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Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de V?rus Respirat?rios e do Sarampo. Rio de Janeiro, RJ, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Refer?ncia Regional em Enteroviroses. Ananindeua, PA, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Imunologia. Rio de Janeiro, RJ, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Imunologia. Rio de Janeiro, RJ, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Imunologia. Rio de Janeiro, RJ, Brasil. Due to the advanced stage of polio eradication, the possible role of non-polio enteroviruses (NPEVs) associated to acute flaccid paralysis (AFP) cases has been highlighted. In this study, we described epidemiological aspects of NPEVs infections associated to AFP and explore the viral genetic diversity, information still scarce in Brazil. From 2005 to 2017, 6707 stool samples were collected in the scope of the Brazilian Poliomyelitis Surveillance Program. NPEVs were isolated in 359 samples (5.3%) and 341 (94.9%) were genotyped. About 46 different NPEV types were identified with the following detection pattern EV-B?>?EV-A?>?EV-C. The major EV-types were CVA2, CV4, EV-A71, CVB3, CVB5, E6, E7, E11, CVA13 and EV-C99, which corresponds to 51.6% of the total. Uncommon types, such as CVA12, EV-90 and CVA11, were also identified. Different E6 genogroups were observed, prevailing the GenIII, despite periods of co-circulation, and replacement of genogroups along time. CVA2 sequences were classified as genotype C and data suggested its dispersion in South-American countries. CVA13 viruses belonged to cluster B and Venezuelan viruses composed a new putative cluster. This study provides extensive information on enterovirus diversity associated with AFP, reinforcing the need of tailoring current surveillance strategies to timely monitor emergence/re-emergence of NPEVs.

Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil.

Wild polioviruses still remain endemic in three countries (Afghanistan, Pakistan, and Nigeria) and re-emergency of wild polio has been reported in previously polio-free countries. Environmental surveillance has been used as a supplementary tool in monitoring the circulation of wild poliovirus (PVs) and/or vaccine-derived PVs even in the absence of acute flaccid paralysis cases. This study aimed to monitor the presence of polioviruses in wastewater samples collected at one wastewater treatment plant located in the municipality of Rio de Janeiro, Brazil. From December 2011 to June 2012 and from September to December 2012, 31 samples were collected and processed. RD and L20B cell cultures were able to isolate PVs and non-polio enteroviruses in 27/31 samples. Polioviruses were isolated in eight samples (type 1 Sabin = 1, type 2 Sabin = 5, and type 3 Sabin = 2). Vaccine-derived polioviruses were not detected nor evidence of recombination with other PVs or non-polio enterovirus serotypes were observed among the isolates. The Sabin-related serotypes 2 and 3 presented nucleotide substitutions in positions associated with the neurovirulent phenotype at the 5′-UTR. Changes in important Amino acid residues at VP1 were also observed in the serotypes 2 and 3. Environmental surveillance has been used successfully in monitoring the circulation of PVs and non-polio enteroviruses and it is of crucial importance in the final stages of the WHO global polio eradication initiative. Our results show the continuous circulation of Sabin-like PVs and non-polio enteroviruses in the analyzed area during the study period.

The human enteroviruses (HEV) are responsible for a large diversity of infections affecting humans. Most infections are asymptomatic, but these viruses can cause a wide spectrum of diseases, including severe cases involving the central nervous system. The aim of this study was to isolate and identify human enteroviruses in natural circulation in children less than 15 years of age admitted at the Mavalane General Hospital in Maputo City, Mozambique. In this study, 178 stool samples were processed, obtained during November 2011 to February 2012. Samples were inoculated onto cell culture and the isolates were identified as enterovirus by conventional RT-PCR in the 5’ non-coding region followed by partial VP1 sequence. Twenty-six (26) out of the 45 cell-culture positive samples were constituted by Enterovirus (14.6% of the total 178 samples). EV-29 was the serotype most prevalent. The results show the importance of maintaining the cell line Hep2C in the diagnosis and Enterovirus circulating in the Maputo city, Mozambique.

