Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

Tumour necrosis factor plays a deleterious role in the pathogenesis of chikungunya virus infection.

Arthralgia is a hallmark of chikungunya virus (CHIKV) infection and can be very debilitating and associated with a robust local inflammatory response. Many pathophysiological aspects associated with the disease remain to be elucidated. Here, we describe a novel model of CHIKV infection in immunocompetent mice and evaluate the role of tumour necrosis factor in the pathogenesis of the disease. C57BL/6 wild type (WT) or TNF receptor 1 deficient (TNFR1-/- ) mice were inoculated with 1 × 106 PFU of CHIKV in the paw. Alternatively, etanercept was used to inhibit TNF in infected WT mice. Hypernociception, inflammatory and virological analysis were performed. Inoculation of CHIKV into WT mice induced persistent hypernociception. There was significant viral replication in target organs and local production of inflammatory mediators in early time-points after infection. CHIKV infection was associated with specific humoral IgM and IgG responses. In TNFR1-/- mice, there was a decrease in the hypernociception threshold, which was associated with a milder local inflammatory response in the paw but delayed viral clearance. Local or systemic treatment with etanercept reduced CHIKV-induced hypernociception. This is the first study to describe hypernociception, a clinical correlation of arthralgia, in immunocompetent mice infected with CHIKV. It also demonstrates the dual role of TNF in contributing to viral clearance but driving tissue damage and hypernociception. Inhibition of TNF may have therapeutic benefits but its role in viral clearance suggests that viral levels must be monitored in CHIKV-infected patients and that TNF inhibitors should ideally be used in combination with anti-viral drugs.

Detection of DENV-2 and ZIKV coinfection in southeastern Brazil by serum and urine testing.

PURPOSE: Aedes aegypti mosquito-borne diseases have a significant impact on public health in Brazil. In this study, we investigated the presence of the Zika virus (ZIKV) and dengue virus (DENV) in serum and urine samples from symptomatic participants who attended an Emergency Care Unit located in a city in the northwestern region of São Paulo between February 2018 and April 2019. METHODS: Serum and urine samples were collected from participants suspected of having arbovirus infection. After the extraction of viral RNA, viral detection was performed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) (One-Step RT-qPCR). RESULTS: A total of 305 participants participated in this study. A total of 283 blood and 270 urine samples were collected. Of 305 patients, 36.4% (111/305) were positive for ZIKV, 43.3% (132/305) for DENV2, and 0.3% (1/305) for DENV1. Coinfection with ZIKV/DENV2 was observed in 13.1% of participants. If only serum samples were used, ZIKV detection would have decreased to 23.3% (71/305). Of all the participants included in the study, only one was suspected of having ZIKV infection based on clinical diagnosis, and the remaining participants were suspected of having DENV. CONCLUSION: By testing serum and urine samples, we increased the detection of both viruses and detected considerable levels of ZIKV and DENV-2 coinfection when compared to other studies. Additionally, we detected an unnoticed ZIKV outbreak in the city. These findings highlight the importance of the molecular diagnosis of arboviruses to aid public health surveillance and management strategies.

Identification of Potential Antiviral Hops Compounds against Chikungunya Virus.

Chikungunya virus (CHIKV) is an arthropod-borne virus that belongs to the genus Alphavirus (family Togaviridae). CHIKV causes chikungunya fever, which is mostly characterized by fever, arthralgia and, sometimes, a maculopapular rash. The bioactive constituents of hops (Humulus lupulus, Cannabaceae), mainly acylphloroglucinols, known as well as α- and ß-acids, exerted distinct activity against CHIKV, without showing cytotoxicity. For fast and efficient isolation and identification of such bioactive constituents, a silica-free countercurrent separation method was applied. The antiviral activity was determined by plaque reduction test and was visually confirmed by a cell-based immunofluorescence assay. All hops compounds demonstrated a promising post-treatment viral inhibition, except the fraction of acylphloroglucinols, in mixture. ß-acids fraction of 125 µg/mL expressed the strongest virucidal activity (EC50 = 15.21 µg/mL), in a drug-addition experiment on Vero cells. Hypothesis for mechanism of action were proposed for acylphloroglucinols based on their lipophilicity and chemical structure. Therefore, inhibition of some steps of the protein kinase C (PKC) transduction cascades was also discussed.

