PubMed Health⌕ Search

Biomedical subjects

R F Silva

Publications and source records attributed to R F Silva.

At least 37 records · Page 2Linked to original sources
PubMed1994

Integration of multiple chicken retroviruses into multiple chicken herpesviruses: herpesviral gD as a common target of integration.

Integration of two different avian retroviruses, reticuloendotheliosis virus (REV) and avian leukosis virus (ALV), into the genome of two different avian herpesviruses, the herpesvirus of turkeys (HVT) and Marek's disease virus (MDV), was investigated. Integration events occurred by the fourth and sixth in vitro passage of cells coinfected with REV/HVT and ALV/MDV, respectively. In order to further characterize the integration events, integrated proviruses and surrounding herpesviral genetic material were cloned and analyzed. In the REV/HVT coinfection experiment, one of the three unique integrated proviruses was found to have integrated into the HVT gD gene, resulting in disruption of the coding region of this gene. The two additional unique integrations were localized to the UL and IRL border regions of HVT, two previously described common sites of REV integration into MDV. Interestingly, one of the integrated proviruses in the HVT genome appeared to be full length, was infectious when transfected into CEF cells, and therefore could potentially function to produce infectious REV from an HVT infectious platform. In the ALV/MDV coinfection experiment, one of two unique integrated proviruses was found to have integrated into the gD gene, resulting in disruption of the coding region of this gene. The second unique integration site was in the polyadenylation site of the SORF2 gene at the boundary of the IRS and US, once again a common site of REV integration into MDV. These results demonstrate that the U/IR-TR border regions of herpesviruses are common sites of retroviral integration. In addition gD, in the US region of the herpesvirus, is a common site of retroviral integration in multiple herpesvirus, indicating a possible selective advantage for disruption of this gene in the in vitro growth of a herpesvirus. Finally, this is the first instance of a full-length provirus found integrated into a herpesvirus genome, indicating that a retrovirus could alter its route of infection by being carried in a herpesvirus genome.

Amino Acid Sequence↗
PubMed1993

A double-cos-site vector containing a multiple cloning site flanked by T7 and T3 RNA polymerase promoters.

A double-cos-site cosmid vector, c2XMCS, contains a multiple cloning site (MCS) with fifteen restriction sites flanked by phage T7 and T3 RNA polymerase promoters. The two cos sites in the cosmid vector enable the construction of cosmid libraries from unfractionated partial digests of genomic DNA. This simple construct combines the efficiency of a double-cos-site cosmid with the versatility provided by the MCS from pBluescript.

Animals↗
PubMed1993

The number of copies of an a-like region in the serotype-3 Marek's disease virus DNA genome is variable.

A 1.2-kilobase-pair DNA region (a-like) in the serotype-3 Marek's disease virus was directly repeated at the ends of the long (L) and short (S) components of the genome and repeated in the opposite orientation at the junction between the L and S components. Four variants from plaque-purified virus that varied in the number of copies of the a-like region were identified. The predominant variant contained one copy at both ends of the genome and at the L-S junction. Two other variants contained either one or two copies at the L end and one to three copies at the L-S junction. The fourth variant lacked the a-like region at the L end and the L-S junction. The fourth variant became the predominate species as the virus was serially passaged in cell culture.

Animals↗
PubMed1993

Selection of Marek's disease virus recombinants expressing the Escherichia coli gpt gene.

We developed a positive selection method for recovering Marek's disease virus (MDV) recombinants. The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt), under the control of the major immediate-early promoter from cytomegalovirus, was inserted into the inverted repeats flanking the unique long (UL) region of a non-pathogenic serotype 2 MDV strain 281MI/1. In a second demonstration of the usefulness of the positive selection system, the gpt gene was inserted into the inverted repeats flanking the unique short (US) region of the turkey herpesvirus (HVT) strain FC126. The targeted insertion site in 281MI/1 was in a previously established nonessential site for virus replication. The targeted insertion site for FC126, at the junction of the UL and US regions, is a nonessential site for in vitro replication of herpes simplex virus. Recombinant viruses were easily selected by incubating the transfected cells in mycophenolic acid (MPA)-containing medium. Purification of recombinants resulted from a series of trypsinization and sonication steps combined with the culturing of virus in MPA-containing medium to inhibit wild-type virus replication. This simple technique for recovering MDV and HVT recombinants should increase the efficiency of identifying nonessential sites and gene function analysis by insertional mutagenesis.

