Eliane Veiga da Costa

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil

As in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. The objective of the present paper was to assess the current circulation of arboviruses in the Nhecolândia sub-region of South Pantanal, Brazil. Sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus(WEEV) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (RT-PCR) and neutralization tests for Saint Louis encephalitis virus (SLEV), EEEV, WEEV and Mayaro virus (MAYV). No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for SLEV in equines regardless of vaccine status, and 36.4% for WEEV and 47.7% for EEEV in unvaccinated horses. There was no evidence of MAYV infections. The serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the Nhecolândia sub-region in South Pantanal is an important area for detection of silent activity of arboviruses in Brazil.

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

Acute Hemorrhagic Conjunctivitis and Coxsackievirus A24v, Rio de Janeiro, Brazil, 2004

An outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Rio de Janeiro in 2004. Coxsackievirus A24v (CA24v) was identified as the etiologic agent, and partial sequences from the VP1 gene show that the isolates are closely related to CA24v viruses that previously caused AHC epidemics in South Korea and French Guiana.

RT-PCR based analysis of cell culture negative stools samples from poliomyelitis suspected cases.

BACKGROUND Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several serotypes of enterovirus which possess the potential to cause neurological disorders are not easily isolated in the cell culture systems used for the AFP diagnosis. OBJECTIVES Our goal was to look into the presence of enterovirus genomes in fecal suspensions previously considered negative by cell culture procedures, using RT-PCR. STUDY DESIGN One hundred and seventy-three fecal samples collected from AFP cases and contacts occurring in Brazil, Peru and Bolivia and tested negative regarding viral isolation, after inoculation in the cell lines RD and Hep2C, were analyzed by RT-PCR using a pair of primers which specifically detects enterovirus genome RNAs. RESULTS Twenty-six samples (15%) showed amplicons compatible with those observed for enterovirus RNA amplification. The identity of these amplicons were confirmed by nucleotide sequencing. By using RT-PCR directly in the fecal suspensions we were able to detect enterovirus RNAs in twenty-six additional samples. These samples would be considered as negative if only the standard cell-culture-based methodology had been utilized. No polioviruses were detected among the positive samples.

Molecular Characterization and Phylogenetic Study of Coxsackievirus A24v Causing Outbreaks of Acute Hemorrhagic Conjunctivitis (AHC) in Brazil

Background Coxsackievirus A24 variant (CA24v) is the most prevalent viral pathogen associated with acute hemorrhagic conjunctivitis (AHC) outbreaks. Sixteen years after its first outbreak in Brazil, this agent reemerged in 2003 in Brazil, spread to nearly all states and caused outbreaks until 2005. In 2009, a new outbreak occurred in the northeast region of the country. In this study, we performed a viral isolation in cell culture and characterized clinical samples collected from patients presenting symptoms during the outbreak of 2005 in Vitória, Espírito Santo State (ES) and the outbreak of 2009 in Recife, Pernambuco State (PE). We also performed a phylogenetic analysis of worldwide strains and all meaningful Brazilian isolates since 2003. Methods and Findings Sterile cotton swabs were used to collect eye discharges, and all 210 clinical samples were used to inoculate cell cultures. Cytopathic effects in HEp-2 cells were seen in 58 of 180 (32%) samples from Vitória and 3 of 30 (10%) samples from Recife. Phylogenetic analysis based on a fragment of the VP1 and 3C gene revealed that the CA24v causing outbreaks in Brazil during the years 2003, 2004 and 2005 evolved from Asian isolates that had caused the South Korean outbreak of AHC during the summer of 2002. However, the 2009 outbreak of AHC in Pernambuco was originated from the reintroduction of a new CA24v strain that was circulating during 2007 in Asia, where CA24v outbreaks has been continuously reported since 1970. Conclusions This study is the first phylogenetic analysis of AHC outbreaks caused by CA24v in Brazil. The results showed that Asian strains of CA24v were responsible for the outbreaks since 1987 and were independently introduced to Brazil in 2003 and 2009. Phylogenetic analysis of complete VP1 gene is a useful tool for studying the epidemiology of enteroviruses associated with outbreaks.

