RT Region Research Articles

Genetic characteristics of HIV-1 primary drug resistance-associated mutations in treatment-naive individuals in Liaoning province, 2004-2008

To investigate the HIV-1 drug resistance associated mutations and examine the susceptibility of HIV-1 with these mutations to antiretroviral in treatment-naive individuals in Liaoning province from 2004 to 2008. RNA was extracted from 20 plasma samples of diagnosed untreated HIV-1-infected treatment-naive patients by drawing method. After the viral loading (VL) test, the protease and nucleoside reverse transcriptase coding regions were amplified by RT-PCR, nested PCR and sequence analysis directly. Levels of resistance and prevalence were evaluated according to the Stanford University HIV Drug Resistance Database's algorithm (http://hivdb.stanford.edu). Among the 20 plasma samples, 13 got PCR products because of their VL values higher than 1000 copies/ml.Meanwhile, the 13 samples got 65 sequences by using 5 primers each. Polymorphisms in subtype H and circulating recombinant forms (CRFs) CRF10_CD sequences were identified. An overall prevalence of 30.8% (4/13) resistance to NNRTIs, 7.7% (1/13) to PI and no NRTIs mutations were found. The most frequent substitutions (4/13) in the RT region at positions P225H, K238S, V179D, K238T and a major position I54S in PR implied to a multiple drug-resistance. A71V or L10V only, respectively, substitution in PR was found in 3 samples, but no any worse with drug sensitivity. HIV-1 polymorphisms in subtype H and CRFs CRF10_CD sequences were identified circulating in Liaoning. A major mutation position I54S in PR implied that it would be the time to commence a higher level drug regimen.

Detection of lamivudine resistance-associated hepatitis B virus mutations by multi-analyte suspension array

Objective To develop and establish a rapid, convenient method with high-throughput, high sensitivity and specificity for detection of lamivudine (LAM) resistance-associated mutations in the hepatitis B virus. Methods HBV DNA was extracted as PCR templates. The target sequence at liT region was amplified using primers with 5'-biotin label. The biotin-labelled target PCR products interacted with those bead-bound probes in aqueous suspension, and then streptomycin and avidin-modified straptavidin phycoerythrin (SAPE) were added to the aqueous suspension after the free HBV DNA was washed out, which had not hybridized with probes on the beads. Finally, mutations were tested in luminex analyzer using the bicolor laser (red and green laser). The mutated type and relative quantity were identified according to the different ratios of red fluorophores incorporated into the beads (for qualitative analysis) and green fluorophores imparted by a specific reporter molecule SAPE (for quantitative analysis). Subsequently, the multi-analyte suspension array (MASA) was applied to detect plasmids containing fnll-length HBV genome with LAM-resistance associated HBV mutations and 25 serum specimens from patients with chronic hepatitis B, who were treated with LAM and experienced virological breakthrough due to LAM-resistanee. The specificity of this method was compared with direct DNA sequencing. Meanwhile, we also determined the sensitivity of this method by mixing the wild and mutant type plasmids in different proportions. Results MASA can rapidly detect mutations at rt180 and rt204 positions in RT region simultaneously. 25 samples with LAM-resistant mutations confirmed by DNA sequencing were found to contain consistent mutations by this method, with 15 cases hybridizing to a particular 20411 probe type( negative and positive median signal values were 5 and 106 respectively), 11 cases hybridizing to 204V probe( negative and positive median signal values were 9 and 114 respectively) (Z value =-4.588 and -4.520, P<0.05), and only 1 specimen hybridizing to 20412 probe. However, two samples were found harboring mixed mutated types (rtM204V and rtM204I). More importantly,MASA was able to detect 5% mutation frequency with up to 100% of specificity. Conclusions MASA could be used for early detection of LAM resistance-associated mutations in hepatitis B virus. This method has high sensitivity, specificity and high-throughput in detection of LAM resistance-associated mutations compared with direct DNA sequencing. This method may also significantly save the time of diagnosis and facilitate the clinical application of large samples. Key words: Hepatitis B virus; Lamivudine; Drug resistance; viral; Microarray analysis

