Silvia Ines Sardi

Outbreak of Exanthematous Illness Associated with Zika, Chikungunya, and Dengue Viruses, Salvador, Brazil

To the Editor: Zika virus (ZIKV) has been recognized as an emerging mosquito-borne flavivirus since outbreaks were reported from Yap Island in 2007 (1), French Polynesia in 2013 (2), and Cook Island and New Caledonia in 2014 (3). It has joined dengue virus (DENV) and chikungunya virus (CHIKV) as global public health threats (4). ZIKV infection typically causes a self-limited dengue-like illness characterized by exanthema, low-grade fever, conjunctivitis, and arthralgia, and an increase in rates of Guillain-Barre syndrome have been observed during ZIKV outbreaks (5). In Brazil, clusters of cases of acute exanthematous illness have been reported from various regions since late 2014, and in April 2015, ZIKV was identified as the etiologic agent (6). In May 2015, the Brazilian Ministry of Health recognized circulation of ZIKV in Brazil. We report epidemiologic findings for an ongoing outbreak of acute exanthematous illness in the population of Salvador, the third largest city in Brazil. The Salvador Epidemiologic Surveillance Office (ESO) was first alerted to cases of an acute exanthematous illness early in 2015. Reporting of cases increased during March, and in April the ESO established 10 public emergency health centers in Salvador as sentinel units for systematic surveillance of patients with acute exanthematous illness of unknown cause. The units searched retrospectively for suspected cases by review of medical charts of patients treated since February 15, continued with prospective case detection, and submitted weekly reports of identified cases to the ESO. During February 15−June 25, a total of 14,835 cases of an indeterminate acute exanthematous illness were reported from the 12 sanitary districts in Salvador. The overall attack rate was 5.5 cases/1,000 persons (4.6 cases/1,000 men and 6.3 cases/1,000 women, 8.2 cases/1,000 children 40 years of age). The epidemic curve peaked in the first week of May, which was 1 week after molecular diagnosis of ZIKV in 8 patients residing ≈50 km from Salvador and during a period of intense media coverage of the outbreak (Figure) (6). Reporting of suspected dengue cases in Salvador did not vary substantially from that in other years and was >5 times lower: 2,630 cases, of which 165/366 (45.1%) were positive for dengue IgM, 20/590 (3.4%) positive for dengue virus nonstructural protein 1, and 1/11 (9.1%) positive for dengue virus by reverse transcription PCR (Figure). During the same period, 58 cases of suspected chikungunya were reported and 24 patients with suspected Guillain-Barre syndrome were hospitalized. Figure Reported cases of indeterminate acute exanthematous illness and suspected dengue fever in Salvador, Brazil, by date of medical care, February 15−June 25, 2015. Letters indicate specific events. A) February 15: systematic reporting of cases of ... The median age of case-patients was 26 years (interquartile range 11–39 years), but all age groups were affected, which is a pattern typical of spread of new microorganisms (or subtypes) in a susceptible population. Median duration of symptoms at time of medical attention was 1 day (interquartile range 0–3 days). All patients had exanthema and most (12,711/14,093 [90.2%]) had pruritus. Fever (4,841/13,786, 35.1%), arthralgia (278/1,048 [26.5%]), headache (3,446/13,503 [25.6%]), and myalgia (223/1,033 [21.6%]) were less common. Serum samples from some patients were examined for rubella IgM (2/200, 1.0% positive), rubella IgG (15/18, 83.3% positive), measles IgM (0/11, 0% positive), dengue nonstructural protein 1 (3/185, 1.6% positive), dengue IgM (17/80, 21.3% positive), parvovirus B19 IgM (0/1, 0% positive), and parvovirus B19 IgG (1/1, 100% positive). Reverse transcription PCR was performed on 58 serum samples stored at −20°C and confirmed ZIKV in 3 (5.2%) samples, CHIKV in 3 (5.2%) samples, DENV type 3 in 1 (1.7%) sample, and DENV type 4 in 1 (1.7%) sample. Identification of ZIKV, CHIKV and DENV as etiologic agents of acute exanthematous illness suggests that these 3 Aedes spp. mosquito−transmitted viruses were co-circulating in Salvador and highlights the challenge in clinically differentiating these infections during outbreaks. Although we were not able to determine the specific incidence of each virus, the low frequency of fever and arthralgia, which are indicators of dengue and chikungunya, point to ZIKV as the probable cause of several of the reported cases. Furthermore, laboratory-confirmed cases of infection with ZIKV were simultaneously identified in other cities within metropolitan Salvador (6,7) and in other states in Brazil (8). Low diagnosis of ZIKV infection is likely because viremia levels among infected patients appear to be low (9). The spread of ZIKV represents an additional challenge for public health systems, particularly because of the risk for concurrent transmission of DENV and CHIKV by the same vectors, Ae. aegypti and Ae. albopictus mosquitoes, which are abundant throughout tropical and subtropical regions. To date, the largest outbreak of chikungunya in Brazil occurred in 2014 in Feira de Santana, Bahia, ≈100 km from Salvador, where dengue is also prevalent (10). This report illustrates the potential for explosive simultaneous outbreaks of ZIKV, CHIKV, and DENV in the Western Hemisphere and the increasing public health effects of Aedes spp. mosquitoes as vectors. The apparent increase in reports of Guillain-Barre syndrome during the outbreak deserves further investigation to elucidate whether this syndrome is associated with ZIKV infection. Public health authorities in Brazil and neighboring countries should plan accordingly.

