Prevalence of oral hairy leukoplakia and epithelial infection by Epstein-Barr virus in pregnant women and diabetes mellitus patients: cytopathologic and molecular study

Oral hairy leukoplakia (OHL) is generally reported in patients with severe immunosuppression, except for a few cases in individuals with moderate degree of immunodeficiency. It is a white lesion that appears mainly in the lateral border of the tongue, caused by Epstein-Barr virus (EBV). The nuclear changes caused by EBV (Cowdry A inclusion, ground glass and nuclear beading), observed in cytopathology, are specific and enough for the definitive diagnosis of OHL, independent of the identification of the virus. Here we investigated the prevalence of OHL and the presence of EBV-DNA in the lateral borders of the tongue from 90 pregnant women, 90 diabetes mellitus (DM) patients, 30 healthy individuals (negative group) and 30 HIV+ with OHL (positive group). Smears were analyzed by cytopathology and polymerase chain reaction (PCR). A case of subclinical OHL and candidiasis was identificated in a DM patient by cytopathologic analysis. PCR results demonstrated EBV-DNA in 65% of the pregnant women, in 35% of DM patients, and in 20% of the healthy individuals. We concluded that DM patients can develop OHL with a low prevalence. Furthermore, the prevalence of the EBV in lateral border of the tongue is larger in pregnant women than in healthy individuals.

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.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Ultrastructural description of rabies virus infection in cultured sensory neurons

Primary cultures were made from adult mouse spinal ganglia for depicting an ultrastructural description of rabies virus (RABV) infection in adult mouse sensory neuron cultures; they were infected with rabies virus for 24, 36, and 48 h. The monolayers were processed for transmission electron microscopy and immunochemistry studies at the end of each period. As previously reported, sensory neurons showed great susceptibility to infection by RABV; however, in none of the periods evaluated were assembled virions observed in the cytoplasm or seen to be associated with the cytoplasmic membrane. Instead, fibril matrices of aggregated ribonucleoprotein were detected in the cytoplasm. When infected culture lysate were inoculated into normal animals via intra-cerebral route it was observed that these animals developed clinical symptoms characteristic of infection and transmission electron microscopy revealed assembled virions in the cerebral cortex and other areas of the brain. Sensory neurons infected in vitro by RABV produced a large amount of unassembled viral ribonucleoprotein. However, this intracellular material was able to produce infection and virions on being intra-cerebrally inoculated. It can thus be suggested that the lack of intracellular assembly in sensory neurons forms part of an efficient dissemination strategy.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

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.

Molecular evidence that human immunodeficiency virus type 1 dissemination in a small Brazilian city was already taking place in the early 1990s

We recently performed a molecular epidemiology survey of human immunodeficiency virus type 1 (HIV-1) infection in Miracema, a small city in Southeast Brazil, and found multiple monophyletic clusters, consistent with independent introductions and spread of different viral lineages in the city. Here we apply Bayesian coalescent-based methods to the two largest subtype B clusters and estimate that the most recent common ancestors that gave rise to these two transmission chains were in circulation around 1991-1992. The finding that HIV-1 spread in this Brazilian small city was already taking place at a time Aids was considered a problem restricted to large urban centers may have important public health implications.

Comparison of the epidemiology, profile of mutations, and clinical response to antiretrovirals among subtypes B and F of the human immunodeficiency virus type 1

The authors compared demographic aspects and profile of mutations in 80 patients with subtypes B and F of human immunodeficiency type 1 (HIV-1). Genotyping of the pol region of the reverse transcriptase was performed using the ViroSeqTM Genotyping System. A total of 61 (76.2%) patients had subtype B and 19 (23.8%) subtype F of the HIV-1. Subtype F tended to be more frequent in heterosexuals and women with a low educational level, but without statistical significance. The frequency of mutations related to nucleoside reverse transcriptase inhibitors and protease inhibitors (PI) was the same in the two subtypes, but mutations related to PI at the codons 63, 77, and 71 were more frequent in subtype B, while mutations at the codons 36 and 20 predominated in subtype F. Sixty-two of the 80 patients infected with subtypes B and F were submitted to antiretroviral therapy for an average of 18-22 months. Undetectable viral loads at the end of follow-up were similar in the two groups, representing 63.8% of subtype B and 73.3% of subtype F (p = 0.715). CD4 lymphocyte counts before and after treatment were similar in the two groups. This study, despite pointing to possible epidemiological and genetic differences among subtypes B and F of HIV-1, suggests that the use of highly active antiretroviral therapy is equally effective against these subtypes.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

Antiretroviral resistance in individuals presenting therapeutic failure and subtypes of the human immunodeficiency virus type 1 in the Northeast Region of Brazil

This study aimed to analyze human immunodeficiency virus (HIV) mutation profiles related to antiretroviral resistance following therapeutic failure, and the distribution of hiv subtypes in the Northeast Region of Brazil. A total of 576 blood samples from AIDS patients presenting therapeutic failure between 2002 and 2004 were analyzed. The genotyping kit viroSeq® was used to perform viral amplification in order to identify mutations related to hiv pol gene resistance. An index of 91.1% of the patients presented mutations for nucleoside reverse transcriptase inhibitors (nrti), 58.7% for non-nucleoside reverse transcriptase inhibitors (nnrti), and 94.8% for protease inhibitors (pi). The most prevalent mutations were 184V and 215E for nrti, 103N and 190A for nnrti. Most mutations associated with PIs were secondary, but significant frequencies were observed in codons 90 (25.2%), 82 (21.1%), and 30 (16.2%). The resistance index to one class of antiretrovirals was 14%, to two classes of antiretrovirals 61%, and to three classes 18.9%. Subtype B was the most prevalent (82.4%) followed by subtype F (11.8%). The prevalence of mutations related to nrti and nnrti was the same in the two subtypes, but codon analysis related to PI showed a higher frequency of mutations in codon 63 in subtype B and in codon 36 in subtype F. The present study showed that there was a high frequency of primary mutations, which offered resistance to nrti and nnrti. Monitoring patients with treatment failure is an important tool for aiding physicians in rescue therapy.

