Viral etiology among the elderly presenting acute respiratory infection during the influenza season

INTRODUCTION: Acute respiratory tract infections are the most common illness in all individuals. Rhinoviruses have been reported as the etiology of more than 50% of respiratory tract infections worldwide. The study prospectively evaluated 47 elderly individuals from a group of 384 randomly assigned for acute respiratory viral infections (cold or flu) and assessed the occurrence of human rhinovirus (HRV), influenza A and B, respiratory syncytial virus and metapneumovirus (hMPV) in Botucatu, State of São Paulo, Brazil. METHODS: Forty-nine nasal swabs collected from 47 elderly individuals following inclusion visits from 2002 to 2003 were tested by GenScan RT-PCR. HRV-positive samples were sequenced for phylogenetic analysis. RESULTS: No sample was positive for influenza A/B or RSV. HRV was detected in 28.6% (14/47) and hMPV in 2% (1/47). Of 14 positive samples, 9 isolates were successfully sequenced, showing the follow group distribution: 6 group A, 1 group B and 2 group C HRVs. CONCLUSIONS: The high incidence of HRV during the months of the influenza season requires further study regarding HRV infection impact on respiratory complications among this population. Infection caused by HRV is very frequent and may contribute to increasing the already high demand for healthcare during the influenza season.

Clinical and epidemiological profile of blood donors with positive serology for viral hepatitis in southern Brazil

INTRODUCTION: Positive serological tests for hepatitis viruses B and C at blood banks are an important reason for blood deferral. Additionally, high residual risk for transfusing hepatitis-contaminated blood has been estimated in southern Brazil. This study aimed to identify risk factors for positive serological tests for viral hepatitis (VH) in blood donors (BD). METHODS: A case-control study included consecutive BD with positive serology for VH, between 2008 and 2009. Cases and controls (BD with negative serology for VH) were paired 1:1 by sex and donation date. Assessment of clinical and epidemiological characteristics related to viral hepatitis was conducted. RESULTS: Among 1,282 blood donors (641 cases and 641 controls), those with positive serology for viral hepatitis had higher mean age (p<0.001); higher proportion of replacement donation (p<0.001); first donation (p<0.001); and interviewer deferment (p=0.037), compared to controls. Furthermore, donors with positive tests were less regular donors (p<0.001), had less previous history of rejection (p=0.003) and showed lower hematocrit median before donation (p=0.019). Multivariate analysis demonstrated that age (OR=1.056, 95%CI 1.042-1.069, p<0.001), replacement donation (OR=1.545, 95%CI 1.171-2.038, p=0.002) and first donation (OR=9.931, 95%CI 7.486-13.173, p<0.001) were independently associated with positivity of serological tests for viral hepatitis. CONCLUSIONS: Specific characteristics of blood donors were associated with positive serology for viral hepatitis. These peculiarities should be taken into account when assessing candidates for blood donation.

Improving downstream processing for viral vectors and viral vaccines

Viral vectors are playing an increasingly important role in the vaccine and gene therapy elds. The broad spectrum of potential applications, together with expanding medical markets, drives the e orts to improve the production processes for viral vaccines and viral vectors. Developing countries, in particular, are becoming the main vaccine market. It is thus critical to decrease the cost per dose, which is only achievable by improving the production process. In particular advances in the upstream processing have substantially increased bioreactor yields, shifting the bioprocess bottlenecks towards the downstream processing. The work presented in this thesis aimed to develop new processes for adenoviruses puri cation. The use of state-of-the-art technology combined with innovative continuous processes contributed to build robust and cost-e ective strategies for puri cation of complex biopharmaceuticals.(...)