Antiviral Activity of Quercetin Hydrate against Zika Virus.

Zika virus (ZIKV) has re-emerged in recent decades, leading to outbreaks of Zika fever in Africa, Asia, and Central and South America. Despite its drastic re-emergence and clinical impact, no vaccines or antiviral compounds are available to prevent or control ZIKV infection. This study evaluated the potential antiviral activity of quercetin hydrate against ZIKV infection and demonstrated that this substance inhibits virus particle production in A549 and Vero cells under different treatment conditions. In vitro antiviral activity was long-lasting (still observed 72 h post-infection), suggesting that quercetin hydrate affects multiple rounds of ZIKV replication. Molecular docking indicates that quercetin hydrate can efficiently interact with the specific allosteric binding site cavity of the NS2B-NS3 proteases and NS1-dimer. These results identify quercetin as a potential compound to combat ZIKV infection in vitro.

Type 1 Innate Lymphoid Cell and Natural Killer Cells Are Sources of Interferon-γ and Other Inflammatory Cytokines Associated With Distinct Clinical Presentation in Early Dengue Infection.

BACKGROUND: Increased levels of inflammatory cytokines are associated with severe dengue evolution, but the source of such hypercytokinemia is elusive. We investigated the contribution of innate lymphocytes, innate lymphoid cells (ILCs), and natural killer (NK) cells in cytokine production in early dengue infection. METHODS: Peripheral blood mononuclear cells of individuals with dengue without warning signs (DWS-) and dengue with warning signs and severe dengue (SD) presentation combined (DWS+) were obtained between 2 and 7 days since fever onset and submitted to flow cytometry without specific antigen stimulation to evaluate cytokines in ILC and NK cell subpopulations. RESULTS: ILCs and NK cells were found to be important sources of cytokines during dengue. ILCs of the DWS+/SD group displayed higher production of interferon gamma (IFN-γ) and interleukin (IL) 4/IL-13 when compared to DWS- individuals. On the other hand, NK Eomes+ cells of DWS- patients displayed higher IFN-γ production levels compared with the DWS+/SD group. Interestingly, when NK cells were identified by CD56 expression, DWS+/SD displayed higher frequency of IL-17 production compared with the DWS- group. CONCLUSIONS: These results indicate that ILCs and NK cells are important sources of inflammatory cytokines during acute dengue infection and display distinct profiles associated with different clinical forms.

HLA-G, LILRB1 and LILRB2 Variants in Zika Virus Transmission from Mother to Child in a Population from South and Southeast of Brazil.

During the 2015-2016 epidemic, Brazil was the country with the highest rate of Zika virus (ZIKV) infection in the Americas. Twenty-nine percent of pregnant women positive for ZIKV exhibited ultrasound scans with fetus anomalies. Human leukocyte antigen-G (HLA-G) exerts immunoregulatory effects by binding to inhibitory receptors, namely LILRB1 and LILRB2, thus preventing mother-fetus rejection and vertical pathogen transmission. The binding of HLA-G to one of its receptors modulates both innate and adaptive immunity. However, in a viral infection, these molecules may behave as pathogenic mediators shifting the pregnancy environment from an anti-inflammatory profile to a pro-inflammatory phenotype. Genetic mutations might be associated with the change in phenotype. This study aimed to explore the possible role of polymorphic sites in HLA-G, LILRB1 and LILRB2 in mother-fetus ZIKV transmission. Polymorphisms were detected by direct sequencing. Differences in allele and/or genotype frequencies for each SNP analyzed among ZIKV non-transmitting and transmitting mother-child pairs, among ZIKV-transmitting and non-transmitting mothers and between ZIKV-infected and non-infected children were compared by Mid-P exact test or Yates' correction. Significant susceptibility of ZIKV vertical transmission is suggested in ZIKV-transmitting and non-transmitting mothers and ZIKV-infected and non-infected children for LILRB1_rs1061684 T/T (p = 0.03, Pc = 0.06, OR = 12.4; p = 0.008, Pc = 0.016, OR = 16.4) and LILRB1_rs16985478 A/A (p = 0.01, Pc = 0.02, OR = 19.2; p = 0.008, Pc = 0.016, OR = 16.4). HLA-G_rs1710 (p = 0.04, Pc = 0.52, OR = 4.30) was also a susceptibility factor. LILRB2_rs386056 G/A (p = 0.02, Pc = 0.08, OR = 0.07), LILRB2_rs7247451 G/G (p = 0.01, Pc = 0.04, OR = 0.04) and HLAG_rs9380142 T/T (p = 0.04, Pc = 0.52, OR = 0.14) were suggested as protective factors against vertical transmission. The current study suggests that polymorphic sites in the LILRB1 and HLA-G genes might be associated with mother-to-child ZIKV transmission while LILRB2 might be associated with protection against ZIKV transmission in the womb in a population from the south and southeast of Brazil.

