Zika virus infection among symptomatic patients from two healthcare centers in Sao Paulo State, Brazil: prevalence, clinical characteristics, viral detection in body fluids and serodynamics

Zika virus (ZIKV) clinical presentation and frequency/duration of shedding need further clarification. Symptomatic ZIKV-infected individuals identified in two hospitals in Sao Paulo State, Brazil, were investigated regarding clinical characteristics, shedding in body fluids, and serodynamics. Ninety-four of 235 symptomatic patients (Site A: 58%; Site B: 16%) had Real-Time PCR-confirmed ZIKV infection; fever, headache and gastrointestinal symptoms were less frequent, and rash was more frequent compared to ZIKV-negative patients. Real-Time PCR in serum had worse performance compared to plasma, while urine had the highest sensitivity. Shedding in genital fluids and saliva was rare. IgM positivity was the highest <14 days after the symptoms onset (86%), decreasing >28 days (24%); IgG positivity increased >14 days (96%) remaining positive in 94% of patients >28 days. ZIKV prevalence varied importantly in two neighboring cities during the same transmission season. Urine Real-Time PCR can improve diagnostic sensitivity; serum testing is less useful. Accurate serological tests are needed to improve diagnosis and surveillance

Zika virus infection among symptomatic patients from two healthcare centers in Sao Paulo State, Brazil: prevalence, clinical characteristics, viral detection in body fluids and serodynamics.

Zika virus (ZIKV) clinical presentation and frequency/duration of shedding need further clarification. Symptomatic ZIKV-infected individuals identified in two hospitals in Sao Paulo State, Brazil, were investigated regarding clinical characteristics, shedding in body fluids, and serodynamics. Ninety-four of 235 symptomatic patients (Site A: 58%; Site B: 16%) had Real-Time PCR-confirmed ZIKV infection; fever, headache and gastrointestinal symptoms were less frequent, and rash was more frequent compared to ZIKV-negative patients. Real-Time PCR in serum had worse performance compared to plasma, while urine had the highest sensitivity. Shedding in genital fluids and saliva was rare. IgM positivity was the highest 28 days (24%); IgG positivity increased >14 days (96%) remaining positive in 94% of patients >28 days. ZIKV prevalence varied importantly in two neighboring cities during the same transmission season. Urine Real-Time PCR can improve diagnostic sensitivity; serum testing is less useful. Accurate serological tests are needed to improve diagnosis and surveillance

Design and application of a microarray for fluorescence and and Surface Plasmon Resonance imaging analysis of peptide-antibody interactions.

International audienceIn the past few years, protein and peptide microarrays have shown a great potential in fundamental research, medical diagnostics and drug discovery. We have developed a new method based on covalent immobilization of peptides on a microarray gold surface for detecting antibodies by either fluorescence or surface plasmon resonance imaging (SPRi). The fluorescence-based revelation approach allows for an indirect analysis of protein-antibody binding via labeled molecules, whereas the SPRi-based assay is a real-time and label-free detection system suitable for the direct analysis of biological interactions. We illustrate our peptide chip's efficiency in specifically binding serum antibodies and compare the fluorescence and SPRi approaches to detect peptide-antibody interactions. Moreover, we used a monoclonal antibody against the hepatitis C virus nucleocapsid protein to provide evidence that our system is a powerful tool to identify which epitope is recognized by a given antibody. This novel technology therefore provides a promising tool for both antibody screening and epitope mapping

Role of the Hepatitis C Virus Core+1 Open Reading Frame and Core cis-Acting RNA Elements in Viral RNA Translation and Replication▿ †

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo

Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules

The Relationship between DUGBE Virus Infection and Autophagy in Epithelial Cells

International audienceDugbe orthonairovirus (DUGV) is a tick-borne arbovirus within the order Bunyavirales. Although displaying mild pathogenic potential, DUGV is genetically related to the Crimean–Congo hemorrhagic fever virus (CCHFV), another orthonairovirus that causes severe liver dysfunction and hemorrhagic fever with a high mortality rate in humans. As we previously observed that CCHFV infection could massively recruit and lipidate MAP1LC3 (LC3), a core factor involved in the autophagic degradation of cytosolic components, we asked whether DUGV infection also substantially impacts the autophagy machinery in epithelial cells. We observed that DUGV infection does impose LC3 lipidation in cultured hepatocytes. DUGV infection also caused an upregulation of the MAP1LC3 and SQSTM1/p62 transcript levels, which were, however, more moderate than those seen during CCHFV infection. In contrast, unlike during CCHFV infection, the modulation of core autophagy factors could influence both LC3 lipidation and viral particle production: the silencing of ATG5 and/or ATG7 diminished the induction of LC3 lipidation and slightly upregulated the level of infectious DUGV particle production. Overall, the results are compatible with the notion that in epithelial cells infected with DUGV in vitro, the autophagy machinery may be recruited to exert a certain level of restriction on viral replication. Thus, the relationship between DUGV infection and autophagy in epithelial cells appears to present both similarities and distinctions with that seen during CCHFV infection

RSV Infection in Human Macrophages Promotes CXCL10/IP-10 Expression during Bacterial Co-Infection