HIV-1 Plasma Variants Encoding Truncated Reverse Transcriptase (RT) in a Patient With High RT-Specific CD8+ Memory T-Cell Response

During replication, HIV-1 reverse transcriptase lacks proof reading activity and is error prone. In addition APOBEC-driven hypermutation of HIV-1 Gag and Pol genes may generate replication-deficient viral variants with in-frame stop codons. Virus variants with several stop codons in the RT gene were identified in a subject with residual HIV-1 replication during antiretroviral treatment. A role for the T-cell response in the selection of replication-deficient variants was hypothesized. Clonal analysis of HIV-1 DNA and RNA sequences from three sequential blood samples was performed. Moreover, the HIV-1-specific memory CD8(+) T-cell response was investigated using a peptide-based cultured ELISPOT assay. The accumulation of HIV-1 variants with stop codons in the Pol gene (from 0% to 100%) was observed in sequential plasma samples. The cultured ELISPOT showed a sustained response to a Pol region downstream from the last stop codon. A more detailed analysis of the Pol region encompassing the detected stop codons showed a strong response to a peptide at the end of the RT region containing stop codons (positions 206 to 220) and including the last stop codon (212). These results suggest a role for a peptide-specific immunologic response in the positive selection of cells expressing the truncated HIV-1 RT and the accumulation of replication-deficient viral variants in plasma. The antigen-specific CD8(+) T-cell response could be exploited to redirect the response to HIV-1 infection toward in vivo selection of viral variants with reduced or abolished pathogenicity.

Mutation patterns in the RT region of hepatitis B virus P gene in patients treated with nucleoside/nucleotide analogs

To analyze mutation patterns in the RT region of hepatitis B virus (HBV) P gene in patients treated with nucleoside or nucleotide analogs. Viral DNA was extracted from 227 serum samples of chronic hepatitis B patients from September, 2005 to March, 2007. The RT region of HBV P gene was PCR-amplified and sequenced. Known resistant mutations were found in 111 cases (48.9%) among 227 samples, 75 cases with clear therapy history. Novel mutations, including A222T, L229V and S256C, were also found. The incidence of multi-drug resistance in patients sequentially treated with lamivudine and adefovir was 25% (4/16), and none of the patients treated with lamivudine plus adefovir in combination shown multi-drug resistance. The patterns of mutation is complex in nucleotide analogue treated patients. Switching to adefovir in lamivudine resistant patients may lead to multi-drug resistance. Novel mutations identified in this study need further investigation.

Genetic characterization of HIV-1 BC recombinants and evolutionary history of the CRF31_BC in Southern Brazil

To evaluate the recombination profiles and evolutionary history of HIV-1 BC recombinants in Southern Brazil, 81 isolates collected in the city of Porto Alegre (Rio Grande do Sul State) from 1998 to 2006 previously subtyped as C ( env- gp120/C2V3) were screened in the protease-reverse transcriptase ( pr/ rt), integrase and gp41 genomic regions. Detailed phylogenetic, bootscan and informative site analyses were performed to trace the subtype classification. The evolutionary rate and divergence time of the Brazilian CRF31_BC epidemic were estimated using a Bayesian Markov Chain Monte Carlo framework. Analysis of the four target regions identified: 43 isolates as “pure” subtype C, 23 as CRF31_BC, and 15 as unique BC recombinant forms (URFs_BC). Recombination breakpoints were mainly localized in the rt gene and 100% of the recombinant samples could be detected analyzing only this region. Most URFs_BC (86.7%) contained small subtype B fragments (≤160 nt) in the rt region and shared one of the recombination breakpoints with CRF31_BC. In conclusion, despite the high co-prevalence of subtypes B and C in Porto Alegre, the diversity of BC recombinant forms circulating in this region was extremely low. Most BC recombinants were CRF31_BC and URFs_BC that appeared to be second generation recombinants derived from CRF31_BC and subtype C strains, confirming the importance of this CRF in this region. The emergence of the CRF31_BC was estimated to be around 1988 (1982–1992).

