Identification of Human T-lymphotropic Virus Type I (HTLV-I) Subtypes Using Restrited Fragment Length Polymorphism in a Cohort of Asymptomatic Carriers and Patients with HTLV-I-associated Myelopathy/tropical Spastic Paraparesis from São Paulo, Brazil

Although human T-lymphotropic virus type I (HTLV-I) exhibits high genetic stability, as compared to other RNA viruses and particularly to human immunodeficiency virus (HIV), genotypic subtypes of this human retrovirus have been characterized in isolates from diverse geographical areas. These are currently believed not to be associated with different pathogenetic outcomes of infection. The present study aimed at characterizing genotypic subtypes of viral isolates from 70 HTLV-I-infected individuals from São Paulo, Brazil, including 42 asymptomatic carriers and 28 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), using restricted fragment length polymorphism (RFLP) analysis of long terminal repeat (LTR) HTLV-I proviral DNA sequences. Peripheral blood mononuclear cell lysates were amplified by nested polymerase chain reaction (PCR) and amplicons submitted to enzymatic digestion using a panel of endonucleases. Among HTLV-I asymptomatic carriers, viral cosmopolitan subtypes A, B, C and E were identified in 73.8%, 7.1%, 7.1% and 12% of tested samples, respectively, whereas among HAM/TSP patients, cosmopolitan A (89.3%), cosmopolitan C (7.1%) and cosmopolitan E (3.6%) subtypes were detected. HTLV-I subtypes were not statistically significant associated with patients' clinical status. We also conclude that RFLP analysis is a suitable tool for descriptive studies on the molecular epidemiology of HTLV-I infections in our environment.

Polymorphism of the Human Immunodeficiency Virus Type 1 in Brazil: Genetic Characterization of the nef Gene and Implications for Vaccine Design

Most of the Brazilian HIV-1 samples have been characterized based on the structural genes (env, gag and pol) and no data concerning the variability of the accessory genes such as nef have been available so far. Considering the role of the nef on virus biology and the inclusion of this region in some HIV/AIDS vaccine products under testing, the purpose of this study was to document the genetic diversity of the nef gene in third-four HIV-1 Brazilian samples previously subtyped based on the env C2-V3 region. Although only few non-subtype B samples have already been analyzed so far, the cytotoxic Tlymphocyte epitopes encoded in this region were relatively conserved among the subtypes, with some amino acid signatures mainly in the subtype C samples. Considering the increasing of the non-B HIV-1 subtypes worldwide, in special the subtype C, more data should be generated concerning the genetic and antigenic variability of these subtypes, as well as the study of the impact of such polymorphism in HIV/AIDS vaccine design and testing.

Dengue virus type 3 isolation from Aedes aegypti in the municipality of Nova Iguaçu, State of Rio de Janeiro

In a prospective field study conducted from July 2000 to June 2001, adult Aedes aegypti and Ae. albopictus mosquitoes were caught from the municipality of Nova Iguaçu, State of Rio de Janeiro, Brazil. Virus isolation in Ae. albopictus clone C6/36 cell line and a semi-nested reverse transcription-polymerase chain reaction detected only dengue virus type 3 in three pools of Ae. aegypti, despite the co-circulation of DEN-1, DEN-2 and DEN-3 serotypes in that area. No viruses were detected in Ae. albopictus mosquitoes. This virological surveillance consists in a sentinel system alerting for dengue outbreaks.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells

Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.

Isolation of respiratory syncytial virus from nasopharyngeal aspirates stored at 20ºC from one to fifteen months after collection

Cell culture isolation is used for recovering respiratory syncytial virus (RSV) from respiratory specimens. As RSV is a thermolabile virus, specimens destined for inoculation into cell culture require special transport, handling, and storage. The isolation rate of RSV from nasopharyngeal aspirates (NPA) stored at 20ºC for one to 15 months after collection was investigated. A total of 126 samples considered positive for RSV by indirect fluorescence-antibody were tested by virus isolation in HEp-2 cell culture. RSV was isolated from 47/126 specimens (37.3%). These results show that RSV may be recovered from NPA stored at 20ºC by cell culture.

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.

