Purification and Characterization of Viral Agents Isolated

@ Corresponding author

Purification and Characterization of Viral Agents IsolatedDuring 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 Más1

1Dpto. de Virología, Instituto de Medicina Tropical �Pedro Kourí�, IPK, Habana, Cuba.Fax: (53-7) 204 6051; E-mail: [email protected]

2Lab. de Microscopía Electrónica, Centro Nacional de Investigaciones Científicas, CNIC, Habana, Cuba.3Dpto Anatomía Patológica, Instituto de Medicina Tropical � Pedro Kourí �, IPK Habana, Cuba.

ABSTRACTDuring the neuropathy epidemic in Cuba, two types of antigenically-related cytopathic agents were isolated from thecerebrospinal fluid (CSF) of patients diagnosed with neuropathy. Different strains were studied. A group of theseshowed a cytopathogenic effect (CPE) typical of the enterovirus and was identified as coxsackievirus A9. The othergroup produced a slowly progressive CPE, this pattern was referred to as light CPE (CPE-L). Previous results obtainedduring the characterization of these agents had suggested the presence of more than one agent in the same samplesince an agent with a CPE typical of the enterovirus was obtained from a CPE-L strain. Two standard methods for viruspurification, 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 wasassayed. Viral size and morphology were studied by electron microscopy and our results point at the presence of twovariants of the same agent. No virus particle was observed in the case of CPE-L strains. These observations related toCPE-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, fueronaislados dos tipos de agentes citopatogénicos relacionados antigénicamente. Diferentes cepas fueron estudiadas. Ungrupo de éstas mostró efecto citopatogénico (ECP) típico de enterovirus y se identificó como coxsackievirus A9. El otrogrupo producía un ECP de progresión lenta, al que se denominó ligero (ECP-L). Resultados previos, obtenidos en lacaracterización de estos agentes han sugerido la presencia de más de un agente en la misma muestra, ya que unagente de ECP típico de enterovirus fue obtenido a partir de una cepa de ECP-L. En este trabajo se desarrollaron dosmétodos convencionales de purificación viral: gradiente de centrifugación y clonaje por placas. Se describen algunascaracterísticas físico-químicas de los agentes purificados. Se realizaron ensayos de sensibilidad a: cloroformo, ácidofosfonoacético, cloruro de guanidina y bromodeoxiuridina. Por microscopía electrónica se estudiaron talla y morfologíaviral. Nuestros resultados sugieren la presencia de dos variantes del mismo agente. En las muestras correspondientesa cepas de ECP-L, no se encontraron partículas virales. Estas observaciones relacionadas con las cepas de ECP-Lconcuerdan con otros estudios moleculares realizados, que muestran alteraciones estructurales en el virus.

Palabras claves: microscopía electrónica, neuropatía epidémica, purificación, virus

IntroductionAn epidemic of more than 50 000 cases of optic and/orperipheral neuropathy occurred in Cuba from late 1991through 1993; 52 406 cases were reported by the endof 1995. The disease was associated with dietary limi-tations. In particular, an impairment of the protectiveantioxidant pathways was suggested. Smoking was arelated risk factor, which intensified the effects of thenutritional deficiencies and was thought to cause in-jury due to oxidative damage. In addition to the clini-cal and epidemiological studies, virological researchwas 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 epidemicneuropathy 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 incell cultures from mice, strongly evidence that a viruswas present in the CSF of patients. Also, its in vivopersistence for more than 1 month was shown by itsreisolation in the CSF of 24 patients. This result fur-ther suggests that the virus may be important in thepathophysiology of this disease [2].

The CPE-L producing agents were not identifiedby the Lim Benyesh-Melnick (LBM) pools of antis-era [3] but the specific antisera developed in rabbitsagainst these agents neutralized the CA9 strain in vitro.During the isolation process the typical EnterovirusCPE was identified in samples that had been showingCPE-L for several days. The results of the physico-chemical characterization of the CPE-L agents werenot conclusive enough to classify the strains. Enterovi-rus in general show no sensitivity to chloroform andtheir replication is inhibited by guanidinium chloride

1. Más P, Pelegrino JL, Guzmán MG, CapóV, 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. Viralisolation from cases of epidemic neuro-pathy in Cuba. Arch Pathol Lab Med1997;121:825-33.

3. Lim KA, Benyesh-Melnick M. Typing ofviruses by combinations of antiserumpools application to typing of enteroviruses(Coxsackie and Echo). J Immunol 1960;84:309-17.