SARS-COV-2 genomic monitoring in the state of São Paulo unveils two emerging AY.43 sublineages.

Delta VOC is highly diverse with more than 120 sublineages already described as of November 30, 2021. In this study, through active monitoring of circulating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants in the state of São Paulo, southeast Brazil, we identified two emerging sublineages from the ancestral AY.43 strain which were classified as AY.43.1 and AY.43.2. These sublineages were defined by the following characteristic nonsynonymous mutations ORF1ab:A4133V and ORF3a:T14I for the AY.43.1 and ORF1ab:G1155C for the AY.43.2 and our analysis reveals that they might have a likely-Brazilian origin. Much is still unknown regarding their dissemination in the state of São Paulo and Brazil as well as their potential impact on the ongoing vaccination process. However, the results obtained in this study reinforce the importance of genomic surveillance activity for timely identification of emerging SARS-CoV-2 variants which can impact the ongoing SARS-CoV-2 vaccination and public health policies.

Detection of Zika virus in urine from randomly tested individuals in Mirassol, Brazil.

PURPOSE: Studies show that around 80% of Zika virus (ZIKV) infections are asymptomatic. The present study tested urine samples from volunteers, unsuspected of arboviral infection, which attended an emergency care unit (ECU) in Mirassol, Brazil, from March 2018 to April 2019. METHODS: The volunteers were divided into two groups. The first group was composed of outpatients who were not suspected to have an arbovirus infection. This first group was subdivided into two subgroups: outpatients with and without arbovirus-like symptoms. The second group consisted of companions of outpatients treated at the ECU. The second group was also subdivided into two subgroups: totally asymptomatic individuals and those who had arbovirus-like symptoms. RNA was extracted from urine samples, followed by RT-qPCR for ZIKV. RESULTS: We found that 11% (79/697) of the samples tested positive for ZIKV-RNA. Among the ZIKV-RNA-positive individuals, 16.5% (13/79) were companions, of which 61.5% (8/13) were totally asymptomatic and 38.5% (5/13) reported symptoms that could be suggestive of arbovirus infection. In addition, 83.5% (66/79) of the ZIKV-RNA-positive individuals were outpatients without a clinical diagnosis of arbovirus. Of these undiagnosed ZIKV-RNA-positive outpatients, 47% (31/66) had no arbovirus-related symptoms. CONCLUSION: Our study shows the effectiveness of urine as a non-invasive sample to detect the incidence of ZIKV infection. We also highlight the importance of ZIKV molecular diagnosis to aid public health surveillance and prevention of congenital Zika syndrome and other ZIKV-associated diseases.

Lack of serological and molecular evidences of Zika virus circulation in non-human primates in three states from Brazil.