Quantification of HIV-1 viral RNA in the blood in needles used for venous puncture in HIV-infected individuals

INTRODUCTION: Occupational HIV infection among healthcare workers is an important issue in exposures involving blood and body fluids. There are few data in the literature regarding the potential and the duration of infectivity of HIV type 1 (HIV-1) in contaminated material under adverse conditions. METHODS: We quantified HIV-1 viral RNA in 25×8mm calibre hollow-bore needles, after punctures, in 25 HIV-1-infected patients selected during the sample collection. All of the patients selected were between the ages of 18 and 55. Five samples were collected from 16 patients: one sample for the immediate quantification of HIV-1 RNA in the plasma and blood samples from the interior of 4 needles to be analyzed at 0h, 6h, 24h, and 72h after collection. In nine patients, another test was carried out in the blood from one additional needle, in which HIV-1 RNA was assessed 168h after blood collection. The method used to assess HIV-1 RNA was nucleic acid sequence-based amplification. RESULTS: Up to 7 days after collection, HIV-1 RNA was detected in all of the needles. The viral RNA remained stable up to 168h, and there were no statistically significant differences among the needle samples. CONCLUSIONS: Although the infectivity of the viral material in the needles is unknown, the data indicate the need to re-evaluate the practices in cases of occupational accidents in which the source is not identified.

Epidemiology of the viral hepatitis B and C in female prisoners of Metropolitan Regional Prison Complex in the State of Goiás, Central Brazil

INTRODUCTION: Little information regarding hepatitis B virus (HBV) and hepatitis C virus (HCV) infections among Brazilian female prisoners exists. This study investigated the prevalence and risk factors associated with HBV and HCV infections and identified viral genotypes among female prisoners in Goiás, Central Brazil. METHODS: Women incarcerated in the largest prison in the State of Goiás were invited to participate in the study. All female prisoners were interviewed and tested for the detection of hepatitis B surface antigen (HBsAg), antibodies against HBsAg (anti-HBs), against hepatitis B core antigen (anti-HBc), and antibody against HCV (anti-HCV) by ELISA. HBsAg and anti-HCV positive samples were tested for HBV DNA and HCV RNA and genotyped, respectively. RESULTS: Participants (n=148; 98.6%) completed the study with an overall HBV prevalence of 18.9%. Age >30 years, a low education level, sex with a sexually transmitted diseases carrier, and a male sexual partner serving in the same penitentiary were associated with HBV infections. Only 24% of the women were anti-HBs positive suggesting previous HBV vaccination. Nine female prisoners (6.1%) were anti-HCV positive. Age >40 years, injecting drug use and length of incarceration were statistically associated with anti-HCV antibodies. Five samples were HCV RNA positive and classified as genotypes 1 (subtypes 1a; n=3 and 1b; n=1) and 3 (subtype 3a; n=1). The HBsAg-reactive sample was HBV DNA positive and genotype A. CONCLUSIONS: These findings highlight the necessity of public policies to control hepatitis B and C infections and emphasize the importance of hepatitis B vaccination in prison environments.

Positive serology for viral hepatitis and donor self-exclusion in Southern Brazil

Introduction Despite the great advances in serological testing for transfusion-transmitted infections, the selection of blood donors by blood bank operators remains the only way to avoid transmission within the testing window period. Part of this selection is the self-exclusion form, on which the donors can exclude their blood from donation without any explanation. This study assessed the clinical and epidemiological characteristics related to positivity for viral hepatitis and to the use of the confidential self-exclusion (CSE) form. Methods This transversal study analyzed the data collected from blood donors' files in a hospital in Southern Brazil. Univariate and multivariate analyses identified the clinical and epidemiological variables related to positive serologies of viral hepatitis and to whether the donor was self-excluded. Results Of the 3,180 donors included in this study, 0.1% tested positive for HBsAg, 2.1% for anti-HBc, and 0.9% for anti-HCV. When the 93 donors with positive serologies for viral hepatitis were compared with those who were negative, a greater proportion of the positive serology group was found to have had a history of blood transfusions (OR=4.908; 95%CI=1.628 - 14.799; p<0.01), had repeatedly donated (OR=2.147; 95%CI=1.236 - 3.729; p<0.01), and used the CSE form for self-exclusion (OR=7.139; 95%CI=2.045 - 24.923; p<0.01). No variables were independently associated with self-exclusion. Conclusions A history of blood transfusion, repeated donations, and self-exclusion are factors that should be considered during viral hepatitis screenings in blood banks.