Predominance of Rotavirus Genotype G9 during the 1999, 2000, and 2002 Seasons among Hospitalized Children in the City of Salvador, Bahia, Brazil: Implications for Future Vaccine Strategies

ABSTRACT Two hundred eight of 648 (32%) diarrheal stool samples collected from hospitalized children under 5 years of age during a 3-year period (1999, 2000, and 2002) in the city of Salvador, in the state of Bahia, Brazil, were rotavirus positive. One hundred sixty-four of 208 (78.8%) rotavirus-positive samples had genotype G9 specificity, predominantly in association with P[8]. Other specificities detected were G1 (12.0%) and G4 (1.4%). Viruses with G2, G3, or P[4] specificity were not detected. Rotavirus genotype G9 predominated during each of the three seasons studied; it represented 89.2% of rotavirus strains detected in 1999, 85.3% in 2000, and 74.5% in 2002. G1 viruses (the globally most common G type) have a unique epidemiological characteristic of maintaining predominance during multiple consecutive rotavirus seasons. We have shown in this study for the first time that the G9 viruses also have a similar epidemiological characteristic, albeit for a shorter period of surveillance. The next generation of rotavirus vaccines will need to provide adequate protection against disease caused by G9 viruses.

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

ABSTRACT Metagenomic next-generation sequencing (mNGS) of samples from 15 patients with documented Zika virus (ZIKV) infection in Bahia, Brazil, from April 2015 to January 2016 identified coinfections with chikungunya virus (CHIKV) in 2 of 15 ZIKV-positive cases by PCR (13.3%). While generally nonspecific, the clinical presentation corresponding to these two CHIKV/ZIKV coinfections reflected infection by the virus present at a higher titer. Aside from CHIKV and ZIKV, coinfections of other viral pathogens were not detected. The mNGS approach is promising for differential diagnosis of acute febrile illness and identification of coinfections, although targeted arbovirus screening may be sufficient in the current ZIKV outbreak setting.

Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection, we used human neural progenitor cells (NPC), derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation, ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2+ cells was demonstrated by activation of caspases 3/7, 8 and 9, and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2+ cells was prevented by incubation with the pan-caspase inhibitor, Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities, including multipolar spindle, chromosome laggards, micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy, such as monosomy, trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain, including microcephaly.

Distinct Zika Virus Lineage in Salvador, Bahia, Brazil.

Sequencing of isolates from patients in Bahia, Brazil, where most Zika virus cases in Brazil have been reported, resulted in 11 whole and partial Zika virus genomes. Phylogenetic analyses revealed a well-supported Bahia-specific Zika virus lineage, which indicates sustained Zika virus circulation in Salvador, Bahia’s capital city, since mid-2014.

Molecular detection and genetic diversity of norovirus in hospitalized young adults with acute gastroenteritis in Bahia, Brazil

The molecular epidemiology of a recent norovirus (NoV) outbreak in Brazil performed by comparative analysis with Genebank NoV sequences showed that the GII.4 strain was responsible for 72.5% of all NoV-positive cases (58/80). Other detected NoV strains included GII.3 (7/80; 8.8%) and GII.9 (8/80; 10%). This is the first outbreak reported in Bahia state, Brazil, during June–July of 2006, where NoV was identified as the principal etiologic agent in hospitalized young adults with acute gastroenteritis symptoms. These findings suggest that GII.4 is a predominant circulating genotype in NoV outbreaks in Brazil.

Neonatal encephalitis due to Chikungunya vertical transmission: First report in Brazil

Purpose To report the first case of Chikungunya encephalitis acquired in the perinatal period during the current outbreak in Brazil Methods Case report. Results A male neonate with 3500 g developed macular erythematous rash, hypoactivity and fever progressing to generalized seizures. His mother had experienced a disseminated rash and fever before delivery. EEG showed diffuse slowing and cranial NMR was suggestive of encephalitis. Rt-PCR for Chikungunya virus (CHIKV) was positive in cerebrospinal fluid (CSF), blood, urine and saliva. The newborn was discharged home with neurological improvement. Conclusion We report the first case of a perinatal CHIKV infection associated with a rapidly evolving encephalitis and an extensive dissemination of the virus as documented by positive rt-PCR results in CSF, blood, urine and saliva in the present outbreak in Brazil. In countries experiencing outbreaks of CHIKV infections, clinicians and neonatologists must be familiar with the possibility of the occurrence of neurologic complications and its possible consequences.

Serological survey for bovine herpesvirus 1 in cattle from different regions in the state of Bahia, Brazil

A presenca de anticorpos contra Herpesvirus Bovino tipo 1 (HBV-1) foi avaliada em bovinos jovens provenientes de quinze municipios diferentes, distribuidos nas regioes Norte, Sul e Central do estado da Bahia. Foram coletadas amostras de sangue de bovinos de 1 a 4 anos de idade, nao vacinados contra HBV-1 e sem previo diagnostico de infeccao pelo virus HBV-1. O total de amostras correspondeu a 558 soros, equivalente a 10% da populacao bovina de cada fazenda. Os 558 soros foram submetidos a tecnica de Microssoroneutralizacao (MSN) em microplacas, utilizando-se monocamadas de celulas MDBK e a cepa de HBV-1 Los Angeles, e ao teste de ELISA comercialmente adquirido: HerdCheck-IDDEX. Os resultados da amostragem feito pelo teste de ELISA mostraram que 56% (314/558) dos soros foram positivos e 44% (244/558), negativos. A tecnica de MSN mostrou que 48% (269/558) dos soros foram negativos e 52% (289/558) foram positivos. Destes soros positivos, 54% tiveram titulos soroneutralizantes entre 4-16, 35% entre 32-64, e 11% ³ 64. Os resultados sugerem que o HBV-1 esteja circulando ativamente na populacao bovina jovem. Por outra parte, a alta difusao do virus em animais jovens representaria uma fonte continua de infeccao viral para o rebanho.

Molecular characterization of group A rotavirus isolates obtained from hospitalized children in Salvador, Bahia, Brazil

Rotavirus is a major cause of infectious diarrhea in infants and young children. The objective of this study was to characterize the genotypes of Human Rotavirus found in children hospitalized with acute diarrhea in the Pediatric Hospital Prof. Hosannah de Oliveira of the UFBA in Salvador, Bahia, Brazil, during the years of 1999, 2000 and 2002. Fecal samples were analyzed (n=358) by methods EIARA and SDS-PAGE for detection of Rotavirus. Positive samples of one or two of these methods (n=168) were submitted to RT-PCR and Multiplex-Nested PCR to determine genotypes G and P. A hundred sixty-eight (46.9%) samples were positive and 190 (53.1%) negative. Only 17 (4.7%) samples had divergent results. The distribution of genotypes G during the first year, showed that the genotype G9 was present in 96,8% of the analyzed samples, in the second year, it was responsible for 96% and in the third year, 88,1%. The characterization of genotypes P demonstrated that the genotype P1A[8] was the most outstanding in all years. In this study we discuss the benefit to control the genotypes of Rotavirus through the molecular characterization for the development of potential vaccines.

N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection

ABSTRACT Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration. IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo. These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment. IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo. These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.