Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005-2017).

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.

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

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.

Complete genome sequence of the last representative genotype of wild indigenous poliovirus type 1, which circulated in Brazil.

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.

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.

Genetic variation of foot-and-mouth disease virus isolates recovered from persistently infected water buffalo (Bubalus bubalis).

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 0.44s/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.

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.

Enterovirus meningitis in Brazil, 1998-2003.

Acute viral infections of the central nervous system (CNS) such as acute flaccid paralysis, meningitis, and encephalitis, are responsible for a high morbidity, particularly in children. Non-Polio enteroviruses (NPEV) are known to be responsible for over 80% of viral meningitis in which the etiologic agent is identified. In the present study, we show the frequency of enterovirus meningitis in Brazil from December 1998 to December 2003. Enterovirus were isolated from 162 (15.8%), of a total of 1,022 cerebrospinal fluid (CSF) specimens analyzed. Echovirus 30 was identified in 139 of these isolates (139/162-85.2%). Other identified enteroviruses were: Coxsackievirus B5 (3.7%), Echovirus 13 (3.7%), Echovirus 18 (3%), Echovirus 6 (1.2%), Echovirus 25 (1.2%), Echovirus 1 (0.6%), and Echovirus 4 (0.6%). Patients's age ranged from 28 days to 68 years old. The most frequent symptoms were fever (77%), headache (69.5%), vomiting (71.3%), neck stiffness (41.3%), convulsion (7.1%), and diarrhea (3.7%). Although, the majority of the patients recovered without any complication or permanent squeal, five deaths occurred. Throughout the surveillance period, five viral meningitis outbreaks were confirmed: four in the Southern Brazil and one in the Northeast Brazil. Echovirus 30 was responsible for four out of the five outbreaks while Echovirus 13 caused the fifth one. Besides the outbreaks, 734 sporadic cases were also identified during the study period and 59 of these were positive for virus isolation (8%). Echovirus 30 accounted for 70% of the isolates. Our results showed that Echovirus 30 was the most prevalent etiological agent of viral meningitis in Brazil, causing both outbreaks and sporadic cases.

Characterization of species B adenoviruses isolated from fecal specimens taken from poliomyelitis-suspected cases.

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.

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 RNA's. 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 RNA's 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.