Daniele Freitas Henriques

Zika virus in the Americas: Early epidemiological and genetic findings

Zika virus genomes from Brazil The Zika virus outbreak is a major cause for concern in Brazil, where it has been linked with increased reports of otherwise rare birth defects and neuropathology. In a phylogenetic analysis, Faria et al. infer a single introduction of Zika to the Americas and estimated the introduction date to be about May to December 2013—some 12 months earlier than the virus was reported. This timing correlates with major events in the Brazilian cultural calendar associated with increased traveler numbers from areas where Zika virus has been circulating. A correlation was also observed between incidences of microcephaly and week 17 of pregnancy. Science, this issue p. 345 Virus sequencing indicates that Zika arrived in Brazil during the middle of 2013, coincident with a surge in air travelers. Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects

BACKGROUND Zika virus (ZIKV) was first detected in Brazil in May 2015 and the country experienced an explosive epidemic. However, recent studies indicate that the introduction of ZIKV occurred in late 2013. Cases of microcephaly and deaths associated with ZIKV infection were identified in Brazil in November, 2015. OBJECTIVES To determine the etiology of three fatal adult cases. STUDY DESIGN Here we report three fatal adult cases of ZIKV disease. ZIKV infection in these patients was confirmed by cells culture and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and by antigen detection using immunohistochemical assay. Samples of brain and other selected organs taken at autopsy from three patients were also analyzed by histopathological and immunohistological examination. RESULTS The first patient, a 36-year-old man with lupus and receiving prednisone therapy, developed a fulminant ZIKV infection. At autopsy, RT-qPCR of blood and tissues was positive for ZIKV RNA, and the virus was cultured from an organ homogenate. The second patient, a previously healthy female, 16 years of age, presented classic symptoms of Zika fever, but later developed severe thrombocytopenia, anemia and hemorrhagic manifestations and died. A blood sample taken on the seventh day of her illness was positive RT-PCR for ZIKV RNA and research in the serum was positive for antinuclear factor fine speckled (1/640), suggesting Evans syndrome (hemolytic anemia an autoimmune disorder with immune thrombocytopenic purpura) secondary to ZIKV infection. The third patient was a 20-year-old woman hospitalized with fever, pneumonia and hemorrhages, who died on 13days after admission. Histopathological changes were observed in all viscera examined. ZIKV antigens were detected by immunohistochemistry in viscera specimens of patients 1 and 3. These three cases demonstrate other potential complications of ZIKV infection, in addition to microcephaly and Guillain-Barre syndrome (GBS), and they suggest that individuals with immune suppression and/or autoimmune disorders may be at higher risk of developing severe disease, if infected with ZIKV.

