Rafael F. O. Franca

Initial Description of the Presumed Congenital Zika Syndrome.

OBJECTIVES To provide an initial description of the congenital syndrome presumably associated with infection by Zika virus compared with other syndromes including congenital infections of established etiologies. METHODS We provide an overview of a published case series of 35 cases, a clinical series of 104 cases, and published and unpublished reports of clinical and laboratory findings describing cases diagnosed since the beginning of the epidemic of microcephaly in Brazil. RESULTS About 60% to 70% of mothers report rash during pregnancy; mainly in the first trimester. Principal features are microcephaly, facial disproportionality, cutis girata, hypertonia/spasticity, hyperreflexia, and irritability; abnormal neuroimages include calcifications, ventriculomegaly, and lissencephaly. Hearing and visual abnormalities may be present. CONCLUSIONS Preliminary data suggest that severe congenital abnormalities are linked to Zika virus infection. Cases have severe abnormalities, and although sharing many characteristics with congenital abnormalities associated with other viral infections, abnormalities presumably linked to the Zika virus may have distinguishing characteristics. These severe neurologic abnormalities may result in marked mental retardation and motor disabilities for many surviving offspring. POLICY IMPLICATIONS Affected nations need to prepare to provide complex and costly multidisciplinary care that children diagnosed with this new congenital syndrome will require.

Establishment and cryptic transmission of Zika virus in Brazil and the Americas

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

Background The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. Methodology/Principal findings We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. Conclusions/Significance The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.

Zika virus replication in the mosquito Culex quinquefasciatus in Brazil

Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated.

First International Workshop on Zika Virus Held by Oswaldo Cruz Foundation FIOCRUZ in Northeast Brazil March 2016 – A Meeting Report

