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Relationship between apoptosis and the BH2 domain sequence of the VP5 peptide of infectious pancreatic necrosis virus/Relacion entre apoptosis y secuencia del dominio BH2 del peptido VP5 del virus de la necrosis pancreatica infecciosa.

INTRODUCTION

The infectious pancreatic necrosis virus (IPNV) causes a contagious globally distributed disease that mainly affects young salmonids (1); the species, age and health condition of animals affected and the virulence of the strain are factors that influence the clinical signs and mortality level (2, 3).

The IPNV genome consists of two double-stranded RNA segments (dsRNA). Segment A exhibits two open reading frames (URFs); the highest URF encodes a polyprotein 5'-pVP2-NS-VP3', which is cotranslationally divided by the nonstructural protease (NS) or VP4, generating structural proteins VP2 and VP3, which respectively comprise the external and internal surface of the viral capsid; the lower URF encodes an NS peptide known as VP5 which is not part of the virion and has been implicated in the inhibition of apoptosis, thus promoting viral survival. The Segment B shows a single URF encoding a polymerase RNA dependent on RNA (RdRp), known as VP1 (3, 4).

The role of IPNV proteins as virulence factor is not fully determined, but it is suggested that through any of its products, the virus could modulate certain cellular processes to favor the survival of its progeny, in addition, to alter defense mechanisms for establishing the infection (5, 6). Sano et al (7), related the virulence of IPNV with products of the genome of segment A, and subsequently, Song et al (4) identified that variations in residues 217 and 221 in the amino acid sequence of the VP2 protein influence the virulence level of strains. The function of VP5 is uncertain, but since it shows four homologous domains with anti-apoptotic cellular proteins Bcl-2 (BH) it is considered as virulence factor that could modulate this form of cell death (6, 8, 9), associated with the amino acid sequence of the BH2 domain (10).

Apoptosis is a genetically controlled cell death process, involved in regulating the development and homeostasis of tissues; it is also a defense mechanism that can be stimulated by immune reactions or when there is cell damage by disease or toxic agents. This process can be initiated by external (extrinsic or receptor-mediated) or internal (intrinsic or mitochondrial) cell stimulation; both involve a preserved group of specific aspartate cysteine-proteases called caspases that are synthesized as zymogens, which must undergo proteolytic fragmentation to activate. The activation of the initial caspase triggers a sequential death program, where executing caspases alter proteins of the cytoskeleton and nuclear matrix. The activation of caspase 3 releases the inhibitor of a cytoplasmic DNase (CAD) that gives rise to the internucleosomal fragmentation of DNA (9, 11, 12). In viral infections, the modulation of apoptosis has been associated with products of the virus, which can manipulate certain cells processes to allow the survival of their progeny (13-15).

Hong et al (5) reported that the cytopathic effect (CPE) showed by cells infected with IPNV is a post apoptotic process, related to the suppression of the gene of the survival factor Mcl-1, a member of the antiapoptotic protein family Bcl-2; in contrast, Espinoza et al (13), reported that only 12% of the cell population infected with a strain of serotype VR-299 showed apoptosis, with cell death by necrosis taking precedence. Santi et al (10), observed no differences in the percentage of apoptosis in cells infected with recombinant strains of Spjarup serotype (Sp), including a strain that did not express VP5, suggesting that replacements or absences of certain residues of the sequence of the BH2 domain or other factor(s) of the virus could influence the development of this form of cell death. With this background, this study was undertaken to relate the induction level of apoptosis with the BH2 domain sequence and with the level of infectivity of three field strains of IPNV.

MATERIALS AND METHODS

Cells and virus. Chinook salmon embryo (CHSE-214) cells were used for the propagation of IPNV and in assays to assess apoptosis. Cells were cultured at 20[degrees]C in a Minimal Essential Medium with Eagle (E-MEM) salts, supplemented with 100 IU of penicillin/ml, 100 pg of streptomycin/ml and 10% (v/v) of fetal bovine serum (FBS). Once the monolayer has been formed, cells were kept in E-MEM supplemented with 2% (v/v) of FBS.

The virus strains used were obtained from clinical cases of infectious pancreatic necrosis (IPN). The V33/98 virus was isolated in 1998 from juvenils of Atlantic salmon (Salmo salar) the infection of which caused a mortality of 80%; the V70/06 virus was obtained in 2006 from juvenils of S. salar in which high initial mortality was observed with evolution to low mortality and chronicity, while the V112/06 virus was isolated from rainbow trout fries (Oncorhynchus mykiss) where a mortality of 40% was observed (16). The virus was reactivated by inoculation in pre-confluent CHSE-214 cells in E-MEM with 2% (v/v) of FBS; when showing an extensive cytopathic effect (CPE), the culture was frozen for 24 h and then thawed and centrifuged for 10 min at 4, 000 rpm at 4[degrees]C; the viral titer was calculated by the Reed-Muench method (17).

The following access codes to the GenBank were used for the alignment and comparison of the VP5 protein of IPNV: AAK71697 VP5 Ab/E1S; AY379740 VP5 of 12.1 kDa/Sp; Q69CI1 VP5 15 Kda IPNV/Sp; AAA92630 VP5 IPNV/VR-299; P10415 Bcl2 Humana-Apoptosis regulator.

Amplification of VP2 y VP5 genomes by RTPCR. The viral RNA was isolated from cell culture supernatants by column extraction according to specifications of the Viral RNA mini Kit QIAamp[R] (Cat. No. 52904; QiAGEN Germany). The RNA diluted in 50 [micro]l of sterile deionized water treated with diethyl pyrocarbonate was quantified by spectrophotometry and frozen at -80[degrees]C until use.

The obtaining and amplification of cDNA was carried out using 0.25 pg of Random primers (Promega) in 20 [micro]l of reagent solution. The amplification of a gene product of 1180 pb corresponding to a portion of the VP2 coding sequence was carried out in a thermal cycler Gene Amp PCR System 2400[R] (Perkin Elmer) in a volume of 50 [micro]l; the primers used for the amplification of VP2 and cycle parameters were the same as described in Ortega et al (16). The amplification of the VP5 gene was carried out with quantities of reagents and similar cycles as for VP2, replacing specific primers to amplify a product of 450 pb: Fwd-5 ' ATGGCGAAGCCCTTTCTAAC-3 ' and Rev-5 ' ACAGACTTCCTTCGAAGGTG-3'; both products were subjected to electrophoresis in agarose gel at 1% with ethidium bromide, applying a current of 80 mA for 30 min in a camera SUB-CELL GT[R] and power supply POWER-Cap 200[R] (BIO-RAD). The gels were analyzed in a transilluminator TFX20.M[R] (VILBER LOURMAT), detecting fragments of 1.180 bp and 450 bp, corresponding to VP2 and VP5, respectively.

