The spectrum of neurological disease associated with Zika and chikungunya viruses in adults in Rio de Janeiro, Brazil: A case series.

BACKGROUND: During 2015-16 Brazil experienced the largest epidemic of Zika virus ever reported. This arthropod-borne virus (arbovirus) has been linked to Guillain-Barré syndrome (GBS) in adults but other neurological associations are uncertain. Chikungunya virus has caused outbreaks in Brazil since 2014 but associated neurological disease has rarely been reported here. We investigated adults with acute neurological disorders for Zika, chikungunya and dengue, another arbovirus circulating in Brazil. METHODS: We studied adults who had developed a new neurological condition following suspected Zika virus infection between 1st November 2015 and 1st June 2016. Cerebrospinal fluid (CSF), serum, and urine were tested for evidence of Zika, chikungunya, and dengue viruses. RESULTS: Of 35 patients studied, 22 had evidence of recent arboviral infection. Twelve had positive PCR or IgM for Zika, five of whom also had evidence for chikungunya, three for dengue, and one for all three viruses. Five of them presented with GBS; seven had presentations other than GBS, including meningoencephalitis, myelitis, radiculitis or combinations of these syndromes. Additionally, ten patients positive for chikungunya virus, two of whom also had evidence for dengue virus, presented with a similar range of neurological conditions. CONCLUSIONS: Zika virus is associated with a wide range of neurological manifestations, including central nervous system disease. Chikungunya virus appears to have an equally important association with neurological disease in Brazil, and many patients had dual infection. To understand fully the burden of Zika we must look beyond GBS, and also investigate for other co-circulating arboviruses, particularly chikungunya.

Study on the persistence of Zika virus (ZIKV) in body fluids of patients with ZIKV infection in Brazil.

BACKGROUND: Zika virus (ZIKV) has been identified in several body fluids of infected individuals. In most cases, it remained detected in blood from few days to 1 week after the onset of symptoms, and can persist longer in urine and in semen. ZIKV infection can have dramatic consequences such as microcephaly and Guillain-Barré syndrome. ZIKV sexual transmission has been documented. A better understanding of ZIKV presence and persistence across biologic compartments is needed to devise rational measures to prevent its transmission. METHODS: This observational cohort study will recruit non-pregnant participants aged 18 years and above with confirmed ZIKV infection [positive reverse transcriptase-polymerase chain reaction (RT-PCR) test in blood and/or urine]: symptomatic men and women in ZIKV infection acute phase, and their symptomatic or asymptomatic household/sexual infected contacts. Specimens of blood, urine, semen, vaginal secretion/menstrual blood, rectal swab, oral fluids, tears, sweat, urine and breast milk (if applicable) will be collected at pre-established intervals and tested for ZIKV RNA presence by RT-PCR, other co-infection (dengue, Chikungunya, HIV, hepatitis B and C, syphilis), antibody response (including immunoglobulins M and G), plaque reduction neutralization test (if simultaneously positive for ZIKV and dengue), and ZIKV culture and RNA sequencing. Data on socio-demographic characteristics and comorbidities will be collected in parallel. Participants will be followed up for 12 months. DISCUSSION: This prolonged longitudinal follow-up of ZIKV infected persons with regular biologic testing and data collection will offer a unique opportunity to investigate the presence and persistence of ZIKV in various biologic compartments, their clinical and immunological correlates as well as the possibility of ZIKV reactivation/reinfection over time. This valuable information will substantially contribute to the body of knowledge on ZIKV infection and serve as a base for the development of more effective recommendation on the prevention of ZIKV transmission. TRIAL REGISTRATION: NCT03106714 . Registration Date: April, 7, 2017.

Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus.

Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

Maternal Zika Virus Disease Severity, Virus Load, Prior Dengue Antibodies, and Their Relationship to Birth Outcomes.

