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Vaccination and the emergence of SARS-CoV-2 variants mark the second year of the pandemic. Variants have amino acid mutations at the spike region, a viral protein central in the understanding of COVID-19 pathogenesis and vaccine response. Variants may dominate local epidemics, as Gamma (P.1) in Brazil, emerging in 2020 and prevailing until mid-2021. Different obstacles hinder a wider use of Next-Generation Sequencing for genomic surveillance. We describe Sanger based sequencing protocols: i) Semi-nested RT-PCR covering up to 3.684 kb (>96 %) spike gene; ii) One-Step RT-PCR for key Receptor Binding Domain (RBD) mutations (codons 417–501); iii) One-Step RT-PCR of partial N region to improve genomic capability. Protocols use leftovers of RNA extracted from nasopharyngeal swabs for quantitative RT-PCR diagnosis; with retro-transcribed DNA sequenced at ABI 3500 using dye termination chemistry. Analyses of sequences from 95 individuals (late 2020/early 2021) identified extensive amino acid variation, 57 % with at least one key mutation at the Receptor Binding Domain, with B.1.1.28 lineage most prevalent, followed by Gamma and Zeta variants, with no Delta variant observed. The relatively low cost and simplicity may provide an accessible tool to improve surveillance of SARS-CoV-2 evolution, monitor new variants and vaccinated breakthroughs.

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Human sapoviruses (HuSaV) are associated with acute gastroenteritis (AGE), causing sporadic cases and outbreaks in patients worldwide. In Brazil, however, there are few reports describing the prevalence of HuSaV in patients with AGE. Objective Describing the diversity of HuSaV in Brazil by detecting and molecularly characterizing HuSaV among patients with AGE during an 8-year period (2010–2017). Study design A total of 3974 stool samples, testing negative for rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV), were selected and screened for the presence of HuSaV. Nested RT-PCR were performed for a partial region of VP1, sequenced and genetic analyzed for genotyping the positive samples. Results In the current study, the HuSaV prevalence was determined to be 3.7% (149/3974). A higher prevalence, 5.7% (118/2074), was observed in children under 2 years of age. During the surveillance period, 13 outbreaks were detected: 12 outbreaks in children under 3 years old and one outbreak in adults. Among the 149 HuSaV positive cases, 106 samples (71%) were successfully sequenced. The most prevalent genotype found was GI.1 (44.3%), followed by GI.2 (21.7%), GI.3 (3.8%), GI.6 (2.8%), GII.1 (5.7%), GII.2 (8.5%), GII.3 (2.8%), GII.4 (2.8%), GII.5 (5.7%) and GIV.1 (1.9%). Two GIV.1 strains characterized in this study are, to date, the only strains of this genotype reported in Brazil. Conclusions The present study elucidated the circulation of HuSaV in Brazil and highlight that HuSaV has not assumed an epidemiological importance in the country after the introduction of the RVA vaccine.

The aim of this study was to improve flavivirus field monitoring in Brazil using a reliable probe-based RT-qPCR assay. Standard flavivirus strains were employed to evaluate the performance of the assay, and its applicability was evaluated using 235 stored pools of Culicidae samples collected between 1993 and 1997 and in 2016. Flavivirus species were identified by sequencing. Sixteen (6.8%) samples tested positive: Ilheus virus, Iguape virus, and Saint Louis encephalitis virus were identified in historical specimens from 1993-1994, while insect-specific flaviviruses were detected in the samples from 2016. This approach was demonstrated to be accurate for flavivirus detection and characterization, and it can be successfully applied for vector surveillance and for monitoring and discovery of insect specific flaviviruses.

In São Paulo metropolitan area, Brazil, Amblyomma aureolatum ticks are the main vector of Rickettsia rickettsii, which causes Brazilian spotted fever. In 2013, a boy in São Paulo died of Brazilian spotted fever associated with household dogs and A. aureolatum ticks. Prompt recognition and treatment of this illness might prevent deaths.

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.