OBJETIVO: Avaliar características clínicas, bioquímicas, hormonais e genéticas de familiares de duas pacientes portadoras de lipodistrofia parcial familiar (FPLD) tipo 2. MATERIAIS E MÉTODOS: Foram avaliados 50 indivíduos de duas famílias brasileiras não relacionadas a partir de dois propósitos com fenótipo de FPLD. Foi confirmada a mutação no éxon 8 do gene LMNA em 18 destes e identificada a substituição em heterozigose no códon 482, resultando na mutação p.R482W. Com base na presença ou não da mutação, os indivíduos foram separados em afetados e não afetados, e comparados quanto a parâmetros clínicos, bioquímicos e hormonais. RESULTADOS: Indivíduos afetados tiveram 2,8 vezes mais chance de manifestar diabetes e síndrome dos ovários policísticos (SOP), maiores índices HOMA-IR, níveis de insulina e de triglicérides e menores níveis de leptina. Essas alterações precedem o início do diabetes, pois foram evidenciadas nos afetados diabéticos e não diabéticos. Foi constatada heterogeneidade fenotípica entre os portadores da mutação. CONCLUSÃO: A mutação no gene da LMNA é determinante de alterações clínicas, bioquímicas e hormonais que implicam deterioração metabólica nos portadores da mutação. OBJECTIVE: To evaluate clinical, biochemical, hormonal and genetic characteristics of relatives of two patients with familial partial lipodystrophy (FPLD) type 2. MATERIALS AND METHODS: Fifty subjects, members of two non-related Brazilian families from two different probands with FPLD phenotype, were evaluated. A mutation in exon 8 of LMNA gene was confirmed in 18 of them, and a heterozygous substitution at codon 482 was identified, predicting a p.R482W mutation. Based on the presence or absence of the mutation, subjects were classified in affected and unaffected, and compared in terms of clinical, biochemical and hormonal parameters. RESULTS: Affected subjects were 2.8 times more likely to manifest diabetes and PCOS, higher HOMA-IR, insulin and triglyceride levels, and lower levels of leptin. These changes preceded the onset of diabetes, because they were observed in diabetic and non-diabetic affected patients. A phenotypic heterogeneity was found among mutation carriers. CONCLUSION: A mutation in the LMNA gene is a determinant of clinical, biochemical and hormonal changes that imply in metabolic deterioration in mutation carriers.

Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Enterov?rus. Rio de Janeiro, RJ, Brazil. 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.

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.

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 ∼6–8 days.

Genetic variation of foot-and-mouth disease virus (FMDV) isolates, serotype O, recovered serially over a 1-year period from persistently infected buffalos was assessed. The persistent state was established experimentally with plaque-purified FMDV, strain O 1 Campos, in five buffalos ( Bubalus bubalis ). Viral isolates collected from esophageal–pharyngeal (EP) fluids for up to 71 weeks after infection were analyzed at different times by nucleotide sequencing and T 1 RNase oligonucleotide fingerprinting to assess variability in the VP1-coding region and in the complete genome, respectively. Genetic variation increased, although irregularly, with time after infection. The highest values observed for the VP1-coding region and for the whole genome were 2.5% and 1.8%, respectively. High rates of fixation of mutations were observed using both methodologies, reaching values of 0.65 substitutions per nucleotide per year (s/nt/y) and 0.44 s/nt/y for nucleotide sequencing and oligonucleotide fingerprinting, respectively, when selected samples recovered at close time periods were analyzed. The data herein indicate that complex mixtures of genotypes may arise during FMDV type O persistent infection in water buffalos, which can act as viral reservoirs and also represent a potential source of viral variants. These results fit within the quasi-species dynamics described for FMDV, in which viral populations are constituted by related, non-identical genomes that evolve independently from each other, and may predominate at a given time.

