Flaviviruses as agents of childhood central nervous system infections in Brazil.

Flaviviruses are agents of a major emerging human public health issue and members of this genus have been associated with central nervous system (CNS) infections. In Brazil, a country endemic for some arboviruses, the most clinically relevant neurotropic flaviviruses include Dengue virus and Zika virus. Flaviviruses cause diseases ranging from mild or sub-clinical infections to severe cases as CNS infections. There is a lack of data about the incidence of flaviviruses in the CNS of children in Brazil. In this review, we provide a general overview of several flaviviruses that cause CNS infections in Brazilian children and explore the importance of epidemiological surveillance of CNS infections in cases of flavivirus infections.

Flaviviruses as agents of childhood central nervous system infections in Brazil.

Flaviviruses are agents of a major emerging human public health issue, and members of the Flavivirus genus have been associated with central nervous system (CNS) infections. In Brazil, a country endemic for some arboviruses, the most clinically relevant neurotropic flaviviruses include dengue virus and Zika virus. Flaviviruses cause diseases ranging from mild or subclinical infections to severe cases as CNS infections. There is a lack of data about the incidence of flaviviruses in the CNS of children in Brazil. In this review, we provide a general overview of several flaviviruses that cause CNS infections in Brazilian children and explore the importance of epidemiologic surveillance of CNS infections in cases of Flavivirus infections.

Detection and Molecular Characterization of Yellow Fever Virus, 2017, Brazil.

At the end of 2016, Brazil experienced an unprecedented yellow fever (YF) outbreak. Clinical, molecular and ecological aspects of human and non-human primate (NHP) samples collected at the beginning of the outbreak are described in this study. Spatial distribution analyses demonstrated a strong overlap between human and NHP cases. Through molecular analyses, we showed that the outbreak had a sylvatic origin, caused by the South American genotype 1 YFV, which has already been shown to circulate in Brazil. As expected, the clusters of cases were identified in regions with a low vaccination coverage. Our findings highlight the importance of the synchronization of animal surveillance and health services to identify emerging YF cases, thereby promoting a better response to the vulnerable population.

Dairy production practices and associated risks for bovine vaccinia exposure in cattle, Brazil.

A cross-sectional serosurvey was performed to identify environmental features or practices of dairy farms associated with risk for exposure to vaccinia-like viruses in dairy cattle in Brazil. Sera from 103 cows from 18 farms in Minas Gerais state were examined for Orthopoxvirus-neutralizing antibodies. A database of 243 binary or multiple-selection categorical variables regarding the physical features and surrounding ecology of each property was obtained. Thirteen of 46 presumptive predictor variables were found to be significantly associated with Orthopoxvirus serostatus by univariate logistic regression methods. Use of teat sanitizer and having felids on the property were independently associated with virus exposure by multivariable analysis. Rodents have long been suspected of serving as maintenance reservoirs for vaccinia-like viruses in Brazil. Therefore, domestic felids are not only effective predators of small rodent pests, but also their urine can serve as a deterrent to rodent habitation in buildings such as stables and barns. These results corroborate previous evidence of the high significance of rodents in the Vaccinia virus transmission cycle, and they also raise questions regarding the common use of teat sanitizers in dairy production areas.

Daily ingestion of the probiotic Lactobacillus paracasei ST11 decreases Vaccinia virus dissemination and lethality in a mouse model.

Vaccinia virus (VACV) is an important pathogen. Although studies have shown relationships between probiotics and viruses, the effect of probiotics on VACV infection is unknown. Therefore, this work aims to investigate the probiotics effects on VACV infection. Mice were divided into four groups, two non-infected groups, one receiving the probiotic, the other one not receiving it, and two groups infected intranasally with VACV Western Reserve (VACV-WR) receiving or not receiving the probiotic. Viral titres in organs and cytokine production in the lungs were analysed. Lung samples were also subjected to histological analysis. The intake of probiotic results in reduction in viral spread with a significant decrease of VACV titer on lung, liver and brain of treated group. In addition,treatment with the probiotic results in attenuated mice lung inflammation showing fewer lesions on histological findings and decreased lethality in mice infected with VACV. The ingestion of Lactobacillus paracasei ST11 (LPST11) after VACV infection resulted in 2/9 animal lethality compared with 4/9 in the VACV group. This is the first study on probiotics and VACV interactions, providing not only information about this interaction, but also proposing a model for future studies involving probiotics and other poxvirus.