Blotting, Southern↗
PubMed1993

Defective Marek's disease virus DNA contains a gene encoding a potential nuclear DNA binding protein and a HSV a-like sequence.

Four RNA transcripts from chicken embryo fibroblast cells infected with Marek's disease virus (MDV) strain 281Ml/1 hybridized to the 4-kbp MDV replicon DNA. In an attempt to identify open reading frames coding for the four transcripts, we determined the nucleotide sequences of 4-kbp replicon DNA (represents a single monomeric repeat unit of defective MDV genome). Computer analysis indicates that the 4-kbp MDV replicon DNA contains two intact open reading frames (ORFs) with common promoter regulatory elements. ORF-A codes for a putative 204 amino acid protein that shares 21 and 36% amino acid sequence identity to nuclear DNA binding proteins such as the EBNA-1 of Epstein-Barr virus and galline, a chicken sperm histone protein, respectively. ORF-B encodes for a potential 350 amino acid protein, which did not show any significant amino acid sequence identity to known protein sequences within Swiss-Protein data base. ORF-B may, therefore, encode a MDV specific protein. The 5'-region of MDV replicon DNA revealed seven reiterated copies of an 11-bp motif sharing 8 out of 11 nucleotide sequence identity to DR2 elements of the herpes simplex virus strain USA-8 a sequence.

Amino Acid Sequence↗
PubMed1993

Cosmid library of the turkey herpesvirus genome constructed from nanogram quantities of viral DNA associated with an excess of cellular DNA.

A protocol was designed for the rapid and efficient construction of cosmid libraries from cell-associated viral genomes available in very low quantities. Purification of viral DNA from cellular DNA was unnecessary. The vast excess of cellular DNA compensated for the limited amount of available viral DNA, enabling titration of the restriction endonuclease partial digest. A cosmid library of the turkey herpesvirus DNA genome was constructed from 1.5 micrograms of cellular DNA containing approximately 6 nanograms of viral DNA.

Animals↗
PubMed1992

Genetic mechanisms of antigenic variation in infectious bursal disease virus: analysis of a naturally occurring variant virus.

The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222-334) lies a highly divergent area that we have termed the variable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284-288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman beta turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.

Amino Acid Sequence↗
PubMed1992

Plasmid-associated effects on test gene expression and Marek's disease virus plaque formation during recombination trials.

Marek's disease virus (MDV), a herpesvirus of avian origin, is being examined for suitability as a vector for expressing foreign genes. We observed that plasmids encoding the LacZ gene of E. coli under the control of either the herpes simplex virus alpha 4 immediate-early promoter or the cytomegalovirus major immediate-early promoter inhibited MDV plaque formation. Plaque numbers were decreased by one-third, and transient expression of the beta-galactosidase reporter gene was increased by up to 6-fold, when the plasmids were linearized. Sequences associated with the heterologous promoter were identified as being responsible for inhibiting MDV replication.

Cytomegalovirus↗
PubMed1991

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

Animals↗
PubMed1990

Cell culture amplification of a defective Marek's disease virus.

A highly amplified 4-kb EcoRI fragment was present in DNA isolated from high cell culture passaged stocks (greater than 93 passages) of 281MI/1, a serotype 2 Marek's disease virus (MDV). The isolated 4-kb fragment is amplified in the presence of MDV, replicating as a high molecular weight, head-to-tail concatemer. When the 4-kb fragment was cloned into pUC18 and cotransfected with MDV DNA into chicken embryo fibroblast cells, the plasmid clone also replicated as a high molecular weight concatemer.

Cloning, Molecular↗
PubMed1990

Transactivation of the Rous sarcoma virus long terminal repeat promoter by Marek's disease virus.