HIV‐1 diversity among inmates of Italian prisons

In Italy, the prevalence of non-B HIV-1 subtypes ranges reportedly from 5.4% to 12.6%, yet there are no data on their circulation in prisons, where the prevalence of HIV infection is high. A retrospective study was conducted to evaluate the circulation of non-B subtypes and to characterize their determinants in five Italian prisons. To this end an aliquot of samples of blood was taken in the period 2001-2006 from all 262 HIV-positive inmates in whom antiretroviral treatment had failed. Complete HIV-1 PR and RT regions were sequenced for all samples and subjected to phylogenetic analysis; 250 (95.4%) sequences clustered with subtype B. The non-B subtype was found in 4% of Italian prison inmates and 16.7% of non-Italian prison inmates; the overall percentage increased from 1.8% for inmates infected in 1982-1990 to 4.4% in 1991-1999 and 21.9% in 2000-2006. Factors significantly associated with non-B subtypes were an exposure to other than injecting drug use and a first positive HIV test in 2000-2006. Non-B subtypes were distributed within five monophyletic clades. In all cases but one, it was possible to correlate the history of HIV-exposure to the origin of the clade, with high bootstrap values. In conclusion, although the sample may not be representative of the prison inmate population in Italy, the data suggest strongly that the circulation of non-B subtypes has apparently increased. Non-B subtypes were found to have been associated with heterosexual contact and time of the first HIV-positive test. Knowledge of the different subtypes circulating in prisons may be useful for tracking the epidemiology of HIV infection and for choosing antiretroviral therapy.

Drug Resistance-Associated Genotypic Alterations in the pol Gene of HIV Type 1 Isolates in ART-Naive Individuals in North India

ABSTRACT We genotyped the RT and PI regions of the pol gene of HIV-1 from treatment-naive infected individuals in North India and evaluated their possible physiological relevance and association with drug resistance. Plasma samples from 52 newly diagnosed HIV-1-infected drug-naive individuals were subjected to CD4(+) cell count and plasma viral load. For genotyping, the protease and RT regions of the pol gene were amplified from cDNA reverse transcribed from plasma viral RNA by single or nested polymerase chain reaction (PCR). Sequences of amplified products were analyzed for mutations using the Stanford DR and REGA database. Two out of 49 amplicons showed mutations at known "major" subtype B drug resistance positions (one each in protease and RT). In the protease region it showed a major drug resistance mutation at M46I as well as "minor" positions F53L and T74P. In the RT gene, one patient showed a mutation at major NNRTI position G190V. Forty-nine percent had mutations in the hinge (M36I, R41K, H69K) and alpha-helix (L89M) regions of the C-virus protease, which has been linked to increased catalytic activity. Our study indicates that a number of major mutations associated with resistance to PIs, NNRTIs, and NRTIs do exist, though at a low frequency, among HIV-1 isolates from treatment-naive individuals in North India. Many minor or accessory mutations related to resistance to PIs and NRTIs are also present as the variants. These results point to the greater biochemical fitness of subtype C protease and faster decrease in drug sensitivity.

Subtype Assignment and Phylogenetic Analysis of HIV Type 1 Strains in Patients from Swaziland

Aim of this study was to assess HIV-1 subtype distribution and prevalence of transmitted mutations related to antiretroviral drugs in Swaziland. According to the WHO guidelines, 47 plasma samples from naive patients stored at HIV/AIDS National Reference Laboratory in Mbabane between 2002 and 2003, before the introduction of antiretroviral therapy in the country, were studied. HIV-1 RNA was extracted from the plasma samples, RT and protease regions of pol gene were amplified and sequenced. The mutations associated to drug resistance were defined as major or minor on the basis of the recommendations of the International AIDS Society-USA panel. A mutation associated to non nucleoside inhibitors (Y181I) was found in one case showing a prevalence of transmitted drug resistance <5% in Swaziland. No major mutations conferring resistance to protease and nucleoside RT inhibitors were found. Clade assignment was performed by phylogenetic analysis of pol gene. The general time-reversible model of substitution was used to study the phylogenetic relationships between sequences obtained from Swazi patients and sequences from neighbor countries. All patients were found to carry a C subtype. No phylogenetic relationships were detected within Swazi sequences, indicating the absence of epidemiological relationships among patients in study. Although local variants of subtype C have been recently recognized, phylogenetic analysis did not reveal the presence of significant cluster of Swaziland sequences within African variants. This finding may be explained by multiple introduction of C strains.

