Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 1, 2002, pp. 47-49

Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(1) 2002, pp. 47-49
Neutralizing Antibodies to 
  Enterovirus 71 in Belém, Brazil  
Maria de Lourdes C Gomes/+, Ceyla 
  Maria O de Castro**, Maria José C Oliveira*, Edson Elias da Silva**  

Seção de Virologia, Instituto Evandro 
  Chagas, Funasa, Av. Almirante Barroso 492, 66090-000 Belém, PA, Brasil 
  *Fusam, Secretaria de Estado de Saúde, Recife, PE, Brasil **Instituto 
  Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil 

  +Corresponding author. Fax:+55-91-214.2005. E-mail: lourdesgomes@iec.pa.gov.br 
  
Received 16 March 2001 

  Accepted 10 September 2001 
Code Number: oc02007 
Non-polio enteroviruses (Coxsackievirus A, 
  Coxsackievirus B, Echovirus and EV 68-72) which belong to the enterovirus (EV) 
  genus, Picornaviridae 
  family, may be responsible for acute flaccid paralysis, aseptic meningitis, 
  myocarditis, hepatitis, pleurodinia, neonatal sepsis, hand, foot and mouth disease 
  (HFMD) even though 50-80% of infections are asymptomatic. EV 71 has been responsible 
  for outbreaks and epidemics of HFMD and acute neurologic disease justifying 
  its study in our country. The aim of this study was to detect neutralizing antibodies 
  (NtAb) to EV 71 in individuals up to 15 years of age living in Belém, 
  State of Pará, northern Brazil. Serum samples from 238 patients attending 
  the Virology Sector of Evandro Chagas Institute in Belém, Brazil, were 
  analyzed using microneutralization tests that included RD cells and BrCr strain. 
  Overall 40.8% (97/238) of tested samples had NtAb to EV 71. Regarding the distribution 
  per age group, 85.2% (92/108) of patients aged 0-3 years had no NtAb to this 
  virus and 69.2% of those 12 to15 years of age were seropositive. These results 
  confirm that EV 71 infection occurs in the city of Belém; and that a 
  high rate of individuals in this study were infected aged 3 years and over and, 
  when aged 15 years nearly 70% had EV 71 NtAb.  
Key words: enterovirus 71- neutralizing antibodies 
  to EV 71 - seroepidemiology study - Belém - Brazil  
At the beginning of this century, motor deficiency 
  cases could be related to pathogens such as viruses and particularly to polioviruses, 
  which belong to the enterovirus (EV) genus, Picornaviridae family. Besides 
  the poliovirus (3 serotypes), EV includes Coxsackievirus A (23 serotypes: types 
  1-17, 19-22 and 24), Coxsackievirus B (6 serotypes), Echovirus (30 serotypes): 
  1-7, 9, 11-27, 29-33), and EV 68-72 (Wiedbrank & Johnston 1993).  

