Camila Malta Romano

Molecular characterization of the Hepatitis B virus genotypes in Colombia: A Bayesian inference on the genotype F

Hepatitis B is a worldwide health problem affecting about 2 billion people and more than 350 million are chronic carriers of the virus. Nine HBV genotypes (A to I) have been described. The geographical distribution of HBV genotypes is not completely understood due to the limited number of samples from some parts of the world. One such example is Colombia, in which few studies have described the HBV genotypes. In this study, we characterized HBV genotypes in 143 HBsAg-positive volunteer blood donors from Colombia. A fragment of 1306 bp partially comprising HBsAg and the DNA polymerase coding regions (S/POL) was amplified and sequenced. Bayesian phylogenetic analyses were conducted using the Markov Chain Monte Carlo (MCMC) approach to obtain the maximum clade credibility (MCC) tree using BEAST v.1.5.3. Of all samples, 68 were positive and 52 were successfully sequenced. Genotype F was the most prevalent in this population (77%) - subgenotypes F3 (75%) and F1b (2%). Genotype G (7.7%) and subgenotype A2 (15.3%) were also found. Genotype G sequence analysis suggests distinct introductions of this genotype in the country. Furthermore, we estimated the time of the most recent common ancestor (TMRCA) for each HBV/F subgenotype and also for Colombian F3 sequences using two different datasets: (i) 77 sequences comprising 1306 bp of S/POL region and (ii) 283 sequences comprising 681 bp of S/POL region. We also used two other previously estimated evolutionary rates: (i) 2.60 × 10(-4)s/s/y and (ii) 1.5 × 10(-5)s/s/y. Here we report the HBV genotypes circulating in Colombia and estimated the TMRCA for the four different subgenotypes of genotype F.

Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic

Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the re-introduction of DENV2 in the coast of São Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the São Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of São Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas.

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in São Paulo state, Brazil. We show that different viral genotypes entered São Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in São Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.

Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of São Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Molecular epidemiology and genetic diversity of hepatitis B virus genotype E in an isolated Afro-Colombian community.

Hepatitis B virus (HBV) infection is a significant public health concern with 350 million chronic carriers worldwide. Eight HBV genotypes (A-H) have been described so far. Genotype E (HBV/E) is widely distributed in West Africa and has rarely been found in other continents, except for a few cases in individuals with an African background. In this study, we characterized HBV genotypes in Quibdó, Colombia, by partial S/P gene sequencing, and found, for the first time, HBV/E circulating in nine Afro-Colombian patients who had no recent contact with Africa. The presence of HBV/E in this community as a monophyletic group suggests that it was a result of a recent introduction by some Afro-descendent contact or, alternatively, that the virus came with slaves brought to Colombia. By using sequences with sampling dates, we estimated the substitution rate to be about 3.2 x 10(-4) substitutions per site per year, which resulted in a time to the most recent common ancestor (TMRCA) of 29 years. In parallel, we also estimated the TMRCA for HBV/E by using two previously estimated substitution rates (7.7 x 10(-4) and 1.5 x 10(-5) substitutions per site per year). The TMRCA was around 35 years under the higher rate and 1500 years under the slower rate. In sum, this work reports for the first time the presence of an exclusively African HBV genotype circulating in South America. We also discuss the time of the entry of this virus into America based on different substitution rates estimated for HBV.

Molecular characterization of human erythrovirus B19 strains obtained from patients with several clinical presentations in the Amazon region of Brazil

