Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera hydroeletric plant, São Paulo, Brazil

A febre amarela (FA) é doença infecciosa aguda de origem viral transmitida por mosquitos. No ciclo silvestre, o vírus é mantido por meio da infecção de macacos e da transmissão transovariana nos vetores. A vigilância sobre populações de primatas não humanos torna-se necessária para detectar a circulação viral, quando ainda está restrito a epizootias, e para determinar sua presença em regiões indenes ou de transição para a doença. Padronizou-se a técnica ELISA (Enzyme Linked Immunosorbent Assay) para determinar a prevalência de anticorpos da classe IgG contra o vírus da FA em soros de bugios (Alouatta caraya) da região do reservatório da Usina Hidrelétrica de Porto Primavera, SP. Foram testados soros de 570 macacos sendo que nenhuma amostra mostrou-se reativa para a presença de anticorpos contra o vírus da FA. Os resultados são coerentes com a epidemiologia da FA na região. Mesmo sendo área de transição, não se conhece, até o momento, ocorrência de epizootia ou surto de FA em humanos e investigações entomológicas não apontaram a presença de vetores para esta arbovirose. A técnica mostrou-se sensível, rápida e útil à vigilância epidemiológica como instrumento de busca ativa permitindo desencadear ações preventivas, como vacinação, antes mesmo do surgimento de epizootias

Molecular basis for porcine parvovirus detection in dead fetuses

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; N = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (P < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (P < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background: Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

A Novel Hepatitis C Virus Genotyping Method Based on Liquid Microarray

The strategy used to treat HCV infection depends on the genotype involved. An accurate and reliable genotyping method is therefore of paramount importance. We describe here, for the first time, the use of a liquid microarray for HCV genotyping. This liquid microarray is based on the 5'UTR - the most highly conserved region of HCV - and the variable region NS5B sequence. The simultaneous genotyping of two regions can be used to confirm findings and should detect inter-genotypic recombination. Plasma samples from 78 patients infected with viruses with genotypes and subtypes determined in the Versant (TM) HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany) were tested with our new liquid microarray method. This method successfully determined the genotypes of 74 of the 78 samples previously genotyped in the Versant (TM) HCV Genotype Assay LiPA (74/78, 95%). The concordance between the two methods was 100% for genotype determination (74/74). At the subtype level, all 3a and 2b samples gave identical results with both methods (17/17 and 7/7, respectively). Two 2c samples were correctly identified by microarray, but could only be determined to the genotype level with the Versant (TM) HCV assay. Genotype ""1'' subtypes (1a and 1b) were correctly identified by the Versant (TM) HCV assay and the microarray in 68% and 40% of cases, respectively. No genotype discordance was found for any sample. HCV was successfully genotyped with both methods, and this is of prime importance for treatment planning. Liquid microarray assays may therefore be added to the list of methods suitable for HCV genotyping. It provides comparable results and may readily be adapted for the detection of other viruses frequently co-infecting HCV patients. Liquid array technology is thus a reliable and promising platform for HCV genotyping.

Detection of Human Bocavirus mRNA in Respiratory Secretions Correlates with High Viral Load and Concurrent Diarrhea

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (> 10(8) copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections.

Yellow Fever Virus in Haemagogus leucocelaenus and Aedes serratus Mosquitoes, Southern Brazil, 2008

Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil In October 2008 a yellow fever outbreak was reported there with nonhuman primate deaths and human cases This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths Mosquitoes were collected at ground level identified and processed for virus isolation and molecular analyses Eight YFV strains were isolated (7 from pools of Hg leucocelaenus mosquitoes and another from Aedes serratus mosquitoes) 6 were sequenced and they grouped in the YFV South American genotype I The results confirmed the role of Hg leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae serratus mosquitoes may have a potential role as a secondary vector

Varicella zoster virus in bell`s palsy: a prospective study

Although Bell`s palsy is the major cause of acute peripheral facial palsy, its pathogenesis remains unknown. Reactivation of the varicella zoster virus has been implicated as one of the main causes of Bell`s palsy, however, studies which investigate the varicella zoster virus reactivation in Bell`s palsy patients are mostly Japanese and, therefore, personal and geographic characteristics are quite different from our population. Aims: To determine varicella zoster virus frequency in saliva samples from patients with Bell`s palsy, using PCR. Material and Method: One hundred seventy one patients with acute peripheral facial palsy were prospectively enrolled in this study. One hundred twenty were clinically diagnosed with Bell`s palsy, within one week of onset of the disease and no previous anti-viral therapy. We had 20 healthy adults as controls. Three saliva samples were collected from patients and controls at initial examination and at one and two weeks later. The detection of the varicella zoster virus DNA was performed using PCR. Results: Varicella zoster virus was detected in two patients (1.7%). The virus was not identified in saliva samples from the controls. Conclusions: Varicella zoster virus was detected in 1.7% of saliva samples from patients with Bell`s palsy, using PCR.

