RESUMEN
Human immunodeficiency virus 1 type vpr, vpu and V3 sequences from 15 homosexual men and 19 intravenous drug users in the Amsterdam Cohort studies were analysed. Previously, we reported that V3 domains of viruses from drug users are distinguishable from those of homosexual men on the basis of two silent mutations. Phylogenetic analysis of vpr, vpu and V3 shows that differences in all three regions correlate with risk group. Two positions in both vpr and vpu were found to differ significantly between the risk groups. The distinguishing positions were confirmed for sequences from 11 Scottish and four German samples. The three regions show relatively independent evolution patterns; they resulted in different phylogenies, the only stable clustering being that based on the risk group distinction. Pairwise differences between sequences of the genes were moderately correlated (around 0.30). Surprisingly, when only silent changes are counted, the correlations dropped almost to zero, indicating that the evolution towards independence was more advanced in the silent than in the non-silent positions. This suggests that selection at the amino acid level is not the primary driving force for the independent evolutionary behaviour of the genes. Recombination, combined with restrictions on certain amino acids because of epistatic interactions between the genes, could be an alternative explanation of this phenomenon.
Asunto(s)
Productos del Gen vpr/genética , Variación Genética , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Fragmentos de Péptidos/genética , Proteínas Reguladoras y Accesorias Virales/genética , Secuencia de Bases , Estudios de Cohortes , ADN Viral , Europa (Continente)/epidemiología , Evolución Molecular , Genes Virales , Infecciones por VIH/epidemiología , Homosexualidad , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Masculino , Datos de Secuencia Molecular , Filogenia , Factores de Riesgo , Homología de Secuencia de Ácido Nucleico , Abuso de Sustancias por Vía Intravenosa/complicaciones , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
OBJECTIVE: To investigate genotypes and serotypes of HIV-1 variants in Russia, Byelorussia and Lithuania. PATIENTS AND METHODS: Sera from 20 HIV-1-infected individuals were tested in an enzyme-linked immunosorbent assay (ELISA) with 19 V3 synthetic peptides, and serum HIV-1 V3 RNA was amplified and sequenced. RESULTS: Sequence comparison of the envelope V3 region among specimens tested revealed a 2-29% range nucleotide divergence, with a mean of 19%. Phylogenetic analysis from the homosexual men were shown to belong to subtype B, and all of the heterosexually infected individuals to subtype C. Sequences from the parenterally infected individuals were more heterogeneous. IOn the peptide ELISA three reactivity patterns were found. Serum samples from six out of seven homosexual men showed reactivity to peptides p108 or p110 representing V3 amino-acid sequences found in US/West European HIV-1 isolates. Serum samples from six of seven individuals who had acquired HIV-1 through heterosexual contacts were reactive to peptide p169. Four out of six parenterally infected patients had peak reactivity to p168. CONCLUSION: Distinct HIV-1 variants were found in Russia, Byelorussia and Lithuania, which were introduced simultaneously in the mid-1980s. This diversity was shown to be associated with the route of transmission. Homosexual men appeared to be infected with subtype B and heterosexually infected individuals with subtype C HIV-1 variants. HIV-1 subtypes A, C, D and G were found among parenterally infected individuals.
PIP: HIV-1 variants show a relatively high level of genomic and antigenic diversity. This heterogeneity is particularly high in the V3 domain of envelope glycoprotein gp120, which is implicated in a number of biological properties of HIV-1, including cell tropism, infectivity, and cytopathogenicity; it is also a target for both humoral and cellular immune response. At least nine subtypes of HIV-1 have been identified. Subtypes A-H are phylogenetically equidistant and shown to be geographically associated with different subcontinents. Subtype B is most prevalent in North and South America and western Europe. Subtypes A, C, D, G, and H are found frequently in sub-Saharan countries, while subtype C is also found in India. Subtype E sequences have been found in patients from Thailand and Central Africa, and subtype F has been described in Romania and Brazil. This study reports findings from an investigation of genotypes and serotypes of HIV-1 variants in Russia, Byelorussia, and Lithuania. Sera from 15 HIV-1-infected men and 5 HIV-1-infected females were tested by ELISA with 19 V3 synthetic peptides with amplified and sequenced serum HIV-1 V3 RNA. Sequence comparison of the envelope V3 region among specimens tested revealed a 2-29% range of nucleotide divergence, with a mean of 19%. Distinct variants of HIV-1 were found in Russia, Byelorussia, and Lithuania, which were introduced simultaneously in the mid-1980s. The diversity was shown to be associated with the route of transmission. Homosexual men appeared to be infected with subtype B compared to heterosexually infected individuals with subtype C HIV-1 variants. HIV-1 subtypes A, C, D, and G were found among parenterally-infected individuals. These findings are based upon the three peptide reactivity patterns identified by ELISA. Serum samples from six out of seven homosexual men showed reactivity to peptides p108 or p110 representing V3 amino-acid sequences found in US/West European HIV-1 isolates. Serum samples from six out of seven individuals who had acquired HIV-1 through heterosexual contacts were reactive to peptide p169, and four out of six parenterally infected patients had peak reactivity to p168.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/virología , VIH-1/clasificación , VIH-1/genética , Síndrome de Inmunodeficiencia Adquirida/sangre , Síndrome de Inmunodeficiencia Adquirida/transmisión , Secuencia de Aminoácidos , Secuencia de Consenso , Ensayo de Inmunoadsorción Enzimática , Femenino , Variación Genética , Genotipo , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Humanos , Lituania/epidemiología , Masculino , Datos de Secuencia Molecular , Fenotipo , Filogenia , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , República de Belarús/epidemiología , Federación de Rusia/epidemiología , SerotipificaciónRESUMEN
