RESUMEN
The eleven rotavirus mRNAs contain 5'-cap structures and most end with the 3'-consensus sequence 5'-UGACC-3'. The UGACC functions as a common translation enhancer (3'-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5'-and 3'-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3'-truncations showed that a highly active enhancer was present near the 5'-end of the 139-nucleotide 3'-UTR of the gene 6 mRNA (3'-TEg6). The 3'-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3'-TEg6 differs significantly from the 3'-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3'-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3'-TEg6) and the other is common to nearly all rotavirus genes (3'-TE-con). The activity of the 3'-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.
Asunto(s)
Regiones no Traducidas 3'/genética , Antígenos Virales , Proteínas de la Cápside/genética , Regulación Viral de la Expresión Génica , Genes Virales/genética , Biosíntesis de Proteínas/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Rotavirus/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Línea Celular , Genes Reporteros/genética , Estabilidad del ARN , ARN Viral/genética , Eliminación de Secuencia/genéticaRESUMEN
The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector toxicity. To decrease adenoviral vector immunogenicity and toxicity, we attenuated viral gene expression by eliminating E1, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVIII) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4DeltaE4FVIII) was deficient in the entire E4 coding region and the second vector contained a deletion of the E4 region, except for open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following intravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, the Av4DeltaE4FVIII vector could not be scaled-up for in vivo studies. Both Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVIII vector no longer expressed FVIII while Av3H8101-treated mice displayed persistent FVIII expression. Liver enzyme analyses of plasma samples revealed that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the adenoviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgene expression was reduced.
Asunto(s)
Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Proteínas E3 de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Adenovirus Humanos/genética , Factor VIII/genética , Vectores Genéticos , Animales , Técnicas de Cultivo de Célula , Clonación Molecular , Eliminación de Gen , Expresión Génica , Terapia Genética , Vectores Genéticos/aislamiento & purificación , Hemofilia A/terapia , Humanos , Hígado/enzimología , Hígado/metabolismo , Ratones , Regiones Promotoras Genéticas , Recombinación GenéticaRESUMEN
Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.
Asunto(s)
Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Proteínas E3 de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Adenovirus Humanos/genética , Vectores Genéticos/genética , Animales , Clonación Molecular , Femenino , Eliminación de Gen , Expresión Génica , Humanos , Hígado , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis , Plásmidos , Recombinación Genética , TransgenesRESUMEN
Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.
Asunto(s)
Factor VIII/genética , Terapia Genética/métodos , Hemofilia A/terapia , Adenoviridae/genética , Animales , Anticuerpos/análisis , Factor VIII/inmunología , Factor VIII/metabolismo , Expresión Génica , Vectores Genéticos , Humanos , Ratones , Ratones Noqueados , Proteínas Recombinantes , Factores de TiempoRESUMEN
A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Replicación del ADN , Eliminación de Gen , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Replicación ViralRESUMEN
Mirizzi syndrome is a rare variant of obstructive jaundice due to compression of the hepatic duct caused by a stone inserted in the cystic duct or in the Hartmann recess and it is referred with a prevalence of 0.05-1% of patients with cholelithiasis. These percentages are, nevertheless, unreliable because only an accurate preoperative cholangiography allow to detect a Mirizzi syndrome and so, very often, the real cause of the jaundice remains unacknowledged. Early diagnosis of the syndrome is particularly important because it suggests an accurate and prudential surgical approach considering the frequent fibrotic adherences caused by chronic inflammation. In this paper the authors present a clinical case quickly and successfully cured operative endoscopy, followed by traditional surgery. The authors believe that the study of obstructive jaundices must include an ERCP either for the diagnosis or because operative endoscopy could ameliorate clinical feature and hepatic performance in order to allow a safer surgical operation.
Asunto(s)
Colangiopancreatografia Retrógrada Endoscópica , Colelitiasis/diagnóstico , Colestasis Extrahepática/diagnóstico , Conducto Hepático Común/diagnóstico por imagen , Colecistectomía , Colelitiasis/complicaciones , Colelitiasis/cirugía , Colestasis Extrahepática/etiología , Colestasis Extrahepática/cirugía , Femenino , Conducto Hepático Común/cirugía , Humanos , Persona de Mediana Edad , Reoperación , Esfinterotomía Endoscópica , SíndromeRESUMEN
After surgery for hepatic injury as a result of blunt abdominal trauma from a motorcycle accident, an external biliary fistula developed in a young patient. The authors describe the rapid and complete healing of the fistula by use of a nasobiliary catheter. These findings emphasize the importance of endoscopic operative technique for postoperative and traumatic external biliary fistulas.