Identification of two new CRF_BF in Rio de Janeiro State, Brazil

Identification of two new CRF_BF in Rio de Janeiro State, Brazil

Accommodating Uncertainty in a Tree Set for Function Estimation

Multiple branching trees have been used to model the acquisition of HIV drug resistance mutations, and several different algorithms have been developed to construct the tree set that best describes the data. These algorithms have mainly focused on the structure of the tree set. The focal point of this paper is estimation of functions of the tree set parameters that incorporate uncertainty in the tree set. The functions of interest are the state probabilities, the co-occurrence of mutations and the order of acquisition. Such functions are of interest because they help characterize the genetic pathways that lead to multi-drug resistance. We propose a bootstrap technique to account for the additional variability in estimates due to uncertainty in the tree set. The methods are applied to genetic sequences of patients from a database compiled by the Forum for Collaborative HIV Research in an effort to characterize genetic pathways to resistance to drugs from the nucleoside reverse transcriptase inhibitor (NRTI) class. The main results were that patients with a 211K mutation in the RT region of the viral genome were more likely to have a 215Y mutation and less likely to have a 70R mutation compared to patients without a 211K mutation.

Study on gag-pol region characteristics of HIV-1 strains epidemic in Liaoning province

To investigate the genetic subtypes and sequence characteristics of gag-pol region of HIV-1 epidemic in Liaoning province. 99 whole blood samples of HIV-1 infected individuals of Liaoning province were collected. Provirus DNA was extracted from anti-coagulated peripheral whole blood, and then the sequences of gag-pol region were amplified through nested PCR and then sequenced. Genetic subtypes were identified by phylogenetic analysis, controlled with new version subtype reference sequences of HIV sequence database. The genetic distances of different coding regions were calculated by MEGA2.0 with Kimura 2-parameter model. The synonymous (Ks) and anonymous (Ka) as well as the ratio of Ks/Ka were analyzed by SNAP tool in HIV sequence database. There are many HIV-1subtypes epidemic in Liaoning province. We found five subtype: A, Thai B, B, C and G, five CRFs: CRF01, CRF03, CRF06, CRF07, CRF08, and one inter CRF recombinant ICR07/08 in 99 HIV-1 infected individuals of Liaoning province. Thai B subtype was the main subtype epidemic in Liaoning, next were CRF01_AE. In gag-pol region, the genetic distances of p17, p2-p6 were higher than p24, protease and RT coding region. The genetic distance of p24 region in B subtype strains were relative high compared with other subtypes. The genetic distance and the value of Ks and Ka were rather low in each region of CRF07 and CRF08. The ratio of Ks/Ka of p2-p6 region was the lowest in each subtype, the mean value was below 1. The ratio of Ks/Ka of RT region was as high as 10.45. The HIV-1 genetic subtypes in Liaoning province were rather complex. The p24, protease and RT coding region coding region were more conserved in gag-pol region. P24 region of B subtype strains received more positive selective pressure, while the RT region of CRF01_AE strains received more negative selective pressure. CRF07_BC and CRF08_BC were the youngest strains transmitted to Liaoning province and showed strong founder effect.