First fatal case of CNS infection caused by Enterovirus A in Brazil.

We describe what is to our knowledge the first fatal case of central nervous system Enterovirus infection in Brazil. Molecular and phylogenetic characterization revealed that Enterovirus A was the aetiologic agent of this case.

Evaluation of the effectiveness of mass trapping with BG-sentinel traps for dengue vector control: a cluster randomized controlled trial in Manaus, Brazil.

The objective of this study was to assess the effectiveness of BG-Sentinel (BGS) traps for mass trapping at the household level to control the dengue vector, Aedes aegypti (L.), in Manaus (Brazil) by performing a cluster randomized controlled trial. After an initial questionnaire and baseline monitoring, 6 out of 12 clusters were randomly allocated to the intervention arm, where participating premises received one BGS trap for mass trapping. The other six clusters did not receive traps and were considered as the control arm. Biweekly monitoring with BGS in both arms assessed the impact of mass trapping. At the end of the study, a serological survey was conducted and a second questionnaire was conducted in the intervention arm. Entomological monitoring indicated that mass trapping with BGS traps significantly reduced the abundance of adult female Ae. aegypti during the first five rainy months. In the subsequent dry season when the mosquito population was lower, no effect of mass trapping was observed. Fewer Ae. aegypti females were measured in the intervention arm during the next rainy period, but no significant difference between arms was observed. The serological survey revealed that in participating houses of mass trapping areas recent dengue infections were less common than in control areas, although this effect was not statistically significant. The majority of participants responded positively to questions concerning user satisfaction. Our results suggest that BGS traps are a promising tool which might be deployed as part of dengue control programs; however, further investigations and larger scale studies are necessary.

Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV.

Looking back: a genetic retrospective study of Brazilian Orf virus isolates.

Orf virus (ORFV), the prototype of the genus Parapoxvirus, is the aetiological agent of contagious ecthyma (CE), a pustular dermatitis that afflicts domestic and wild small ruminants. CE is one of the most widespread poxvirus diseases in the world, causing public health impacts. Outbreaks of ORFV have been observed in all geographical regions of Brazil, affecting ovine and caprine herds. The origins, epidemiology and identity of Brazilian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In the present study, we revisited CE outbreaks which occurred until 32 years ago, and we assessed, genetically, five viral isolates. We performed the sequencing and analysis of the three ORFV molecular markers: B2L gene, virus interferon resistance gene (VIR) and the vascular endothelial growth factor gene. Nucleotide and amino acid analysis of the analysed genes demonstrated that Brazilian ORFVs do not form a unique cluster, and presented more similarity to other worldwide ORFV samples than with each other. These data raise the questions of whether there are different worldwide ORFVs circulating in Brazil, or if all the Brazilian ORFV samples are of the same virus taken at distinct time points.

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil.

AIMS: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. METHODS AND RESULTS: The antiviral activity was evaluated against human herpesvirus type 1 (HSV-1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV-WR) and dengue virus 2 (DENV-2) by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti-HSV-1 [EC(50) 142.8 ± 5.3 µg ml(-1); selectivity index (SI) 2.0] and anti-DENV-2 activity (EC(50) 9.8 ± 1.3 µg ml(-1) ; SI 1.5). The pectolinarin (1) isolated from LEE was less active against HSV-1 and DENV-2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV-1. A mixture of pectolinarin (1) and acacetin-7-O-rutinoside (2) was isolated from FEE and has presented anti-DENV-2 activity (EC(50) 11.1 ± 1.6 µg ml(-1) ; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. CONCLUSIONS: These data shows that D. elongata has antiviral activity mainly against HSV-1 and DENV-2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. SIGNIFICANCE AND IMPACT OF THE STUDY: Distictella elongata might be considered a promising source of anti-dengue fever phytochemicals.

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1.