RESUMEN
A cDNA clone representing the VP4-encoding gene of human rotavirus strain 69M(VP7 serotype 8) was constructed and inserted into a baculovirus expression vector. Baculovirus recombinants that expressed the 69 M VP4 protein in Spodoptera frugiperda (Sf9) cells were screened by immunofluorescence with hyperimmune antiserum to the 69M strain and purified by terminal dilution. The expressed VP4 was detected by Coomassie blue staining of PAGE-separated proteins. The antigenic relationships between the VP4 of the 69M strain and those of various human and other animal rotavirus strains representing ten established VP4 serotypes were examined by plaque reduction neutralization. Hyperimmune antiserum produced in guinea pigs following immunization with a lysate of Sf9 cells infected with a 69M gene 4-baculovirus recombinant neutralized the infectivity of the homologous human rotavirus 69M strain as well as heterologous equine rotavirus H-2 strain to a high titer. The anti-69M VP4 hyperimmune antiserum did not neutralize significantly other rotavirus strains of human, simian, porcine, bovine, or murine origin. It thus appears that the human rotavirus 69M strain has a distinct VP4 serotype (designated as P serotype 4) which is closely related antigenically to equine rotavirus H-2 VP4.
Asunto(s)
Proteínas de la Cápside , Cápside/biosíntesis , Rotavirus/clasificación , Rotavirus/genética , Animales , Baculoviridae , Cápside/análisis , Línea Celular , Caballos , Humanos , Proteínas Recombinantes/biosíntesis , Serotipificación , Especificidad de la Especie , Spodoptera , Transfección , Ensayo de Placa ViralRESUMEN
Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Hemaglutinación/genética , Hemaglutininas Virales/genética , Rotavirus/genética , Animales , Células Cultivadas , Clonación Molecular , Humanos , Nucleopoliedrovirus/genética , Proteínas Recombinantes de Fusión/genética , SpodopteraRESUMEN
Growing interest in adenoviral gene-transfer vectors, stimulated by efforts to develop in vivo gene therapy for cystic fibrosis, has led to an evaluation of their use in many other applications of in vivo gene therapy. Studies are beginning to define strategies for the efficient, albeit transient, expression of the transferred gene and have further identified and partially characterized important host responses to in vivo gene transfer that modulate the duration of expression of the transgene. Ongoing work is actively exploring these issues, with a view to the design of the next generation of adenoviral vectors.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Vectores Genéticos , Animales , Ensayos Clínicos como Asunto , Fibrosis Quística/terapia , Técnicas de Transferencia de Gen , HumanosRESUMEN
A template-dependent, in vitro rotavirus RNA replication system was established. The system initiated and synthesized full-length double-stranded RNAs on rotavirus positive-sense template RNAs. Native rotavirus mRNAs or in vitro transcripts, with bona fide 3' and 5' termini, derived from rotavirus cDNAs functioned as templates. Replicase activity was associated with a subviral particle containing VP1, VP2, and VP3 and was derived from native virions or baculovirus coexpression of rotavirus genes. A cis-acting signal involved in replication was localized within the 26 3'-terminal nucleotides of a reporter template RNA. Various biochemical and biophysical parameters affecting the efficiency of replication were examined to optimize the replication system. A replication system capable of in vitro initiation has not been previously described for Reoviridae.
