Isolation and biological characterization of human immunodeficiency virus type 1 from Cubans asymptomatic patients
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1
AIDS Research Laboratory, Cuba
The natural history of infection with the human immunodeficiency virus type 1 (HIV-1) varies widely among infected patients. The factors responsible for this diversity are determined by a complex interaction of viral, environmental and host factors. Large differences have been described amongst humans in their susceptibility to infection with HIV-1 as well as rates of disease progression. After infection some individuals progress slowly in their disease course some progress rapidly and develop disease in as little as 2–5 years (fast progressors), whilst a small minority show no progression over 20 years of infection (elite controllers). Isolation and characterization of primary strains of human immunodeficiency virus (HIV) is a fundamental tool for the evaluation of the properties of viral replication in patients infected with HIV-1. In asymptomatic patients infected with HIV-1 have been reported elevated levels of viral load and immune response induction, this has been linked to multiple viral and host factors that may determine the rate of disease progression and transmission. The aim of this study was to relate the immunological and virological markers of Cuban asymptomatic patients with the phenotypic and genotypic characteristics of the viral isolation. Peripheral blood with anticoagulants was obtained from fifty asymptomatic patients. All patients acquired the infection by sexual contact, of which thirty eight had sexual behavior of men who have sex with men (MSM) and twelve heterosexual (HT). Sixteen patients were classified in A1 clinical categories (CD4+ cell count >500 cell/mL), twenty-one as A2 (CD4+ cell count 200- 499 cell/mL) and thirteen as A3 (CD4+ cell count <200 cell/mL), according to the 1993 revised Classification System for HIV Infection and Expanded Surveillance Case Definition for AIDS among Adolescents and Adults. The median age of the patients at the time of the study was 39.8 years with a range from 21 years to 65 years. Thirty eight patients were antiretroviral-naive, while twelve received antiretroviral therapy (ART). The viral load assay was run using the COBAS Ampliprep/COBAS Taqman HIV-1 Test (Roche Diagnostics GmbH, Mannheim, Germany). For the genotypic analysis, viral RNA was isolated from 200 μL of plasma using the High Pure Viral Acid Nucleic Acid kit (Roche Diagnostics, GmbH, Mamheim, Germany) and the subtype determination was performed by sequence analysis of the pol gene. Virus isolation was performed by standard coculture technique as previously described by WHO, 2002. The cultures were maintained for 28 days and supernatants were assayed for P24 antigen using the P24 antigen detection kit (DAVIH Ag P24, Laboratorios DAVIH, Cuba), the culture was considered positive where antigen level above the 2000 pg/mL. The biological phenotype determination was performed by the capacity of syncytium inducing (SI) in MT2 cells and the co-receptors usage prediction by genotypic analysis of the V3 loop with the application of bioinformatics methods. The molecular characterization of HIV-1 isolation was carried out on the PBMCs infected with the HIV-1 primary isolates. The ADN was extracted from the cells using phenol-chloroform and ethanol precipitation. A part of the HIV-1 group M env gene covering the C2-V3 region was amplified by nested PCR. A chi square test was used to evaluate the correlation between the viral load and CD4 cell count which HIV isolation (P24 antigen positivity in culture). Descriptive analysis of virus phenotype of isolates from patients on ART and those that are naïve was made. HIV-1 was successfully isolated from thirty-one of the patients (62%) as determined by p24 ELISA. Greater positivity was obtained in clinical category A3 with 92.3% followed by A2 with 76.19% and finally A1 with 18.75%. In correspondence of viral load, the virus was isolation from 90.47% with values greater than 5x104 copies/ mL and only 15.38% from patients which viral load lower than 1x104 copies/ mL. A strong association was found between CD4 counts, viral load and virus isolation (p<0.0001). Positive isolation was achieved in four of the twelve heterosexual patients with 33% of positivity and in twenty seven patients of the MSM with 71% of positivity. Association between HIV-1 positive isolation and transmission routes was found (p<0.01). Five of the twelve ART patients were isolates positives, while twenty six isolations were obtained from naive patients. There was no relationship between the detectable HIV-1 isolation (p24 antigen detection) and treatment status. The isolates were evaluated for SI capability in the MT2 cell line, no syncytium inducing (NSI) phenotype was detected in twenty strains (64.5%) and SI in eleven (35.5%). By analyzing the sequences of env V3 regions of each HIV-1 isolate, theoretical phenotypes including the syncytium-inducing (SI) effects and co-receptor usage of each isolate were performed. According to the PSSM prediction indicated that eighteen isolates were CCR5-tropic (R5), five were CXCR4-tropic (X4) and four dual tropic (R5X4). All R5 isolates were NIS, while X4 and R5X4 had the ability to form syncitium in the MT-2 cell line (SI). Of the twelve isolates from patients with AIDS, one not amplified, five had tropism X4 or R5X4 and the remaining six had R5 tropism. The percentage of isolates with X4 or R5X4 tropism from AIDS patients was 41.6%. A phylogenetic tree constructed using sequences from the C2-V3 region of isolates included in the study. Our analysis resulted that twenty seven (48.2 %) isolates were grouped with reference sequences of the subtype B, while that the 51.8% were grouped with reference sequences of the non B subtypes (CRF19_cpx: 25.9 %, CRF20, 23, 24_BG: 22.2 %, CRF18_cpx: 3.7 %). There was no relationship between the detectable HIV-1 isolation (p24 antigen detection) and the subtype. The growth properties, co-receptor usage and syncitium inducing ability of these isolates were found to be related to patients' clinical statuses. The immunologic and virology characteristic of patients determinates the progression of the disease in Cubans asymptomatic patients independent of viral genotype
Keywords:
HIV-1,
Asymptomatic patients,
Biological characterization,
Viral genotype,
Immunological markers
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Oral Presentation
Topic:
Infectious and parasitic diseases
Citation:
Noa Romero
E,
Navea
L,
Machado
L,
Dubed
M,
Blanco
M,
Enriquez Puertas
JM,
Valdés
N and
Díaz
H
(2015). Isolation and biological characterization of human immunodeficiency virus type 1 from Cubans asymptomatic patients.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00313
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Received:
16 May 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
PhD. Enrique Noa Romero, AIDS Research Laboratory, San José de las Lajas, Mayabeque, 32700, Cuba, enrnoa@infomed.sld.cu