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.

To the Editor: Human enteroviruses (polioviruses, coxsackievirus A, coxsackievirus B, echoviruses, enterovirus 71, and newer recognized serotypes) belong to the Picornaviridae family, Enterovirus genus (1). They are common viral agents associated with a diversity of clinical manifestations, including respiratory illness; nonspecific rashes; hand, foot, and mouth disease; myocarditis; acute hemorrhagic conjunctivitis; and central nervous system (CNS) syndromes (2). Acute viral infections of the CNS are the source of a group of globally distributed diseases, which affect the population in a sporadic, endemic, or epidemic way. These infections cause a number of illnesses, particularly in children, and may result in serious sequelae; in severe cases, they can be fatal (3). Meningitis, encephalitis, acute flaccid paralysis (poliomyelitis), mononeuritis, polyneuritis, and Reye syndrome constitute most of the illnesses (4). Nonpolio enteroviruses are responsible for >80% of viral meningitis cases in which the etiologic agent is identified (2). Several of the 28 currently recognized serotypes of echovirus are found in association with these infections (3). We describe an outbreak of aseptic meningitis that occurred in southern Brazil in 2003 with echovirus 13 (E13) virus as the etiologic agent. This is the first meningitis outbreak due to E13 reported in the country. From March to April 2003, 17 children and young adults from Horizontina City (population 16,800), Rio Grande do Sul State, southern Brazil, with symptoms of meningitis, sought medical attention at the local hospital. Seven of these case-patients were linked to each other either by school or domiciliary contact. Lumbar puncture showed clear cerebrospinal fluid (CSF), which suggests a viral cause. The following symptoms were associated with patients: fever (92%), headache (84%), vomiting (79%), diarrhea, stiff neck, and fatigue (7.69% each). Patients' ages ranged from 1 to 19 years of age, with the age peak incidence in children 5–9 years of age (46%). Fifty-eight percent of patients were male. All patients recovered, and no sequelae or deaths were identified. The pattern of meningitis associated with E13 in this outbreak was clinically similar to those observed in aseptic meningitis due to other enteroviruses in previous outbreaks. For diagnostic purposes, 12 CSF and 8 fecal specimens were collected from the 17 patients with clinically suspected viral meningitis. For viral diagnosis, RD and HEp2 cells were injected with 0.2 mL of each clinical specimen (clarified fecal specimens and CSF) and examined daily for at least 7 days postinoculation. Enterovirus characteristic cytopathic effect was observed in 6 (50%) of 12 CSF samples and in 5 (62.5%) of 8 fecal samples. All isolates were typed as echovirus 13 by a reverse transcription–PCR and nucleotide sequencing of a portion of the VP1 gene (5). Before 2000, echovirus 13 was considered a rare serotype of enterovirus (6) and had never been reported in association with outbreaks (7). In the United States, before 2001, this enterovirus accounted for only 65 of the 45,000 reported enteroviral isolates (6). However, the incidence of E13 is increasing; several meningitis outbreaks have been recently reported in England, Germany, Belgium, Spain, France, Israel, and Japan (8). In spite of the temporal clustering and close contact of 7 patients, the causes of the outbreak were not completely defined and remain speculative. The sudden emergence of E13 as a prominent enterovirus associated with viral meningitis in many countries, including Brazil, demonstrates the potential of enteroviruses to circulate widely and to unpredictably cause diseases, which underscores the continued need for enterovirus surveillance. Although this specific outbreak was restricted both geographically and in terms of magnitude (only 17 cases), E13 seemed to be widely distributed in Brazil and has been detected in fecal specimens obtained from patients with acute flaccid paralysis since 1998 (C. Blal, unpub. data). Epidemiologic surveillance plays a crucial role in understanding the changing patterns of enterovirus infection and disease associations. Such knowledge may help in the control of diseases (9,10). Although identifying the enterovirus serotype does not contribute substantially to patient management, establishing the dominant virus each year or in each outbreak is essential for epidemiologic purposes.