Glaucoma and Congenital Zika Syndrome

Figure 1. Severe corneal edema (blue-eye) associated with an enlarged right eye (buphthalmos) typically associated with congenital glaucoma. Ocular lesions are a prominent feature of congenital Zika virus (ZIKV) infection, in addition to microcephaly and severe central nervous system defects. Such manifestations, which include chorioretinal atrophy, focal pigmented mottling, and optic nerve abnormalities, have been for the most part restricted to the posterior segment. Glaucoma is a rare sequel of congenital infections and at present, has not been described among infants exposed to ZIKV during gestation. Herein, we report a 3-month-old male infant who was born during the microcephaly outbreak in the city of Salvador, Brazil, and presented with an enlarged right eye, photophobia, and persistent tearing. During the fourth week of pregnancy, the mother had an acute illness characterized by cutaneous rash, fever, and arthralgia that lasted 3 days. An infant weighing 1.892 kg was delivered by caesarian section at 38 weeks of gestation who had severe microcephaly ( 4.54 standard deviation below the InterGrowth standard), bilateral lower extremity arthrogryposis, and according to cranial computed tomography, ventriculomegaly, diffuse parenchymal calcifications, dysgenesis of the corpus callosum, and a simplified gyral pattern. An ophthalmologic evaluation, performed 3 days after birth, found chorioretinal atrophy and focal pigmented mottling in both eyes and optic nerve hypoplasia in the right eye, signs consistent with glaucoma were not identified. Sera obtained at birth tested positive for anti-ZIKV immunoglobulin M antibodies and negative for anti-dengue virus immunoglobulin M antibodies, as well as for other causes of congenital infection. Real-time reverse transcriptase polymerase chain reaction testing of newborn blood did not detect ZIKV RNA. During an outpatient visit 95 days after birth, the infant was found to have an enlarged right eye and persistent tearing. The mother did not report additional ocular symptoms before the evaluation. The infant displayed significant irritability and severe photophobia. The right eye had an increased horizontal corneal diameter in comparison to the left eye (13 mm in the right eye vs 10 mm in the left eye; Fig 1) and an increased intraocular pressure (30 mm Hg in the right eye vs 14 mm Hg in the left eye). The right cornea was severely edematous, but the angle was gonioscopically unremarkable. The left eye demonstrated posterior embryotoxon and gonioscopy showed a white membrane in the peripheral iris extending through Schwalbe’s line. The angle was open without evidence of inflammation. The patient underwent trabeculectomy of the right eye 114 days after birth, which resulted in normalization of the ocular pressure (15 mmHg) and significant reduction of the corneal edema, tearing, and photophobia. Examination indicated mild atrophy of the iris in the right eye and retinal lesions bilaterally unchanged from the examination performed at birth. Real-time reverse transcriptase polymerase chain reaction did not detect ZIKV-specific RNA in samples of the aqueous humor and vitreous obtained during surgery. To our knowledge, this is the first report that describes glaucoma as a manifestation of congenital ZIKV infection. Clinicians should be aware of the possibility given the morbidity associated with glaucoma among infants. Furthermore, this case suggests that ZIKV may mediate damage to the anterior segment in addition to the posterior chamber of the eye. We reported a case of an infant with congenital ZIKV infection who presented with iris coloboma and lens subluxation at birth. Recently, uveitis has been reported as a manifestation of acute ZIKV infection in adults. With an infection occurring many months before birth, we did not detect viral RNA in the aqueous humor; however, further study is needed to determine whether glaucoma and anterior segment lesions are owing to the direct or indirect effects of the virus during gestation or postpartum, as well as the risk that these sequelae pose for newborn infants with congenital ZIKV infection.

Epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon region and in the State of Mato Grosso do Sul, Brazil: elevated prevalence of antibodies in horses

O presente trabalho recebeu apoio financeiro do Instituto Evandro Chagas/SVS/MS, OPAS (Carta Acordo 109/2005) e do Conselho Nacional de Desenvolvimento Cientifico e Tecnologico – CNPq (processo no. 300460/2005-8)The immunity of horses (n = 1401) against Saint Louis encephalitis virus (SLEV) was investigated in the Brazilian Amazon region (Braganca/Para, Salvaterra/Para, Macapa/Amapa and Rio Branco/Acre) and Maracaju, State of Mato Grosso do Sul, by the hemagglutination inhibition (HI) and plaque reduction neutralization (PRNT) tests. HI and neutralizing antibodies specific (monotypic reactivity, MR) for SLEV and other flaviviruses included in the tests were detected, as was cross-reactivity (CR) against flaviviruses. In the HI test, MR was observed in 248 (17.7%) serum samples, 137 of which were (55.2%) against SLEV; CR was detected in 380 (27.1%). The frequency of MR against SLEV was significantly higher in Macapa and CR was significantly higher in Salvaterra. In the PRNT, neutralization of SLEV was observed in 713 (50.9%) samples, and the prevalence of neutralizing antibodies was significantly higher in Macapa than in Salvaterra (p = 0.0083). This study adds new data regarding the immunity of horses against SLEV in Brazil, and it confirms the wide distribution of SLEV and the diversity of flaviviruses in the country, as well as the apparent absence of disease in SLEV-infected horses. Encephalitis, St. Louis; Horses; Serologic Tests; Encephalitis, Arbovirus.

Seroprevalence of flaviviruses antibodies in water buffaloes (Bubalus bubalis)in Brazilian Amazon

BackgroundThe state of Pará encompasses 26% of Brazilian Amazon where an enormous diversity of arboviruses has been found. This study aimed to assess the prevalence and distribution of hemagglutination-inhibition antibodies against antigens of six Flavivirus (yellow fever virus, Ilheus virus, Saint Louis encephalitis virus, Cacipacore virus, Bussuquara virus and Rocio virus) in water buffaloes in Pará state, Brazil. The prevalence of antibodies in these farm animals is important to determine the circulating arboviruses.FindingsAll investigated arboviruses were detected in the species studied and our results indicate that water buffaloes are susceptible to Flavivirus infection. Furthermore, there is solid evidence of active circulation of these viruses in the Brazilian Amazon.ConclusionsWater buffaloes showed higher prevalence of heterotypic antibody reactions and we hypothesized that they can serve as sentinels to detect the movement of such arboviruses in the Brazilian Amazon.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Epidemiologia do vírus da Encefalite de Saint Louis na Amazônia brasileira e no Estado do Mato Grosso do Sul, Brasil: elevada prevalência de anticorpos em equinos

O presente trabalho recebeu apoio financeiro do Instituto Evandro Chagas/SVS/MS, OPAS (Carta Acordo 109/2005) e do Conselho Nacional de Desenvolvimento Cientifico e Tecnologico – CNPq (processo no. 300460/2005-8)The immunity of horses (n = 1401) against Saint Louis encephalitis virus (SLEV) was investigated in the Brazilian Amazon region (Braganca/Para, Salvaterra/Para, Macapa/Amapa and Rio Branco/Acre) and Maracaju, State of Mato Grosso do Sul, by the hemagglutination inhibition (HI) and plaque reduction neutralization (PRNT) tests. HI and neutralizing antibodies specific (monotypic reactivity, MR) for SLEV and other flaviviruses included in the tests were detected, as was cross-reactivity (CR) against flaviviruses. In the HI test, MR was observed in 248 (17.7%) serum samples, 137 of which were (55.2%) against SLEV; CR was detected in 380 (27.1%). The frequency of MR against SLEV was significantly higher in Macapa and CR was significantly higher in Salvaterra. In the PRNT, neutralization of SLEV was observed in 713 (50.9%) samples, and the prevalence of neutralizing antibodies was significantly higher in Macapa than in Salvaterra (p = 0.0083). This study adds new data regarding the immunity of horses against SLEV in Brazil, and it confirms the wide distribution of SLEV and the diversity of flaviviruses in the country, as well as the apparent absence of disease in SLEV-infected horses. Encephalitis, St. Louis; Horses; Serologic Tests; Encephalitis, Arbovirus.

Encephalitis associated with Zika virus infection and reactivation of the varicella-zoster virus in a Brazilian child

We report a case of encephalitis associated with Zika virus infection and reactivation of varicella-zoster virus in the central nervous system of a Brazilian child. This case raises the possibility that reactivation of the latent varicella-zoster virus may be a mechanism of neurological impairment induced by acquired Zika virus infection.

Potential role of dengue virus, chikungunya virus and Zika virus in neurological diseases

This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

Seizures as a Complication of Congenital Zika Syndrome in Early Infancy

Zika virus transmission in Brazil was linked to a large outbreak of microcephaly but less is known about longer term anthropometric and neurological outcomes. We studied a cohort of infants born between October 31, 2015, and January 9, 2016, in a state maternity hospital, followed up for 101 ± 28 days by home visits. Microcephaly (< 2 standard deviations, Intergrowth standard) occurred in 62 of 412 (15%) births. Congenital Zika syndrome (CZS) was diagnosed in 29 patients. Among CZS patients, we observed a significant gain in anthropometric measures (P < 0.001) but no significant gain in percentile for these measures. The main neurological outcome was epilepsy, occurring in 48% of infants at a rate of 15.6 cases per 100 patient-months, frequently requiring multiple anti-seizure medications. The cumulative fatality rate was 7.4% (95% confidence interval: 2.1-23.4%). Health-care professionals should be alerted on the high risk of epilepsy and death associated with CZS in early infancy and the need to actively screen for seizures and initiate timely treatment.