Genetic diversity of Echovirus 30 involved in aseptic meningitis cases in Brazil (1998-2008)

Aseptic meningitis is one of the most common neurological disorders caused by enteroviruses. Among them, Echovirus 30 (E30) is described as the main etiological agent of many outbreaks and sporadic cases. This study investigated the genomic variability of E30 isolated from the cerebrospinal fluid (CSF) of aseptic meningitis cases that occurred from 1998 to 2008 in Brazil. Over a 10‐year period (1998–2008), 302 non‐polio enteroviruses were isolated, of which 177 were identified as E30 (58.6%). Phylogenetic analysis of the complete VP1 gene (876 nt) of 48 E30 isolates was performed and compared with additional Brazilian and foreign strains. E30 VP1 sequences segregated into three distinct major groups and seven subgroups, which were linked to the isolation year. In general, sequence divergence among E30 strains ranged from 0.2% to 13.8%. A common direct ancestor for this set of E30 strains was not defined. Brazilian isolates from Group I were related genetically to a 1997 USA isolate and both may have a common origin. Group III representatives showed close relationship to the 2007 Argentinean isolates. The present results complement existing data on the molecular characterization and genetic variability of E30 and may contribute to the understanding of the epidemiology of aseptic meningitis in the region. J. Med. Virol. 83:2164–2171, 2011.

Description of a widespread outbreak of aseptic meningitis due to echovirus 30 in Rio de Janeiro state, Brazil

Echovirus 30 belongs to the genus Enterovirus and is widely associated with aseptic meningitis (AM) outbreaks. In Brazil epidemics due to this serotype were reported in several states but in Rio de Janeiro, before this study, it was only involved in sporadic episodes. We retrospectively collected data from AM notifications charts and enterovirus isolation database from Rio de Janeiro State Health Department (RJSHD) and Enterovirus Reference Laboratory in the year of 2005. An outbreak of AM was detected during March, April and May associated with a high cell culture isolation rate for echovirus 30 (17.4%). Male children with ages varying from 1 to 9 years were more affected. Of the 22 patients with confirmed echovirus 30 disease, clinical information was available in eight; fever, headache and vomiting were the most common manifestations. CSF analysis showed a typical pattern of viral infection with median of cellularity of 100 cells/mm(3) and mononuclear cell predominance in 64.7% of the cases. The median of protein and glucose levels of 49 mg/dL and 56.5 mg/dL. The fatality rate was null. Despite its benign course and the lack of treatment options, aseptic meningitis surveillance is crucial for early identification of causative agents of outbreaks, which helps to avoid additional testing and inappropriate use of antimicrobials.

Evaluation of a protocol for rapid diagnosis of enterovirus associated with acute flaccid paralysis cases

BACKGROUND The virological surveillance of acute flaccid paralysis (AFP) is a critical component of the initiative of the World Health Organization (WHO) to eradicate poliomyelitis worldwide. Furthermore rapid methods are needed either to detect or rule out the presence of polioviruses during the late stages of eradication, especially in polio-free areas. OBJECTIVES The aim of this study was to evaluate a fast protocol combining one passage (5 days) in cell culture followed by RT-PCR and molecular typing in order to detect and type poliovirus (PV) and other enteroviruses associated with AFP cases. STUDY DESIGN A total of 216 fecal suspensions from AFP suspected cases were tested by using this approach and compared with the WHO gold standard. RESULTS Using the WHO protocol enterovirus was detected in 12 out of the 216 AFP samples (5.55%) while with the proposed protocol enterovirus was detected in 15 out of the 216 AFP samples (6.94%). The additional positive samples detected by the proposed method were classified as non-polio enteroviruses (NPEV). CONCLUSIONS The proposed protocol showed higher sensitivity than the WHO gold standard, reducing the entire process of identification and typing of the isolates from the typically 14-21 days to only approximately 6-8 days.

Acute flaccid paralysis laboratorial surveillance in a polio-free country: Brazil, 2005–2014

ABSTRACT The last case of paralytic poliomyelitis caused by wild poliovirus in Brazil occurred in 1989. The interruption of the indigenous poliovirus transmission was obtained through mass immunization campaigns to eligible children and an active epidemiological and laboratorial surveillance of all cases of acute flaccid paralysis (AFP) among children under 15 y of age. This paper describes and evaluates the performance of the AFP surveillance system in different geographic areas of Brazil between 2005 and 2014, using indicators recommended by WHO. AFP surveillance indicators as well as virological investigation of polio and non-polio enteroviruses in stool samples received in the laboratory were assessed from 2005–2014. During the period, 5463 cases of AFP were investigated. Of these, 55% were males and 45% were females. Those under 5 y of age represented 48% of all cases reported and investigated. AFP notification rate was within the acceptable values with mean value of 1.3 (North), 1.4 (Northeast), 1.1 (Southern), 1.0 (Southeast) and 1.4 (Midwest) cases of AFP per 100.000 population aged 15 y as well as the adequacy of fecal specimens received in the laboratory. Sabin- related polioviruses accounted for 1.7% of the isolates while, 6.7% were non-polio enterovirus with the values ranging from 5.0% to 8.9 %. No wild-type polio was found. The AFP epidemiological and laboratorial surveillance activities have been kept at appropriate levels in Brazil. These data are a very strong indication, which supports the status of country free of polio.