Morphological studies in a model for dengue-2 virus infection in mice

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

Histopathological and ultrastructural aspects of mice lungs experimentally infected with dengue virus serotype 2

Histological and ultrastructural alterations in lung tissue of BALB/c mice infected with dengue virus serotype 2 (non-neuroadapted), by intraperitoneal and intravenous routes were analyzed. Lung tissues were processed following the standard techniques for photonic and electron transmission microscopies. Histopathological and ultrastructural studies showed interstitial pneumonia, characterized by the presence of mononuclear cells. In the mouse model, the dengue virus serotype 2 seems to led to a transient inflammatory process without extensive damage to the interalveolar septa, but caused focal alterations of the blood-exchange barrier. Endothelial cells of blood capillaries exhibited phyllopodia suggesting activation by presence of dengue virus. Morphometrical analysis of mast cells showed an expressive increase of the number of these cells in peribronchiolar spaces and adjacent areas to the interalveolar septa. Alveolar macrophages showed particles dengue virus-like inside rough endoplasmic reticulum and Golgi complex, suggesting viral replication. The tissue alterations observed in our experimental model were similar to the observed in human cases of dengue fever and dengue hemorrhagic fever. Our results show that BALB/c mice are permissive host for dengue virus serotype 2 replication and therefore provides an useful model to study of morphological aspects of dengue virus infection.

Abnormal humoral immune response to influenza vaccination in pediatric type-1 human immunodeficiency virus infected patients receiving highly active antiretroviral therapy

Given that highly active antiretroviral therapy (HAART) has been demonstrated useful to restore immune competence in type-1 human immunodeficiency virus (HIV-1)-infected subjects, we evaluated the specific antibody response to influenza vaccine in a cohort of HIV-1-infected children on HAART so as to analyze the quality of this immune response in patients under antiretroviral therapy. Sixteen HIV-1-infected children and 10 HIV-1 seronegative controls were immunized with a commercially available trivalent inactivated influenza vaccine containing the strains A/H1N1, A/H3N2, and B. Serum hemagglutinin inhibition (HI) antibody titers were determined for the three viral strains at the time of vaccination and 1 month later. Immunization induced a significantly increased humoral response against the three influenza virus strains in controls, and only against A/H3N2 in HIV-1-infected children. The comparison of post-vaccination HI titers between HIV-1+ patients and HIV-1 negative controls showed significantly higher HI titers against the three strains in controls. In addition, post vaccination protective HI titers (defined as equal to or higher than 1:40) against the strains A/H3N2 and B were observed in a lower proportion of HIV-1+ children than in controls, while a similar proportion of individuals from each group achieved protective HI titers against the A/H1N1 strain. The CD4+ T cell count, CD4/CD8 T cells ratio, and serum viral load were not affected by influenza virus vaccination when pre- vs post-vaccination values were compared. These findings suggest that despite the fact that HAART is efficient in controlling HIV-1 replication and in increasing CD4+ T cell count in HIV-1-infected children, restoration of immune competence and response to cognate antigens remain incomplete, indicating that additional therapeutic strategies are required to achieve a full reconstitution of immune functions.

The role of triple infection with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus (HIV) type-1 on CD4+ lymphocyte levels in the highly HIV infected population of North-Central Nigeria

We set out to determine the seroprevalence of hepatitis B and C among human immunodeficiency virus type-1 (HIV-1) infected individuals in North-Central Nigeria to define the influence of these infections on CD4+ lymphocytes cells among our patients as access to antiretroviral therapy improves across the Nigerian nation. The CD4+ values of 180 confirmed HIV-1 infected individuals were enumerated using a superior fluorescence-activated cell sorter system. These patients were tested for the presence of hepatitis B surface antigen and anti-hepatitis C virus (HCV) using third generation enzyme-linked immunosorbent assays. Fifty (27.8%) patients had active hepatitis B virus (HBV) infection while 33 (18.3%) tested positive for anti-HCV antibody. Of these infections, 110 (61.1%), 37 (20.6%), and 20 (11.1%) had HIV only, HBV/HIV-only, and HCV/HIV-only respectively. A HBV/HCV/HIV coinfection prevalence of 7.2% (13 patients) was recorded. Patients coinfected with HIV/HBV/HCV appeared to have lower CD4+ counts (mean = 107 cells/µl; AIDS defining) when compared to HBV/HIV-only (mean = 377 cells/µl), HCV/HIV-only (mean = 373 cells/µl) and patients with mono HIV infection (mean = 478 cells/µl). Coinfection with HBV or HCV is relatively common among HIV-infected patients in Nigeria and should be a big consideration in the initiation and choice of therapy.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.