Segments of cDNA with a portion of the VP2 and VP5 coding sequence were extracted from the agarose gel according to the instructions of the supplier of the E.Z.N.A.[R] Gel Extraction Kit (OMEGA BIO-TEK) and once purified, were sent to be sequenced. The nucleotide sequences obtained were translated using ExPASy and the amino acid sequence deduced of both proteins was compared with sequences obtained from NCBI, using the Molecular Evolutionary Genetics Analysis software version 4.0 MEGA4 (4, 16).

Determination of the viral titer in cell culture supernatant. For determining differences in the level of infectivity and replication of the viruses used, CHSE-214 cells were inoculated to a infection multiplicity MDI=0.1 in 25 [cm.sup.2] culture bottles and after 12, 24, 36 and 45 h of incubation at 15[degrees]C, the bottles were frozen and thawed twice; the content was centrifuged at 4.000 rpm for 10 min at 4[degrees]C and the supernatants were tittered in accordance with the Reed and Muench method (17). The titers obtained were represented by the average of three samples with a standard deviation indicator bar.

Detection of apoptosis induction in CHSE-214 cells. CHSE-214 cells with a confluence of 90% in circular glass coverslips were infected by triplicate at MDI = 10 with each of the strains; three coverslips were not infected to be used as negative control. After 8, 12 and 18 h of incubation at 15[degrees]C, the samples were washed in PBS pH 7.2 and fixed for 15 min in paraformaldehyde at 4%. Apoptosis was detected by the identification of the DNA fragmentation by TUNEL assay (Terminal Transferase Uridyl Nick End Labeling) according to instructions from the supplier Apoptosis Detection Kit (USBiological T9162-180), and also by detection of caspase 3 by immuno-histochemical assay, following the recommendations of the manufacturer of the product Anti-ACTIVE[R] Caspase-3 pAb. Samples were analyzed under a confocal microscope; the number of positive cells in a sample of approximately 300 cells was obtained, and was represented as the average of three independent samples [+ or -] standard error of the mean (SEM). Data were analyzed by the Friedman test, with statistical significance defined by p<0.05 values.

RESULTS

Thirty-six hours after the infection (hpi), the three virus strains caused multiple cytopathic effect foci (CPE) in infected cells; the presence and intracytoplasmic distribution of the viral antigen (VP3 protein) was diffusely detected by indirect immunofluorescence (IFAT) showing areas of aggregation or inclusion bodies. The detection of the antigen occurred later in V112/06. Non-infected cells used as negative control showed no fluorescence (Figure 1).

The gene fragments of 1180 and 450 pb, corresponding to the VP2 and VP5 proteins of IPNV, were detected by RT-PCR in cells infected with the three strains (not shown). The amino acid sequence of VP2 was compared with the sequence of the Sp (Genbank AJ829474) and VR-299 strains (L40584); the sequence of the V70/06 (Genbank GU072916) and V33/98 strains (Genbank GU072915) corresponded to genogroup Sp, which show identity percentages of 98 and 96%, respectively; the sequence of the V112/06 strain (Genbank GU072914) showed a identity percentage of 96% with genogroup VR-299.

The analysis of the VP2 sequence shows that the residues involved in the virulence or infectivity of the IPNV in the V70/06 strain corresponded to Pro-217 and Ala-221; Ala-217 and Thr-221 for V112/06, and finally Pro-217 and Thr-221 for V33/98 (Table 1).

Regarding VP5, the genes of the three strains showed the four homology domains with Bcl-2, and initiated translation at codon 16. However, the VP5 ORF in V112/06 was 394 pb, coding 131 aa (15 kDa); while in V70/06 and V33/98 the ORF was composed of 351 pb, coding a truncated protein of 116 aa (13 kDa).

The multiple alignment of VP5 sequences of V70/06 (JQ247979), V112/06 (JQ253903) and V33/98 (JQ253902), with the partial sequence of human protein Bcl-2 (P10415) and VP5 of strains E1S, 12 kDa and 15 kDa (10), was similar in BH4, BH1, and BH3 domains. However, the BH2 domain was only complete in V112/06, and partially truncated in V70/06 and V33/98. Residues 115 and 122 of VP5, which are homologous to residues Trp188 and Trp195 of Bcl-2 and that are considered critical to the antiapoptotic function of this protein, were Trp115 and Arg122 for V112/06; while V70/06 and V33/98 only showed Arg115 (Table 2).

In general, the viral titers of the three strains were increased as the infection spread. At 12 hpi, cells infected with V70/06 and V112/06 showed a viral titer < 1x[10.sup.0.66] TCID50/mL, while in those infected with V33/98 it was 1x[10.sup.3.5]. At 24 and 36 hpi, V70/06 showed more titers than the other two strains, but at 45 hpi its titer was similar to V33/98 (1x[10.sup.7.5]). The titers in V112/06 were lower at 36 and 45 hpi (Figure 2).

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The percentage of apoptosis obtained in cells infected with the three strains was similar (p>0.05); there was no gradual increase between the times analyzed, and in any case the percentage was not greater than 12%. In cells infected with V70/06, the percentage of apoptosis increased between 8 and 12 hpi, without further variation (Figure 3). Cells used as negative control and not infected showed a lower percentage of apoptosis with respect to infected samples.

Figure 4 shows positive samples to the reaction of the TUNEL assay and positive cells to the IPNV infection.

The activation profile of caspase 3 examined by immunoassay (Figure 5) was similar among the three strains; despite the fact that at 18 hpi it is possible to observe a slight increase with respect to the previous reading, there were no differences between evaluated times (Figure 6), and the percentage of cells in apoptosis did not exceed 12% in the analyzed times (p>0.05).

DISCUSSION

Several products of RNA virus genes affecting fish have been reported as regulators of apoptosis (14); since that VP5 protein of IPNV has four homology domains with proteins of the subfamily Bcl-2 (BH) with antiapoptotic activity, it has been suggested that during replication the virus could inhibit the apoptosis to permit the viral replication.