Enteroviruses are non-enveloped small viruses 
  (20-30 nm) of icosahedral symmetry. Their RNA genomes have 7.5 kb, positive 
  sense and single strand. They are responsible for an extensive variety of diseases, 
  although 50 to 80% of the infections are asymptomatic. They cause hepatitis, 
  pleurodinia, stomatitis and neonatal sepsis in a significant number of patients 
  every year. In developing countries, the poliovirus is clinically the most significant 
  member of the genus EV causing paralysis diseases in every 4 out of 1,000 children 
  in school age. The non-polio enteroviruses (NPEV) are the main responsible for 
  aseptic meningitis, myocarditis and nonspecific febrile exanthematous illnesses. 
  Approximately 75% of infections by EV occur in children under 15 years of age 
  and the attack rates are highest in children under 1 year of age (Wiedbrank 
  & Johnston 1993).  
EV 71, one of the last NPEV studied, was described 
  for the first time by NJ Schimidt et al. (da Silva et al. 1990) in 1974, in 
  California, associated with cases of the central nervous system (CNS) diseases. 
  In the same year Kennett et al. (1974) mentioned the occurrence in Melbourne, 
  Australia, of cases of aseptic meningitis, cutaneous eruption, acute respiratory 
  tract infections and infective polyneuritis caused by EV 71. In 1975, an epidemic 
  was observed in Bulgaria with clinical symptoms of poliomyelitis, encephalitis, 
  encefalomyocarditis and aseptic meningitis. Ninety-two strains of EV 71 were 
  isolated from the 65 cases with the same symptomatology of the poliomyelitis, 
  including 37 strains from brain and medulla, 1 from cerebrospinal fluid, 10 
  from mesenterial lymph nodes and tonsils and 44 from feces (Chumakov et al. 
  1979). An outbreak of infections caused by EV 71 occurred in Australia during 
  the winter of 1986. Of the 114 studied patients, 65 were hospitalized and 33 
  frequently had CNS involvement associated to severe symptoms (Gilbert et al. 
  1988). During an outbreak of hand, foot and mouth disease (HFMD) in Malaysia, 
  in 1997, 4 children developed cardiopulmonary collapse and neurological problems. 
  All the children received cardiopulmonary resuscitation but died. Postmortem 
  studies showed infection by EV 71, with extensive damage to the medulla and 
  pons (Lum et al. 1988). More recently the EV 71 was responsible for an epidemic 
  occurred in Taiwan. Most of the patients had HFMD with or without complications. 
  Many enteroviruses were isolated from the 238 cases, approximately half of them 
  were EV 71 (Wang et al. 1998). In Brazil the first evidence of infections by 
  EV 71 was mentioned by da Silva et al. (1990); these authors studied cases of 
  acute flaccid paralysis (AFP) and finding neutralizing antibodies (NtAb) for 
  EV 71 in 32.1% of serum samples. In another study developed by da Silva et al. 
  (1996) positive IgM to EV 71 was detected in 20 (21%) of 92 children living 
  in different areas of Brazil that presented clinical symptoms of AFP over a 
  3 year-old period (1988 to 1990). Takimoto et al. (1998) detected positivity 
  for EV 71 in 5.6% of the 426 samples of children's feces with symptoms of acute 
  neurological disease (AND), residents in the State of São Paulo and in 
  the city of Brasília. Despite the significant variability of clinical 
  and epidemic manifestations, there is close antigenic similarity among strains 
  of EV 71 isolated in different countries (da Silva et al. 1990), justifying 
  its study in several aspects including seropreva-lence. On this subject Hagiwara 
  et al. (1979) in the 70s showed interesting results including reference to circulation 
  of EV 71 in Japan in 1966. It is important to mention that the presence of this 
  virus in the community is concerning, considering the evaluation of the poliomyelitis 
  control program (Blal et al. 1998 ).  
The aim of this work was to detect NtAb to the 
  EV 71 in serum samples from patients up to 15 years of age living in the city 
  of Belém, Brazil. 
MATERIALS 
  AND METHODS 
Patients - Serum samples from 238 patients 
  of both sexes aged up to 15 years and residents in the city of Belém, 
  were included in this study with symptoms of fever and exanthem. Patients received 
  care from the Virology Section of the Evandro Chagas Institute during the year 
  of 1998.  
Cell culture - RD cell (rabdomiosarcoma 
  of human origin) was used, and maintained in Eagle's MEM (Minimum Essential 
  Medium) with Earle's salts, without sodium bicarbonate and without phenol red. 
  The preparation of this medium was as follows: MEM powder was added with (9.7 
  g, sodium bicarbonate 1.5 g , fetal bovine serum 100 ml, L-glutamine 200 mM 
  2 ml, HEPES 1 M 10 ml, penicillin 100,000 U/ml, and streptomycin 100,000 µg/ml, 
  1 ml of each) in 1,000 ml of distilled and purified water. This mixture was 
  filtered in membranes of 0.22 µm, distributed in small volumes and maintained 
  at 4°C. The RD cells were maintained weekly using Trypsin 0.25% plus EDTA.  

Virus - An aliquot of EV 71, BrCr strain, 
  provided by EE da Silva, was inoculated in RD cells to obtain a large volume. 
  When the cellular layer presented 75% of cytopathic effect, the cultivation 
  was frozen and thawed three times, centrifuged at 3,000 rpm for 30 min; the 
  supernatant was distributed in aliquots of 500 µl and frozen at -20°C. 
   
Neutralization microtechnique - Aliquots 
  of 100 µl of serum were separated, inactivated at 56°C for 30 min and 
  diluted 1:8 in Eagle's MEM (prepared equal to the growth medium except in the 
  bovine serum concentration that decreased to 2% and the increase of 0.06% of 
  anfothericin B 5,000 µg/ml), which was used as diluent of the test. The 
  protocol utilized in this test was the same used for polioviruses (WHO 1996) 
  with some modifications. Initially 50 µl of the diluent was put into all 
  wells of the microplate (polystyrene, 96 flat bottom wells) except for the first 
  row, then 50 µl of 1:8 dilution serum was put in the first and second 
  rows. With the aid of the multichannel pipette, serum samples were diluted starting 
  from the second row, removing 50 µl and placing it into the third row 
  and so forth until the last dilution. In the following stage, 50 ml of virus 
  contends the challenge dose (100 TCD50 with variations from 31.5 to 315) was 
  added. Two incubation periods were used: 2 h at 37°C in CO2 incubator, 
  and overnight at 4°C. The next day 100 µl of the suspension of RD cells 
  with 3 days of growth was added, in the concentration of 2x105 cells/ml. 
  They were maintained at 35°C in CO2 incubator. Microscopic observation 
  was made on the 5th day. 
RESULTS 
  
