Objective To screen the level of novel drug resistance mutations in subtype B' in China. Methods 451 pol sequences collected from the previous study, which including 354 AIDS patients who had received antiretroviral treatment(ART)and 97 the untreated patients. Entire protease gene(codous 1-99)and full-length reverse transcriptase gene(codons 1-560)were included.Variation of mutations between the treated and the untreated patients with consensus/ancestral sequences were compared and the mutations with higher frequencies in the treated patients than in the untreated patients were screened before submitting the mutations to the Stanford HIV Drug Resistance Database(SHDB)(http://hivdb.stanford.edu/). Relation between the mutations and resistance preliminarily was then analyzed, according to the information including SHDB. Results Frequencies of 7 mutations at 6 positions, DI23E, V292I, K366R, T369A, T369V, A371V and 1375V, 2 at DNA polymerase domain and 5 at connection domain of reverse transcriptase(RT)were higher in the treated patients than in the untreated patients. The information of 7 mutations including the SHDB showed that 7 mutations were major variants at corresponding positions, and theirs frequencies were higher in the treated patients using some drugs, than in the untreated patients. Conclusion 7mutations being screened from the China subtype B were possibly associated with the resistance,which called for the construction of mutated viruses by site-directed mutagenesis to identify their effects on the susceptivity of different drugs.

Objective To determine whether morphine having the ability to influence the antiviral effect of lamivudine(3TC)in vitro study.Methods MT2 cells were randomly assigned into morphine+3TC treatment group,morphine+naloxone+3TC treatment group,naloxone+3TC treatment group.Both 3TC and virus control groups were set up.The corresponding MT2 cells were treated with opiates antagonist(naloxone)for 0.5 hours before the 24-hours morphine treatment program was implemented while all of the groups were then infected with equal amounts of cell-free HIV-1 ⅢB strain and 3TC.HIV-1 p24 antigen in culture supernatants collected at days 3,4,5 and 6after infection status was tested and the inhibition of 3TC anti-HIV-1 p24 antigen of various treatment groups calculated.Results Inhibition of 3TC anti-HIV-1 p24 antigen of Morphine+3TC treatment group was the lowest when HIV-1 infected cells at 3rd and 4th day and showed significant difierence (P<0.05)when compared to the 3TC control.However,there was no statistically significant difference among them(P>0.05),when virus was infected the cells at 5th and 6th day.The difference of 3TC anti-HIV-1 p24 antigen inhibition between the morphine+naloxone+3TC treatment group and the naloxone+3TC treatment group was not significant(P>0.05).Similar results were obtained when these two groups were compared to the 3TC control group(P>0.05),respectively.The 3TC anti-HIV-1 p24 antigen inhibition of each treatment group reduced as the time of infection prolonged,showing a significant and time-course effbct.Conclusion The 3TC antiviral effect was reduced by morphine in the early stage of infection,and could be blocked by naloxone.

Objective To elucidate the prevalence and the mutation pattern of N348I that related to the resistance seen in the AIDS patients, in China. Methods Partial pol gene of HIV-1 comprising of full protease (PR) and reverse transeriptase (RT) was obtained from plasma samples of therapy-failure individuals (n=614) and therapy-naive individuals (n=619) by using reverse transcription polymerase chain reaction(RT-PCR). 1233 sequences were then submitted to the HIV-1 drug resistance database of the Stanford University to analyze the prevalence and the emergence pattern of N348I. Results The prevalence of N348I was 6.5％ in the therapy-failure patients and 0.8％ in the naive individuals, respectively. The prevalence of N348I was more popular among those patients whose ART regimens containing zidovudine (AZT or ZDV) than those without AZT in regimens( 14.1％ vs. 4.7％, x2=10.21, P＜0.01 ). N3481 always emerged, and combined with others mutations among patients of ART, whose frequencies were: 85.0％ in combination with thymidine analog mutations (TAMs) and 52.5％ with M184V/I, respectively. Conclusion N348I was somehow prevalent in the therapy-failure patients when using the first-line antiretroviral drugs,and it emerged as unique patterns. This study laid the ground in improving the techaology of drug resistance genotypes detection and providing theoretical basis to study the mechanism of resistance and the law of molecular evolution.