In this study, the VP5 protein of the strains used showed the four BH domains characteristic of Bcl-2 antiapoptotic proteins. However, the BH2 domain, which is considered critical to the antiapoptotic activity, was truncated at V70/06 and V33/98, and was complete in the V112/06 strain; despite this, there were no differences in the induction level of apoptosis between strains, and the average did not exceed 12% in the times evaluated. This indicates that the size of VP5 has no influence on the induction of apoptosis and is consistent with what was reported by Santi et al (10), who observed that IPNV may induce apoptosis regardless of the size and expression of the VP5 protein. A similar situation has been reported with the virus of the African swine fever that contains a homologous protein to the inhibitor of apoptosis of baculovirus (IAP) that is not necessary for in vitro viral growth, does not modulates virulence and has no influence on the induction of apoptosis.

As a possible explanation for the above, Cuconati and White (18), pointed out that certain viral Bcl-2 proteins may have antiapoptotic activity even lacking some of the BH domains, but that the BH2 domain is a key element for heterodimerization with the BAX and BAK death promoters. In the case of IPNV, this interaction would take place through the residue Trp155 of VP5 that would correspond to residue Trp188 of Bcl-2, considered a critical residue for fulfilling the antiapoptotic function; accordingly, strains that express Trp-155 in VP5 should inhibit apoptosis. In this regard, Santi et al (10) proposed that the fact of not observing differences in the induction level among IPNV strains used in her study, could be due to the absence of the Trp155 residue. However, although in the present study the VP5 of V112/06 showed the Trp-115 residue in BH2, there was no difference in terms of induction with respect to other strains, so the sequence or substitution of this residue is not a critical factor in the modulation of apoptosis.

The results obtained in this study showed that the IPNV VP5 protein would not participate in the modulation of apoptosis, contrary to what was observed in the infection with the infectious bursa disease virus (IBDV), that encodes a homologous protein containing a transmembrane region, but lacks homology domains with Bcl-2, shows a PEST domain that is usually present in short-lived and early-expression proteins that fulfill regulatory activities (19). During the replication of IBDV, VP5 accumulates in the cell membrane, alters its morphology and causes subsequent rupture, acting as death factor allowing the release of its progeny (12, 20, 21).

The involvement of the VP2 protein in the virulence or infectivity of IPNV is associated with its amino acid sequence; strains that show Thr-217 and Ala-247 residues produce more viral titer during a replication cycle, and are considered of greater virulence (4). In this study, it was observed that the infectivity of the strains used was unrelated to the induction level of apoptosis in CHSE-214 cells; strains V70/06 and V33/98 corresponded to average virulence strains by having the residues Pro-217 and Ala-247, and Pro-217 and Thr221 respectively, while in V112/06 sequence was Ala-217 and Ala-247, related to avirulent strains (4). While overall the replication curves of the three strains showed a similar growth pattern, V112/06 produced less titers, confirming it as a strain with lower infectivity.

The percentage of apoptosis obtained through TUNEL and immunoreaction assay does not show differences in cells infected with the strains V70/06 and V33/98 of the Sp genotype and strain V112/06 of genotype VR-299, evidencing that the genotype of the strain is not related to this form of cell death. It has been indicated that the IPNV dsRNA genome can trigger apoptosis by the activation of the PKR pathway that inhibits the translation of cellular proteins, and the RNase L pathway that degrades the mRNA (22-24); more recently, Chiu et al (25), observed that the VP3 protein induces the expression of BAD stimulating apoptosis through the mitochondrial pathway. These assertions would support the fact that apoptosis observed during infection by IPNV may develop for independent reasons from the characteristics of the strain involved in the infection (14, 26). In support of the foregoing, Imajoh et al (14) reports that Salmo salar cells positive to IPNV infection do not frequently express this form of death; suggesting that apoptosis corresponds to an autonomous suicide process for eliminating infected cells preventing the viral spread.

After infecting CHSE-214 cells with Ab serotype IPNV, considered of low virulence with respect to Sp and VR-299 strains, Hong and Wu (27) determined that apoptosis is related with the expression of the BAD death gene, as an early stress factor induced through TNF receptors. In relation to this, Espinoza et al (13) propose that if apoptosis is initiated via the receptor, the viral serotype does not affect the activation of the process. This proposal is consistent with that observed in this study, where there are no differences in the average of apoptosis obtained in cells infected with V70/06 and V33/98 Sp serotype and V112/06 VR-299 serotype strains. However, the receptor that could modulate the process is unknown.

Taking into account that apoptosis is an early defense mechanism, the level obtained with the three strains of this study evidences that IPNV is a poor inducer of this death mechanism, which is consistent with Espinoza et al (13), who observed that the percentage of apoptotic cells was less than 12%, while at 15 hpi 75% of the cell population showed necrosis; the results are also consistent with that reported by Hong and Wu (27), who observed that although practically all cells were positive to the infection, only a low percentage developed apoptosis.

In this study it was observed that the percentage of apoptosis during infection was unrelated to the presence of the Trp-155 residue in the sequence of the IPNV BH2 domain; this result is consistent with researches indicating that apoptosis occurs irrespective of the presence or sequence of certain viral peptides with Bcl-2 homology (14, 18, 26). Likewise, since neither the differences in the VP2 and VP5 sequence nor the length of the latter influence the degree of apoptosis, probably other variables such as the type of tissue and host susceptibility would have greater relevance, as observed in the case of ISAV (28). However, the actual participation of the viral components in the induction of this form of cell death and pathogenesis is yet to be determined.

In conclusion, differences in the BH2 sequence of the VP5 protein and the infectivity in the sequence of the VP2 protein are not associated with the modulation of apoptosis.

Acknowledgements

To The National Science and Technology Council (Conacyt) project 99736 and project PROMEP 103.5/09/4196; to the Graduate School and the Biotechnology and Aquatic Pathology Laboratory of the Animal Pathology Institute, Faculty of Veterinary Sciences of Universidad Austral de Chile.

INTRODUCCION

El virus de la necrosis pancreatica infecciosa (IPNV) ocasiona una enfermedad contagiosa de distribucion mundial que afecta principalmente a salmonidos jovenes (1); la especie, la edad y el estado de salud de los animales afectados y la virulencia de la cepa son factores que influyen sobre la signologia clinica y el nivel de mortalidad (2, 3).