Asunto(s)
ARN Viral/metabolismo , Rotavirus/fisiología , Replicación Viral , Animales , Baculoviridae/genética , Secuencia de Bases , Células Cultivadas , Datos de Secuencia Molecular , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Virión/fisiologíaRESUMEN
Rotavirus strain A253, isolated from the faeces of a diarrhoeic piglet in Venezuela, was classified as serotype G11 by cross-neutralization studies and by comparison of the deduced amino acid sequence of the VP7 surface protein. The epitopes involved in neutralization of the two G11 porcine rotavirus strains A253 and YM were analysed using neutralization-resistant mutants selected with seven neutralizing monoclonal antibodies (MAbs), monotype-specific (M-) MAbs and serotype-specific (S-) MAbs, produced against VP7 of strain A253. Cross-neutralization tests and sequence analysis of the escape mutants selected from strains A253 and YM indicated the presence of two antigenic sites, one common to both M-MAbs and S-MAbs in region A (positions 87, 91 and 96) and the other defined by one S-MAb in region C (position 223). All A253 variants selected with M-MAbs and two S-MAbs, although having different amino acid substitutions, had a change at amino acid position 87, whereas YM variants involved residues 91 and 96, part of the same antigenic site. Compared to strain A253, the YM stain presents an amino acid substitution at position 87 and was not recognized by M-MAbs. These results suggest that in the VP7 of G11 serotype specificity, the amino acid at position 87 is an important component of a neutralization site associated with region A and the intraserotypic variation between strains A253 and YM may account for the selection of mutations at different positions by a single MAb.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales , Proteínas de la Cápside , Cápside/inmunología , Epítopos/inmunología , Rotavirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Datos de Secuencia Molecular , Pruebas de Neutralización , Mutación Puntual , Rotavirus/clasificación , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido , Serotipificación , Especificidad de la Especie , PorcinosRESUMEN
The nucleotide and deduced amino acid sequence of the gene 4 of murine rotavirus strain Eb were determined. The gene is 2359 nucleotides in length and encodes for a protein of 775 amino acids. Comparison of the VP4 amino acid sequence of the Eb strain with several human and animal rotavirus strains which represent all of the currently recognized distinct VP4 genotypes revealed amino acid identities of from 55.7-75.1% for VP4, 37.1-63.3% for VP8, and 23.9-52.1% for the B region (amino acids 84-180). In addition, antisera to recombinant VP4s of five distinct rotavirus serotypes and two subtypes failed to react significantly by neutralization assay with the Eb strain. Thus, it appears that the Eb strain should be considered a new VP4 genotype and/or serotype.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Genes Virales/genética , Rotavirus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cobayas , Humanos , Ratones , Datos de Secuencia Molecular , Rotavirus/clasificación , SerotipificaciónRESUMEN
Two neutralizing monoclonal antibodies (N-mAbs) were utilized to locate amino acid (aa) residues involved in the formation of serotype-cross-reactive epitopes on the VP7 of selected group A rotaviruses. N-mAb 954/159/13 neutralized G serotype 3 as well as porcine G serotype 4 rotaviruses, whereas N-mAb 57/8 neutralized G serotype 3, 4, 6, 9, and 10 strains. Neutralization-resistant variants of each serotype were selected in the presence of these two monoclonal antibodies. Sequence analysis of the gene encoding VP7 of such variants revealed: (i) variable regions VR-5 (aa 88-100), VR-8 (aa 209-223), and VR-9 (aa 235-242) are involved in cross-reactive neutralization; (ii) an aa substitution can occur at the same position on the VP7 of different serotypes selected by a given N-mAb; (iii) the location of an aa substitution on the variant VP7 selected by a given N-mAb can vary depending on the rotavirus serotype; (iv) a substituted single aa species at a specific position on the variant VP7 selected by a single N-mAb can vary, resulting in variants which exhibit antigenic differences; and (v) the VP7 of a porcine rotavirus Gottfried strain has a unique antigenic mosaic of serotype 3 and serotype 4.