Molecular evidence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections in HTLV seroindeterminate individuals from Sao Paulo, Brazil

Background: Using enzyme immunoassays and Western blot (Wb) tests, HTLV serodiagnosis yields indeterminate results in a significant number of cases. Objective: To determine the prevalence of HTLV infection among HTLV-seroindeterminate individuals. Study design: We studied peripheral blood mononuclear cells from 65 anti-HTLV Wb-seroindeterminate individuals by attempting to amplify proviral DNA sequences (tax and pot) to identify HTLV-I and HTLV-2 infections. Results: These 65 specimens exhibited predominantly (43%) anti-HTLV antibodies to gag-coded antigens in the absence of anti-p24 on Wb analysis. Tax proviral sequences were detected in 6 (9.2%) samples. According to restricted fragment polymorphism analysis (RFLP), we identified HTLV-1 proviral DNA in 4 samples. HTLV-2 in one and sequences from both in another. Nested PCR for the pot region was positive in 3 (4.6%) specimens, which were also positive for tax sequences. After hybridization HTLV-1 infection was confirmed in 2 samples (3.1%) and HTLV-2 in another (1.5%). Detection of a single HTLV DNA sequence may be due to infection by defective provirus, but its significance remains undefined. In this cohort, no Wb reactivity pattern was predictive of proviral detection. HTLV-I infection was demonstrated in an individual who had Wb reactivity to gag-coded antigens only. Conclusions: This emphasizes the importance of clinical and laboratory follow-up of HTLV-seroindeterminate individuals from endemic areas. (c) 2009 Elsevier B.V. All rights reserved.

Neither Molecular Diversity of the Envelope, Immunosuppression Status, Nor Proviral Load Causes Indeterminate HTLV Western Blot Profiles in Samples From Human T-Cell Lymphotropic Virus Type 2 (HTLV-2)-Infected Individuals

Although human T-cell lymphotropic virus type 2 (HTLV-2) is considered of low pathogenicity, serological diagnosis is important for counseling and monitoring. The confirmatory tests most used are Western blot (WB) and PCR. However, in high-risk populations, about 50% of the indeterminate WB were HTLV-2 positives by PCR. The insensitivity of the WB might be due to the use of recombinant proteins of strains that do not circulate in our country. Another possibility may be a high level of immunosuppression, which could lead to low production of virus, resulting in low stimulation of antibody. We found one mutation, proline to serine in the envelope region in the position 184, presented at least 1/3 of the samples, independent the indeterminate WB profile. In conclusion, we found no correlation of immune state, HTLV-2 proviral load, or env diversity in the K55 region and WB indeterminate results. We believe that the only WB kit available in the market is probably more accurate to detect HTLV-1 antibodies, and some improvement for HTLV-2 detection should be done in the future, especially among high-risk population. J. Med. Virol. 82:837-842,2010. (C) 2010 Wiley-Liss, Inc.

GB virus type C infection modulates T-cell activation independently of HIV-1 viral load