Respiratory syncytial virus (RSV), a major etiologic agent of acute lower respiratory infection constitutes the most important cause of death in young children worldwide. Viral/bacterial mixed infections are related to severity of respiratory inflammatory diseases, but the underlying mechanisms remain poorly understood. We have previously investigated the intracellular mechanisms that mediate the immune response in the context of influenza virus/Streptococcus pneumoniae (Sp) co-infection using a model of human monocyte-derived macrophages (MDMs). Here, we set up and characterized a similar model of MDMs to investigate different scenarios of RSV infection and co-infection with Sp. Our results suggest that Sp contributes to a faster and possibly higher level of CXCL10/IP-10 expression induced by RSV infection in human MDMs

Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo.: HCV protein expression in intestine

International audiencePrevious work on hepatitis C virus (HCV) led to the discovery of a new form of virus particle associating virus and lipoprotein elements. These hybrid particles (LVP for lipo-viro-particles) are enriched in triglycerides and contain at least apolipoprotein B (apoB), HCV RNA and core protein. These findings suggest that LVP synthesis could occur in liver and intestine, the two main organs specialized in the production of apoB-containing lipoprotein. To identify the site of LVP production, the genetic diversity and phylogenetic relationship of HCV quasispecies from purified LVP, whole serum and liver biopsies from chronically infected patients were studied. HCV quasispecies from LVP and liver differed significantly, suggesting that LVP were not predominantly synthesized in the liver but might also originate in the intestine. The authors therefore searched for the presence of HCV in the small intestine. Paraffin-embedded intestinal biopsies from 10 chronically HCV-infected patients and from 12 HCV RNA-negative controls (10 anti-HCV antibody-negative and two anti-HCV antibody-positive patients) were tested for HCV protein expression. HCV NS3 and NS5A proteins were stained in small intestine epithelial cells in four of the 10 chronically infected patients, and not in controls. Cells expressing HCV proteins were apoB-producing enterocytes but not mucus-secreting cells. These data indicate that the small intestine can be infected by HCV, and identify this organ as a potential reservoir and replication site. This further emphasizes the interaction between lipoprotein metabolism and HCV, and offers new insights into hepatitis C infection and pathophysiology

False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV

Chronic hepatitis B liver disease in patients living in the Amazon region: S gene mutations and genotypes characterization

Submitted by EMERSON LEAL ([email protected]) on 2016-06-20T18:39:08Z No. of bitstreams: 1 Chronic hepatitis B liver.pdf: 233232 bytes, checksum: 9a815d9a6920f8d928f82785cc1aba7f (MD5)Approved for entry into archive by EMERSON LEAL ([email protected]) on 2016-07-14T15:31:45Z (GMT) No. of bitstreams: 1 Chronic hepatitis B liver.pdf: 233232 bytes, checksum: 9a815d9a6920f8d928f82785cc1aba7f (MD5)Made available in DSpace on 2016-07-14T15:31:45Z (GMT). No. of bitstreams: 1 Chronic hepatitis B liver.pdf: 233232 bytes, checksum: 9a815d9a6920f8d928f82785cc1aba7f (MD5) Previous issue date: 2013Made available in DSpace on 2016-07-15T18:40:32Z (GMT). No. of bitstreams: 3 Chronic hepatitis B liver.pdf.txt: 25259 bytes, checksum: b784c3498783414824a7e653920c297d (MD5) Chronic hepatitis B liver.pdf: 233232 bytes, checksum: 9a815d9a6920f8d928f82785cc1aba7f (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Research Center for Tropical Medicine. Tropical Pathology Research Institute-CEPEM. Porto Velho, RO, Brasil.Fondation Mérieux. Emerging Pathogens Laboratory. IFR 128 BioSciences. Gerland, Lyon, France.Fundação Oswaldo Cruz. Research Center for Tropical Medicine. Tropical Pathology Research Institute-CEPEM. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Research Center for Tropical Medicine. Tropical Pathology Research Institute-CEPEM. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Research Center for Tropical Medicine. Tropical Pathology Research Institute-CEPEM. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Research Center for Tropical Medicine. Tropical Pathology Research Institute-CEPEM. Porto Velho, RO, Brasil.Fondation Mérieux. Emerging Pathogens Laboratory. IFR 128 BioSciences. Gerland, Lyon, France.Fondation Mérieux. Emerging Pathogens Laboratory. IFR 128 BioSciences. Gerland, Lyon, France.Fundação Oswaldo Cruz. Research Center for Tropical Medicine. Tropical Pathology Research Institute-CEPEM. Porto Velho, RO, Brasil.The Amazon region is considered to be a high endemic area for Hepatitis B Virus (HBV) infections, Rondônia state having the highest prevalence. The aim of this study was to identify molecular genotypes and mutations in the S gene region of HBV viral genomes from 20 patients using a DNA microarray. Results: Serological tests showed that 88% of patients were HBeAg negative, 82% had anti-HBe antibodies and 33% were co-infected with Hepatitis Delta Virus. Sixteen percent of the patients were considered cirrhotic, and 11% have been transfused. The microarray technique identified the genotypes A (4 patients), D (7 patients) and F (7 patients) in 18 samples. Mutations were detected in all 3 genotypes and, overall, A159G, which has been associated with a reduced antigenicity of the virus, was detected most frequently. In genotype A, G119E was the most frequently detected mutation followed by mutations A159G, F134Y, W172C, Y161F and T143S. A159G was detected in all genotype D and F samples followed by mutations T143S, Y161F, N131T, T114S and G119E in genotype D and mutations T143S, Y161F, N131T, T114S and G119E in genotype F. Conclusion: The analysis of mutations repartition among genotypes suggests that some of them are preferentially or exclusively associated with genotype A, D or F. This type of tool is adapted for clinical and therapy monitoring of patient as well as for molecular epidemiology research on HBV