, there is no large variation of the EV71 sequences isolated from four adult HFMD patients in Guangzhou this time. The adult HFMD patients should be isolated for treatment to avoid them transmitting the virus and causing disease spreading.
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Objective To identify the sequence of hepatitis B virus S gene"a"determinant in patients with positive HBsAg and anti-HBs.Methods Nested-PCR Was used to amplify the HBV S gene in 4 patients with positive HBsAg and anti-HBs,and the PCR products were sequenced directly or after cloning.The sequences of"a" determinants were then analyzed by sequence alignment.Results Direct sequencing of PCR products showed that there was one amino acid (aa)residue in"a"determinant less conserved region emerging polymorphism in all 4 patients.Clone sequencing showed that aa residue at 126 of "a"determinant in patient 1 miSht be Thr,Ile and Set,at 134 might be Phe and Set;the aa at 126 in patient 2 misht be Ala and Thr.and in patient 3 might be Ile and Asn;aa polymorphism was not found in patient 4.Conclusion The polymorphism of"a"determinant in HBV S gene might be associated with positivity of both HBsAg and anti-HBs in hepatitis B patients.
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<p><b>OBJECTIVE</b>To investigate the changes of subgroups of peripheral blood T lymphocytes in patients with severe acute respiratory syndrome (SARS) and its clinical significance.</p><p><b>METHODS</b>Subgroups of blood T lymphocytes in 93 patients with SARS were detected by flow cytometer. The results detected in 64 normal subjects and 50 patients with AIDS served as controls.</p><p><b>RESULTS</b>The numbers of CD(3)(+), CD(4)(+), and CD(8)(+) lymphocytes all significantly decreased in acute phase of patients with SARS [(722 +/- 533)/microliter, (438 +/- 353)/microliter, (307 +/- 217)/microliter] compared with those in normal controls [(1527 +/- 470)/microliter, (787 +/- 257)/microliter, (633 +/- 280)/microliter, all P <0.01], which was different from what we observed in patients with AIDS who had decreased CD(4)(+) [(296 +/- 298)/microliter] but increased CD(8)(+) [(818 +/- 566)/microliter] counts. The counts of CD(3)(+), CD(4)(+), and CD(8)(+) lymphocytes decreased more apparently in patients with severe SARS. All the five patients who died had CD(4)(+) counts less than 200/microliter. As the patients' condition improved, CD(3)(+), CD(4)(+), and CD(8)(+) counts gradually returned to normal ranges.</p><p><b>CONCLUSION</b>The damage of cellular immunity is probably an important mechanism of pathogenesis of SARS.</p>