El genoma de IPNV consiste de dos segmentos de ARN de doble cadena (dsRNA). El segmento A presenta dos marcos de lectura abiertos (uRFs); el ORF mayor codifica una poliproteina 5'-pVP2-NSVP3', que es escindida co-traduccionalmente por la proteasa no estructural (NS) o VP4, generando las proteinas estructurales VP2 y VP3, que respectivamente integran la superficie externa e interna de la capside viral; el ORF menor codifica un peptido NS conocido como VP5 el cual no forma parte del virion y ha sido implicado en la inhibicion de apoptosis, favoreciendo asi la supervivencia viral. El segmento B presenta un solo ORF que codifica una ARN polimerasa dependiente de ARN (RdRp), conocida como VP1 (3, 4).

El papel de las proteinas de IPNV como factor de virulencia no esta plenamente determinado, pero se sugiere que a traves de alguno de sus productos, el virus podria modular ciertos procesos celulares para favorecer la supervivencia de su progenie, ademas, de alterar mecanismos de defensa para establecer la infeccion (5, 6). Sano et al (7), relacionaron la virulencia de IPNV con productos del genoma del segmento A, y posteriormente, Song et al (4) identificaron que variaciones en los residuos 217 y 221 en la secuencia aminoacidica de la proteina VP2 influyen en el nivel de virulencia de las cepas. La funcion de VP5 es incierta, pero debido a que presenta cuatro dominios homologos con proteinas celulares anti-apoptoticas Bcl-2 (BH) se considera factor de virulencia que podria modular esta forma de muerte celular (6, 8, 9), asociado a la secuencia aminoacidica del dominio BH2 (10).

La apoptosis es un proceso de muerte celular geneticamente controlado, implicado en regular el desarrollo y la homeostasis de los tejidos; tambien es un mecanismo de defensa que puede estimularse por reacciones inmunes o cuando existe dano celular por enfermedad o agentes toxicos. Este proceso puede iniciarse mediante estimulacion celular externa (via extrinseca o mediada por receptor) o interna (via intrinseca o mitocondrial); en ambas vias participan un grupo conservado de cisteina-proteasas especificas de aspartato denominadas caspasas que se sintetizan como zimogenos, los cuales deben sufrir fragmentacion proteolitica para activarse. La activacion de la caspasa iniciadora desencadena un programa de muerte secuencial, donde las caspasas ejecutoras alteran proteinas del citoesqueleto y la matriz nuclear. La activacion de caspasa 3 libera el inhibidor de una DNAasa citoplasmatica (CAD) que da lugar a la fragmentacion internucleosomal del ADN (9, 11, 12). Ante infecciones virales, la modulacion de apoptosis se ha asociado a productos del virus, que pueden manipular ciertos procesos de las celulas para permitir la supervivencia de su progenie (13-15).

Hong et al (5), reportaron que el efecto citopatico (CPE) que muestran celulas infectadas con IPNV es un proceso post apoptotico, relacionado con represion del gen del factor de supervivencia Mcl-1, un miembro de la familia de proteinas antiapoptoticas Bcl-2; en contraste, Espinoza et al (13), reportan que solo el 12% de la poblacion celular infectada con una cepa de serotipo VR-299 presento apoptosis, prevaleciendo la muerte celular por necrosis. Santi et al (10), no observaron diferencias en el porcentaje de apoptosis en celulas infectadas con cepas recombinantes de serotipo Spjarup (Sp), incluyendo una cepa que no expresaba VP5, sugiriendo que sustituciones o ausencias de ciertos residuos de la secuencia del dominio BH2 u otro(s) factor(es) del virus podrian influir en el desarrollo de esta forma de muerte celular. Ante estos antecedentes, este estudio se realizo para relacionar el nivel de induccion de apoptosis con la secuencia del dominio BH2 y con el nivel de infectividad de tres cepas de campo de IPNV.

MATERIALES Y METODOS

Celulas y virus. Se utilizaron celulas Chinook salmon embryo (CHSE-214) para propagacion de IPNV y en los ensayos para evaluar apoptosis. Las celulas se cultivaron a 20[degrees]C en medio de cultivo Minimal Essential Medium con sales de Eagle (E-MEM), suplementado con 100 UI de penicilina/ml, 100 pg de estreptomicina/ ml y 10% (v/v) de suero fetal bovino (SFB). Una vez formada la monocapa, las celulas se mantuvieron en E-MEM suplementado con 2% (v/v) de SFB.

Las cepas de virus utilizadas fueron obtenidas de casos clinicos de necrosis pancreatica infecciosa (IPN). El virus V33/98 fue aislado en 1998 a partir de juveniles de salmon del Atlantico (Salmo salar) cuya infeccion ocasiono un 80% de mortalidad; el virus V70/06 se obtuvo en 2006 de juveniles de S. salar en los que se observo mortalidad inicial alta con evolucion a baja mortalidad y cronicidad, mientras que el virus V112/06, se aislo de crias de trucha arcoiris (Oncorhynchus mykiss) donde se observo un 40% de mortalidad (16). Los virus se reactivaron por inoculacion en celulas CHSE-214 pre-confluentes en E-MEM con 2% (v/v) de SFB; al presentar extenso efecto citopatico (CPE), el cultivo se congelo por 24 h y posteriormente se descongelo y centrifugo durante 10 min a 4000 rpm a 4[degrees]C; el titulo viral se calculo por el metodo de Reed y Muench (17).

Los siguientes codigos de acceso al GenBank se utilizaron para alineamiento y comparacion de la proteina VP5 de IPNV: AAK71697 VP5 Ab/E1S; AY379740 VP5 de 12.1 kDa/Sp; Q69CI1 VP5 15 Kda IPNV/Sp; AAA92630 VP5 IPNV/VR-299; P10415 Bcl2 Humana-Apoptosis regulator.

Amplificacion de los genomas de VP2 y VP5 por RT-PCR. El RNA viral, fue aislado de sobrenadantes de cultivo celular mediante extraccion en columna segun especificaciones del Viral RNA mini Kit Q1Aamp[R] (Cat. No. 52904; QiAGEN Alemania). El ARN eluido en 50 [micro]l de agua desionizada esteril tratada con dietil pirocarbonato se cuantifico por espectrofotometria y se congelo a -80[degrees]C hasta su utilizacion.