BACKGROUND Human erythrovirus B19, endemic in the Amazon region since 1990, is associated with a wide spectrum of clinical presentations. OBJECTIVES To assess the prevalence of erythrovirus B19 infection and the relative frequency of erythrovirus B19 genotypes in patients in the Amazon region with various clinical presentations. STUDY DESIGN A total of 487 clinical samples obtained from patients with symptoms suggestive of erythrovirus infection were tested using specific IgM and IgG antibody assays (ELISA) and PCR for viral DNA detection. Partial VP1 and VP2 regions were sequenced and genotyped by phylogenetic reconstruction. RESULTS B19 DNA was detected in 117 (24%) of 487 samples. Of these, 106 (91%) isolates were genotype 1 and 11 (9%) were genotype 3. No genotype 2 was found. Genotype 1 had three clusters (A1, A2 and B) and all genotype 3 sequences were subtype 3b. All patients with hematological disorders within cluster B of genotype 1 were infected by the same B19 lineage, suggesting that this lineage of B19 may have been transmitted via transfusion of blood products. CONCLUSION We reported two genotypes, 1 and 3b, with three genotype 1 clusters co-circulating in the Amazon region during the past 10 years.

Tempo and mode of ERV-K evolution in human and chimpanzee genomes

Summary.Several families of endogenous retrovirus (ERV) exist in copious numbers in the genomes of primate species. Therefore, we undertook a systematic search for endogenous retrovirus sequences from the ERV-K family, comparing across both human (Homo sapiens) and chimpanzee (Pan troglodytes) genomes. Using conserved motifs of the ERV-K as query we identified and characterized 76 complete ERV-K elements, 54 in human (HERV-K), 34 of which were described previously, and 21 in the chimpanzee (CERV-K). Phylogenetic analysis using coding regions and LTRs showed the existence of two main branches. Group I was the most heterogeneous and had an average integration time of 18.3 MYBP (million years before present), using rates ranging from 1.5 to 4.0 × 10−9 s/s/y (substitution per site per year). Group O/N integrated around 19.4 MYBP and nested Group N integrated about 14 MYBP. We found evidence for strong positive selection on the gag, pol and env coding regions and for A/T hypermutation. Our data suggest that the endogenous elements were possibly involved in chromosomal rearrangements and retained a great deal of information from their active stage, most likely as a consequence of host interactions. This study also contributes to the annotation effort of both human and chimpanzee genomes.

Persistence of Zika Virus in Breast Milk after Infection in Late Stage of Pregnancy

We detected Zika virus in breast milk of a woman in Brazil infected with the virus during the 36th week of pregnancy. Virus was detected 33 days after onset of signs and symptoms and 9 days after delivery. No abnormalities were found during fetal assessment or after birth of the infant.

Low Sensitivity of NS1 Protein Tests Evidenced during a Dengue Type 2 Virus Outbreak in Santos, Brazil, in 2010

ABSTRACT In 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.

Serum angiopoietin-2 and soluble VEGF receptor 2 are surrogate markers for plasma leakage in patients with acute dengue virus infection.

BACKGROUND Endothelial cell dysfunction is believed to play an important role in the pathogenesis of plasma leakage in patients with acute dengue virus (DENV) infection. Several factors, produced by activated endothelial cells, have been associated with plasma leakage or severe disease in patients with infectious diseases. OBJECTIVES The aim of this study was to investigate which of these markers could serve as a surrogate marker for the occurrence of plasma leakage in patients with acute DENV infection. STUDY DESIGN A case-control study was performed in patients with acute DENV infection in Santos, Brazil. Plasma leakage was detected with X-ray and/or ultrasound examination at admission. Serum levels of soluble endoglin, endothelin-1, angiopoietin-2, VEGF, soluble VEGFR-2, MMP-2, MMP-9, TIMP-1 and TIMP-2 were determined using commercially available ELISAs. RESULTS Increased levels of angiopoietin-2, endothelin-1 and MMP-2 and decreased levels of soluble VEGFR-2 were significantly associated with the occurrence of plasma leakage. An unsupervised cluster analysis confirmed that angiopoietin-2 and soluble VEGFR-2 were strongly associated with clinical apparent vascular leakage. CONCLUSION Angiopoietin-2 and soluble VEGFR-2 can serve as surrogate markers for the occurrence of plasma leakage in patients with acute DENV infection.