RESUMO
The needs for development and/or improvement of molecular approaches for microorganism detection and characterization such as polymerase chain reaction (PCR) are of high interest due their sensitivity and specificity when compared to traditional microbiological techniques. Considering the worldwide importance of human immunodeficiency virus type 1 (HIV-1) infection, it is essential that such approaches consider the genetic variability of the virus, the heterogeneous nature of the clinical samples, the existence of contaminants and inhibitors, and the consequent needs for standardization in order to guarantee the reproducibility of the methods. In this work we describe a nested PCR assay targeting HIV-1 virus gag and env genes, allowing specific and sensitive diagnosis and further direct characterization of clinical samples. The method described herein was tested on clinical samples and allowed the detection of HIV-1 presence in all samples tested for the gag gene and 90.9% for the env gene, revealing sensitivities of 1 fg and 100 fg, respectively. Also, no cross-reactions were observed with DNA from infected and noninfected patients and the method allowed detection of the env and gag genes on an excess of 10(8) and 10(4) of human deoxyribonucleic acid (DNA), respectively. Furthermore, it was possible to direct sequence all amplified products, which allowed the sub typing of the virus in clinical samples.
Assuntos
Produtos do Gene env/análise , Produtos do Gene env/genética , Produtos do Gene gag/análise , Produtos do Gene gag/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Eletroforese em Gel de Ágar , Etídio , Genes Virais/genética , Humanos , Sensibilidade e EspecificidadeRESUMO
SIVsm chronically infected cultures were obtained after infection of CEMX174 cells with either SIVsmH3 or SIVsmE660. These phenotypically CD4 cells, formed syncytia but only when cocultivated with CD4+ cells. Single cell clones were derived from these cultures and examined for the production of virus-specific proteins. The majority of the clones expressed SIV p27 antigen and low levels of virus reverse transcriptase activity. Western blot analysis, performed with either monoclonal or polyclonal sera, showed that a chronically infected clone (B7) produced particles which contained envelope (gp135 and gp43), gag precursors and gag proteins (p27, p16 and p8). However, these particles (SIVsmB7) lacked detectable levels of vpx and of integrase, and contained several fusion proteins which expressed viral protease antigens. This defective virus failed to infect established CD4+ cell lines, as well as primary cultures of macrophages and of peripheral blood lymphocytes, obtained both from humans and from rhesus macaques. Lack of infection correlated with lack of viral DNA detection by PCR amplification of genomic DNA extracted from these cell cultures. In addition, SIVsmB7 virus lacked infectivity in vivo. Rhesus macaques inoculated with high concentrations of SIVsmB7 showed no viremia and their PBMC were PCR negative. Thus, B7 cells produced stable, non-infectious virus mutants, which contained env and gag proteins, but lacked detectable amounts of vpx and of enzymes required for virus replication. Due to the high constitutive expression of this virus-like particle, we are now testing this preparation as a vaccine.
Assuntos
Linfócitos T CD4-Positivos/virologia , Células Clonais/virologia , Vírus Defeituosos/fisiologia , Corpos de Inclusão Viral/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Sequência de Bases , Fusão Celular , Efeito Citopatogênico Viral , DNA Viral/análise , Vírus Defeituosos/genética , Vírus Defeituosos/isolamento & purificação , Produtos do Gene gag/análise , Genes Virais , Humanos , Macaca mulatta/virologia , Macrófagos/virologia , Microscopia Eletrônica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Proteínas dos Retroviridae/biossíntese , Proteínas dos Retroviridae/genética , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/isolamento & purificação , Replicação ViralRESUMO
A modified Western blot (WB) that includes both shared (r21e) and unique recombinant envelope proteins from human T-lymphotropic virus (HTLV) type I (rgp46I) and type II (rgp46II) was compared to conventional HTLV serologic tests in 379 United States blood donors and individuals residing in diverse geographic regions, and the specimens were categorized as positive (n = 158), indeterminate (n = 158), or negative (n = 63) for HTLV infection. Of the 158 HTLV-I/II-positive specimens (66 requiring radioimmunoprecipitation assay [RIPA] for confirmation), 156 reacted concordantly with r21e, gag, and either rgp46I or rgp46II, thus eliminating the need for RIPA in all but two specimens and yielding a test sensitivity of 98.7 percent. Of the 158 indeterminate and 63 negative specimens, none reacted with r21e and rgp46I or rgp46II, yielding a test specificity of 100 percent. Furthermore, analysis of an additional 184 consecutive specimens from a retrovirology reference laboratory demonstrated that the modified WB correctly identified 27 of 28 HTLV-I specimens and all 13 HTLV-II specimens, with a test sensitivity of 97.6 percent. None of specimens that were indeterminate or nonreactive in conventional WB and/or RIPA and none of the screening enzyme immunoassay-negative specimens reacted with r21e and either rgp46I or rgp46II, for a test specificity of 100 percent. Thus, the modified WB appears to be highly sensitive and specific for simultaneous detection and discrimination of HTLV-I from HTLV-II and has the advantage of being a one-step assay that is easily performed in all types of laboratory settings and allows rapid, reliable, and standardized testing for HTLV-I/II infection.