La obtencion y amplificacion de cDNA se realizo utilizando 0.25 [micro]g de Random primers (Promega) en 20 [micro]l de solucion de reaccion. La amplificacion de un producto genico de 1180 pb correspondiente a parte de la secuencia codificante de VP2 se realizo en un termociclador Gene Amp PCR System 2400[R] (Perkin Elmer) en un volumen de 50 [micro]l; los primers utilizados para amplificacion de VP2 y los parametros de ciclos fueron iguales a lo descrito en Ortega et al (16). La amplificacion del gen VP5 se realizo con cantidades de reactivos y ciclos similares como para VP2, sustituyendo los primers especificos para amplificar un producto de 450 pb: Fwd-5'ATGGCGAAGCCCTTTCTAAC-3' y Rev-5 'ACAGACTTCCTTCGAAGGTG-3'; ambos productos se sometieron a electroforesis en gel de agarosa al 1% con bromuro de etidio, aplicando corriente de 80 mA por 30 min en camara SUB-CELL GT[R] y fuente de poder POWER-PAC 200[R] (BIORAD). Los geles se analizaron en transiluminador TFX-20.M[R] (VILBER LOURMAT), detectando fragmentos de 1180 pb y 450 pb, correspondientes a VP2 y VP5, respectivamente.

Los segmentos de cDNA con parte de la secuencia codificante de VP2 y VP5 se extrajeron desde el gel de agarosa de acuerdo a instrucciones del proveedor de E.Z.N.A.[R] Gel Extraction Kit (OMEGA BIO-TEK) y una vez purificados, se enviaron a secuenciar. Las secuencias nucleotidicas obtenidas se tradujeron mediante ExPASy y la secuencia aminoacidica deducida de ambas proteinas se comparo con secuencias obtenidas en NCBI, utilizando el software Molecular Evolutionary Genetics Analysis version 4.0 MEGA4 (4, 16).

Determinacion del titulo viral en sobrenadante de cultivo de celulas. Para determinar diferencias en el nivel de infectividad y de replicacion de los virus utilizados se inocularon celulas CHSE-214 a una multiplicidad de infeccion MDI=0.1 en botellas de cultivo de 25 [cm.sup.2] y despues de 12, 24, 36 y 45 h de incubacion a 15[degrees]C las botellas se congelaron y descongelaron en dos ocasiones; el contenido fue centrifugado a 4.000 rpm por 10 min a 4[degrees]C y los sobrenadantes se titularon de acuerdo con el metodo de Reed y Muench (17). Los titulos obtenidos se representaron por el promedio de tres muestras con barra indicadora de desviacion estandar.

Deteccion de induccion de apoptosis en celulas CHSE-214. Celulas CHSE-214 con confluencia de 90% en cubreobjetos circulares de vidrio fueron infectadas por triplicado a MDI=10 con cada una de las cepas; tres cubreobjetos no fueron infectados para usarse como control negativo. Despues de 8, 12 y 18 h de incubacion a 15[degrees]C, las muestras se lavaron en PBS pH 7.2 y se fijaron durante 15 min en paraformaldehido al 4%. La apoptosis fue detectada por identificacion de la fragmentacion de ADN mediante ensayo TUNEL (Terminal Transferase Uridyl Nick End Labelling) de acuerdo a instrucciones del proveedor Apoptosis Detection Kit (USBiological T9162-180), y tambien por deteccion de caspasa 3 mediante ensayo inmuno-histoquimico, siguiendo recomendaciones del fabricante del producto Anti-ACTIVE[R] Caspase-3 pAb. Las muestras se analizaron bajo microscopio confocal; el numero de celulas positivas en una muestra de aproximadamente 300 celulas fue obtenido, y se represento como el promedio de tres muestras independientes [+ or -] error estandar de la media (SEM). Los datos se analizaron mediante test de Friedman, con significancia estadistica definida en valores de p<0.05.

RESULTADOS

A 36 horas post-infeccion (hpi) las tres cepas de virus ocasionaron multiples focos de efecto citopatico (CPE) en las celulas infectadas; mediante inmunofluorescencia indirecta (IFAT) se demostro la presencia y distribucion intracitoplasmatica del antigeno viral (proteina VP3) en forma difusa con areas de agregacion o cuerpos de inclusion. La deteccion del antigeno fue mas tardia en V112/06. Las celulas sin infectar utilizadas como control negativo no mostraron fluorescencia (Figura 1).

Los fragmentos genicos de 1180 y 450 pb, correspondientes a las proteinas VP2 y VP5 de IPNV fueron detectados mediante RT-PCR en las celulas infectadas con las tres cepas (No mostrado). La secuencia aminoacidica de VP2 fue comparada con la secuencia de las cepas de referencia Sp (Genbank AJ829474) y VR-299 (L40584); la secuencia de las cepas V70/06 (Genbank GU072916) y de V33/98 (Genbank GU072915) correspondieron al genogrupo Sp, con el que presentan porcentajes de identidad de 98 y 96%, respectivamente; la secuencia de la cepa V112/06 (Genbank GU072914) mostro un porcentaje de identidad de 96% con el genogrupo VR-299.

El analisis de la secuencia de VP2 demuestra que los residuos implicados en la virulencia o infectividad de IPNV en la cepa V70/06 correspondieron a Pro-217 y Ala-221; Ala-217 y Thr-221 para V112/06, y finalmente Pro-217 y Thr-221 para V33/98 (Tabla 1). Con respecto a VP5, los genes de las tres cepas presentan los cuatro dominios de homologia con Bcl-2, e inician la traduccion en el codon 16. Sin embargo, el ORF de VP5 en V112/06 fue de 394 pb, codificando 131 aa (15 kDa); mientras que, en V70/06 y V33/98 el ORF estuvo compuesto de 351 pb, codificando una proteina truncada de 116 aa (13 kDa).

El alineamiento multiple de las secuencias de VP5 de V70/06 (JQ247979), V112/06 (JQ253903) y V33/98 (JQ253902), con la secuencia parcial de la proteina Bcl-2 humana (P10415) y con VP5 de las cepas E1S, 12 kDa y 15 kDa (10), fue similar en los dominios BH4, BH3 y BH1. Sin embargo, el dominio BH2 unicamente fue completo en V112/06, y fue parcialmente truncado en V70/06 y V33/98. Los residuos 115 y 122 de VP5, que son homologos a los residuos Trp188 y Trp195 de Bcl-2 y que se consideran criticos para la funcion antiapoptotica de esta proteina, fueron Trp115 y Arg122 para V112/06; mientras que V70/06 y V33/98 solo presentaron Arg115 (Tabla 2).