Clinical and virological features of non-breakthrough and severe exacerbation due to lamivudine-resistant hepatitis B virus mutants

Patients who develop YMDD mutant during lamivudine therapy for hepatitis B virus (HBV) infection exhibit various clinical courses. Some patients show normal ALT levels, whereas others develop severe hepatitis exacerbations (SHEs) due to YMDD mutants. We studied 136 patients with YMDD mutant among 362 Japanese adult patients on lamivudine therapy. Clinical and virological features of patients without elevated HBV DNA after emergence of YMDD mutant (non-elevated group) were investigated. Moreover, virological analysis was also performed in patients with SHE due to YMDD mutants. Patients in the non-elevated group were characterized by HBeAg-negative pretreatment, HBeAg loss during therapy, a longer duration from commencement of therapy until emergence of YMDD mutant, and no mixed-type YMDD mutants. Patients with SHE had more substitutions in the reverse transcriptase (rt) region within the polymerase gene at the time of exacerbation than those without SHE, although no specific substitutions were noted. Sequence analysis of full-length HBV genome showed more substitutions in X, rt, and surface proteins in patients with SHE than in those without elevated HBV DNA level. In conclusion, negativity for HBeAg at commencement of therapy or before emergence of YMDD mutant was an important factor among non-elevated group. More substitutions in the rt region and the other proteins may be related to the emergence of severe hepatitis caused by lamivudine-resistant virus.

Changes in viral loads of lamivudine-resistant mutants and evolution of HBV sequences during adefovir dipivoxil therapy

The addition of adefovir dipivoxil (ADV) to ongoing lamivudine therapy is effective against lamivudine-resistant virus in patients with hepatitis B virus (HBV) infection. We studied 39 patients who received ADV added to lamivudine for breakthrough hepatitis. We determined early viral changes (12 weeks) in YMDD mutants (rtM204I [YIDD sequence], rtM204V [YVDD]) and rtL180M in all 39 patients as well as amino acid changes in the polymerase reverse transcriptase (rt) region and precore/core promoter mutations in 15 patients who received long-term treatment (more than 1 year). Changes in rtM204I and rtL180M viral loads were greater than that of the rtM204V, albeit statistically insignificant. Moreover, the greatest change in viral load was seen for rtM204I without hepatitis B e antigen (HBeAg). The precore mutant was replaced with wild-type virus in three of eight patients after 1 year of added ADV therapy. Compared to baseline with lamivudine therapy only, new amino acid mutations were seen in the rt region at baseline with ADV in seven patients. At 1 year after ADV coadministration, the YMDD motif was replaced with wild-type (rt204M) in two patients, in whom mutations were fewer and of a different type. We conclude that the rtM204I may be more sensitive to ADV in vivo. ADV tended to select wild-type virus from precore mutants. Moreover, viruses that were wild-type in the rt region reappeared after 1 year of ADV coadministration in some patients.

Analysis of drug resistance‐associated mutations in treatment‐naïve individuals infected with different genetic forms of HIV‐1 circulating in countries of the former Soviet Union

There are few data on drug resistance-associated mutations in the former Soviet Union since, studies have usually been focused on the env or gag genes for subtype information. This study examines the prevalence and patterns of resistance-associated mutations to reverse transcriptase and protease inhibitors (RTI, PRI) in 278 HIV-1-infected treatment-naïve subjects from countries of Eastern Europe, and defines characteristic polymorphisms of RT and PR sequences in HIV-1 subtype A viruses. Blood samples were collected between 1997 and 2004. Plasma RNA was used for PR-RT amplification by reverse transcription coupled with nested PCR and sequencing. Phylogenetic analysis was done with neighbor-joining trees and bootscanning. Analysis of drug resistance mutations, with Stanford University HIV Drug Resistance Database's algorithm, resulted in an overall prevalence of 12.9% resistance to RTI and 3.9% to PRI. The most frequent substitutions in the RT region were at positions 62 and 236. V77I substitution in PR was found in 47.8% of samples. Polymorphisms in subtype A sequences were identified. This is the first study reporting the prevalence and patterns of both PRI and RTI resistance-associated mutations in naïve HIV-1 infected patients from the former Soviet Union. These data underline the importance of genotypic resistance testing of chronically HIV-1-infected patients before initiating treatment, in order to select the most suitable drug regimen.