In the analysis of 238 serum samples it was verified 
  that the largest percentage of samples tested against EV 71 were located in 
  the age group up to 3 years, with 45.4%, followed by the 4 to 7 years-old with 
  24.4%. The rest were divided into the 8 to 11 years-old (19.3%) and 12 to 15 
  years-old (10.9%) age groups. In terms of gender, 54.2% were female and 45.8% 
  male.  
Fig. 1 relates 
  the levels of antibodies less (<) than 1:8 and higher or equal to (³) 
  1:8, with the patients' age group. It was observed that in 59.2% the level of 
  antibodies was < 1:8 and in 40.8% ³ 1:8. 
  In the age group from 0 to 3 years which included 108 cases, 85.2% showed a 
  level of < 1: 8 while 14.8% ³1:8. In the 
  4 to 7 years-old group, an inversion of percentage was noted because in the 
  dilution ³ 1: 8, the percentage increased to 
  53.4% while at < 1:8 it decreased to 46.6%. The 8 to 11 and 12 to 15 years-old 
  groups showed a pattern similar to the previous one.  
Fig. 2 shows 
  the four groups of levels of NtAb detected in the samples of these patients. 
  Of the 238 samples tested, 141 (59.2%) presented level of < 1: 8.The two 
  following groups 1:8-1:32 and 1:64-1:256 contained 39 (16.4%) and 36 (15.1%) 
  samples respectively. The last group, > 1: 256 included 22 (9.2%) samples. 
  
DISCUSSION 
  
Studies involving detection of NtAb to EV 71 
  are scarce and when existent they are related to the cases of HFMD, AND in general, 
  and AFP in particular. As the main objective of this work was to verify the 
  seroprevalence in a group of individuals with less than 15 years of age presenting 
  different symptomatology, it was difficult to discuss our results.  
Hagiwara et al. (1979) analyzing 137 samples 
  of healthy children from 0 to 10 years of age, living in the city of Kawasaki, 
  in Japan detected 6.6% with NtAb to EV 71. In our study the percentage corresponding 
  to the patients up to 11 years of age was 37.3%. The use of different cells 
  lines, Cynomolgus monkey cell line (CMK1-S1) in the Japanese study and RD in 
  our study could have influenced these results. It is also possible that in the 
  period in which the Japanese evaluation was done, circulation of EV 71 was extremely 
  low and perhaps restricted to a determined country. In Brazil studies involving 
  EV 71 were carried out by da Silva et al. (1990) and Takimoto et al. (1998) 
  both relating that this viruses to motor disorder occurred in the states of 
  Bahia, Goiás, Piauí, São Paulo and Federal District, different 
  from our study that involved patients with fever and exanthem. These authors 
  detected positivity to EV 71 in 32.1% (9/28) and 5.6% (24/426) of the cases 
  respectively. Our results show a percentage of 40.8% (97/238). It is evident 
  that a great number of people were infected by EV 71 in Belém in contrast 
  to the low number of individuals infected in São Paulo. Our percentage 
  of infection is closer to the percentage found by da Silva et al. (1990) in 
  the other localities. In relation to clinical symptoms the patients studied 
  by Takimoto et al. (1998) and da Silva et al. (1990) presented disease with 
  motor involvement while our patients presented fever and exanthem. Probably 
  the strain of EV 71 that has circulated in Belém is genetically different 
  from the strain that has circulated in the localities studied by that authors. 
  According to our results the infection by EV 71 in Belém occurred with 
  great frequency after 3 years of age considering that the individuals in the 
  age group 0-3 years with NtAb > 1:8 was 14.8% (16/238). Another point is 
  that a large number of individuals when reaching the age of 15 years posses 
  NtAb to EV 71 considering the percentage of 69.2% (18/26) detected. In relation 
  to the children with AND studied by Takimoto et al. (1998) the majority was 
  infected later because until 5 years of age the percentage of individuals infected 
  was 7.3% (17/231).  
Although the importance of EV 71 has already 
  been defined, more studies will be necessary in order to increase the knowledge 
  on the several aspects related to this virus in Brazil. The data obtained in 
  this study will be used for comparison with other studies in different localities 
  of Brazil where this virus can be associated to other clinical disorders. 
ACKNOWLEDGEMENTS 
   
To Dr Yvone Gabbay for critically reviewing this 
  manuscript. 
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© 2002  
  Instituto Oswaldo Cruz - Fiocruz

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