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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 90, Num. 4, 1995, pp. 523-524
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol.
90(4): 523-524 Jul/Aug. 1995
RESEARCH NOTE
Absence of Antibody-dependent Enhancement (ADE) of Viral
Infectivity in the Epidemic Neuropathy in Cuba
Maria G Guzman, Maritza Soler, Pedro Mas, Luis Morier, Aida
Castillo, Sonia Resik, Mayling Alvarez, Gustavo Kouri
Departamento de Virologia, Instituto de Medicina Tropical
"Pedro Kouri", Apartado Postal 601, Habana, Cuba
Code Number: OC95104
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Key words: antibody-dependent enhancement - enterovirus -
epidemic neuropathy
During 1992-1993, a new disease called Epidemic Neuropathy
(EN) was reported in Cuba. The patients complained of weight
loss, blurred vision, sensitivity to light and gradual loss of
visual activity. Other patients presented complaints of pain
in the upper and lower limbs, dysesthesia and paresthesia,
burning sensation on the feet soles and other symptoms. More
than 50,000 cases were reported from the entire country. The
disease was observed mainly in adults. The etiology of EN
appears to be related to several factors, including
nutritional deficiencies and a probable neurotoxic factor. In
addition, an enterovirus identified as coxsackie A9 and an
agent that produced a slow progressive CPE, were isolated from
some of the patients' CFS samples (G Llanos et al. 1993
Epidemiol Bul PAHO 14: 1-4). Since the etiopathogenesis of EN
is not known, and some patients had evidence of immune complex
formation (Grupo Operativo Nacional, Ministerio de Salud
Publica de Cuba, Informe sobre Neuropatia Epidemica, julio de
1993), we study whether or not an ADE phenomenon was related
to this disease.
The aim of this study is twofold. First to study the possible
immunoamplification of the strain 47/93/IPK (identified as
Coxsackie A9) in peripheral blood lymphocytes (PBL) from
healthy donors in the presence of subneutralizing
concentrations of antibodies against this virus; and second,
to study the possible presence in sera of EN patients of
immunenhancing antibodies to the same strain grown in U-937
cells.
For the first experiment, PBLs from ten healthy donors were
obtained by the Ficoll-Hypaque method (A Boyum 1968 Scand J
Clin Lab Investig 21: suppl 97). Cells were resuspended in
RPMI at 10^6 cells/ml. Volumes of strain 47/93/IPK (moi=0.01)
were mixed with different dilutions (10^3-10^6) of a serum of
EN patient with 1/160 neutralizing titer to this virus. After
1 hr incubation at 37 C, mixtures were added to cell
suspensions previously dispensed in 24 wells Costar tissue
culture plates. These cells were incubated at 37 C in a
humidifed incubator with 5% CO2 for three days after which
were frozen and thawed three times. Virus was titered in Vero
cells and the titers calculated by Reed and Muench method.
For the second study volumes of strain 47/93/IPK (moi=0.01)
were mixed with serum dilutions of EN patients
(1/50-1/500000). After 1 hr incubation at 37 C, mixtures were
added to U937 cell suspensions in RPMI (5x10^6 cells/ml). Cell
incubation and virus titration were done as described above.
In both studies each combination was tested in duplicated and
a virus control with PBS was included. The immunenhancement
response was considered positive when viral titer (in
enhancing conditions) was two or more log ID50 greater than
the control viral titer.
Table 1. Enhancing activity to 47/93/IPK (cox A9) strain in
health donors
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Case Sex Age Enhancing rate^a
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1 F 41 2
2 F 38 0
3 F 27 2
4 F 35 1
5 F 41 1
6 M 31 0
7 M 34 0
8 M 25 1
9 M 35 2
10 M 41 1
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^a viral titer in enhancing conditions/control viral titer
The immunenhancement activity of the strain 47/93/IPK is shown
in Table I. Only in three (30%) of donors the immunenhancement
reaction at 1/1000-1/10000 serum dilutions was positive. No
immunenhancement antibodies were detected in sera studied in
the second experiment (Table II). The ability of strain
47/93/IPK to replicate in U-937 cells was previously confirmed
(data not shown).
Table II. Immunenhancement antibody detection study in EN sera
patients
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caso Nt titer^a enhancing rate^b
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152 160 0
153 >320 1
164 >320 0
167 >320 0
192 >320 1
245 - 1
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^a: titer of neutralizing antibodies to 47/93/IPK strain
^b: viral titer in enhancing conditions/control viral titer
Antibody dependent enhancement results from the interaction of
three components: virus, antibody and Fc receptor. This
phenomenon was not previously reported in enteroviruses
although there is a large number of viruses in which in vitro
virus enhancement has been observed (M Bendinelli, H Friedman
Plenum Press 1988, I Kurane et al. 1991 Rev Med
Virology 1: 211-221, SB Halstead 1988 Science 239: 476-481).
During the EN outbreak, immune complexes were detected in some
EN patients suggesting the possibility that an ADE phenomenom
could be related (Grupo Operativo Nacional, loc. cit.). Our
preliminary results suggest that, at least in the PBL of some
of healthy donors studied, it was possible to observe an
increase of viral replication under immunenhancement
conditions. Although this finding indicates the possibility of
this phenomenon, it does not mean that it occurs in vivo.
Furthermore, no enhancing antibodies were detected in sera
patients under the conditions of this experiment.
The EN outbreak ceased in September 1993. So far the
etiopathogenesis of the disease is not known although toxic,
metabolic and nutritional factors have been implicated. The
finding of an agent, in addition to Coxsackie A9 virus, in the
CSF of a high number of patients does not allow us to
eliminate the possible infection etiology of this disease.
The role of this agent is not clear at this moment, however
this preliminary results do not suggest an immunenhancement
mechanism involving Coxsackie A9 infection. More studies are
needed in order to clarify the etiology and the pathogenesis
of the disease.
Copyright 1995 Fundacao Oswaldo Cruz
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