Association between histological findings, aminotransferase levels and viral genotype in chronic hepatitis C infection

Introduction: The genomic heterogeneity of hepatitis C virus (HCV) influences liver disorders. This study aimed to determine the prevalence of HCV genotypes and to investigate the influence of these genotypes on disease progression. Methods: Blood samples and liver biopsies were collected from HCV-seropositive patients for serological analysis, biochemical marker measurements, HCV genotyping and histopathological evaluation. Results: Hepatitis C virus-ribonucleic acid (HCV-RNA) was detected in 107 patients (90.6% with genotype 1 and 9.4% with genotype 3). Patients infected with genotype 1 exhibited higher mean necroinflammatory activity and fibrosis. Conclusions: HCV genotype 1 was the most prevalent and was associated with greater liver dysfunction.

Viral epidemiology of respiratory infections among children at a tertiary hospital in Southern Brazil

Introduction This study reports the pediatric epidemiology of respiratory syncytial virus (RSV), influenza (IF), parainfluenza (PIV), and adenovirus (ADV) at Hospital de Clínicas de Porto Alegre. Methods Cases of infection, hospitalizations in intensive care units (ICUs), nosocomial infections, and lethality rates were collected from 2007 to 2010. Results RSV accounted for most nosocomial infections. Intensive care units admission rates for ADV and RSV infections were highest in 2007 and 2010. During 2008-2009, H1N1 and ADV had the highest ICU admission rates. ADV had the highest fatality rate during 2007-2009. Conclusions Each virus exhibited distinct behavior, causing hospitalization, outbreaks, or lethality.

Evolutionary dynamics of Chlamydia trachomatis genome and identification of molecular patterns of hypothetical protein coding genes

The obligate intracellular bacterium Chlamydia trachomatis is a human pathogen of major public health significance. Strains can be classified into 15 main serovars (A to L3) that preferentially cause ocular infections (A-C), genital infections (D-K) or lymphogranuloma venereum (LGV) (L1-L3), but the molecular basis behind their distinct tropism, ecological success and pathogenicity is not welldefined. Most chlamydial research demands culture in eukaryotic cell lines, but it is not known if stains become laboratory adapted. By essentially using genomics and transcriptomics, we aimed to investigate the evolutionary patterns underlying the adaptation of C. trachomatis to the different human tissues, given emphasis to the identification of molecular patterns of genes encoding hypothetical proteins, and to understand the adaptive process behind the C. trachomatis in vivo to in vitro transition. Our results highlight a positive selection-driven evolution of C. trachomatis towards nichespecific adaptation, essentially targeting host-interacting proteins, namely effectors and inclusion membrane proteins, where some of them also displayed niche-specific expression patterns. We also identified potential "ocular-specific" pseudogenes, and pointed out the major gene targets of adaptive mutations associated with LGV infections. We further observed that the in vivo-derived genetic makeup of C. trachomatis is not significantly compromised by its long-term laboratory propagation. In opposition, its introduction in vitro has the potential to affect the phenotype, likely yielding virulence attenuation. In fact, we observed a "genital-specific" rampant inactivation of the virulence gene CT135, which may impact the interpretation of data derived from studies requiring culture. Globally, the findings presented in this Ph.D. thesis contribute for the understanding of C.trachomatis adaptive evolution and provides new insights into the biological role of C. trachomatishypothetical proteins. They also launch research questions for future functional studies aiming toclarify the determinants of tissue tropism, virulence or pathogenic dissimilarities among C. trachomatisstrains.