JB25 and JB26 are new HIV-1 nonnucleoside reverse transcriptase inhibitors, and show potent anti-HIV activities. Sequential passage experiments with wild-type virus were performed to select and identify mutations induced by these two compounds in vitro. For the initial passage, compounds were present at approximately 2-fold IC50 in MT-2 cells. When cytopathic effect (CPE) was observed in more than 75% of the cells, the culture supernatants were collected. For the subsequent passages, fresh MT-2 cells were infected with 1 mL supernatants from the previous passage (regardless of the virus titer) and cultured in the presence of the compounds at concentrations that were increased 2-fold compared with that in the previous passage. This procedure was repeated with increasing concentrations for 12 passages. JB25 had amino acid substitution L100I (TTA-->ATA) at passage 6, and then changed into 100 M (ATA-->ATG) at passage 12, which was rare mutation form and had not been reported. At the same time, Y188C (TAT-->TGT) mutation appeared at passage 10. For JB26, there was a L100I (TTA-->ATA) mutation at passage 10. In a word, JB25 and JB26 showed a low genetic barrier to the development of resistance, and the resistance to JB26 developed slower than JB25. The mutations selected by JB25 and JB26 were mainly associated with codons 188 and 100 of HIV-1 reverse transcriptase.
Amino Acid Sequence ; Amino Acid Substitution ; Anti-HIV Agents ; pharmacology ; Cell Line ; Codon ; Drug Resistance, Viral ; HIV Reverse Transcriptase ; antagonists & inhibitors ; genetics ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; Reverse Transcriptase Inhibitors ; pharmacology

BACKGROUNDIt is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.

METHODSWe developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method.

RESULTSThe sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.

CONCLUSIONSDrug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

Alleles ; Drug Resistance, Viral ; HIV-1 ; drug effects ; genetics ; Humans ; Mutation ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo compare the clinical results of additional screws fixation on fractured vertebrae versus only short-segment posterior transpedicular instrumentation for A3 thoracolumbar fracture without neurologic deficit.

METHODSClinical data of 52 cases of thoracolumbar burst fracture without neurologic deficit were retrospectively analyzed. All patients were divided into 2 groups due to different instrumentation and all fractures were classified as type A3 according to AO Classification.From January 2005 to December 2006, 23 cases in group A were treated by short-segment posterior instrumentation combined with additional screws fixation on fractured vertebrae. There were 18 male and 5 female with a mean age of (35.3+/-8.3) years. The fracture segment included 1 in T11, 9 in T12, 11 in L1 and 2 in L2. From January 1999 to December 2004, 29 cases in group B were treated only by conventional short-segment posterior transpedicular instrumentation. There were 20 male and 9 female with a mean age of (37.3+/-6.8) years. The fracture segment included 1 in T11, 7 in T12, 20 in L1 and 1 in L2. The clinical effect and radiographic measurements were respectively compared preoperatively, immediate and 2 years postoperatively.

RESULTSAll patients were followed up and the mean follow-up time was (37.4+/-10.9) months (from 24 to 48 months). There was no statistic difference of mean JOA and VAS score between 2 groups preoperatively, immediate and 2 years postoperatively (P>0.05). The average immediate postoperative correction of Cobb's angle was 13.7 degrees+/-7.7 degrees in group A, which was statistically significantly higher than that of 8.8 degrees+/-5.0 degrees in group B (P<0.01). The mean kyphosis correction loss of 2.9 degrees+/-1.5 degrees in group A was statistically significantly lower than that of 5.0 degrees+/-2.9 degrees in group B 2 years postoperatively (P<0.01). The average restoration of anterior height of fractured vertebral body immediate postoperatively was (29.4+/-6.0)% and (21.7+/-6.9)% respectively. The mean correction loss of anterior height 2 years postoperatively was (3.1+/-0.8)% and (6.6+/-3.0)% respectively. The average restoration of posterior height of fractured vertebral body immediate postoperatively was (8.5+/-3.2)% and (6.1+/-1.8)% respectively. The mean correction loss of posterior height 2 years postoperatively was (2.0+/-0.8)% and (3.4+/-1.0)% respectively. There were significant differences in average restoration of anterior/posterior height immediate postoperatively and correction loss of anterior/posterior height 2 years postoperatively between the 2 groups (P<0.01). According to fracture fragments protruded into the spinal canal on immediate postoperative CT image, there were complete reduction in 11 cases (47.8%) and partial reduction in 12 cases (52.2%) in group A, which was statistically significantly better than those in group B (P<0.01). There was no severe neurologic complications and no other complications related to additional screws fixation postoperatively. Pedicle screw breakage occurred in 2 cases in group B and none in group A.

CONCLUSIONSBetter initial kyphosis correction and less loss of correction 2 years after operation can be obtained by using additional screws fixation on fractured vertebra for thoracolumbar A3 fracture without neurologic deficit.

Adult ; Female ; Follow-Up Studies ; Fracture Fixation, Internal ; methods ; Humans ; Lumbar Vertebrae ; injuries ; Male ; Middle Aged ; Retrospective Studies ; Spinal Fractures ; surgery ; Thoracic Vertebrae ; injuries ; Treatment Outcome

BACKGROUNDThe adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.

METHODSMost circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.

RESULTSResponse of gp120-specific antibody was relatively low after DNA primes (mean titer = 10(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer = 10(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P < 0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.

CONCLUSIONPolyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.