We studied the relationship between the rate of disease progression after HIV-1 seroconversion and the level of IgG antibody response to HIV-1 envelope and core epitopes. This was done by comparing a group of fast-progressing individuals and a group of slow-progressing individuals for serum IgG titers to peptides from the gp120-V3 neutralization domain, to a peptide from the immunodominant gp41 epitope (residues 590 to 607), and to recombinant gp120 and p24. The two groups displayed a large overlap in titers to the envelope epitopes, which precluded their differentiation at most time points after seroconversion. Low responsiveness to envelope antigens was not only found in a few fast-progressors but also in one individual who remained asymptomatic for at least 92 months after seroconversion. The only significant differences between the groups were found in the first months after seroconversion when the responses to the V3 domain and the gp41 epitope were more vigorous in the group of fast-progressors. Furthermore, on evaluating ratios of anti-V3 antibody titers to anti-gp120 antibody titers we found no indication that fast disease progression was associated with a restriction in antibody response to the V3 epitope. We did confirm the finding that fast disease progression is associated with low levels of p24-directed antibodies, both early after seroconversion and at later stages. These data demonstrate that levels of IgG antibodies to envelope epitopes are poor predictors of rapid disease progression and suggest that the role of V3-directed neutralizing antibodies in preventing subversion of the immune system is not decisive in natural HIV-1 infection.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Seropositividad para VIH/inmunología , VIH-1/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Células CHO , Estudios de Cohortes , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Anticuerpos Anti-VIH/biosíntesis , Seropositividad para VIH/fisiopatología , Humanos , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/inmunología , Homología de Secuencia de AminoácidoRESUMEN
By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between high-risk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patients will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.
Asunto(s)
Cuello del Útero/patología , Infecciones Tumorales por Virus/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Biopsia , Cuello del Útero/microbiología , ADN Viral/análisis , Diagnóstico Diferencial , Femenino , Humanos , Papillomaviridae/química , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Infecciones Tumorales por Virus/patología , Neoplasias del Cuello Uterino/patologíaRESUMEN
We have mapped using the RNA PCR the viral-cellular junctions of HPV16 viral-cellular cotranscripts expressed in CaSki cells and a cervical carcinoma to nt 3728 and 881, respectively. Both junctions were located within the E1-E2 region. Examination of the cellular sequences of the cotranscripts showed the presence of a polyadenylation signal in each of the transcripts. In CaSki cells and in the cervical carcinoma transcripts derived from the full-length early region including the E2 transcript were also detected. Our results suggest that the utilization of a cellular polyadenylation site could be important in the development of cancer by HPV.
Asunto(s)
Carcinoma/microbiología , Regulación Viral de la Expresión Génica , Papillomaviridae/genética , ARN Viral/genética , Neoplasias del Cuello Uterino/microbiología , Secuencia de Bases , Carcinoma/genética , Línea Celular , Femenino , Genes Virales , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Empalme del ARN , ARN Mensajero/genética , Neoplasias del Cuello Uterino/genética , Proteínas Estructurales Virales/genéticaRESUMEN
We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs.
Asunto(s)
Adenocarcinoma/patología , Papillomaviridae/clasificación , Infecciones Tumorales por Virus/tratamiento farmacológico , Neoplasias del Cuello Uterino/microbiología , Tipificación de Bacteriófagos , Secuencia de Bases , Southern Blotting , Carcinoma de Células Escamosas/patología , Mapeo Cromosómico , Sondas de ADN de HPV , Femenino , Humanos , Immunoblotting , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Homología de Secuencia de Ácido NucleicoRESUMEN
We utilized the RNA polymerase chain reaction (PCR) to analyse the transcripts of the E6/E7 open reading frames of human papillomavirus type 16 (HPV-16). Total RNA was isolated from 14 cervical squamous carcinomas, nine cervical intraepithelial neoplasias and from human fibroblasts transformed with different HPV-16 constructs. In all specimens two spliced transcripts were detected. Sequence analysis of the cloned PCR products showed that both transcripts were generated by splicing out an intron in E6, from nucleotides (nt) 226 to 409 in one transcript and from nt 226 to 526 in the other. The major transcript present in all RNA specimens had the smallest intron in E6. The RNA PCR described here is the method of choice for analysing splice and donor sites in tissue specimens where a limited amount of RNA is available. Results obtained with transformed cells revealed no difference in splicing whether HPV-16 was controlled by its homologous promoter or by a heterologous promoter, the Rous sarcoma virus long terminal repeat.