In the first two days of March 2016, an International Workshop on Zika Virus was held in the city of Recife, Brazil, hosted by the Oswaldo Cruz Foundation (Fiocruz). The workshop took place at the Research Institute Aggeu Magalhaes (IAM), the state of Pernambuco research branch of Fiocruz, responsible for hosting the meeting and its organization. Representatives from several institutions working on Zika virus science attended, including researchers and academics from several other Fiocruz research entities (based in Rio de Janeiro, Belo Horizonte and Bahia), from major Brazilian Universities (University of Sao Paulo, Federal University of Rio de Janeiro and others) and from other Brazilian research centers (such as the Evandro Chagas Institute—IEC). Foreign participants included representatives from the National Institute of Allergy and Infectious Diseases NIAID-USA and the University of Glasgow, among others. The Workshop brought together more than 600 participants and was accessible, live, through the web (over four thousand accessions from 26 countries). The purpose of the meeting was to provide an updated view on Zika virus in Brazil and to discuss recent advances and challenges on its research. With the recent Zika virus outbreak in Northeast Brazil and especially the remarkable increase in newborns suffering a congenital disorder termed microcephaly—a rare neurological condition in which an infants head is significantly smaller than the heads of other children of the same age—recently linked to Zika virus infection during pregnancy [1–5], the Brazilian public health authorities declared a National Public Health Emergency on Nov 11, 2015. This was followed by the recognition by the World Health Organization—WHO, on February 1, 2016, of the cluster of microcephaly cases and other neurological disorders as a health emergency, declaring a Global Emergency and the spread of the Zika virus an “extraordinary event” with a public health threat to other parts of the world (source: http://www.who.int/emergencies/zika-virus/en/). During the meeting, special sessions were organized by experts in the following research fields: Arboviruses, Zika and vaccines–discussed data regarding different circulating arthropod borne viruses in Brazil (such as chikungunya and dengue), new data about Zika virus biology, in vitro models of infection and also strategies for vaccine development; Biology of the virus vector interaction–focused on new findings on virus vector studies, including an evaluation of potential vectors for Zika virus and countermeasures to mosquito control; Clinical–with the intention of discussing the last clinical findings on Zika virus infection and major birth congenital malformations; Diagnosis—plotted new strategies to improve laboratory tests, development of technological products and also a historical update of Zika virus identification in Brazil; Epidemiology—this session was designed to provide an update on the clinical epidemiological studies under development in Brazil, with a presentation of the data on a case-control investigation of microcephaly cases and cohort studies. The first day the meeting opened with a talk from the IEC team on Zika virus infection and the global challenge to control its spread. Subsequent talks covered the clinical manifestations presented by Zika infected individuals, from a medical perspective. Medical doctors from Northeast Brazil reported that the major clinical findings on microcephaly confirmed cases were calcifications (approximately 98%), ventriculomegalia and disorder of cortical development, including simplification of gyrus and lissencephaly with a lower proportion of other abnormalities such as pachygyria. Official data from the Brazilian Ministry of Health confirmed that currently Zika virus is circulating in 22 of the 27 states in Brazil, with two confirmed deaths (Fig 1). By March 1, a total of 5.909 cases of microcephaly were reported and are currently under investigation. From 641 confirmed microcephaly cases, the number of newborn deaths reached 139 (stillbirths and/or newborn deaths) and from the total number of microcephaly cases investigated, 82 were confirmed to be Zika by laboratory tests (RT-qPCR and/or serology). Most of the cases were reported from the state of Pernambuco (1.672 cases) (source: Brazilian Ministry of Health). Fig 1 Brazilian federative units with Zika documented autochthonous cases (triangles) and number of microcephaly confirmed cases in each federal state (color shading of the affected Brazilian states) (source: Brazilian Ministry of Health and Secretary of Vigilance ... On the Diagnosis panel, the discussions were mainly on the development of new laboratory tools. Given the extensive cross reactivity with other circulating arboviruses in Brazil, the lack of reliable and specific serologic tests for Zika virus represents a major challenge. Currently, only two serological tests are licensed for Zika virus diagnosis in Brazil (Anti-Zika Virus ELISA IgG/IgM and IIFT Arboviral Fever Mosaic 2 IgG/IgM, both from EUROIMMUN). In addition to serological tests, Fiocruz is providing a recently licensed multiplex Dengue/Zika and Chikungunya assay based on NAT technology (nucleic acid test or nucleic acid amplification test). Discussions about development and characterization of monoclonal antibodies and recombinant proteins, as biotechnological products to be applied on different platforms for Zika virus diagnostic, occurred during the event, however no new kits are being licensed at the moment, which could be explained, in part, by the lack of reliable serum panels for kits validation. Researchers from IAM investigating the microcephaly cases reported the detection of Zika virus specific IgM in roughly 90% of the samples of cerebrospinal fluid (CSF) tested from microcephalic newborns, which strongly correlates Zika virus infection with brain malformation. The team from the Oswaldo Cruz Institute (IOC—a Fiocruz branch from Rio de Janeiro) provided results on Zika virus identification from several body fluids such as urine, semen (up to 2 months after symptoms), saliva and CSF. The data showed that Zika virus found in unusual samples (urine, saliva, semen) can persist for longer time periods than in the blood. So far, however, only sexual, perinatal and vector transmissions have been documented for Zika virus. Therefore, is concerning the fact that Zika virus detection on semen for long periods after the initial infection has serious implications on sexual transmission. Although organ transplantation and blood transfusion are probable transmission routes, there are no reports yet in the literature [6–11]. Despite the fact that new Zika candidates vaccines may not enter clinical trials until the next 2 or 3 years, scientific efforts to achieve a safe and efficient Zika vaccine are crucial to counteract this new emerged viral disease. Vaccine development is still very preliminary and to the moment only pre clinical studies are in course. On vaccine discussion topic, scientists presented different strategies for vaccine development, based on several immunization platforms, such as viral chimeras using the vaccinal Yellow Fever 17D strain, virus attenuation by reverse genetics (infectious clones) and subunit vaccines (DNA and/or recombinant proteins). Mosquitoes from the genus Aedes (Ae. albopictus and Ae. Aegypti) are the main species responsible for Zika virus transmission in Brazil [12,13]. During the Biology of the Virus Vector Interaction session, researchers from IAM reviewed the evidence regarding Zika virus transmission and presented results derived from laboratory experiments that implicate other mosquito species as potential vectors involved in the Zika virus life cycle. Using mosquitos artificially fed with Zika virus infected blood, they were able to detect the virus in the salivary glands of Culex quinquefasciatus 7 and 15 days post-feeding, confirming a high infection rate of 100% and 67%, respectively. In addition, advances and new approaches for mosquito control were presented in the session and challenging aspects for the implementation of control measures discussed, taking into account the socio-economical and environmental conditions of urban areas in Brazil. Since its introduction in Brazil, Zika virus infection has spread rapidly through the tropical Americas and we hypothesize that sequential flaviviruses exposures can represent a complicating factor for its spread, especially in Brazil where rates for prior dengue infection are extremely high. Currently, a number of independent groups are developing projects on Zika virus investigation in Brazil. Recently, Fiocruz (the largest, health related, research institute in Latin America, with a total of 18 units scattered throughout different regions in Brazil) established a National Network Zika Committee through the integration of efforts carried out by several of its research branches. These efforts are focusing on the development of better diagnostic tools, vaccine research, virus genetic studies, immunopathologic studies on Zika patients and research on vector populations and control. Currently, Zika virus is still expanding within the Brazilian territory and in other countries from Central and South America, probably due to the availability/abundance of vectors and the challenges in achieving efficient levels of vector reduction with a consequent impact on virus transmission. It is currently very difficult to determine how and to where will the virus spread over time. Assumptions by field specialists testify that when herd immunity reaches a level of ~80%, Zika virus transmission could then drop, however this is only speculative and based on previous data from recent outbreaks of dengue and chikungunya [14]. Serious funding and science investments are thus critical in order to face this newly emerged disease.