Asunto(s)
Antígenos Virales/inmunología , Proteínas de la Cápside , Cápside/inmunología , Rotavirus/inmunología , Secuencia de Aminoácidos , Animales , Variación Antigénica , Reacciones Cruzadas , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Rotavirus/clasificación , SerotipificaciónRESUMEN
We isolated five stable SA11 clones (TN-S1, TN-S2, TN-L1, TN-L2, and BN-S4) with different plaque sizes from two SA11 stocks. In polyacrylamide gel electrophoresis, the mobilities of the fourth, fifth, and seventh RNA segments of SA11 clones with large plaque size (TN-L1 and TN-L2) were faster than those of clones with small plaque size (TN-S1, TN-S2, and BN-S4). Nucleotide sequence determination of the fourth RNA segment identified a five-amino-acid difference in VP4s between the clones with large and small plaque sizes. The VP4 sequences of two clones with small plaque size (TN-S1 and BN-S4) were the same as the sequence of SA11-SEM reported by K. Nishikawa et al. (1988, J. Virol. 62, 4022-4026), while the VP4 sequences of the clones with large plaque size (TN-L1 and TN-L2) were similar, but not identical, to that reported by D. B. Mitchell and G. W. Both (1989, Nucleic Acids Res., 17, 2122). A single-gene reassortant, K8-L2.4, in which RNA segment 4 was derived from clone TN-L2 of SA11 virus and the other RNA segments were from strain K8, produced large plaques like TN-L2, suggesting that the five-amino-acid difference in VP4 between the clones with large and small plaque sizes might be associated with the difference in plaque size.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Rotavirus/crecimiento & desarrollo , Ensayo de Placa Viral , Animales , Variación Genética , Haplorrinos , Datos de Secuencia Molecular , ARN Viral/genética , Recombinación Genética , Rotavirus/química , Rotavirus/genéticaRESUMEN
The antigenic relationships of the VP4 serotype of porcine rotavirus Gottfried strain with other rotaviruses were determined by using antiserum to Gottfried VP4-baculovirus recombinant. This antiserum failed to react significantly with virus of serotypes P1A, P1B, P3, and OSU; however, it reacted with P2 strains. In the reciprocal assay, antiserum to VP4 of an asymptomatic strain (P2) failed to neutralize the Gottfried strain virus to a significant level. It thus appears that the Gottfried strain should be considered a subtype of P2.
Asunto(s)
Proteínas de la Cápside , Cápside/inmunología , Rotavirus/inmunología , Porcinos/microbiología , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Cápside/genética , Cobayas , Sueros Inmunes/inmunología , Datos de Secuencia Molecular , Pruebas de Neutralización , Rotavirus/clasificación , SerotipificaciónRESUMEN
The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.
Asunto(s)
Antígenos Virales/inmunología , Proteínas de la Cápside , Cápside/inmunología , Epítopos/análisis , Rotavirus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Variación Antigénica , Antígenos Virales/análisis , Antígenos Virales/genética , Codón , Femenino , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Rotavirus/genética , Análisis de Secuencia de ADN , Porcinos/microbiologíaRESUMEN
Recombinant OSU VP4 protein, and outer capsid antigen of porcine rotavirus, was purified to a high level from the spent broth of baculovirus-infected Spodoptera frugiperda insect cells. Initial clarification of the broth with a 0-60% ammonium sulfate cut retained 93% of the total VP4. Q-sepharose ion exchange chromatography performed at pH 6.5 yielded 67% of the initial amount of VP4 in the pooled fractions, with more than four times the purity of the original sample. Gel filtration chromatography followed ion exchange. VP4 eluted from this column at a volume corresponding to a protein of a molecular weight of approximately 85 kDa, the single chain molecular weight of VP4. This step retained 34% of the initial VP4, with a 28-fold purification. Affinity chromatography, using heparin and glycophorin as ligands, was chosen as a final polishing step. The selective binding of VP4 to the two ligands suggested that VP4 may play a role during in vivo rotaviral infection. These specific interactions based on the biological properties of rotaviruses achieved a VP4 purity level of 85-95%. The overall purification scheme recovered about 1.5 mg per liter of VP4 spent broth from about 50 mg/liter (5% of total protein) present initially in the broth.
Asunto(s)
Proteínas de la Cápside , Cápside/biosíntesis , Cápside/aislamiento & purificación , Rotavirus/genética , Animales , Baculoviridae/genética , Cápside/genética , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Mariposas Nocturnas/citología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Porcinos/microbiologíaRESUMEN
The nucleotide and deduced amino acid sequence of G serotype 3 equine rotavirus strain H-2 was determined. A predicted 776-amino-acid H-2 VP4 shows less than or equal to 85.3% identity to other rotavirus VP4 types sequenced to date and thus represents a new P serotype. A PCR-generated probe derived from a cDNA clone of H-2 gene 4 hybridized to gene 4 of several tissue-culture-adapted equine rotavirus isolates, demonstrating that the gene 4 allele present in the H-2 strain is present in the equine rotavirus population.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Genes Virales/genética , Rotavirus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cápside/química , Caballos , Datos de Secuencia Molecular , Rotavirus/química , Homología de Secuencia de AminoácidoRESUMEN
Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants.