@ Corresponding author Purification and Characterization of Viral Agents Isolated During the Outbreak of Epidemic Neuropathy in Cuba Rosmari Rodríguez Roche, 1 @ María Guadalupe Guzmán, 1 Odelsa Ancheta, 2 Virginia Capó, 3 Gustavo Kourí, 1 Pedro Más 1 1 Dpto. de Virología, Instituto de Medicina Tropical Pedro Kourí, IPK, Habana, Cuba. Fax: (53-7) 204 6051; E-mail: lupe@ipk.sld.cu 2 Lab. de Microscopía Electrónica, Centro Nacional de Investigaciones Científicas, CNIC, Habana, Cuba. 3 Dpto Anatomía Patológica, Instituto de Medicina Tropical  Pedro Kourí , IPK Habana, Cuba. ABSTRACT During the neuropathy epidemic in Cuba, two types of antigenically-related cytopathic agents were isolated from the cerebrospinal fluid (CSF) of patients diagnosed with neuropathy. Different strains were studied. A group of these showed a cytopathogenic effect (CPE) typical of the enterovirus and was identified as coxsackievirus A9. The other group produced a slowly progressive CPE, this pattern was referred to as light CPE (CPE-L). Previous results obtained during the characterization of these agents had suggested the presence of more than one agent in the same sample since an agent with a CPE typical of the enterovirus was obtained from a CPE-L strain. Two standard methods for virus purification, gradient centrifugation and viral cloning by plaque assay, were performed. Some physicochemical char- acteristics of the purified agents are described in this paper. Sensitivity to chloroform, PAA, GHCL and BRDU was assayed. Viral size and morphology were studied by electron microscopy and our results point at the presence of two variants of the same agent. No virus particle was observed in the case of CPE-L strains. These observations related to CPE-L strains are in agreement with other molecular studies that show structural virus alterations. Keywords: electron microscopy, epidemic neuropathy, virus, Biotecnología Aplicada 2002;19:25-29 RESUMEN Purificación y caracterización de los agentes virales aislados durante la neuropatía epidémica en Cuba. Durante la epidemia de neuropatía ocurrida en Cuba, a partir del líquido cefaloraquídeo (LCR) de pacientes, fueron aislados dos tipos de agentes citopatogénicos relacionados antigénicamente. Diferentes cepas fueron estudiadas. Un grupo de éstas mostró efecto citopatogénico (ECP) típico de enterovirus y se identificó como coxsackievirus A9. El otro grupo producía un ECP de progresión lenta, al que se denominó ligero (ECP-L). Resultados previos, obtenidos en la caracterización de estos agentes han sugerido la presencia de más de un agente en la misma muestra, ya que un agente de ECP típico de enterovirus fue obtenido a partir de una cepa de ECP-L. En este trabajo se desarrollaron dos métodos convencionales de purificación viral: gradiente de centrifugación y clonaje por placas. Se describen algunas características físico-químicas de los agentes purificados. Se realizaron ensayos de sensibilidad a: cloroformo, ácido fosfonoacético, cloruro de guanidina y bromodeoxiuridina. Por microscopía electrónica se estudiaron talla y morfología viral. Nuestros resultados sugieren la presencia de dos variantes del mismo agente. En las muestras correspondientes a cepas de ECP-L, no se encontraron partículas virales. Estas observaciones relacionadas con las cepas de ECP-L concuerdan con otros estudios moleculares realizados, que muestran alteraciones estructurales en el virus. Palabras claves: microscopía electrónica, neuropatía epidémica, purificación, virus Introduction An epidemic of more than 50 000 cases of optic and/or peripheral neuropathy occurred in Cuba from late 1991 through 1993; 52 406 cases were reported by the end of 1995. The disease was associated with dietary limi- tations. In particular, an impairment of the protective antioxidant pathways was suggested. Smoking was a related risk factor, which intensified the effects of the nutritional deficiencies and was thought to cause in- jury due to oxidative damage. In addition to the clini- cal and epidemiological studies, virological research was carried out to exclude the possibility of an infec- tious agent. However, 5 isolates of Coxackievirus A9 (CA9) and 100 isolates of the light cytopathic effect (CPE-L) agent were recovered from 105 cerebrospi- nal fluid (CSF) specimens tapped from 125 epidemic neuropathy patients [1]. The viral isolation of anti- genically-related agents by means of independent sys- tems that are confirmed by immunohistochemical techniques and reisolation of the light CPE virus in cell cultures from mice, strongly evidence that a virus was present in the CSF of patients. Also, its in vivo persistence for more than 1 month was shown by its reisolation in the CSF of 24 patients. This result fur- ther suggests that the virus may be important in the pathophysiology of this disease [2]. The CPE-L producing agents were not identified by the Lim Benyesh-Melnick (LBM) pools of antis- era [3] but the specific antisera developed in rabbits against these agents neutralized the CA9 strain in vitro. During the isolation process the typical Enterovirus CPE was identified in samples that had been showing CPE-L for several days. The results of the physico- chemical characterization of the CPE-L agents were not conclusive enough to classify the strains. Enterovi- rus in general show no sensitivity to chloroform and their replication is inhibited by guanidinium chloride 1. Más P, Pelegrino JL, Guzmán MG, Capó V, Rodríguez L, Rodríguez P, et al. Neuro- patía epidémica cubana. Parte I. Aisla- miento viral. Rev Cub Med Trop 1995; 47(1):11-5. 2. Más P, Pelegrino JL, Guzmán MG, Comellas MM, Resik S, Álvarez M, et al. Viral isolation from cases of epidemic neuro- pathy in Cuba. Arch Pathol Lab Med 1997;121:825-33. 3. Lim KA, Benyesh-Melnick M. Typing of viruses by combinations of antiserum pools application to typing of enteroviruses (Coxsackie and Echo). J Immunol 1960; 84:309-17.