Asunto(s)
Transformación Celular Viral , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Empalme del ARN , Neoplasias del Cuello Uterino/microbiología , Secuencia de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/microbiología , Células Cultivadas , Femenino , Fibroblastos , Humanos , Datos de Secuencia Molecular , Proteínas E7 de Papillomavirus , Reacción en Cadena de la Polimerasa , Lesiones Precancerosas/genética , Lesiones Precancerosas/microbiología , ARN Viral/genética , Transcripción Genética , Neoplasias del Cuello Uterino/genéticaRESUMEN
The localization of human papillomavirus type 16 (HPV-16) DNA throughout the cervix uteri of women with cervical intraepithelial neoplasia (CIN) was studied by utilizing the polymerase chain reaction technique directly on histologically defined sections of paraffin-embedded cervical tissue obtained by conizations. HPV-16 DNA was detected only in the sections that contained CIN lesions and/or koilocytes. No HPV-16 DNA was detected in sections that contained only normal epithelium. This is in accordance with HPV-16 playing a role in the development of CIN lesions.
Asunto(s)
Cuello del Útero/microbiología , ADN Viral/análisis , Papillomaviridae/genética , Infecciones Tumorales por Virus/diagnóstico , Neoplasias del Cuello Uterino/microbiología , ADN Viral/genética , Femenino , Humanos , Oligonucleótidos , Parafina , Reacción en Cadena de la PolimerasaRESUMEN
Primers for polymerase chain reaction (PCR) were synthesized from clones derived from a Leptospira hardjo (type hardjobovis) library. One pair of synthetic oligonucleotide primers was selected for further analysis. Under experimental conditions an amplification was obtained with DNA of Leptospira interrogans of some serovars belonging to serogroup sejroe. However, very little or no amplification was observed with DNA from other serovars of this group. No amplification was observed with DNA from other serogroups, other bacteria, or eucaryotic organisms. Cattle urine, seeded with hardjobovis, was processed in several ways and subsequently subjected to PCR. Boiling of the samples or treatment with detergents appeared to be most effective. Urine samples containing fewer than 10 leptospires gave a positive result in the PCR assay. Twenty urine samples obtained from a slaughterhouse or farm cows were investigated using the PCR assay, culture isolation, dot and quick blot hybridization, and serological tests. This comparative study suggests that amplification by PCR may be a valuable method for the detection of leptospires in cattle urine.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Leptospira/aislamiento & purificación , Leptospirosis/veterinaria , Animales , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/orina , ADN Bacteriano/genética , Estudios de Evaluación como Asunto , Leptospira/genética , Leptospirosis/diagnóstico , Leptospirosis/orina , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The sensitivities of dot blot hybridisation and in situ filter hybridisation for the detection of HPV DNA were compared. Dot blot hybridisation was 10-50 times more sensitive than in situ filter hybridisation in detecting HPV 16 DNA in the cervical cancer cell lines SiHa and CaSki. Cervical smears collected from 51 women with a history of one or more abnormal cervical smears were tested by both hybridisation techniques for the presence of HPV 16 DNA; 11 were positive in the in situ filter hybridisation, 35 in the dot blot hybridisation. Thirty-five cervical biopsies available from this group of 51 women were processed for dot blot hybridisation. In 30 of the 35 cases the results of this hybridisation corresponded with the results of the dot blot hybridisation on the smears.
Asunto(s)
Cuello del Útero/microbiología , ADN Viral/análisis , Hibridación de Ácido Nucleico , Papillomaviridae/aislamiento & purificación , Biopsia , ADN Viral/aislamiento & purificación , Femenino , Humanos , Papillomaviridae/genética , Células Tumorales Cultivadas , Frotis VaginalRESUMEN
The structure of the gene coding for ribosomal protein S33, - a protein which escapes the coordinate control of ribosomal protein synthesis in rna 2 mutant cells -, was determined by sequence analysis. The gene comprises an uninterrupted coding region of 204 nucleotides encoding a protein of 8.9 kD. Like for other yeast ribosomal protein genes that have been sequenced so far, a relatively strong codon bias was observed. By S1 nuclease mapping the 5' end of the S33 mRNA was shown to be located at 11 to 15 nucleotides upstream from the initiation codon.