BACKGROUND: Zika virus (ZIKV) was discovered in 1947 with the virus isolation from Rhesus monkey (Macaca mulatta) in Uganda forest, Africa. Old World Primates are involved in a sylvatic cycle of maintenance of this arbovirus, however a limited knowledge about the role of New World primates in ZIKV transmission cycles has been established. OBJECTIVE: This work aimed to investigate the presence of enzootic circulation of ZIKV in New World Primates from three Brazilian states: São Paulo, Paraíba, and Paraná. METHODS: We analyzed 100 non-human primate samples collected in 2018 and 2020 from free-ranging and captive environments from São Paulo (six municipalities belonging to Sorocaba region), Paraíba (João Pessoa municipality), and Paraná (Foz do Iguaçu municipality) using reverse transcriptase quantitative polymerase reaction (RT-qPCR) assays, indirect enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT). FINDINGS: All samples (n = 141) tested negative for the presence of ZIKV genome from tissue and blood samples. In addition, all sera (n = 58) from Foz do Iguaçu' non-human primates (NHPs) were negative in serological assays. MAIN CONCLUSION: No evidence of ZIKV circulation (molecular and serological) was found in neotropical primates. In addition, the absence of antibodies against ZIKV suggests the absence of previous viral exposure of NHPs from Foz do Iguaçu-PR.

Lack of association of the KIR and HLA class I ligands with ZIKV infection in south and southeast of Brazil.

BACKGROUND: Zika virus (ZIKV) is an emerging arbovirus associated with foetal malformations and neurological complications. The infection is usually associated with mild symptoms. The comparison between the allelic frequency of polymorphic genes in symptomatic infected individuals in the population can clarify the pathogenic mechanisms of ZIKV. During ZIKV infection, cytokines are produced and natural killer (NK) cells are recruited, whose activation depends on signaling pathways activated by specific receptors, such as killer cell immunoglobulin-like receptors (KIR). These molecules interact with human leukocyte antigen (HLA) class I ligands and are encoded by polymorphic genes. OBJECTIVES: This study aimed to evaluate the frequency of allelic variants of the genes encoding the KIR receptors and their HLA class I ligands in 139 symptomatic ZIKV-patients and 170 controls negative for the virus, and to evaluate the role of these variants for ZIKV susceptibility. METHODS: KIR and HLA class I genes were genotyped using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique. FINDINGS: No significant differences in the frequency distribution of KIRs and KIR-HLA in patients compared to controls were observed. MAIN CONCLUSIONS: KIR and its HLA ligands might play a minor role in ZIKV infection in the south and southeast Brazilian individuals.

Predicting Antigenic Peptides from Rocio Virus NS1 Protein for Immunodiagnostic Testing Using Immunoinformatics and Molecular Dynamics Simulation.

The mosquito-borne disease caused by the Rocio virus is a neglected threat, and new immune inputs for serological testing are urgently required for diagnosis in low-resource settings and epidemiological surveillance. We used in silico approaches to identify a specific antigenic peptide (p_ROCV2) in the NS1 protein of the Rocio virus that was theoretically predicted to be stable and exposed on its surface, where it demonstrated key properties allowing it to interact with antibodies. These findings related to the molecular dynamics of this peptide provide important insights for advancing diagnostic platforms and investigating therapeutic alternatives.

Methylprednisolone as Adjunctive Therapy for Patients Hospitalized With Coronavirus Disease 2019 (COVID-19; Metcovid): A Randomized, Double-blind, Phase IIb, Placebo-controlled Trial.

BACKGROUND: Steroid use for coronavirus disease 2019 (COVID-19) is based on the possible role of these drugs in mitigating the inflammatory response, mainly in the lungs, triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the efficacy of methylprednisolone (MP) among hospitalized patients with suspected COVID-19. METHODS: A parallel, double-blind, placebo-controlled, randomized, Phase IIb clinical trial was performed with hospitalized patients aged ≥18 years with clinical, epidemiological, and/or radiological suspected COVID-19 at a tertiary care facility in Manaus, Brazil. Patients were randomly allocated (1:1 ratio) to receive either intravenous MP (0.5 mg/kg) or placebo (saline solution) twice daily for 5 days. A modified intention-to-treat (mITT) analysis was conducted. The primary outcome was 28-day mortality. RESULTS: From 18 April to 16 June 2020, 647 patients were screened, 416 were randomized, and 393 were analyzed as mITT, with 194 individuals assigned to MP and 199 to placebo. SARS-CoV-2 infection was confirmed by reverse transcriptase polymerase chain reaction in 81.3%. The mortality rates at Day 28 were not different between groups. A subgroup analysis showed that patients over 60 years old in the MP group had a lower mortality rate at Day 28. Patients in the MP arm tended to need more insulin therapy, and no difference was seen in virus clearance in respiratory secretion until Day 7. CONCLUSIONS: The findings of this study suggest that a short course of MP in hospitalized patients with COVID-19 did not reduce mortality in the overall population. CLINICAL TRIALS REGISTRATION: NCT04343729.