Rosmari Rodríguez Roche et al. Purification and characterization of viral antigens

Biotecnología Aplicada 2002; Vol.19, Nos. 1 y 226

(GHCl), however the M26 IG strain is sensitive tochloroform, it is not sensitive to GHCl or tophosphonoacetic acid (PAA) [4]. These results sug-gest the presence of more than one agent in the samesample. The purification of the prototype strains mayprovide important information for the characteriza-tion of these agents.

In this report, CPE-L strains were characterized,through sucrose gradient purification and viral cloningby plaque assay. These results allow us to proposethe participation of defective interfering particles inthe course of the disease.

Materials and Methods

CellsVero cells (ATCC) were grown at 37 ºC in a Parkermedium 199 plus 10 % fetal bovine serum (FBS) and1.2 g/L sodium bicarbonate.

VirusIsolates from the CSF of epidemic neuropathy pa-tients that were comprehensively studied includedone typical strain of CA9 (CPE-E): 47/93 IPK andtwo strains of the agent producing CPE-L: 44/93IPK and M26 IG. The later was isolated at the Cen-ter for Genetic Engineering and Biotechnology inHavana.

Hyperimmune seraThe hyperimmune sera were produced in New Zealandrabbits immunized with viral antigens obtained fromthe inoculated Vero cells supernatant.

Purification of viruses by gradientcentrifugationVero cell cultures were seeded into plastic rollers andimmediately infected at a multiplicity of infection of 1.The cultures were incubated at 37 ºC until cells wereharvested. The virus was aggregated by adding 7%PEG 6000 and centrifuged at 10 000 rpm. Precipita-tion was enhanced by adding 0.5 M/L NaCl. The con-centrated virus was centrifuged through a sucrosegradient (15-60%) in an SW40 rotor at 28 000 rpm for2 h at 4 ºC. The band formed was recovered for theelectron microscopy study.

Viral cloning by plaque assayRapid virus plaque assay was performed as previ-ously described [5] with certain modifications. A sus-pension of 2.5 x 105 Vero cells/mL was seeded into24-well plastic plates. Plates were inoculated withthe 100-fold serial dilutions of virus strains and incu-bated at 37 ºC in a 5% carbon dioxide atmosphere.After 4 h, a media overlay was added, consisting of a2x Parker medium 199 containing 10 % FBS, 1.2 g/Lsodium bicarbonate and 1.5% carboxymethyl Cellu-lose. Cultures were reincubated until the plaques weredeveloped. Individual plaques were chosen, expandedand cloned twice.

Characterization of cloned virusSensitivity to chloroform, PAA, GHCl and 5- bro-modeoxyuridine (BDUR) were assayed following theprocedure described by Guzmán et al. [4].

Immunoelectron microscopyFifty milliliters from each band were mixed with 50 mLof the corresponding hyperimmune serum at a 1:20dilution, and incubated overnight at 4 ºC. The virus-antibody suspension was centrifuged for 30 min(100 000 xg). The pellet was resuspended in distilledwater and stained with 2% uranyl acetate [6]. Thegrids were examined in a JEOL JEM 100S electronmicroscope (Jeol, Canada) at 80 kV.

Electron microscopyThe infected Vero cell monolayers were washed withPBS and immediately fixed using 2.5% glutaraldehyde at4 ºC, post fixed with osmium tetroxide, dehydrated andembedded in spurr resin (Polyscience, Inc., USA). Thinsections were cut on an LKB Ultratome, Nova (LKB,Sweden), placed on uncoated grids, stained with uranylacetate and lead citrate and examined with a JEOL JEM100S electron microscope (Jeol, Canada) at 80 kV [7].

ResultsFigure 1 shows two standard methods of viral puri-fication that were used to separate the different par-ticle populations that may be present in the samesample.

For the characterization of the purified strains westudied the sensitivity to chloroform, BDUR, PAAand GHCl, as well as the morphology, size and timeof appearance of the plaques during cloning. The mor-phology and size of the particles were determinedthrough electron microscopy (Tables 1 and 2).

4. Guzmán MG, Rodríguez R, Resik S,Álvarez M, Morier L, Castillo A, et al. Ca-racterización físico-química de los agen-tes aislados durante el brote de neuropatíaepidémica en Cuba. Parte I. Rev Cub MedTrop. 1995a;47(1 ):32-5.

5. Morens DM, Halstead S. Simplifiedplaque reduction neutralisation assay fordengue viruses by semimicro methods inBHK-21 cells. Comparison of the BHKsuspension test with standard plaquereduction neutralisation. J Clin Microbiol1985;22:250.

6. Hayat MA, Miller SE. Negative staining:applications and methods. New York:McGraw Hill; 1990.

7. Miller SE. Electron microscopy of tissueculture. In: Jones BR, editor. ElectronMicroscopy: 41 Exercises by 17 scientists.Monroe, NY: Library Research Associates;1985. p.293-315.

Figure 1 Flow chart to indicate the procedure followed with the 44/93, M26 IG and 47/93 strains andpart of the results obtained according to the different ways of virus purification.

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Strain 47/93 IPKStrain 47/93 IPK identified as a CA9 was purified bysucrose gradient showing virus-like particles of 28 nmin the negative staining of the purification band. Vi-rus-like particles of 28 nm were observed in the cyto-plasm of infected cells studied by transmission electronmicroscopy (TEM) Figure 2.

Strain M26 IGStrain M26 IG showed similar characteristics beforeand after purification by sucrose gradient and cloning(Tables 1 and 2). However, when the fraction obtainedfrom the purification gradient of this strain (Fraction11, F11/94) was treated with PAA at a low multiplic-ity, its CPE-L changed to a typical enterovirus CPE.The new agent was named F11PAA/94. After cloning,the plaque size it produced increased, and 28 nm par-ticles were detectable after purification in the sucrosegradient (Figure 3).

The purified F11PAA/94 strain (Fraction 20) showednot only a different CPE to F11/94 but also a majorsedimentation coefficient similar to the 47/93 IPKstrain. In addition, the identification of F11PAA/94,using the LBM pools revealed an antigenic relationwith echovirus (EV) 7 and 27 although a precise iden-tification was not posible. At this point we have theCF11PAA/94 strain obtained from a CPE-L strain(M26 IG) that became another agent with a typicalenterovirus CPE.

Strain 44/93 IPKCells infected with 44/93 IPK were studied underTEM. No virus-like particles were seen in the in-fected cells. However, infected cells show an enhance-ment of granular electrodense structures whencompared with the control that may be related withribonucleoproteins (Figure 4). Infected cells in a ter-minal stage of degeneration were observed.

When cloning the 44/93 IPK strain, a slight CPEchange was observed. The C44/94 strain produces theCPE-L in shorter time and more intensely (Figure 5).Plaques formed by C44/94 are similar to the originalstrain and very different to the CPE-E strains (Tables 1and 2 and Figure 6).

After purification by the sucrose gradient the C44/94band, showed a sedimentation coefficient similar toM26 IG (CPE-L), but lower than that of 47/93 andF11PAA/94. No virus-like particles were seen by thenegative staining of the band. In this case, when theC44/94 purification band was inoculated in Vero cellsat a low multiplicity, a typical enterovirus CPE wasobserved after two passages and identified as CA9.

DiscussionThe complete characterization of the 47/93 IPK strainwas accomplished. According to the neutralizationtest using the LBM antiserum pools, this strain maybe classified as a CA9 virus. In this case, the strain isnot sensitive to chloroform, it is sensitive to GHCland particles of 28 nm were seen by electron micros-copy as reported for enteroviruses [8].

It is significant that the majority of the viral iso-lates obtained during the neuropathy epidemic corre-sponded to CPE-L strains. This type of strain can notbe identified with the LBM pools, as described previ-ously by Más et al, 1997 [2], but its relationship withthe enteroviruses has been demonstrated [1, 9].

Figure 4. Transmission electron micrograph of Vero cell infectedby the prototype strains 44/93 IPK with CPE-L at day 7 afterinoculation. n: nucleus, c: cytoplasm. Bars =100 nm.

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Figure 2. Transmission electronmicrograph of Vero cells infected bythe prototype 47/93 IPK strainidentified as coxackievirus A9, 18 hafter inoculation. P: Virus-like particles.C: cytoplasm. Bar =100 nm.

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During the study of these CPE-L agents some pe-culiar characteristics were observed. Contrary to ourexpectation, the CPE-L produced by the M26 IG dif-fers when Vero cells are kept in a maintenance mediacontaining PAA. In the presence of PAA a typicalenterovirus CPE is obtained. Based on the viral DNApolymerase inhibitory action of PAA [10], it is diffi-cult to postulate a molecular mechanism to explainthis change to an RNA virus. However, when the M26IG strain was grown at a low multiplicity in the ab-sence of PAA, a typical enterovirus CPE was observed.Therefore, it is probable that the change may be fa-vored by the low multiplicity of infection and not bythe presence of PAA. In addition, similar results wereobtained in the characterization of the 44/93 IPK strain.A typical enterovirus CPE appears when the purifiedstrain of CPE-L is grown at a low multiplicity withoutPAA. These results may support the theory that thepresence of PAA is not the cause of the change.

