Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Evidence for multiple sylvatic transmission cycles during the 2016-2017 yellow fever virus outbreak, Brazil.

OBJECTIVES: Since December 2016, Brazil has experienced an unusually large outbreak of yellow fever (YF). Whether urban transmission may contribute to the extent of the outbreak is unclear. The objective of this study was to characterize YF virus (YFV) genomes and to identify spatial patterns to determine the distribution and origin of YF cases in Minas Gerais, Espírito Santo and Rio de Janeiro, the most affected Brazilian states during the current YFV outbreak. METHODS: We characterized near-complete YFV genomes from 14 human cases and two nonhuman primates (NHP), sampled from February to April 2017, retrieved epidemiologic data of cases and used a geographic information system to investigate the geospatial spread of YFV. RESULTS: All YFV strains were closely related. On the basis of signature mutations, we identified two cocirculating YFV clusters. One was restricted to the hinterland of Espírito Santo state, and another formed a coastal cluster encompassing several hundred kilometers. Both clusters comprised strains from humans living in rural areas and NHP. Another NHP lineage clustered in a basal relationship. No signs of adaptation of YFV strains to human hosts were detected. CONCLUSIONS: Our data suggest sylvatic transmission during the current outbreak. Additionally, cocirculation of two distinct YFV clades occurring in humans and NHP suggests the existence of multiple sylvatic transmission cycles. Increased detection of YFV might be facilitated by raised awareness for arbovirus-mediated disease after Zika and chikungunya virus outbreaks. Further surveillance is required, as reemergence of YFV from NHPs might continue and facilitate the appearance of urban transmission cycles.

Antibody-based assay discriminates Zika virus infection from other flaviviruses.

Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged recently as a global health threat, causing a pandemic in the Americas. ZIKV infection mostly causes mild disease, but is linked to devastating congenital birth defects and Guillain-Barré syndrome in adults. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to differentially detect ZIKV and dengue virus (DENV) infections, accentuating the urgent need for a specific and sensitive serological test. We previously generated a ZIKV nonstructural protein 1 (NS1)-specific human monoclonal antibody, which we used to develop an NS1-based competition ELISA. Well-characterized samples from RT-PCR-confirmed patients with Zika and individuals exposed to other flavivirus infections or vaccination were used in a comprehensive analysis to determine the sensitivity and specificity of the NS1 blockade-of-binding (BOB) assay, which was established in laboratories in five countries (Nicaragua, Brazil, Italy, United Kingdom, and Switzerland). Of 158 sera/plasma from RT-PCR-confirmed ZIKV infections, 145 (91.8%) yielded greater than 50% inhibition. Of 171 patients with primary or secondary DENV infections, 152 (88.9%) scored negative. When the control group was extended to patients infected by other flaviviruses, other viruses, or healthy donors (n = 540), the specificity was 95.9%. We also analyzed longitudinal samples from DENV-immune and DENV-naive ZIKV infections and found inhibition was achieved within 10 d postonset of illness and maintained over time. Thus, the Zika NS1 BOB assay is sensitive, specific, robust, simple, low-cost, and accessible, and can detect recent and past ZIKV infections for surveillance, seroprevalence studies, and intervention trials.

Aedes aegypti in Brazil: genetically differentiated populations with high susceptibility to dengue and yellow fever viruses.

Aedes aegypti was eliminated from Brazil in 1955, but re-infested the country in the 1970s. Dengue outbreaks have occurred since 1981 and became endemic in several cities in Brazil after 1986. Urban yellow fever has not occurred since 1942, and only jungle yellow fever cases have been reported. A population genetic analysis using isoenzyme variation combined with an evaluation of susceptibility to both yellow fever and dengue 2 viruses was conducted among 23 A. aegypti samples from 13 Brazilian states. We demonstrated that experimental infection rates of A. aegypti for both dengue and yellow fever viruses (YFV) are high and heterogeneous, and samples collected in the endemic and transition areas of sylvatic yellow fever were highly susceptible to yellow fever virus. Boa Vista, a border city between Brazil and Venezuela, and Rio de Janeiro in the Southeast region are considered as the most important entry points for dengue dissemination. Considering the high densities of A. aegypti, and its high susceptibility to dengue and yellow fever viruses, the risk of dengue epidemics and yellow fever urbanization in Brazil is more real than ever.

Outbreak of jaundice and hemorrhagic fever in the Southeast of Brazil in 2001: detection and molecular characterization of yellow fever virus.