Enteric viruses circulating in undiagnosed central nervous system infections at tertiary hospital in São José do Rio Preto, São Paulo, Brazil.

Enterovirus (EV) is commonly associated with central nervous system (CNS) syndromes. Recently, gastroenteric viruses, including rotavirus (RVA), human astrovirus (HAstV), and norovirus (NoV), have also been associated with CNS neurological disorders. The aim of the present study was to investigate the presence of EV, RVA, HAst, and NoV associated to CNS infections with undiagnosed etiology in Northwest region of São Paulo State, Brazil, and to conduct the molecular characterization of the positive samples detected. A total of 288 cerebrospinal fluid samples collected from July to December 2017 were tested for EV and NoV by quantitative real-time polymerase chain reaction (RT-qPCR), HAstV by conventional RT-PCR, and RVA by enzyme-linked immunosorbent assay. Positive-EV samples were inoculated in cells lines, amplified by RT-PCR and sequenced. RVA, NoV, and HAstV were not detected. EV infection was detected in 5.5% (16/288), and five samples successful genotyped: echovirus 3 (E3) (1/5), coxsackie virus A6 (CVA6) (1/5), and coxsackie virus B4 (CVB4) (3/5). Meningitis was the main syndrome observed (12/16; 75%). CVA6, CVB4, and E3 were identified associated with aseptic meningitis. Reports of CVA6 associated with aseptic meningitis are rare, E3 had not been previously reported in Brazil, and epidemiological data on CVB4 in the country is virtually unknown. The present investigation illustrates the circulation of diverse EV types in a small regional sample set and in a short period of time, highlighting the importance of an active EV surveillance system in CNS infections. Enhanced understanding of undiagnosed CNS infections will assist in public health and health care planning.

Alphacoronavirus Detection in Lungs, Liver, and Intestines of Bats from Brazil.

Bats are flying mammals distributed worldwide known to host several types of Coronavirus (CoV). Since they were reported as the probable source of spillover of highly pathogenic CoV into the human population, investigating the circulation of this virus in bats around the world became of great importance. We analyzed samples from 103 bats from two distinct regions in Brazil. Coronavirus from the Alphacoronavirus genus was detected in 12 animals, 11 from São José do Rio Preto-SP region and 1 from Barreiras-BA region, resulting in a prevalence of 17.18% and 2.56% respectively. The virus was detected not only in intestines but also in lungs and liver. Phylogenetic analysis based on nsP12 genomic region suggests that the sequences group according to host family and sampling location. Studies on the circulation of these viruses in bats remain important to understand the ecology and evolutionary relationship of these pathogens.

Applying a pan-flavivirus RT-qPCR assay in Brazilian public health surveillance.

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.

Seroprevalence for dengue virus in a hyperendemic area and associated socioeconomic and demographic factors using a cross-sectional design and a geostatistical approach, state of São Paulo, Brazil.

BACKGROUND: São José do Rio Preto is one of the cities of the state of São Paulo, Brazil, that is hyperendemic for dengue, with the presence of the four dengue serotypes. OBJECTIVES: to calculate dengue seroprevalence in a neighbourhood of São José do Rio Preto and identify if socioeconomic and demographic covariates are associated with dengue seropositivity. METHODS: A cohort study to evaluate dengue seroprevalence and incidence and associated factors on people aged 10 years or older, was assembled in Vila Toninho neighbourhood, São José do Rio Preto. The participant enrolment occurred from October 2015 to March 2016 (the first wave of the cohort study), when blood samples were collected for serological test (ELISA IgG anti-DENV) and questionnaires were administrated on socio-demographic variables. We evaluated the data collected in this first wave using a cross-sectional design. We considered seropositive the participants that were positive in the serological test (seronegative otherwise). We modelled the seroprevalence with a logistic regression in a geostatistical approach. The Bayesian inference was made using integrated nested Laplace approximations (INLA) coupled with the Stochastic Partial Differential Equation method (SPDE). RESULTS: We found 986 seropositive individuals for DENV in 1322 individuals surveyed in the study area in the first wave of the cohort study, corresponding to a seroprevalence of 74.6% (95%CI: 72.2-76.9). Between the population that said never had dengue fever, 68.4% (566/828) were dengue seropositive. Older people, non-white and living in a house (instead of in an apartment), were positively associated with dengue seropositivity. We adjusted for the other socioeconomic and demographic covariates, and accounted for residual spatial dependence between observations, which was found to present up to 800 m. CONCLUSIONS: Only one in four people aged 10 years or older did not have contact with any of the serotypes of dengue virus in Vila Toninho neighbourhood in São José do Rio Preto. Age, race and type of house were associated with the occurrence of the disease. The use of INLA in a geostatistical approach in a Bayesian context allowed us to take into account the spatial dependence between the observations and identify the associated covariates to dengue seroprevalence.