Autochthonous horizontal transmission of a CRF02_AG strain revealed by a human immunodeficiency virus type 1 diversity survey in a small city in inner state of Rio de Janeiro, Southeast Brazil

As part of an ongoing study on the features of AIDS spread towards small cities and rural areas, we present a molecular survey of human immunodeficiency virus type 1 (HIV-1) polymerase sequences recovered between 2004 and 2006 from 71 patients receiving care in the city of Saquarema, inner state of Rio de Janeiro. Phylogenetic reconstructions found the two prevalent lineages in the state (subtypes B [59 strains, 83.1%], F1 [6 strains; 8.4%], and BF1 recombinants [four strains; 5.6%]), as well as two (2.8%) CRF02_AG strains, which seems to be an emerging lineage in the capital. These CRF02_AG sequences were recovered from a married heterosexual couple who never traveled abroad, thus providing the first molecular evidence of autochthonous horizontal transmission of this lineage of major global importance. Also, three phylogenetic clusters of strains recovered from a total of 18.3% of the cohort were uncovered. Their close genetic relatedness suggests they were recovered from patients who probably took part in the same chain of viral spread. In conjunction with our previous surveys from inner Rio de Janeiro, these results suggest that although small cities harbor unique molecular features of HIV-1 infection, they also clearly reflect and may rapidly absorb the diversity recorded in large urban centers.

Anti-human immunodeficiency virus type 1 humoral immune response and highly active antiretroviral treatment

Highly active antiretroviral treatment (HAART) of human immunodeficiency type 1 (HIV-1) infection is very effective in controlling infection, but elimination of viral infection has not been achieved as yet, and upon treatment interruption an immediate rebound of viremia is observed. A combination of HAART with an immune stimulation might allow treatment interruption without this rebounding viremia, as the very low viremias observed with successful HAART may be insufficient to permit maintenance of a specific anti-HIV-1 immune response. The objective of this study was to compare the humoral immune response of individuals undergoing successful HAART (NF=no failure) with that of individuals with evidence of failure of therapy (FT) and to verify if the viremia peaks observed in individuals with therapy failure would act as a specific stimulus for the humoral anti-HIV-1 immune response. Antibodies binding to gp120 V3 genotype consensus peptides were more frequently observed for FT, mainly against peptides corresponding to sequences of genotypes prevalent in the Rio de Janeiro city area, B and F. HIV-1 neutralization of HIV-1 IIIB and of four primary isolates from Rio de Janeiro was less frequently observed for plasma from the NF than the FT group, but this difference was more expressive when plasma from individuals with detectable viremia were compared to that of individuals with undetectable viral loads in the year before sample collection. Although statistically significant differences were observed only in some specific comparisons, the study indicates that presence of detectable viremia may contribute to the maintenance of a specific anti-HIV-1 humoral immune response.

High proportion of hepatitis C virus genotypes 1 and 3 in a large cohort of patients from Southern Brazil

Hepatitis C virus (HCV) isolates have been divided into six genotypes (1 to 6). The duration of hepatitis C standard treatment is 48 weeks for patients infected with HCV genotype 1 vs 24 weeks for those infected with genotypes 2 and 3. A total of 1544 HCV isolates from chronic patients living in the southern Brazilian states of Rio Grande do Sul (RS, n = 627) and Santa Catarina (SC, n = 917) were genotyped by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. In RS, 338 (53.9%; 95% CI 50.0 - 57.8%), 34 (5.4%; 95% CI 3.8 - 7.4%) and, 255 (40.7%; 95% CI 36.9 - 44.6%) samples were from genotypes 1, 2, and 3, respectively. In SC, 468 (51%; 95% CI 47.8 - 54.2%), 26 (2.9%; 95% CI 1.9 - 4.1%) and, 423 (46.1%; 95% CI 42.9 - 49.3%) samples were from genotypes 1, 2, and 3, respectively. Genotyping results were confirmed by direct nucleotide sequencing of PCR products derived from 68 samples, without any discrepancy between PCR-RFLP and nucleotide sequencing methods. In conclusion, almost half of the hepatitis C patients from South of Brazil are infected by genotypes 2 and 3 and, these results have important consequential therapeutic implications as they can be treated for only 24 weeks, not 48.

The dynamics of dengue virus serotype 3 introduction and dispersion in the state of Bahia, Brazil

By 2002, dengue virus serotype 1 (DENV-1) and DENV-2 had circulated for more than a decade in Brazil. In 2002, the introduction of DENV-3 in the state of Bahia produced a massive epidemic and the first cases of dengue hemorrhagic fever. Based on the standardized frequency, timing and location of viral isolations by the state's Central Laboratory, DENV-3 probably entered Bahia through its capital, Salvador, and then rapidly disseminated to other cities, following the main roads. A linear regression model that included traffic flow, distance from the capital and DENV-1 circulation (r² = 0.24, p = 0.001) supported this hypothesis. This pattern was not seen for serotypes already in circulation and was not seen for DENV-3 in the following year. Human population density was another important factor in the intensity of viral circulation. Neither DENV-1 nor DENV-2 fit this model for 2001 or 2003. Since the vector has limited flight range and vector densities fail to correlate with intensity of viral circulation, this distribution represents the movement of infected people and to some extent mosquitoes. This pattern may mimic person-to-person spread of a new infection.