Genome-scale metabolic network reconstruction of Polaromonas sp. strain JS666: analysis of cDCE degradation rates and design of experiments for bioremediation improvement

Release of chloroethene compounds into the environment often results in groundwater contamination, which puts people at risk of exposure by drinking contaminated water. cDCE (cis-1,2-dichloroethene) accumulation on subsurface environments is a common environmental problem due to stagnation and partial degradation of other precursor chloroethene species. Polaromonas sp. strain JS666 apparently requires no exotic growth factors to be used as a bioaugmentation agent for aerobic cDCE degradation. Although being the only suitable microorganism found capable of such, further studies are needed for improving the intrinsic bioremediation rates and fully comprehend the metabolic processes involved. In order to do so, a metabolic model, iJS666, was reconstructed from genome annotation and available bibliographic data. FVA (Flux Variability Analysis) and FBA (Flux Balance Analysis) techniques were used to satisfactory validate the predictive capabilities of the iJS666 model. The iJS666 model was able to predict biomass growth for different previously tested conditions, allowed to design key experiments which should be done for further model improvement and, also, produced viable predictions for the use of biostimulant metabolites in the cDCE biodegradation.

Evaluation of arboviruses of public health interest in free-living non-human primates (Alouatta spp., Callithrix spp., Sapajus spp.) in Brazil

INTRODUCTION: The aim of the present study was to evaluate the presence of arboviruses from the Flavivirus genus in asymptomatic free-living non-human primates (NHPs) living in close contact with humans and vectors in the States of Paraná and Mato Grosso do Sul, Brazil. METHODS: NHP sera samples (total n = 80, Alouatta spp. n = 07, Callithrix spp. n = 29 and Sapajus spp. n = 44) were screened for the presence of viral genomes using reverse transcription polymerase chain reaction and 10% polyacrylamide gel electrophoresis techniques. RESULTS: All of the samples were negative for the Flavivirus genome following the 10% polyacrylamide gel electrophoresis analysis. CONCLUSIONS: These negative results indicate that the analyzed animals were not infected with arboviruses from the Flavivirus genus and did not represent a risk for viral transmission through vectors during the period in which the samples were collected.

Clinical and epidemiological profile of female blood donors with positive serology for viral hepatitis B

ABSTRACTINTRODUCTION:Since women are frequently the minority among blood donors worldwide, studies evaluating this population usually reflect male features. We assessed the features of female blood donors with positive serology for HBV and compared them with those of men.METHODS The study comprised consecutive blood donors referred to a specialized liver disease center to be evaluated due to HBsAg- and/or anti-HBc-positive tests.RESULTS: The study encompassed 1,273 individuals, 219 (17.2%) of whom were referred due to positive HBsAg test and 1,054 (82.8%) due to reactive anti-HBc test. Subjects' mean age was 36.8±10.9 years, and 28.7% were women. Female blood donors referred for positive HBsAg screening tests demonstrated higher prevalence of healthcare workers (9.3% vs 2.5%) and lower prevalence of sexual risk behaviors (15.1% vs 41.1%) and alcohol abuse (1.9% vs 19.8%) compared to men. Women had lower ALT (0.6 vs 0.8×ULN), AST (0.6 vs 0.8×ULN), direct bilirubin (0.2 vs 0.3mg/dL), and alkaline phosphatase (0.5 vs 0.6×ULN) levels and higher platelet count (223,380±50,293 vs 195,020±53,060/mm3). Women also had a higher prevalence of false-positive results (29.6% vs 17.0%). No differences were observed with respect to liver biopsies. Female blood donors referenced for reactive anti-HBc screening tests presented similar clinical, epidemiological, and biochemical characteristics to those reported for positive HBsAg screening tests and similarly had a higher prevalence of false-reactive results.CONCLUSIONS: Compared to men, female blood donors with positive HBsAg and/or anti-HBc screening tests demonstrated higher prevalence of professional risk and false-positive results and reduced alteration of liver chemistry.

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

Engineering modular viral scaffolds for targeted bacterial population editing

Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to precisely manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision this approach accelerating phage biology studies and enabling new technologies for bacterial population editing.