AIDS Vaccines ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Female ; HIV Envelope Protein gp120 ; genetics ; immunology ; Humans ; Immunization ; Immunoglobulin G ; blood ; Phylogeny ; Rabbits ; Vaccines, DNA ; immunology

BACKGROUNDVirus with nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs) resistant mutations show different evolution tendencies when the anti-viral therapies are interrupted. Understanding the replication fitness of drug-resistant virus is important for the study of the prevalence of drug-resistance. For this purpose, we characterized the replication capacity of HIV-1 virus carrying lamivudine (3TC) or nevirapine (NVP) resistant mutations.

METHODS3TC and NVP resistant variants were induced in vitro by selecting wild type virus in the presence of drugs. For the competitive replication assay, drug-resistant variants were cocultured with wild-type virus in the presence or absence of drugs. The ratios of the viral species were determined over time by using a real-time RT-PCR-based assay.

RESULTS3TC-resistant (M184I mutation) and NVP-resistant (Y181I mutation) virus should be selected in vitro in two different ways. The competitive replication assay showed that the ratio of virus carrying a M184I mutation increased from 98.8%, while the wild type virus decreased to 1.2% after 4 passages in the presence of 3TC; the percentage of virus carrying the Y181I mutation increased to 90.5%, while wild type virus decreased to 9.5% in the presence of NVP. In the absence of drugs, the ratio of virus carrying the M184I mutation decreased to 5.3%, while wild type virus increased to 94.7%; the ratio of virus carrying Y181I increased to 75%, while wild type virus decreased to 25% after 4 passages.

CONCLUSIONSThe NVP-resistant virus is fitter than wild type virus even in the absence of NVP that may be the reason that NNRTIs-resistant virus is spreading quickly.

Cell Line ; Drug Resistance, Viral ; genetics ; HIV-1 ; drug effects ; genetics ; growth & development ; physiology ; Humans ; Lamivudine ; pharmacology ; Mutation ; Nevirapine ; pharmacology ; Reverse Transcriptase Inhibitors ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication ; genetics ; physiology

Objective:To evaluate the anti-HIV-1 activity of two new nonnucleoside reverse transcriptase inhibitors (NNRTIs), JB25 and JB26, in combination with 3 approved drugs (AZT, EFV, SQV)in vitro.Methods:The serially diluted 10 concentrations of JB25 and JB26 were combined with 7 serially diluted AZT, EFV and SQV respectively.The combination was added to 384 cell culture plates and then cocultured with HIV-1 ⅢB infected MT-2 cells for 3 days. Finally, the HIV-1 production was determined by measuring the expression of reporter genes of TZM bl cells. The data were analyzed by MacSynergy Ⅱ software.Results:The average capacity of synergism/antagonism of JB25 with AZT, EFV and SQV was 244.45/-5.05(nmol/L)~2%, 119.58/-65.93 (nmol/L)~2% and 145.83/-0.32 (nmol/L)~2% respectively;the average capacity of synergism/antagonism of JB26 with AZT, EFV and SQV was 398.90/0(nmol/L)~2%, 103.62/-0.49(nmol/L)~2% and 138.473/-0.27 (nmol/L)~2% respectively. Conclusion:Two new NNRTIs JB25 and JB26 develop synergism when combined with 3 approved drugs, respectively. MacSynergy Ⅱ software could evaluate the anti-HIV-1 activity of drug combination.

Objective:To investigate the subtype distribution of HIV-1 strains prevalent in four areas of China,and to study the characteristics of gag gene variation and changes in antigen epitopes under the host immune pressures. Methods:The plasma of HIV-1 infected people from Henan, Guangdong, Sichuan and Beijing in China were collected. Virion RNA was extracted directly from plasma after the virion was condensed. The gag gene was amplified by RT-PCR and nested-PCR.Sequences were subtyped by Genotyping Tool software, and phylogenetic analysis of gag gene were performed using the MEGA 4.1 software.The gene distances intra each subtype were calculated by Distance program. The Ks/Ka ratios were calculated using SNAP program. The variation analysis of CTL antigen epitopes restricted by main HLA-Ⅰ specificities in China was performed.Results:Six subtypes or circulating recombinant forms(CRFs)of HIV-1,including B',CRF07_BC,CRF01_AE,B,CRF08_BC and CRF02_AG,were identified in four areas of China.The gene distances intra each subtype were CRF01_AE>B>CRF08_BC> CRF07_BC>B' listed in order of size, meanwhile the order of Ks/Ka ratios was CRF01_AE>B>CRF08_BC>B'>CRF07_BC. Far more diversity of antigen epitopes in P17 region was observed than that in P24.Epitope mutations intra subtypes were CRF01_AE>B>B'>CRF07_BC listed in order of size. Conclusion:Itseems that CRF01_AE is under the strongest immune pressures,and displays the most diversity of gene and variation of epitopes intra subtypes prevalent in China, followed by subtype B, B' and CRF07_BC. The discrepancy of epitope mutations intra the subtypes is significant.