En general, los titulos virales de las tres cepas se incrementaron conforme se extendio la infeccion. A las 12 hpi las celulas infectadas con V70/06 y V112/06 presentaron un titulo viral [less than or equal to] a 1x[10.sup.0.66] TCID50/mL, mientras que en las infectadas con V33/98 fue de 1x[10.sup.3.5]. A 24 y 36 hpi, V70/06 presento mayores titulos que las otras dos cepas, pero a 45 hpi su titulo fue similar a V33/98 (1x[10.sup.7.5]). Los titulos en V112/06 fueron menores a 36 y 45 hpi (Figura 2).

El porcentaje de apoptosis obtenido en las celulas infectadas con las tres cepas fue similar (p>0.05); no se observo incremento progresivo entre los tiempos analizados, y en ningun caso el porcentaje fue mayor a 12%. En las celulas infectadas con V70/06, el porcentaje de apoptosis tuvo un incremento entre las 8 y 12 hpi, sin variacion posterior (Figura 3). Las celulas usadas como control negativo, que no fueron infectadas presentaron menor porcentaje de apoptosis con respecto a las muestras infectadas.

La figura 4 representa muestras positivas a la reaccion del ensayo TUNEL y celulas positivas a la infeccion por IPNV.

El perfil de activacion de caspasa 3 evaluado mediante inmuno-ensayo (Figura 5) fue similar entre las tres cepas; pese a que a las 18 hpi es posible observar un ligero incremento con respecto a la lectura anterior, no se mostraron diferencias entre los tiempos evaluados (Figura 6), y el porcentaje de celulas en apoptosis no sobrepaso el 12% en los tiempos analizados (p>0.05).

DISCUSION

Varios productos de genes de virus ARN que afectan peces han sido reportados como reguladores de apoptosis (14); debido a que la proteina VP5 de IPNV presenta cuatro dominios de homologia con proteinas de la subfamilia Bcl-2 (BH) de actividad antiapoptotica, se ha sugerido que durante su replicacion el virus podria inhibir la apoptosis para multiplicarse.

En este estudio, la proteina VP5 de las cepas utilizadas presentaron los cuatro dominios BH que son caracteristicos de proteinas antiapoptoticas tipo Bcl-2. Sin embargo, el dominio BH2 que es considerado critico para la actividad antiapoptotica fue truncado en V70/06 y V33/98, y fue completo en la cepa V112/06; pese a esto, no se observaron diferencias en el nivel de induccion de apoptosis entre las cepas, y el promedio no supero el 12% en los tiempos evaluados. Esto indica que el tamano de VP5 no influye en la induccion de apoptosis, y esta acorde con lo reportado por Santi et al (10), quienes observaron que IPNV puede inducir apoptosis independientemente del tamano y expresion de la proteina VP5. Una situacion similar se ha reportado con el virus de la peste porcina africana que contiene una proteina homologa al inhibidor de apoptosis de baculovirus (IAP) que no es necesaria para el crecimiento viral in vitro, no modula la virulencia y tampoco influye en la induccion de apoptosis.

Como posible explicacion a lo anterior, Cuconati y White (18), indicaron que algunas proteinas Bcl-2 virales pueden tener actividad antiapoptotica aun careciendo de alguno de los dominios BH, pero que el dominio BH2 es elemento clave para heterodimerizar con los promotores de muerte BAX y BAK. Para el caso de IPNV, dicha interaccion se realizaria a traves del residuo Trp155 de VP5 que corresponderia al residuo Trp188 de Bcl-2, que es considerado un residuo critico para cumplir la funcion anti-apoptotica; de acuerdo con esto, las cepas que expresan Trp-155 en VP5 deberian inhibir la apoptosis. En este sentido, Santi et al (10), propusieron que el hecho de no observar diferencias en el nivel de induccion entre las cepas de IPNV utilizadas en su estudio, pudo deberse a la ausencia del residuo Trp155. Sin embargo, pese a que en el presente estudio, VP5 de V112/06 presento el residuo Trp-115 en BH2, no mostro diferencia de induccion con respecto a las otras cepas, por lo que la secuencia o sustitucion de este residuo no es un factor critico en la modulacion de apoptosis.

El resultado obtenido en este estudio mostro que la proteina VP5 de IPNV no participaria en la modulacion de apoptosis, contrario a lo observado en la infeccion con el virus de la enfermedad infecciosa de la bursa (IBDV), que codifica una proteina homologa que contiene una region transmembrana, pero carece de dominios de homologia con Bcl-2, presenta un dominio PEST que usualmente esta presente en proteinas de vida corta y de expresion temprana que cumplen actividad reguladora (19). Durante la replicacion de IBDV, VP5 se acumula en la membrana celular, altera su morfologia y provoca ruptura posterior, actuando como factor de muerte permitiendo la liberacion de su progenie (12, 20, 21).

La implicacion de la proteina VP2 en la virulencia o infectividad de IPNV, esta asociada a su secuencia aminoacidica; cepas que presentan los residuos Thr-217 y Ala-247 producen mayor titulo viral durante un ciclo de replicacion, y se consideran de mayor virulencia (4). En este estudio, se observo que la infectividad de las cepas utilizadas no tuvo relacion con el nivel de induccion de apoptosis en celulas CHSE-214; las cepas V70/06 y V33/98 correspondieron a cepas de virulencia media por presentar los residuos Pro-217 y Ala-247, y Pro-217 y Thr221 respectivamente, mientras que en V112/06 la secuencia fue Ala-217 y Ala-247, relacionada a cepas avirulentas (4). Pese a que en general las curvas de replicacion de las tres cepas mostraron un patron similar de crecimiento, V112/06 produjo titulos menores, confirmandose como una cepa de menor infectividad.