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.

Polioviruses are the major etiological agents associated with acute flaccid paralysis (AFP). The complete genome sequence of a representative of the last wild poliovirus type 1 genotype isolated in Brazil from a paralytic poliomyelitis case is reported here.

Enteroviruses are the main etiologic agents involved in outbreaks and sporadic cases of aseptic meningitis. Viral isolation in permissive cells is a traditional method for diagnosis, but it is time consuming and may be unsuccessful due to low viral titers and because some enteroviruses do not replicate in routine cell cultures. Therefore, molecular techniques are increasingly being used for detection of these agents. In the present work, 267 cerebrospinal fluid (CSF) samples from aseptic meningitis and meningoencephalitis cases, received between 2008 and 2009, were tested by RT-PCR. These clinical samples were previously negative for virus isolation in cell culture. Enterovirus genome was detected in 59 CSF samples (22.1%) and identification was confirmed by partial nucleotide sequencing. Some demographics of the patients included in the study were analyzed. Direct detection of enterovirus genome from CSF is appropriate to increase sensitivity in aseptic meningitis and meningoencephalitis cases.

Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV), mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

To evaluate clinical, biochemical, hormonal and genetic characteristics of relatives of two patients with familial partial lipodystrophy (FPLD) type 2.Fifty subjects, members of two non-related Brazilian families from two different probands with FPLD phenotype, were evaluated. A mutation in exon 8 of LMNA gene was confirmed in 18 of them, and a heterozygous substitution at codon 482 was identified, predicting a p.R482W mutation. Based on the presence or absence of the mutation, subjects were classified in affected and unaffected, and compared in terms of clinical, biochemical and hormonal parameters.Affected subjects were 2.8 times more likely to manifest diabetes and PCOS, higher HOMA-IR, insulin and triglyceride levels, and lower levels of leptin. These changes preceded the onset of diabetes, because they were observed in diabetic and non-diabetic affected patients. A phenotypic heterogeneity was found among mutation carriers.A mutation in the LMNA gene is a determinant of clinical, biochemical and hormonal changes that imply in metabolic deterioration in mutation carriers.

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.

Background: Human adenoviruses are classified into six species, A–F, and 51 serotypes are recognized. Adenoviruses can cause a broad range of diseases. Serotypes 3, 7 and 21 are most commonly associated with CNS disease. Serotype 21 (specie B) was isolated from brain tissue and CSF of patients with acute flaccid paralysis (AFP) in Malaysia. Objectives: Characterize, by molecular methods, species B adenoviruses isolated from poliomyelitis-suspected cases and investigate the possible etiological role of adenoviruses in acute flaccid paralysis (AFP). Study design: 622 virus isolates, including Sabin-related polioviruses, non-polio enteroviruses (NPEV) and adenoviruses, were recovered from fecal specimens in our laboratory during the period of 1997–2002 from AFP cases occurring in Brazil, Peru and Bolivia. Negative controls consisted of 528 fecal specimens collected from healthy children ≤5 of age. Of these, 478 were contacts of AFP negative cases and 50 were from a day-care center. Results: Sixty-four adenovirus strains isolated in HEp2 (human laryngeal tumor cells) cells were confirmed as such by an adenovirus-group specific PCR. Nucleotide sequencing identified the following adenovirus species: A (3 isolates), B (20 isolates), C (38 isolates), D (2 isolates) and E (1 isolate). The following serotypes belonging to the species B were identified: Ad3 (1 strain), Ad7 (17 strains) and, Ad16 (2 strains). Conclusion: Other viral agents became more recognized in association with CNS diseases in areas where wild polioviruses have been eradicated. The possible role of species B adenoviruses in the etiology of AFP cases similar to that caused by wild poliovirus is discussed.

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.

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.

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.

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.