Failure of the Lamivudine-Resistant rtM204I Hepatitis B Virus Mutants To Efficiently Support Hepatitis Delta Virus Secretion

Hepatitis delta virus (HDV) is encapsidated by the envelope proteins of hepatitis B virus (HBV). The major HBV lamivudine (LMV)-resistant mutations in the polymerase gene within the reverse transcriptase (rt) region at rtM204V or rtM204I are associated with changes in the overlapping envelope gene products, in particular, the gene encoding small envelope protein (s) at sI195M or sW196L/S/Stop. We have demonstrated that the LMV resistance mutations corresponding to sW196L/S inhibited secretion of HDV particles, while changes corresponding to sI195M did not affect secretion. Differential efficiencies of HBsAg proteins expressed by LMV-resistant HBV to support HDV secretion may have consequences for clinical prognosis as coinfected patients are treated with antiviral agents.

HIV-1 resistance genotyping on dried serum spots

To assess the feasibility of HIV-1 group M resistance genotyping on dried serum spots, by testing samples from previously untreated patients, patients on treatment, and patients having stopped treatment, representing a wide genetic diversity panel. Serum samples from 62 HIV-1-infected Caucasian and African patients, with viral load values from 715 copies/ml to more than 750,000 copies/ml, were deposited on filter paper. After elution and RNA extraction, nested RT-PCR was used to amplify the protease and RT regions of the pol gene. Resistance sequencing was performed on all the protease and RT amplicons. The sequences obtained for resistance genotyping were used for subtyping by phylogenetic analysis. Amplification was successful in the protease region in 53/62 cases (85.5%) and in the RT region in 51/62 cases (82.3%). All samples with viral loads of at least 5 Log (17 of 62) were successfully amplified in both the RT and protease regions. Of the 29 samples with viral loads between 4 Log and 5 Log, 28 (97%) were amplified in the RT region and 25 (86%) in the protease region. The detected mutations were in keeping with the treatment status. Marked natural polymorphism was observed in the protease region, but no major consequences were deduced in terms of resistance. The results showed a broad diversity of the panel, including subtype B (n = 36) and non B or recombinant forms (n = 20). Our results show the feasibility of this dried serum spot method for monitoring resistance to antiretroviral drugs and the molecular epidemiology of HIV diversity. The simplicity of sample preparation, storage and transport potentially makes this an importance tool for individual and epidemiological monitoring throughout the world.

Sequence analysis of gag-pol gene of HIV-1 B/C recombinant viruses in China

To characterize full length gag gene and partial pol gene of Chinese prevalent HIV-1 B/C recombinant strains, to explore the genetic difference between parent strains and B/C recombinant strains, and to investigate the mechanism of different biologic phenotype among them. The peripheral blood samples were collected from 138 HIV-positive persons from 12 regions of China respectively. Samples of total DNA were extracted from the peripheral blood mononuclear cells to undergo nested PCR and sequencing. The 117 HIV-1 CRF07-BC strains and 21 HIV-1 CRF08-BC strains were screened with the resulted that 5 CRF07-BC samples from Xinjiang and 1 CRF08-BC sample from Chongqing were regarded as the most potential new type recombinant viruses. The sequences thus obtained underwent phylogenetic tree analysis and amino acid variation analysis. Simplot software was used to analyze the sequence recombination and identify the breakpoints of B/C recombinant strains. To confirm the breakpoints, separate phylogenetic analysis according to the breakpoints was performed with MEGA software. The genetic distances of different gene fragments were calculated by DISTANCE program in GCG software package. The gene dispersion in the gene fragment of a length of 2550 bp of the recombinant HIV-1-B/C and the potential influence of gene recombination on its function were further analyzed. No change of breakpoint was found in the 5 samples from Xinjiang. But a breakpoint shift of 160 nucleotides occurred in RT region of a sample from Congqing city. CRF07-BC and CRF08-BC remain the main prevalent HIV-1 B/C recombinant strains in China. No epidemic of new mosaic recombinant strain is found. The variation of amino acids at the sites 286 and 799 may be the reasons of the transmission dominance of the B/C recombinant strain.

Detection of HIV type 1 non-B subtypes in Sicily, Italy.