Zika virus tropism and interactions in myelinating neural cell cultures: CNS cells and myelin are preferentially affected

The recent global outbreak of Zika virus (ZIKV) infection has been linked to severe neurological disorders affecting the peripheral and central nervous systems (PNS and CNS, respectively). The pathobiology underlying these diverse clinical phenotypes are the subject of intense research; however, even the principal neural cell types vulnerable to productive Zika infection remain poorly characterised. Here we used CNS and PNS myelinating cultures from wild type and Ifnar1 knockout mice to examine neuronal and glial tropism and short-term consequences of direct infection with a Brazilian variant of ZIKV. Cell cultures were infected pre- or post-myelination for various intervals, then stained with cell-type and ZIKV-specific antibodies. In bypassing systemic immunity using ex vivo culture, and the type I interferon response in Ifnar1 deficient cells, we were able to evaluate the intrinsic infectivity of neural cells. Through systematic quantification of ZIKV infected cells in myelinating cultures, we found that ZIKV infection is enhanced in the absence of the type I interferon responses and that CNS cells are considerably more susceptible to infection than PNS cells. In particular, we demonstrate that CNS axons and myelinating oligodendrocytes are especially vulnerable to injury. These results have implications for understanding the pathobiology of neurological symptoms associated with ZIKV infection. Furthermore, we provide a quantifiable ex vivo infection model that can be used for fundamental and therapeutic studies on viral neuroinvasion and its consequences.

Joint production of IL-22 participates in the initial phase of antigen-induced arthritis through IL-1β production

IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neutrophil articular infiltration, joint pain and the progressive destruction of cartilage and bone. IL-22 is a key effector molecule that plays a critical role in autoimmune diseases. However, the function of IL-22 in the pathogenesis of RA remains controversial. In this study, we investigated the role of IL-22 in the early phase of antigen-induced arthritis (AIA) in mice.MethodsAIA was induced in C57BL/6, IL-22−/−, ASC−/− and IL-1R1−/− immunized mice challenged intra-articularly with methylated bovine serum albumin (mBSA). Expression of IL-22 in synovial membranes was determined by RT-PCR. Articular hypernociception was evaluated using an electronic von Frey. Neutrophil recruitment and histopathological analyses were assessed in inflamed knee joint. Joint levels of inflammatory mediators and mBSA-specific IgG concentration in the serum were measured by ELISA.ResultsThe IL-22 mRNA expression and protein levels in synovial tissue were increased during the onset of AIA. In addition, pharmacological inhibition (anti-IL-22 antibody) and genetic deficiency (IL-22−/− mice) reduced articular pain and neutrophil migration in arthritic mice. Consistent with these findings, recombinant IL-22 joint administration promoted articular inflammation per se in WT mice, restoring joint nociception and neutrophil infiltration in IL-22−/− mice. Moreover, IL-22-deficient mice showed reduced synovitis (inflammatory cell influx) and lower joint IL-1β levels, whereas the production of IL-17, MCP-1/CCL2, and KC/CXCL1 and the humoral immune response were similar, compared with WT mice. Corroborating these results, the exogenous administration of IL-22 into the joints induced IL-1β production in WT mice and reestablished IL-1β production in IL-22−/− mice challenged with mBSA. Additionally, IL-1R1−/− mice showed attenuated inflammatory features induced by mBSA or IL-22 challenge. Articular nociception and neutrophil migration induced by IL-22 were also reduced in ASC−/− mice.ConclusionsThese results suggest that IL-22 plays a pro-inflammatory/pathogenic role in the onset of AIA through an ASC-dependent stimulation of IL-1β production.