BACKGROUND: Congenital Zika virus (ZIKV) syndrome is a newly identified condition resulting from infection during pregnancy. We analyzed outcome data from a mother-infant cohort in Rio de Janeiro in order to assess whether clinical severity of maternal ZIKV infection was associated with maternal virus load, prior dengue antibodies, or abnormal pregnancy/infant outcomes. METHODS: A clinical severity assessment tool was developed based on duration of fever, severity of rash, multisystem involvement, and duration of symptoms during ZIKV infection. ZIKV-RNA load was quantified by polymerase chain reaction (PCR) cycles in blood/ urine. Dengue immunoglobulin G (IgG) antibodies were measured at baseline. Adverse outcomes were defined as fetal loss or a live infant with grossly abnormal clinical or brain imaging findings. Regression models were used to study potential associations. RESULTS: 131 ZIKV-PCR positive pregnant women were scored for clinical disease severity, 6 (4.6%) had mild disease, 98 (74.8%) had moderate disease, and 27 (20.6%) severe manifestations of ZIKV infection. There were 58 (46.4%) abnormal outcomes with 9 fetal losses (7.2%) in 125 pregnancies. No associations were found between: disease severity and abnormal outcomes (P = .961; odds ratio [OR]: 1.00; 95% confidence interval [CI]: 0.796-1.270); disease severity and viral load (P = .994); viral load and adverse outcomes (P = .667; OR: 1.02; 95% CI: 0.922-1.135); or existence of prior dengue antibodies (88% subjects) with severity score, ZIKV-RNA load or adverse outcomes (P = .667; OR: 0.78; 95% CI: 0.255-2.397). CONCLUSIONS: Congenital ZIKV syndrome does not appear to be associated with maternal disease severity, ZIKV-RNA load at time of infection or existence of prior dengue antibodies.

Zika Virus Outbreak in Rio de Janeiro, Brazil: Clinical Characterization, Epidemiological and Virological Aspects.

BACKGROUND: In 2015, Brazil was faced with the cocirculation of three arboviruses of major public health importance. The emergence of Zika virus (ZIKV) presents new challenges to both clinicians and public health authorities. Overlapping clinical features between diseases caused by ZIKV, Dengue (DENV) and Chikungunya (CHIKV) and the lack of validated serological assays for ZIKV make accurate diagnosis difficult. METHODOLOGY / PRINCIPAL FINDINGS: The outpatient service for acute febrile illnesses in Fiocruz initiated a syndromic clinical observational study in 2007 to capture unusual presentations of DENV infections. In January 2015, an increase of cases with exanthematic disease was observed. Trained physicians evaluated the patients using a detailed case report form that included clinical assessment and laboratory investigations. The laboratory diagnostic algorithm included assays for detection of ZIKV, CHIKV and DENV. 364 suspected cases of Zika virus disease were identified based on clinical criteria between January and July 2015. Of these, 262 (71.9%) were tested and 119 (45.4%) were confirmed by the detection of ZIKV RNA. All of the samples with sequence information available clustered within the Asian genotype. CONCLUSIONS / SIGNIFICANCE: This is the first report of a ZIKV outbreak in the state of Rio de Janeiro, based on a large number of suspected (n = 364) and laboratory confirmed cases (n = 119). We were able to demonstrate that ZIKV was circulating in Rio de Janeiro as early as January 2015. The peak of the outbreak was documented in May/June 2015. More than half of the patients reported headache, arthralgia, myalgia, non-purulent conjunctivitis, and lower back pain, consistent with the case definition of suspected ZIKV disease issued by the Pan American Health Organization (PAHO). However, fever, when present, was low-intensity and short-termed. In our opinion, pruritus, the second most common clinical sign presented by the confirmed cases, should be added to the PAHO case definition, while fever could be given less emphasis. The emergence of ZIKV as a new pathogen for Brazil in 2015 underscores the need for clinical vigilance and strong epidemiological and laboratory surveillance.

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos.

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.