Background: Many clinical studies have suggested a beneficial effect of GB virus type C (GBV-C) on the course of HIV-1 infection, but the mechanisms involved in such amelioration are not clear. As recent evidence has implicated cellular activation in HIV-1 pathogenesis, we investigated the effect of GBV-C viremia on T-cell activation in early HIV-1 infection. Methods: Forty-eight recently infected HIV-1 patients (23 GBV-C viremic) were evaluated for T-cell counts, expanded immunophenotyping GBV-C RNA detection, and HIV-1 viral load. Nonparametric univariate and multivariate analyses were carried out to identify variables associated with cellular activation, including GBV-C status, HIV-1 viral load, T lymphocyte counts, and CD38 and chemokine (C-C motif) receptor 5 (CCR5) surface expression. Finding: We not only confirmed the positive correlation between HIV-1 viral load and the percentage of T cells positive for CD38(+)CD8(+) but also observed that GBV-C viremic patients had a lower percentage of T cells positive for CD38(+)CD4(+), CD38(+)CD8(+), CCR5(+)CD4(+), and CCR5(+)CD8(+) compared with HIV-1-infected patients who were not GBV-C viremic. In regression models, GBV-C RNA(+) status was associated with a reduction in the CD38 on CD4(+) or CD8(+) T cells and CCR5(+) on CD8(+) T cells, independent of the HIV-1 viral load or CD4(+) and CD8(+) T-cell counts. These results were also supported by the lower expression of CD69 and CD25 in GBV-C viremic patients. Interpretation: The association between GBV-C replication and lower T-cell activation may be a key mechanism involved in the protection conferred by this virus against HIV-1 disease progression to immunodeficiency in HIV-1-infected patients. (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Araraquara Virus Recombinant Nucleocapsid Protein

Laboratory diagnosis of hantavirus cardiopulmonary syndrome (HCPS) in Brazil has been performed mostly by a detection of IgM antibodies to recombinant antigen purified from Sin Nombre virus and Andes Virus (ANDV). Recently, a recombinant nucleocapsid (rN) protein of Argentina virus (ARAV), a Brazilian hantavirus, was Obtained in Escherichia coli. To evaluate ARAV rN as antigen for antibody detection, serum samples from 30 patients front Argentina seropositive for hantavirus were tested. All samples were positive for IgG and IgM by enzyme-linked immunosorbent assay (ELISA) using either ARAV rN or ANDV rN antigens. In Brazil, six of 00 serum samples from patients With suspected HCPS (10%) were positive for IgM by ELISA Using ARAV rN antigen and 7 were positive Using ANDV rN antigen. For results obtained with 90 serum samples analyzed by IgM ELISA with ANDV rN antigen, the sensitivity of the IgM ELISA using ARAV rN antigen was 97.2%,, the specificity was 100%, the positive predictive value was 100% and the negative predictive value was 98.1%. The results show that ARAV rN is a Suitable antigen for diagnosis Of hantavirus infection in Brazil and Argentina.

Haemophilus ducreyi detection by polymerase chain reaction in oesophageal lesions of HIV patients

HIV patients frequently have opportunistic oesophageal infections. We report Haemophilus ducreyi genetic material detected by polymerase chain reaction in biopsies of oesophageal lesions in three HIV-1-infected patients. This finding may be an indication of its aetiopathological role in oesophageal lesions of HIV patients.

Clinical Evaluation of the NS1 Antigen-Capture ELISA for Early Diagnosis of Dengue Virus Infection in Brazil

The fact that the diagnosis of infection with dengue virus is usually made by detecting IgM antibodies during the convalescent phase of the disease interferes with disease management and, consequently, with reducing mortality rates. This study evaluated the sensitivity and specificity of detection of NS1 in samples of patients suspected of acute dengue virus infection in Brazil. The results were used to institute treatment and the sensitivity and specificity of detection of NS1 were compared to the results of detection of IgM, virus isolation, and RT-PCR. Detection of NS1 yielded better results than RTPCR and virus isolation. When considering IgM detection and RT-PCR positive results as ""gold standards,"" the sensitivity and specificity of the NS1 assay were 95.9% and 81.1%, respectively. All patients enrolled in the study were treated promptly and had an uneventful course of the disease. The detection of NS1 provided better results than the diagnostic techniques used currently during the acute phase of disease (RT-PCR and virus isolation). Detection of NS1 is an important tool for the diagnosis of acute dengue infection, particularly in highly endemic areas, allowing for rapid treatment of patients and reduction of disease burden. J. Med. Virol. 82: 1400-1405, 2010. (C) 2010 Wiley-Liss, Inc.

Low Expression of Human Histocompatibility Soluble Leukocyte Antigen-G (HLA-G5) in Invasive Cervical Cancer With and Without Metastasis, Associated With Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class lb molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICC(W))) and absence [ICC without metastasis (ICC(WT))] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICC(WT) (n=52 patients) and ICC(W) (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies. (J Histochem Cytochem 58:405-411, 2010)

SURVEY OF FELINE LEUKEMIA VIRUS AND FELINE CORONAVIRUSES IN CAPTIVE NEOTROPICAL WILD FELIDS FROM SOUTHERN BRAZIL

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refugio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.