On the other hand, it is striking that F11PAA/94(a new agent from the M26 IG strain) is significantlyneutralized by the hyperimmune antiserum againstEV 7 and EV 27. This antigenic relation is not con-tradictory. The major echovirus group is geneticallycoherent and related to the coxackievirus B virus.CA9 also belongs to this same cluster. It seems obvi-ous that the great majority of EVs, CBVs, and alsoCA9 have diverged from each other relatively late inevolution. There are some antigenic epitopes wherethe immunological response of the host has had aneffect in favor of mutation [11].

Certain viruses yield a high proportion of mu-tants on passage in the absence of any known mu-tagen. These spontaneous mutations accumulate inthe genome of viruses and introduce the variation inphenotype which is subject to selection pressureduring the evolution of a virus [12]. In viruses withRNA genomes, high rates of spontaneous mutation,in the order of 10-3-10-4 per incorporated nucleotide,have been found [13]. Data from the RNA phage Qbindicated that at equilibrium a wild type dominatedthe population because it replicated more rapidlythan the spontaneously arising mutant. Unfortunatelyfor the geneticist, there are factors that promote dis-equilibrium in genome populations and these factors

8. Pumarola A. Picornavirus. En: Pumaro-la A, Rodríguez TA, García JA, Piedrola AG.Microbiología y parasitología médica. 3ra

Ed. Barcelona, España: Ediciones Científi-cas y Técnicas S.A.; 1987. p.642-51.

9. Guzmán MG, Resik S, Sariol C, Rodrí-guez R, Álvarez M, Morier L, et al. Carac-terización físico-química de los agentesaislados durante el brote de neuropatíaepidémica en Cuba. Parte II. Rev Cub MedTrop 1995b;47(1):36-40.

10. Shipkowitz NL, Bower RR, Appell RN,Nordeen CW, Overby LR, Roderick WR, etal. Suppression of herpes simplex virusinfection by phosphonoacetic acid. ApplMicrobiol 1973;26:264-7.

11. Huttunen P, Santti J. The major echovi-rus group is genetically coherent andrelated to coxsackie B viruses. J Gen Virol1996;77:715-25.

12. Coen DM, Raming RF. Viral genetics.In: Fields BN, Knipe DM, editors. Fieldsvirology. 3rd ed. Philadelphia: Lippincott-Raven Press; 1996, Vol 1. p.113-29.

13.Ward CD, Stokes MAM, Flanegan JB.Direct measurement of the poliovirus RNApolymerase error frequency in vitro. J Virol1988;62:558-62.

14.Huang AS, Baltimore D. Defectiveinterfering animal viruses. In: Fraenkel-Conrat H, Wagner RR, editors. Compre-hensive virology. New York: Plenum Press;1977, Vol. 16. p.73-116.

15. Kuge S, Saito I, Nomoto A. Primaryptructure of poliovirus Defective-interferingparticle genomes and possible generationmechanisms of the particles. J Mol Biol1986;192:473-87.

16.Kaplan G, Racaniello VR. Constructionand characterization of poliovirus subge-nomic replicons. J Virol 1988;62(5):1687-96.

17. Álvarez M, Rosario D, Sarmiento L, BeckM, Handy J, Shi Q, et al. Proceedings of theV Latin American Congress of Tropical Me-dicine, Havana, Cuba, 1997:168.

often favor the accumulation of mutants in a viruspopulation. Viruses are standardly subcloned at in-tervals to combat this problem, but mutation oftenoccurs during plaque formation or virus growth sothat it is often difficult to produce genetically homo-geneous high-titer virus stocks. In these experiments,a population with CPE-E was obtained from onewith CPE-L (44/93 IPK) that had been previouslycloned. [12]

This type of spontaneous mutation may obvi-ously induce the formation of defective interfering(DI) particles. Several types of interference havebeen noted with animal viruses. Of greatest interestto the geneticist is the homologous interference,which occurs within the cell and is exhibited onlyagainst homologous viruses or closely related vi-ruses. One well-documented form of homologousinterference is that occuring in viruses that have beenpassaged at a high multiplicity [14]. An interferingphenotype may be determined not by the deletedgenes but by mutations in the genes that are presentor by both [12].