IL-33 signaling is essential to attenuate viral-induced encephalitis development by downregulating iNOS expression in the central nervous system

BackgroundViral encephalitis is a common cause of lethal infections in humans, and several different viruses are documented to be responsible. Rocio virus is a flavivirus that causes a severe lethal encephalitis syndrome in humans and also mice, providing an interesting model to study the CNS compartmentalized immune response. Interleukin 33 (IL-33), a member of the IL-1 family, is an immunomodulatory cytokine that is highly expressed in the CNS. However, the role of IL-33 on viral encephalitis remains unclear. Therefore, we aimed to explore how the IL-33/ST2 axis regulates the local immune response during Rocio virus infection.MethodsWild-type (WT), ST2 (ST2−/−), and nitric oxide synthase-deficient mice (iNOS−/−) and Stat6 (Stat6−/−)-deficient mice were infected with different concentrations of the Rocio virus by intraperitoneal route, the cytokine mRNA level in CNS was analyzed by qPCR, and cellular immunophenotyping was performed on infected mice by the flow cytometry of isolated CNS mononuclear cells.ResultsWe have shown that the mRNA expression of IL-33 and ST2 receptors is increased in the CNS of Rocio virus-infected WT mice and that ST2−/− mice showed increased susceptibility to infection. ST2 deficiency was correlated with increased tissue pathology, cellular infiltration, and tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA levels and higher viral load in the CNS, compared with wild-type mice. The increased Th1 cytokine levels released in the CNS acted on infiltrating macrophages, as evidenced by flow cytometry characterization of cellular infiltrates, inducing the expression of iNOS, contributing to brain injury. Moreover, iNOS−/− mice were more resistant to Rocio virus encephalitis, presenting a lower clinical score and reduced mortality rate, despite the increased tissue pathology.ConclusionsWe provide evidences of a specific role for IL-33 receptor signaling in nitric oxide induction through local IFN-γ modulation, suggesting that nitric oxide overproduction might have an important role in the progression of experimental viral encephalitis.

The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks zika virus replication

Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC50) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation.

Expression and activity of NOD1 and NOD2/RIPK2 signalling in mononuclear cells from patients with rheumatoid arthritis

Objectives: The aim of this study was to analyse the expression and function of nucleotide-binding oligomerization domain (NOD)1 and NOD2 in isolated cells of patients with rheumatoid arthritis (RA). Method: mRNA expression levels of NOD1, NOD2, and receptor-interacting serine/threonine kinase 2 (RIPK2) genes were determined by quantitative polymerase chain reaction (qPCR) in peripheral blood mononuclear cells (PBMCs) and synovial fluid T cells (SFTCs) isolated from RA and osteoarthritis (OA) patients. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in plasma and cell culture supernatants. The stimulatory effect of RA SF was assessed by an in-vitro NOD2 activation assay using nuclear factor kappa B (NF-κB) luciferase-transfected cells. Results: A significantly higher level of NOD2 and RIPK2 mRNA expression, but not NOD1, was observed on PBMCs and SFTCs isolated from RA patients compared to the OA control group. In addition, the NOD2 pathway up-regulation was functional, as stimulation of PBMCs with muramyl dipeptide (MDP) induced the production of higher amounts of tumour necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1β compared with OA PBMCs. Incubation of PBMCs from healthy donors with recombinant TNF-α or RA serum induced the expression of NOD2 mRNA. Finally, SF isolated from RA patients is able to activate the NF-κB signalling pathway in HEK293T-transfected cells in a NOD2-dependent manner. Conclusions: Our findings suggest that NOD2/RIPK2 signalling is up-regulated in immune cells of RA patients. Moreover, it seems that there is a NOD2 agonist in the SF of RA patients. Therefore, NOD2/RIPK2 activation can modulate the innate immune response and may play a role in the perpetuation of the inflammatory response in RA.