To evaluate the presence of HIV-1 non-B subtypes in Sicily, we sequenced and genotyped HIV-1 PR and RT regions of the pol gene using plasma from 169 HIV-1-infected adult patients. All samples were obtained from a study of antiretroviral-associated resistance mutations resulting in virological failure during highly active antiretroviral therapy (HAART). Eight (4.7%) patients had the non-B HIV-1 subtype including some circulating recombinant forms (CRFs). All of these individuals acquired the infection by heterosexual transmission. The detection of HIV-1 non-B strains was significantly associated with younger age of HIV-1 acquisition. Our findings indicate, for the first time, the presence of HIV-1 non-B subtypes in Sicily in patients who experienced virological failure during HAART, and highlight the need for implementing a network for the epidemiological surveillance of HIV-1 subtypes in Southern Europe.

Chemical stimulation of the thalamic reticular nucleus inhibits the neuronal activity of the posterior insular cortex in rats.

Extracellular neuronal responses were recorded from the posterior insular cortex following electrical and chemical stimulation of the thalamic reticular nucleus (Rt) regions. In the present study, most neurons (29/32) were first characterized for their responses to electrical stimulation of the superior laryngeal (SL) nerve or glossopharyngeal (IXth) nerve. In the first experiment, 15 neurons in the posterior insular cortex were examined for their responses to electrical stimulation of the Rt regions. It was found that effective stimulation sites to evoke action potentials in the posterior insular cortex were the ventromedial portion of the Rt and its adjacent regions. In the second experiment, 17 neurons in the posterior insular cortex were examined for their responses by pressure injection of glutamate (Glu) into the Rt regions. Of the 17 neurons, 13 were inhibited in the spontaneous discharge rate following injection of Glu into the Rt, and the remaining four were unaffected. Histologically, it was demonstrated that Glu injection sites for the case of inhibition were located near or within the Rt. On the other hand, the injection sites for all four non-responsive neurons were located outside of the Rt. These data suggest that excitation of the Rt (GABAergic neurons) causes depression of the neuronal activity in the thalamic relay nucleus and then this may in turn induce depressed neuronal activity in the posterior insular cortex. The results here indicate that neuronal activity in the posterior insular cortex is controlled by the Rt, which has been reported in other sensory systems.

In Vitro Study of Resistance-Associated Genotypic Mutations to Nucleoside Analogs

In spite of a rather long period of investigations, the problem of HIV drug resistance remains unsolved, and more that, at present HIV-1 mutants resistant to all known nucleoside inhibitors being used in clinical therapy against the human immunodeficiency syndrome are discovered. In this study we selected HIV-1 mutants resistant to the nucleoside inhibitors of HIV reverse transcriptase (NRTI): 3′-azido-2′,3′-dideoxythymidine (AZT), 5′-phosphit 3′-azido-2′,3′- dideoxythymidine (ph-AZT), dideoxyinosine (ddI) and didehydrodeoxythymidine (d4T). Selection of resistant mutants was carried out by gradually increasing of drug concentration in the culture medium during propagation of the HIV-1EVK on fresh MT-4 cells. Phenotypic resistance was defined as an increase in ID50 of 160-fold for AZT, 8 for ph-AZT, 10 for ddI, 7 for d4T. In comparison studies it was determined that the viral resistance to these drugs was appeared variously in a similar conditions and duration of selection. The nucleotide sequences of the RT region of the HIV-1 variants were compared with the HIV-1EVK from “0” passage. For some of selected HIV-1 mutants NRTI resistance mutations were detected. Selected AZT resistant variants contained amino acid substitutions in positions D67A and K70R. Our studies was not revealed substitution at position 75 for ph-AZT resistant variants, whereas substitution at position L214F have been observed in both experiments using AZT and ph-AZT. Selected d4T resistant mutants contained amino acid substitutions in positions N54D and P52R. Selected ddI resistant mutants contained only one amino acid substitution in position P143S. Collection of drug-resistant mutants should prove to be a convenient tool for rapid investigations a new antiretroviral agents on cross drug-resistance.