Assuntos
Doadores de Sangue , Anticorpos Antideltaretrovirus/sangue , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-II/diagnóstico , Proteínas do Envelope Viral/análise , Western Blotting/métodos , Infecções por Deltaretrovirus/sangue , Infecções por Deltaretrovirus/diagnóstico , Diagnóstico Diferencial , Feminino , Produtos do Gene gag/análise , Infecções por HTLV-I/sangue , Infecções por HTLV-II/sangue , Humanos , Técnicas Imunoenzimáticas , Masculino , México , Radioimunoensaio/métodos , Proteínas Recombinantes/análise , Abuso de Substâncias por Via Intravenosa/sangue , Estados Unidos , Proteínas do Envelope Viral/sangueRESUMO
The heterogeneity of immune responsiveness to the immunodominant epitopes of human T lymphotropic virus (HTLV) types I (MTA-1(162-209)) and II (K-55(162-205)) were determined in natural infections with HTLV-I and -II from diverse geographic areas (n = 285). Of the HTLV-I specimens confirmed by polymerase chain reaction (PCR), all North American (n = 37) and Peruvian (n = 19) specimens reacted with MTA-1. Of HTLV-II specimens confirmed by PCR, 44 (96%) of 46 from North American blood donors, 28 (97%) of 29 from native Americans, and all from intravenous drug users (n = 29) reacted with K-55. Specimens from other geographic areas (Peru, 30; Brazil, 4; Mexico, 10; Italy, 5; Somalia, 13; Ethiopia, 17; Japan, 32; and Jamaica, 15) all reacted either with MTA-1 or K-55. By synthetic peptide-based serologic typing, all of these specimens could be typed as HTLV-I or -II. In addition to the direct implications of these findings for diagnostic purposes, these data provide indirect evidence for the conservation of immunodominant HTLVenv epitopes in diverse geographic populations.
Assuntos
Epitopos/análise , Produtos do Gene gag/análise , Produtos do Gene gag/imunologia , Anticorpos Anti-HTLV-I/sangue , Anticorpos Anti-HTLV-II/sangue , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Vírus Linfotrópico T Tipo 2 Humano/imunologia , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/imunologia , África , Doadores de Sangue , Geografia , Humanos , Imunoensaio , Jamaica , México , América do Norte , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/imunologia , América do SulRESUMO
To diagnose infection with the human immunodeficiency virus (HIV) soon after birth in infants born to HIV type 1-infected women, we developed antiviral IgA Western blot and dot blot assays with recombinant HIV-1 proteins. Thirty-three infants born to HIV-1-seropositive mothers and nine infants born to HIV-1-seronegative intravenous drug-abusing mothers were followed prospectively. Infection was documented by positive virus culture. Results with the polymerase chain reaction were used for comparison. Twelve infants were found infected with HIV-1; the earliest age at which cultures became positive ranged from birth to 31 weeks of age. Of the 12 culture-positive infants, 10 had anti-HIV IgA antibodies detectable initially between birth (cord blood) and 27 weeks of age. Anti-HIV IgA was not present in the uninfected infants or in the control subjects, either by Western blot or dot blot assays. Testing for anti-HIV IgA antibodies with recombinant HIV-1 proteins is an effective method for detecting viral infection in newborn and young infants.