AIMS: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. METHODS AND RESULTS: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44.1%) have disclosed antiviral activity against one or more of the viruses assayed with EC(50) values in the range of 23.2 ± 2.5-422.7 ± 10.9 µg ml(-1). CONCLUSIONS: Twelve of the 34 extracts (35.3%) might be considered promising sources of antiviral natural products, as they have shown EC50 ≤ 100 µg ml(-1). The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.

Seroprevalence of orthopoxvirus in an Amazonian rural village, Acre, Brazil.

Vaccinia virus strains from the family Poxviridae have been frequently isolated in Brazil and associated with outbreaks of exanthematic disease affecting cows and humans. An ELISA IgG was applied to evaluate the seroprevalence of orthopoxviruses in a community located in a rural settlement in the Amazon region, where no orthopoxvirus outbreaks have yet been reported. An overall seroprevalence of 27.89% was found, and it was 23.38% in the non-vaccinated population (smallpox vaccination). These results strongly suggest that orthopoxviruses circulate in this population, and it is the first finding of seropositivity for orthopoxviruses in a population without any previously reported outbreaks.

Climbing the steps of viral atomic force microscopy: visualization of Dengue virus particles.

In recent years, the application of atomic force microscopy (AFM) to biological systems has highlighted the potential of this technology. AFM provides insights into studies of biological structures and interactions and can also identify and characterize a large panel of pathogens, including viruses. The Flaviviridae family contains a number of viruses that are important human and animal pathogens. Among them, Dengue virus causes epidemics with fatal outcomes mainly in the tropics. In this study, Dengue virus is visualized for the first time using the in air AFM technique. Images were obtained from a potassium-tartrate gradient-purified virus. This study enhances the application of AFM as a novel tool for the visualization and characterization of virus particles. Because flavivirus members are closely related, studies of the morphologic structure of the Dengue virus can reveal strategies that may be useful to identify and study other important viruses in the family, including the West Nile virus.

Activation/modulation of adaptive immunity emerges simultaneously after 17DD yellow fever first-time vaccination: is this the key to prevent severe adverse reactions following immunization?

Over past decades the 17DD yellow fever vaccine has proved to be effective in controlling yellow fever and promises to be a vaccine vector for other diseases, but the cellular and molecular mechanisms by which it elicits such broad-based immunity are still unclear. In this study we describe a detailed phenotypic investigation of major and minor peripheral blood lymphocyte subpopulations aimed at characterizing the kinetics of the adaptive immune response following primary 17DD vaccination. Our major finding is a decreased frequency of circulating CD19+ cells at day 7 followed by emerging activation/modulation phenotypic features (CD19+interleukin(IL)10R+/CD19+CD32+) at day 15. Increased frequency of CD4+human leucocyte antigen D-related(HLA-DR+) at day 7 and CD8+HLA-DR+ at day 30 suggest distinct kinetics of T cell activation, with CD4+ T cells being activated early and CD8+ T cells representing a later event following 17DD vaccination. Up-regulation of modulatory features on CD4+ and CD8+ cells at day 15 seems to be the key event leading to lower frequency of CD38+ T cells at day 30. Taken together, our findings demonstrate the co-existence of phenotypic features associated with activation events and modulatory pathways. Positive correlations between CD4+HLA-DR+ cells and CD4+CD25high regulatory T cells and the association between the type 0 chemokine receptor CCR2 and the activation status of CD4+ and CD8+ cells further support this hypothesis. We hypothesize that this controlled microenviroment seems to be the key to prevent the development of serious adverse events, and even deaths, associated with the 17DD vaccine reported in the literature.

Use of atomic force microscopy as a diagnostic tool to identify orthopoxvirus.

Atomic force microscopy (AFM) is a versatile technique that permits the imaging of surfaces and generates topographical images from a variety of materials. Due to the fact that AFM requires minimum sample manipulation, it is a valuable tool for studying biological materials such as cells, DNA, bacteria and viruses. The aim of the present study was to standardize the AFM technique as a diagnostic tool for detection of naturally occurring orthopoxviruses. The samples analyzed were collected during natural outbreaks of Vaccinia virus (VACV) in dairy cattle in Brazil. These viruses are zoonotic infections; and therefore safe manipulation of all samples is required. The AFM technique would provide a more secure way to diagnose infection. By using the "in air" AFM technique after purification and inactivation process, relatively crude preparations of viruses were visualized rapidly. Details for efficient sample preparation and AFM imaging are described. The AFM technique provides a rapid and biosecure tool for the diagnosis of emerging orthopoxviruses and has potential as a tool for screening bioterrorism samples.