Transient expression of chloramphenicol acetyltransferase (CAT) was used to study Marek's diseases virus (MDV)-mediated transactivation of the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. Cotransfection experiments in primary avian cells were conducted using MDV high-molecular-weight DNA and plasmid pRSVcat. Increased CAT activity, relative to controls, was consistently observed in the presence of MDV. Enhanced CAT activity, expressed via the RSV-LTR promoter, was strictly dependent on the presence of MDV DNA or virus, suggesting that activation of the RSV-LTR promoter was due to factors expressed in MDV-infected cells. Differences in transactivation efficiency were observed between various strains and the serotypes of MDV. In particular, high- and low-passage pairs of serotype 1 MDV showed marked differences in their ability to increase CAT activity in pRSVcat-transfected cells. Attenuation of viral pathogenicity and decreased expression of some cell surface glycoproteins occur in high-passage MDV strains. Decreased transactivation ability in these same strains suggests that continuous passage in culture and attenuation may perturb a regulatory mechanism operating by transcriptional control. In addition, transactivation of the RSV-LTR promoter suggests that increased incidence of avian leukosis following vaccination by MDV may be due to MDV-mediated transactivation of endogenous ALV proviral LTR promoters. MDV-mediated transactivation was not limited to the RSV-LTR promoter. Serotype 3 MDV (HVT) efficiently transactivated the herpes simplex virus (HSV) alpha 4 (ICP4) and beta-TK promoters as well as the human cytomegalovirus (hCMV) immediate early promoter.

Animals↗
PubMed1986

Monoclonal antibodies against avian reticuloendotheliosis virus: identification of strain-specific and strain-common epitopes.

We report the generation and partial characterization of a panel of 11 monoclonal antibodies (MCA) made against the nondefective strain T reticuloendotheliosis virus (REV). In an enzyme-linked immunosorbent assay (ELISA), nine MCA cross-reacted with both homologous strain T and heterologous strain chick syncytial virus (CS), whereas two MCA were strain specific and reacted with strain T but not with CS. Competitive antibody inhibitory ELISA tests demonstrated that the nine MCA recognized at least two distinct type-common epitopes. By using MCA-mediated immunoprecipitation and polyacrylamide gel electrophoresis analysis, we identified a 62,000 dalton glycoprotein that may contain both type-common epitopes and a 21,000 dalton glycoprotein that contains one of these epitopes. The competitive antibody inhibitory ELISA tests confirmed that the remaining two MCA recognize a strain T-specific epitope. We identified a 54,000 to 72,000 dalton glycoprotein that contains the type-specific epitope. All of the MCA reacted in an ELISA assay with cell-free virus preparations, suggesting that the polypeptides we identified are virion envelope glycoproteins. To identify the nonglycosylated precursor proteins, we treated infected cells with tunicamycin. We found a 48,000 polypeptide that was the nonglycosylated precursor to the 54,000 to 72,000 glycoprotein, and 48,000 and 20,000 dalton proteins that were the nonglycosylated precursors to the 62,000 and 21,000 dalton glycoproteins, respectively. These MCA may be of value in the field. They were able to distinguish in preliminary tests in ELISA between strains T and CS, which were otherwise undistinguishable in assays that made use of conventional polyvalent serum.

Animals↗
PubMed1986

Selective shedding and congenital transmission of endogenous avian leukosis viruses.

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.

Animals↗
PubMed1985

Genomic expansion of Marek's disease virus DNA is associated with serial in vitro passage.

An EcoRI restriction endonuclease pattern of Md11 virus DNA, a very virulent strain of Marek's disease virus (MDV), was obtained by using total cellular DNA from infected cells. With the EcoRI restriction endonuclease pattern and a published BamHI map of MDV (Fukuchi et al., J. Virol. 51:102-109), we constructed a partial EcoRI map of a series of MDV clones (gift from H. J. Kung). The clones were used to identify a region of the Md11 genome which is altered as the oncogenic virus is passaged in vitro. This region was mapped into a 1.8-kilobase segment in the inverted-repeat sequences flanking the long unique region of the virus genome. The alteration appeared to result from multiple DNA insertions that produced an increase of 0.6 to 5.4 kilobases. Although the expansion of this region did not diminish the ability of MDV to replicate in vitro, it may be associated with the loss of Marek's disease oncogenicity.