Origin, tempo, and mode of the spread of DENV-4 Genotype IIB across the state of São Paulo, Brazil during the 2012-2013 outbreak.

BACKGROUND: Dengue virus type 4 (DENV-4) was first reported in Brazil in 1982 and since then no more cases were detected again in Brazil until 2010, when the virus was reintroduced. Over the following years, the virus spread to several Brazilian states and resulted in about 1,400,000 dengue cases, in 2013. The largest number of cases were documented in the Southeast macro-region. OBJECTIVES: To determine the phylogeography of DENV-4 Genotype IIB strains isolated during the epidemics in 2012-2013 in São Paulo, Brazil, we aimed to contextualise the contribution of viruses sampled in different localities across the overall movement of DENV-4 in Brazil. METHODS: Based on the envelope gene sequences retrieved from GenBank, we employed a Bayesian phylogeographic approach to assess the spatiotemporal dynamics of DENV-4 Genotype IIB in São Paulo, Brazil. FINDINGS: The dispersal dynamics of DENV-4 Genotype IIB in Brazil indicated Rio de Janeiro and Mato Grosso states as the most likely routes toward São Paulo before the 2012-2013 outbreak. Likewise, Guarujá and São José do Rio Preto facilitated viral spread and transmission to other localities in the South and Southeast macro-regions in Brazil. CONCLUSIONS: The spread pattern of DENV-4 Genotype IIB strains across the country supports two independent introductions of the virus in São Paulo in a short period of time. Furthermore, São Paulo appears to have played a pivotal role in the dissemination of DENV-4 to other locations in Brazil.

External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil.

We conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is needed to adequately estimate risk for Zika virus-associated disease and determine patient care.

Comparison between the traditional (1997) and revised (2009) WHO classifications of dengue disease: a retrospective study of 30 670 patients.

OBJECTIVE: To compare WHO's traditional (1997) and revised (2009) guidelines for dengue classification, using a large sample of patients of all ages with varying clinical conditions from a dengue-endemic area in Brazil. METHODS: We compared 30 670 laboratory-confirmed dengue cases (1998-2012) using both WHO's dengue classification guidelines. Stereotype ordinal logistic regressions were used to analyse the association between patients' demographics and signs and symptoms related to dengue infection severity, as defined in the 1997 and 2009 guidelines. We then compared the degree of agreement in dengue classification of both guidelines. RESULTS: Dengue signs and symptoms in patients were poorly correlated to disease severity as defined by both guidelines (Cramer's V test <0.2). Hypotensive shock was the exception for both classifications, presenting dependence (Z = 56.42; P < 0.001, and Z = 55.24; P < 0.001) and high agreement (Cramers's V = 1; P < 0.001, and Cramers's V = 0.97; P < 0.001) for WHO 1997 and 2009, respectively. Last, we also found substantial agreement in disease classification between both guidelines (Kendall tau-b = 0.79; P < 0.001), although 2009 guidelines were more sensitive in the detection of severe cases. CONCLUSIONS: We hope our results will inform the debate about dengue classification guidelines, particularly concerning clinical value, study comparability, and ways in which future guidelines can support the clinical management of dengue. Our results suggest that caution should be taken when using WHO guidelines to assess dengue severity to improve clinical management of patients.