Surto de varíola bovina causada pelo vírus Vaccinia na região da Zona da Mata Mineira / Outbreak of exantemal disease caused by Vaccinia virus in human and cattle in Zona da Mata region, Minas Gerais

Relata-se um surto de doença exantemática, caracterizada como varíola bovina, acometendo bovinos e seres humanos na Zona da Mata Mineira. Setenta e duas propriedades, distribuídas em 20 municípios localizados na região, foram visitadas para se levantar os aspectos clínicos e epidemiológicos da doença. Detectaram-se 1020 vacas doentes durante a investigação, quando houve queda na produção do leite associada a infecções bacterianas secundárias. Casos humanos foram registrados em 83 por cento das propriedades visitadas. Espécimes clínicos e amostras de soro foram coletados dos animais doentes ou convalescentes. O diagnóstico de laboratório mostrou o envolvimento de um ortopoxvírus, precisamente o Vaccinia virus como agente etiológico do surto.

The use and misuse of the "impact factor" as a parameter for evaluation of scientific publication quality: a proposal to rationalize its application.

We present a critical analysis of the generalized use of the "impact factor". By means of the Kruskal-Wallis test, it was shown that it is not possible to compare distinct disciplines using the impact factor without adjustments. After assigning the median journal the value of one (1.000), the impact factor value for each journal was calculated by the rule of three. The adjusted values were homogeneous, thus permitting comparison among distinct disciplines.

The use and misuse of the "impact factor" as a parameter for evaluation of scientific publication quality: a proposal to rationalize its application

We present a critical analysis of the generalized use of the "impact factor". By means of the Kruskal-Wallis test, it was shown that it is not possible to compare distinct disciplines using the impact factor without adjustments. After assigning the median journal the value of one (1.000), the impact factor value for each journal was calculated by the rule of three. The adjusted values were homogeneous, thus permitting comparison among distinct disciplines.

Frequency of p12K and p12R alleles of HTLV Type 1 in HAM/TSP patients and in asymptomatic HTLV type 1 carriers.

HTLV-1 has a complex genome, and contains four open reading frames (ORFs) in the 3' region encoding viral and cellular regulatory proteins. p12 is a small, ORF I-encoded hydrophobic protein, the function of which is not well understood. It has been shown that p12 enhances the E5-transforming ability of bovine papillomavirus; and binds to the 16-kDa subunit of the vacuolar ATPase pump, immature forms of the beta and gamma(c) chains of the interleukin 2 receptor, and the free chain of MHC I. p12 carrying a lysine residue (p12K) at position 88 of its sequence may be rapidly degraded in the cell via proteasome, whereas p12 with an arginine residue (p12R) at the same position is severalfold more stable. These alleles are found in proviral DNA of HTLV-1-infected individuals and it was previously observed that the p12K allele was more frequent in HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis) patients and was not found at all in asymptomatic carriers, whereas patients with adult T cell leukemia/lymphoma (ATLL) carry the p12R allele. To extend these observations and verify whether the p12K mutation could be used as a marker of progression to HAM/TSP, we analyzed 37 HAM/TSP patients and 40 asymptomatic carriers at different stages of infection. In our cohort, only one HAM/TSP patient carried the p12K phenotype, which accounted for a frequency of 2.7% (1 of 37). We also found, among the 40 asymptomatic HTLV-1 carriers, one who presented the p12K phenotype, contrasting with previous publications. Thus, p12K does not seem to be universally diagnostic for HTLV-1-associated neurological disease. Further screening of HTLV-1-infected individuals in other populations may elucidate this observation.

A mitogenic signal triggered at an early stage of vaccinia virus infection: implication of MEK/ERK and protein kinase A in virus multiplication.

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.