El porcentaje de apoptosis obtenido mediante pruebas de TUNEL e inmunoreaccion no indica diferencias en las celulas infectadas con las cepas V70/06 y V33/98 de genotipo Sp y la cepa V112/06 de genotipo VR-299, evidenciando que el genotipo de la cepa no tiene relacion con esta forma de muerte. Se ha indicado que el genoma dsRNA de IPNV puede desencadenar apoptosis por activacion de la via PKR que inhibe la traduccion de proteinas celulares, y por la via RNasa L que degrada los mRNA (22-24); mas recientemente, Chiu et al (25), observaron que la proteina VP3 induce la expresion de BAD estimulando apoptosis a traves de la via mitocondrial. Estas aseveraciones respaldarian el hecho de que la apoptosis observada durante la infeccion por IPNV puede desarrollarse por razones independientes a las caracteristicas de la cepa involucrada en la infeccion (14, 26). En respaldo a lo anterior, Imajoh et al (14), informa que celulas de Salmo salar positivas a la infeccion por IPNV, frecuentemente no expresan esta forma de muerte; sugiriendo que la apoptosis corresponde a un proceso de suicidio autonomo para eliminar celulas infectadas impidiendo la diseminacion viral.

Tras infectar celulas CHSE-214 con IPNV serotipo Ab, considerado de baja virulencia con respecto de cepas Sp y VR-299, Hong y Wu (27), determinaron que la apoptosis se relaciona con expresion del gen de muerte BAD, como factor temprano de estres, inducido a traves de receptores de tipo TNF. En relacion a esto, Espinoza et al (13) proponen que si la apoptosis es iniciada via receptor, el serotipo viral no influye en la activacion del proceso. Esta propuesta concuerda con lo observado en este estudio, donde no se muestran diferencias en el promedio de apoptosis obtenido en las celulas infectadas con las cepas V70/06 y V33/98 de serotipo Sp y la cepa V112/06 de serotipo VR-299. No obstante, se desconoce el receptor que pudiera modular el proceso.

Tomando en cuenta que la apoptosis es un mecanismo de defensa temprano, el nivel obtenido con las tres cepas de este estudio hace evidente que IPNV es pobre inductor de este mecanismo de muerte, lo que concuerda con Espinoza et al (13), quienes observaron que el porcentaje de celulas apoptoticas fue menor de 12%, mientras que a 15 hpi el 75 % de la poblacion celular presento necrosis; los resultados tambien estan de acuerdo con lo reportado por Hong y Wu (27), que observaron que aunque practicamente todas las celulas fueron positivas a la infeccion, solo un bajo porcentaje desarrollo apoptosis.

En el presente estudio, se observo que el porcentaje de apoptosis obtenido durante la infeccion no tuvo relacion con la presencia del residuo Trp-155 en la secuencia del dominio BH2 de IPNV; este resultado concuerda con investigaciones que indican que la apoptosis ocurre independientemente de la presencia o secuencia de algunos peptidos virales que tienen homologia con Bcl-2 (14, 18, 26). Asimismo, en razon a que ni las diferencias en la secuencia de VP2 y VP5, ni la longitud de esta ultima influyen en el grado de apoptosis, probablemente otras variables como el tipo de tejido y la susceptibilidad de hospedero tendrian mayor relevancia, como se ha observado en caso de ISAV (28). Sin embargo, falta determinar la real participacion de los componentes virales en la induccion de esta forma de muerte celular y en la patogenesis.

En conclusion las diferencias en la secuencia de BH2 de la proteina VP5, la infectividad y en la secuencia de la proteina VP2 no estan asociadas con la modulacion de apoptosis.

Agradecimientos

Al Consejo Nacional de Ciencia y Tecnologia (Conacyt) proyecto 99736 y proyecto PROMEP 103.5/09/4196; a la Escuela de Graduados y el Laboratorio de Biotecnologia y Patologia Acuatica del Instituto de Patologia Animal, Facultad de Ciencias Veterinarias de la Universidad Austral de Chile.

REFERENCES

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(6.) Santi N, Song H, Vakharia V, Evensen 0. Infectious Pancreatic Necrosis Virus VP5 Is Dispensable for Virulence and Persistence. J Virol 2005; 79:9206-9216.

(7.) Sano M, Okamoto H, Fukuda H, Saneyoshi M, Sano T. Virulence of infectious pancreatic necrosis virus is associated with the larger RNA segment (RNA segment A). J Fish Dis 1992; 15:283-293.

(8.) Hong J, Gong H, Wu J. IPNV VP5, a Novel Anti-apoptosis gene of the Bcl-2 Family, Regulates Mcl-1 and Viral Protein Expression. Virol 2002; 295:217-229.

(9.) Weber S, Fichtner D, Mettenleiter T, Mundt E. Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon. J Gen Virol 2001; 82:805-812.

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(11.) Hong J, Lin T, Hsu Y, Wu J. Apoptosis Precedes Necrosis of Fish Cell Line with Infectious Pancreatic Necrosis Virus Infection. Virol 1998; 250:76-84.

(12.) Galloux M, Libersou S, Morellet N, Bouaziz S, Da Costa B, Ouldali M, Lepault J, Delmas B. Infectious Bursal Disease Virus, a Non-enveloped Virus, Possesses a Capsid-associated peptide That Deforms and Perforates Biological Membranes. J Biol Chem 2007; 282:20774-20784.

(13.) Espinoza J, Cortes M, Kuznar J. Necrosis of infectious pancreatic necrosis virus (IPNV) infected cells rarely is preceded by apoptosis. Virus Res 2005; 109:133-138.

(14.) Imajoh M, Hirayama T, Oshima S. Frequent occurrence of apoptosis is not associated with pathogenic Infectious Pancreatic Necrosis Virus (IPNV) during persistent infection. Fish Shellfish Immunol 2005; 18:163-177.

(15.) O'Brien V. Viruses and Apoptosis. J Gen Virol 1998; 79:1833-1845.

(16.) Ortega C, Rodriguez S, de las Heras A, Romero A, Monras M, Enriquez R. Evaluation of the level of Mx3 protein synthesis induced by infectious pancreatic necrosis virus (IPNV) strains of different infectivity. Vet Immunol Immunopathol 2011; 141:190-200.

(17.) Reed J, Muench H. A simple method for estimating fifty percent end points. Am J Hyg 1938; 27:493-497.

(18.) Cuconati A, White E. Viral homologs of BCL-2: role of apoptosis in the regulation of virus infection. Genes & Dev 2002; 16:465-2478.