Purified DI particles are able to initiate a normalreplication cycle but fail to synthesize capsid proteins.Several studies show that the great majority of thedeletions in these DI RNA preparations are located inthe genome region where capsid proteins are encoded.In addition, the population of individual DI RNAs isnot static but changes during passage [15]. DI par-ticles generated after the deletion of no more than20% of the capsid sequences, interfere with the repli-cation of standard particles by competing for the cel-lular machinery and for viral capsids [16].

The CPE-L strains are antigenically related to en-teroviruses. The CPE-E appears when the CPE-Lstrain grows in tissue culture at a low multiplicity.The CPE-L producing strains show a decrease inplaque size and a minor sedimentation coefficient insucrose. In addition, the interference of 44/93 IPKhad been demonstrated in the CA9 growth (Más, un-published data 1993).

The observations related to the CPE-L strains arein agreement with molecular studies carried out byother authors. The 44/93 IPK strain genome was am-plified by PCR using primers of CA9 and B4. Theresults showed amplification in all regions except forthe region that encodes the structural proteins [17].Western blot assays using 44/93 IPK as an antigenrevealed no bands in the size range expected for en-terovirus structural proteins when rabbit hyperim-mune antiserum grown against the 47/93 IPK strainwas used as the primary antibody [2].

The absence of bands with the correct size rangefor the structural proteins and the results obtained byPCR, might be related to changes in virion structureand morphogenesis. This hypothesis may explain theabsence of virus-like particles in cells infected withthe CPE-L producing agent.

Previous studies indicate the possibility of morethan one agent in the same sample obtained from pa-tients with epidemic neuropathy [9]. Our results pointto the presence of two variants of the same agent,including an incomplete form.

DI particles may have biological significance. Theyare formed during in vivo infections and it has been

44/93 IPK

RC44/94 IPK

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Figure 5. Growth rate of the 44/93 IPK strain in Vero cellsbefore and after cloning (RC44/94 IPK).

Figure 6. Plaque assay for theF11PAA/94 IPK and the 44/94 IPKstrains. Arrows point to the plaques.

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Received in October, 2001. Acceptedfor publication in March, 2002.

postulated that they may play a role in the limitationof the progress of the disease. DI particles have alsobeen shown to play a role in the establishment andmaintenance of persistent infections in tissue cultureand in vivo [18, 19, 12].

In this case, persistency was demonstrated in vivo.A second CSF specimen from 25 patients with a pre-vious CSF positive viral isolation and continuing clini-cal symptoms was obtained. The second samples werecollected 21 to 30 days later and in one case, one yearlater. Virus with CPE- was reisolated from 24 (96%)of these second CSF samples [2].

Over the years, scientists have made several attemptsto understand how incomplete particles moderate vi-rus infection in vivo. Taking into consideration the re-

sults obtained in all the research related with the epi-demic neuropathy outbreak that occurred in Cuba, Máset al. formulated an integral, multifactorial hypothesisto explain a pathophysiologic mechanism by whichisolated viruses could participate in the pathogenesisof the disease. According to the hypothesis, the virusisolated from patients with epidemic neuropathy mightbe the product of a reactivation of the enterovirus RNAwhich was latent in the CNS, as a consequence of anincrease in oxidative stress. In very few cases the ex-pression of viral RNA produced complete virions ca-pable of lytic replication that is characteristic ofenteroviruses. In most of the patients, the reactivationproduced incomplete virions, capable of replicatingwithout a helper virus, and producing CPE-L [20].

18. Holland JJ, Grabau E, Jones CL, SemlerBL. Evolution of multiple genome muta-tions during long term persistent infectionwith vesicular stomatitis virus. Cell 1979;16:495-504.

19.Holland JJ, Kennedy SI, Semler BL, JonesJL, Roux L, Grabau E. Defective interferingRNA viruses and the host cell response. In:Fraenkel-Conrat H, Wagner RR, editors.Comprehensive virology. New York:Plenum Press; 1980, Vol. 16. p.137-92.

20. Más P, Guzmán MG, Sarmiento L, PérezAB, Álvarez M, Capó V, et al. Mechanismof enterovirus involvement in epidemicneuropathy: hypothesis regarding patho-physiology. Medical Hypotheses 2001;56(3):339-47.

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