Animals↗
PubMed1985

Biologically active proviral clone of myeloblastosis-associated virus type 1: implications for the genesis of avian myeloblastosis virus.

A biologically active myeloblastosis-associated virus (MAV) provirus was cloned from a bacteriophage recombinant library constructed from leukemic chicken myeloblast DNA. The restriction endonuclease map of this clone was consistent with that of a type 1 MAV (MAV-1). Interference assays of virus recovered from cultured chicken embryo fibroblasts after DNA transfection established that the provirus was infectious and confirmed that it belonged to avian retrovirus subgroup A (type 1). Antipeptide antibodies raised against the env-encoded carboxyl terminus of p48myb, the transforming protein of avian myeloblastosis virus, specifically immunoprecipitated the gp37env from quail cells transfected with MAV-1 proviral DNA but not from cells infected with MAV-2. This suggests that MAV-1 rather than MAV-2 is the progenitor helper virus from which avian myeloblastosis virus arose by the transduction of cellular proto-oncogene sequences.

Animals↗
PubMed1984

Monoclonal antibody-mediated immunoprecipitation of proteins from cells infected with Marek's disease virus or turkey herpesvirus.

The major immunogenic viral proteins of Marek's disease virus (MDV) and turkey herpesvirus (HVT) share antigenic determinants. The polyacrylamide gel electrophoresis pattern of five viral polypeptides immunoprecipitated with homologous convalescent chicken plasma was identical with the pattern obtained after immunoprecipitation with heterologous convalescent chicken plasma. A panel of monoclonal antibodies was used to identify MDV and HVT polypeptides. Sixteen monoclonal antibodies were positive in an immunofluorescence assay. However, only eight monoclonal antibodies immunoprecipitated a total of seven distinct viral proteins from MDV- and HVT-infected cells. Six of these monoclonals immunoprecipitated multiple viral proteins. None of the monoclonal antibodies recognized the common A antigen of MDV or HVT. Monoclonal antibodies immunoprecipitated three glycoproteins (100,000, 60,000, and 49,000 Da) that comigrate in polyacrylamide gels with three of the five common polypeptides obtained with the convalescent chicken plasma. In addition, a 79,000-Da protein was common to all MDV- and HVT-infected cells. Competition immunoprecipitation and peptide mapping by limited proteolysis confirmed that the three glycoproteins and the 79,000-Da protein contain MDV-HVT common epitopes. MDV-specific antigenic determinants were detected on the three remaining viral proteins (41,000, 38,000, and 24,000 Da).

Animals↗
PubMed1984

Several classes of retroviruses are produced by an AKR mouse T lymphoma cell line.

Characterization of the viruses produced by the spontaneous T lymphoma cell line SL3 is presented. Using supernatant fluids or direct co-cultivation of cells, the SL3 cell line was found to produce replication-defective viruses in excess of replication-competent viruses. The replication-competent viruses released were predominantly those negative in the XC plaque assay (XC-); XC+ viruses represented a minor population. However, when the SL3-derived viruses were passed in mouse embryo fibroblasts, XC- viruses were rarely recovered, and XC+ viruses were readily isolated. These viruses were all ecotropic and lymphomagenic. Viruses with dual host range and non-oncogenic ecotropic viruses were not isolated from the lymphoma cells. Two replication-defective viruses from SL3 cells were studied. Both could be rescued by non-oncogenic retroviruses and were then lymphomagenic. One defective virus appeared related to XC+ viruses. In these studies, the XC+ and XC- viruses appeared to represent two different interference classes using separate cell receptors. Taken together, these experiments show that the SL3 T lymphoma cells replicate a variety of viruses most of which are lymphomagenic. Virus replication and/or virus integration may be the means of maintaining the malignant phenotype of these T lymphoma cells.

Animals↗