(19.) Liu M, Vakharia V. Nonstructural Protein of Infectious Bursal Disease Virus Inhibits Apoptosis at the Early Stage of Virus Infection. J Virol 2006; 80:3369-3377.

(20.) Lombardo E, Maraver A, Espinosa I, Fernandez-Arias A, Rodriguez J. VP5, the Nonstructural Polypeptide of Infectious Bursal Disease Virus, Accumulates within the Host Plasma Membrane and Induces Cell Lysis. Virol 2000; 277:345-357.

(21.) Brandt M, Yao K, Liu M, Heckert R, Vakharia V. Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus. J Virol 2001; 75:11974-11982.

(22.) Wolf K. Fish viruses and fish viral diseases. Ithaca, N.Y Canstock Publishing Associates-Cornell University Press, 1988.

(23.) Lyles D. Cytopathogenesis and Inhibition of Host Gene Expression by RNA Viruses. Microbiol Mol Biol Rev 2000; 64:709-724.

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(25.) Chiu CL, Chou YL, Wu JL, Hong JR. Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells. Apoptosis 2010; 15:653-668.

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(27.) Hong J, Wu J. Induction of apoptotic death in cells via Bad gene expression by Infectious pancreatic necrosis virus infection. Cell. Death Differ 2002; 9:113-124.

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Cesar Ortega S, [1]* Ph.D, Sylvia Rodriguez S, [2] Ph.D, Juan Carlos Espinoza, [3] M.Sc, Juan Kuznar, [3] Ph.D, Alex Romero, [4, 5] Ph.D, Ricardo Enriquez, {4} Ph.D.

[1] Universidad Autonoma del Estado de Mexico, Faculty of Veterinary Medicine and Animal Husbandry, Advanced Animal Health Research and Study Center. Toluca, Mexico, AP. 4-56. [2] Biologic Research Center (High Council for Scientific Research) c/ Ramiro de Maeztu 9 Madrid 28040, Spain. [3] Universidad de Valparaiso, Faculty of Sciences, Biochemistry and Virology Laboratory. Gran Bretana 1111, Valparaiso, Chile. [4] Universidad Austral de Chile, Faculty of Veterinary Sciences, Biotechnology and Aquatic Pathology Laboratory. Valdivia, Chile. [5] Centro FONDAP: Interdisciplinary Center for Aquaculture Research (INCAR), Chile. * Correspondence: cos_mx@hotmail.com

Received: March 2013;

Accepted: September 2013.
Table 1. Multiple alignment of the deduced amino acid sequence of 60
residues of the hypervariable region of the IPNV VP2 protein. Residues
217 and 221 are shown in yellow, residue 243 characteristic of
genotype in blue, residue 247 in green.

Sp        [195]   CTAAIAPRRYEIDLPSQRLPPVPATGALTTLYEGNADIVNSTT     [254]
                  -VTGDINFSLAEQPAIET

V70/06    [195]   CTAAIAPRRYEIDLPSQRLPPVPATGALTTLYEGNADIVNSTT     [254]
                  -VTGDINFSLAEQPAVET

V33/98    [195]   CTAATAPRRYEIDLPSQRLPPVPATGTLTTLYEGNADIVNSTT     [254]
                  -VTGDINFSLAEQPAVET

VR-299    [195]   CTAAIAPRRYEIDLPSERLPTVAATGTPTTIYEGNADIVNST      [254]
                  -AVTGDITFQLEAEPVNET

Jasper    [195]   CTAAIAPRRYEIDLPSERLPTVAATGTPTTIYEGNADIVNST      [254]
                  -AVTGDITFQLEAEPVNET

V112/06   [195]   CTAAIAPRRYEIDLPSERLPTVAATGTPTTIYEGNADIVNST      [254]
                  -TVTGDVTFQLAAEPANET

Ab        [195]   CTAAIAPRRYEXDLPSERLPTVAATGTPTTIYXGXGDIVNST      [254]
                  -TVTGDISFSLANNPTADI

             **** ****** ****:***.*.***: **:* * .******:****:. *.* :*.:

Table 2. Sequence and structural homology of human protein Bcl-2 with
protein VP5 of virus V70/06, V112/06, V33/98 and reference strains.

Cepa               BH3                             BH1

Bcl-2     [97]   LRQAGDDF   104   -133    VVEELFRDGVNWGRIVAFFEF
E1S       [59]   LRSLGLRK   65    --84    LQMECEPDGTRVRPVAGDVTG
12kDa     [45]   LRGLRIRK   52    --70    LQMECEPDGAGVRPVAGDVAG
15kDa     [45]   LRGLRIRK   52    --70    LQMECEPDGAGVRPVAGDVAG
V70/06    [43]   LRGLRIRK   50    --68    LQMECEPDGAGVRPVAGDVAG
V112/06   [45]   LRGLRLRK   52    --70    LQVESEPDGTGIRPVVGNITG
V33/98    [45]   LRGLRIRK   52    --70    LQMECEPDGAGVRPVAGDVAG
VR2 9 9   [60]   LRGIRIRK   67    --85    LQVESEPDGTRIRPVARDVTG

Cepa                         BH2

Bcl-2     153--184   HLHTWIQDNGGWD-AFVELY   [202]
E1S       104--125   HAAGWA-LCPEWDHQCCNLR   [143]
12kDa     90--       -------------------     --
15kDa     90--111    HTTGRS-LCSERDAQRCHLR   [129]
V70/06    88--109    HTTGRS-LCS----------   [117]
V112/06   90--111    EPSRWALLCTQRDTECIHLP   [130]
V33/98    90--111    HTTGRS-L------------   [117]
VR2 9 9   105--126   NPSRWS-VCTQWDPERCHLR   [144]
BH4 domain excluded. The residues deemed critical in the apoptosis
   mechanism of Bcl-2 are shown in blue. Access codes to GenBank for
   VP5 of IPNV AAK71697 (E1S), AAQ75358 (12 kDa), Q69CI1 (15 kDa),
   AAA92630 (VR-299), P10415 (human Bcl-2)
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Title Annotation:ORIGINAL
Author:Ortega, Cesar S.; Rodriguez, Sylvia S.; Espinoza, Juan Carlos; Kuznar, Juan; Romero, Alex; Enriquez,
Publication:Revista MVZ (Medicina Veterinaria y Zootecnia)
Date:Jan 1, 2014
Words:8037
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