The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics., info:eu-repo/semantics/published

Beginning in late 2016 Brazil faced the worst outbreak of Yellow Fever in recent decades, mainly located in southeastern rural regions of the country. In the present study we characterize the Yellow Fever Virus (YFV) associated with this outbreak in São Paulo State, Brazil. Blood or tissues collected from 430 dead monkeys and 1030 pools containing a total of 5,518 mosquitoes were tested for YFV by quantitative RT-PCR, immunohistochemistry (IHC) and indirect immunofluorescence. A total of 67 monkeys were YFV-positive and 3 pools yielded YFV following culture in a C6/36 cell line. Analysis of five nearly full length genomes of YFV from collected samples was consistent with evidence that the virus associated with the São Paulo outbreak originated in Minas Gerais. The phylogenetic analysis also showed that strains involved in the 2016–2017 outbreak in distinct Brazilian states (i.e., Minas Gerais, Rio de Janeiro, Espirito Santo) intermingled in maximum-likelihood and Bayesian trees. Conversely, the strains detected in São Paulo formed a monophyletic cluster, suggesting that they were local-adapted. The finding of YFV by RT-PCR in five Callithrix monkeys who were all YFV-negative by histopathology or immunohistochemistry suggests that this YFV lineage circulating in Sao Paulo is associated with different outcomes in Callithrix when compared to other monkeys.

We report a clinical case of African tick-bite fever in a Brazilian traveler right after his return from South Africa. Definitive diagnosis was supported by seroconversion between acute-phase and convalescent-phase serum samples, detection of rickettsial DNA in skin lesions, and in vitro culture of Rickettsia africae from the patient’s skin. Most of the previous reported cases of African tick-bite fever were confirmed solely by serological or/and molecular methods. Through this first confirmed case of African tick-bite fever in Brazil, it is quite possible that other cases are occurring unnoticed by the health authorities, requiring a greater vigilance in traveler’s medicine in South America.

Brazilian spotted fever (BSF) is caused by the bacterium Rickettsia rickettsii. Because of its high case-fatality rate and apparent increase in areas of transmission, it is considered to be the rickettsial illness of primary public health interest. Cases of this disease have historically occurred in Southeastern Brazil. This article reports the first fatal case of BSF in Southern Brazil. This case high lights the importance of BSF to be considered as a differential diagnosis for acute hemorrhagic fever in areas where cases of BSF may not be expected.

INTRODUCTION Brazilian spotted fever is an infectious disease with a high mortality rate if not treated early. Differential diagnosis is difficult, as the first clinical signs are non-specific and can be confused with other diseases. The aim of the study was to investigate evidence of infection with Rickettsia rickettsii and Rickettsia parkeri in negative sera samples, collected in 2014, from patients with suspected leptospirosis, dengue fever, and meningococcal disease in Atibaia and Bragança Paulista municipalities of the State of São Paulo. METHODS The samples stored at the Institute Adolfo Lutz in Campinas were tested using an indirect immunofluorescence assay (IFA) with IgG and IgM against R. rickettsii and R. parkeri. Real-time polymerase chain reaction (PCR) testing was performed for the sera samples of patients who died (n = 3), those with initial suspicion of meningococcal disease (n = 6), and those with positive IFA results. RESULTS Of 258 samples from Bragança Paulista, 4 (1.6%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. Of 155 samples from Atibaia, 2 (1.3%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. No sample showed positive PCR results. CONCLUSIONS This serological investigation suggests there is evidence of exposure to Rickettsia spp. in residents of areas that have environmental conditions favorable to the spread of bacteria, in which Brazilian spotted fever incidence was not previously confirmed.

Santa Catarina State in southern Brazil is the state with the second highest number of laboratory-confirmed cases of spotted fever illness in Brazil. However, all these cases were confirmed solely by serological analysis (seroconversion to spotted fever group rickettsiae), which has not allowed identification of the rickettsial agent. Here, a clinical case of spotted fever illness from Santa Catarina is shown by seroconversion and molecular analysis to be caused by Rickettsia sp. strain Atlantic rainforest. This is the third confirmed clinical case due to this emerging rickettsial agent in Brazil. Like the previous two cases, the patient presented an inoculation eschar at the tick bite site. Our molecular diagnosis was performed on DNA extracted from the crust removed from the eschar. These results are supported by previous epidemiological studies in Santa Catarina, which showed that nearly 10% of the most common human-biting ticks were infected by Rickettsia sp. strain Atlantic rainforest.

Q fever is a zoonosis of worldwide distribution that is caused by Coxiella burnetii. However, reports of this disease in Brazil are rare. Seroepidemiological studies have shown relatively high frequencies of antibodies against Coxiella burnetii in populations with occupational exposure. In humans, it can be manifested clinically as acute or chronic disease. Endocarditis is the most frequent chronic form of Q fever and the form with the greatest morbidity and mortality. We report a severe case of endocarditis due to Coxiella burnetii acquired in Brazil that had a fatal outcome, despite specific antibiotic therapy and valve surgery treatment.

Rocky Mountain spotted fever (RMSF), a tick-borne zoonosis caused by Rickettsia rickettsii, is among the deadliest of all infectious diseases. To identify the distribution of various genotypes of R. rickettsii associated with fatal RMSF, we applied molecular typing methods to samples of DNA extracted from formalin-fixed, paraffin-embedded tissue specimens obtained at autopsy from 103 case-patients from seven countries who died of RMSF. Complete sequences of one or more intergenic regions were amplified from tissues of 30 (29%) case-patients and revealed a distribution of genotypes consisting of four distinct clades, including the Hlp clade, regarded previously as a non-pathogenic strain of R. rickettsii. Distinct phylogeographic patterns were identified when composite case-patient and reference strain data were mapped to the state and country of origin. The phylogeography of R. rickettsii is likely determined by ecological and environmental factors that exist independently of the distribution of a particular tick vector.

This study presents a new recombinant protein that acts as a powerful antiviral (rAVLO-recombinant Antiviral protein of Lonomia obliqua). It was able to reduce the replication by 10(6) fold for herpes virus and by 10(4) fold for rubella virus. RT-PCR of viral RNA rAVLO treated infected cells also showed similar rate of inhibition in replication. The analysis of this protein by bioinformatics suggests that this protein is globular, secreted with a signal peptide and has the ability to bind to MHC class I. It was found that there are several protein binding sites with various HLA and a prevalence of α-helices in the N-terminal region (overall classified as a α/β protein type). BLAST similarity sequence search for corresponding cDNA did not reveal a similar sequence in Genbank, suggesting that it is from a novel protein family. In this study we have observed that this recombinant protein and hemolymph has a potent antiviral action. This protein was produced in a baculovirus/Sf-9 system. Therefore, these analyses suggest that this novel polypeptide is a candidate as a broad spectrum antiviral.

Fifteen bacterial isolates from spotted fever group rickettsiosis in Brazil were genetically identified as Rickettsia rickettsii . In a phylogenetic analysis with other R. rickettsii isolates from GenBank, the Central/South American isolates showed low polymorphism and formed a clade distinct from two North American clades, with the North American clades having greater in-branch polymorphism.

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cut-off IgG and/or IgM ≥ 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n=86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis.

Brazilian spotted fever (BSF) caused by Rickettsia rickettsii is the most important rickettsiosis and the only reportable tick-borne disease in Brazil. In Brazil, the hard tick Amblyomma cajennense is the most important BSF vector; however, in Sao Paulo State, A. aureolatum was also recognized as a vector species in remaining Atlantic forest areas near the metropolitan area of Sao Paulo city. We analyzed clinical and epidemiological features of BSF cases from two distinct areas where A. cajennense (Area 1) and A. aureolatum (Area 2) are the incriminated vectors. The clinical features demonstrate the same severity pattern of BSF in both endemic areas. Differences in seasonality, patient characteristics (median age and gender), and epidemiological risk factors (animals host contact and vegetation characteristics) were observed and possibly could be attributed to the characteristics of each vector and their typical biological cycle (hosts and environment).

Background Chamydophila pneumoniae (CP) and/or Mycoplasma pneumoniae (MP) are two bacteria detected in vulnerable atheromas. In this study we aimed to analyze whether CP and/or MP aggravates atherosclerosis induced by cholesterol-enriched diet in C57BL/6 apoE KO male mice. Thirty male apoE KO mice aged eight weeks fed by a diet containing 1% cholesterol until 32 weeks of age were divided into four groups: the first was inoculated with CP (n = 7), the second with MP (n = 12), the third with both CP + MP (n = 5), and the fourth with saline (sham n = 6). The animals were re-inoculated at 36 weeks of age, and sacrificed at 40 weeks of age. Two ascending aorta and one aortic arch segments were sampled. In the most severely obstructed segment, vessel diameter, plaque height, percentage of luminal obstruction and the degree of adventitial inflammation were analyzed. The plaque area/intimal surface ratio was obtained by measuring all three segments. The adventitial inflammation was semiquantified (0 absent, 1 mild, 2 moderate, and 3 diffuse). Results The mean and standard deviation of plaque height, % luminal obstruction, external diameter, the plaque area/intimal surface ratio and the adventitial inflammation values are the following for each group: MP (0.20 +/- 0.12 mm, 69 +/- 26%, 0.38 +/- 0.11 mm, 0.04 +/- 0.04 and 0.22 +/- 0.67), CP (0.23 +/- 0.08 mm, 90 +/- 26%, 0.37 +/- 0.08 mm, 0.04 +/- 0.03, and 0.44 +/- 0.53), MP + CP (18 +/- 0.08 mm, 84 +/- 4.0%, 0.35 +/- 0.25 mm, 0.03 +/- 0.03 and 1.33 +/- 0.82) and sham (0.08 +/- 0.09 mm, 42 +/- 46%, 0.30 +/- 0.10 mm, 0.02 +/- 0.03 and 0.71 ± 0.76). A wider area of plaque/intimal surface was observed in MP + CP inoculated groups (p = 0.07 and 0.06) as well as an increased plaque height in CP (p = 0.01) in comparison with sham group. There was also an increased luminal obstruction (p = 0.047) in CP inoculated group in comparison to sham group. Adventitial inflammation in MP + CP inoculated group was higher than MP, CP and the sham groups (p = 0.02). Conclusion Inoculation of CP, MP or both agents in C57BL/6 apoE KO male mice caused aggravation of experimental atherosclerosis induced by cholesterol-enriched diet, with distinct characteristics. CP inoculation increased the plaque height with positive vessel remodeling and co-inoculation of MP + CP caused the highest adventitial inflammation measures.

Q fever is a zoonosis of worldwide distribution that is caused by Coxiella burnetii. However, reports of this disease in Brazil are rare. Seroepidemiological studies have shown relatively high frequencies of antibodies against Coxiella burnetii in populations with occupational exposure. In humans, it can be manifested clinically as acute or chronic disease. Endocarditis is the most frequent chronic form of Q fever and the form with the greatest morbidity and mortality. We report a severe case of endocarditis due to Coxiella burnetii acquired in Brazil that had a fatal outcome, despite specific antibiotic therapy and valve surgery treatment.

Bartonella spp. and Coxiella burnetii are recognized as causative agents of blood culture-negative endocarditis (BCNE) in humans and there are no studies of their occurrences in Brazil. The purpose of this study is to investigate Bartonella spp. and C. burnetii as a causative agent of culture-negative endocarditis patients at a cardiology hospital in Sao Paulo, Brazil. From January 2004 to December 2004 patients with a diagnosis of endocarditis at our Institute were identified and recorded prospectively. They were considered to have possible or definite endocarditis according to the modified Duke criteria. Those with blood culture-negative were tested serologically using the indirect immunofluorescent assay (IFA) for Bartonella henselae, B. quintana, and C. burnetii. IFA-IgG titers >800 for Bartonella spp. and C. burnetii were considered positive. A total of 61 patients with endocarditis diagnosis were evaluated, 17 (27%) were culture-negative. Two have had IgG titer greater than 800 (>/=3,200) against Bartonella spp. and one against C. burnetii (phase I and II>/=6,400). Those with Bartonella-induced endocarditis had a fatal disease. Necropsy showed calcifications and extensive destruction of the valve tissue, which is diffusely infiltrated with mononuclear inflammatory cells predominantly by foamy macrophages. The patient with C. burnetii endocarditis received specific antibiotic therapy. Reports of infective endocartitis due to Bartonella spp. and C. burnetii in Brazil reveal the importance of investigating the infectious agents in culture-negative endocarditis.

We investigated the relationship between NOS3, FGB and PAI-1 polymorphisms and endothelial dysfunction and risk factors for acute myocardial infarction (AMI) in young adults.Endothelial function was measured by response to flow mediated vasodilation (FMV) and induced by nitrate (FMN). Biochemical parameters were measured by standard enzymatic methods and plasma total nitrate was analyzed by the NOA system. NOS3 (T-786C, G894T and intron 4A/B STR), FGB (C-148T and G-455A) and PAI-1 (4G/5G) polymorphisms were determined by PCR-RFLP.Concentrations of total and LDL cholesterol, apo B, triglycerides, nitrate, PAI-1 and fibrinogen were higher and apo AI, HDL cholesterol and FMV were lower in AMI patients than in controls (p0.001). PAI-1 (p0.001) but not nitrate was higher in AMI patients with low response to FMV. NOS3 T-786C and FGB C-148T polymorphisms were associated with AMI (p0.050). NOS3 T-786C was also related to hypertension (p=0.049). NOS3 intron 4A/B STR was associated with increased concentrations of total cholesterol and apo B. NOS3-786TT/894GT haplotype was associated with increased FMV (p=0.018) than the other haplotypes.Our data suggest NOS3 and FGB polymorphisms are associated with AMI. NOS3 is also related to hypertension, endothelial dysfunction and variation on serum cholesterol in young adults with AMI.