Between January and March 2001, an outbreak of jaundice and hemorrhagic fever occurred in the state of Minas Gerais, Southeast region of Brazil, in which a mortality rate of 53% was reported. Seroconversion, virus isolation, histopathological and immunohistochemical findings, and reverse transcription-polymerase chain reaction (RT-PCR) identified yellow fever virus (YFV) as the etiological agent responsible for the outbreak. Partial nucleotide sequence analysis from a fragment of the YFV genome spanning parts of nonstructural (NS) 5 gene and 3' noncoding region (3' UTR) showed that the YFV involved in this outbreak belongs to South American genotype I and differs from the Brazilian virus identified in 1996.

Dengue virus type 3 in Rio de Janeiro, Brazil.

Dengue virus type 3 was isolated for the first time in the country as an indigenous case from a 40 year-old woman presenting signs and symptoms of a classical dengue fever in the municipality of Nova Iguaçu, State of Rio de Janeiro. This serotype has been associated with dengue haemorrhagic epidemics and the information could be used to implement appropriate prevention and control measures. Virological surveillance was essential in order to detected this new serotype.

Dengue in the State of Rio de Janeiro, Brazil, 1986-1998.

This paper presents epidemiological, laboratory, and clinical data on 12 years of dengue virus activity in the State of Rio de Janeiro from the time the disease was first confirmed virologically in April 1986 through April 1998. DEN-1 and DEN-2 viruses are the serotypes circulating in the state and were responsible for the epidemics reported during the last 12 years. The results published here show both the impact of dengue virus infections on the population and laboratory advances that have improved dengue diagnosis.

Ultrastructural and immunocytochemical study on the infection of enterovirus 71 (EV 71) in rhabdomyosarcoma (RD) cells.

Rhabdomyosarcoma monolayers were inoculated with enterovirus 71 (EV 71) at 73 degrees C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy, using routine and immunoelectronmicroscopy with polyclonal antibodies against EV 71. The location of EV 71 or its precursors was followed during the viral replicative cycle. The earliest samples (3 h postinoculation) showed a cell shape change, from elongated to rounded. At 6 h postinoculation, the presence of early virus-induced vesicles developing within the cytoplasm was pointed out, although no virus particles were observed at these stages. At 12 and 20 h postinoculation, virus particles appeared in the cytoplasm. They were found free or in clusters in the cytoplasmic matrix, between the virus-induced vesicles. EV 71 particles were also occasionally observed in the form of paracrystalline arrays situated in the vesiculated areas. The immunolabel (gold beads) was found, initially, over dense cytoplasmic areas and in more advanced process at the vesiculated area and over the virus particles. Therefore the main cellular alterations observed in this infection were the shape change of the cell and the appearance of electron-dense areas (viroplasm) and smooth walled vesicles which are probably the site of the virus replication.

Enteroviruses isolated from patients with acute respiratory infections during seven years in Rio de Janeiro (1985-1991).

Enteroviruses were investigated in respiratory secretions collected from patients with acute respiratory infections (ARI) over a seven year period (1985-1991), as part of a longitudinal study of ARI aetiology. All the viruses that are most commonly associated with ARI were found in this study. Among the virus isolates, enteroviruses were only less frequent than respiratory syncytial viruses, adenoviruses and influenzaviruses. Forty five enterovirus samples were isolated from patients with either upper respiratory tract infections (URTI) or lower respiratory tract infections (LRTI). From these enterovirus isolates, thirty one samples were identified as poliovirus (n = 18) and non polio enterovirus (n = 13) by serum neutralization. Poliovirus were identified as type 1 and 2 and all of them were vaccinal strains. From thirteen non polio enterovirus, twelve were identified as echovirus serotypes 1, 2, 7, 11, 19 and 31. The remainder was identified as coxsackievirus B4.

Intratypic differentiation of polioviruses isolated from suspected cases of poliomyelitis in Brazil during the period of 1990 to 1993.

This study analyzed 3129 fecal samples derived from 1626 patients with sudden onset acute flaccid paralysis clinically compatible with poliomyelitis. The samples were collected in the period ranging from January 1990 to September 1993 in all regions of Brazil. Among the 1626 cases studied, 196 had isolation of poliovirus. Nevertheless, it was observed that some factors influenced the isolation rate and the intratypic characterization of these polioviruses. No cases of acute flaccid paralysis has been found to be etiologically related with wild polioviruses.