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R F Silva

Publications and source records attributed to R F Silva.

At least 55 records · Page 3Linked to original sources
PubMed1982

Avian myeloblastosis provirus cloned in a lambda bacteriophage is leukemogenic.

The avian myeloblastosis virus provirus inserted in a lambda bacteriophage, recombinant clone 11A1-1 (Souza et al., Proc. Natl. Acad. Sci. U.S.A. 77:3004-3008, 1980), was transfected into chicken embryo fibroblasts which had been preinfected with either Rous-associated virus type 61 or the transformation-defective avian sarcoma virus tdB77. Within 4 to 5 h after transfection, the cells were injected into 16-day-old chicken embryos or 1-day-old chicks. Acute myeloblastic leukemia developed after a long latent period. Filtered (0.22-micrometer pores) supernatant of transformed buffy-coat cell cultures from one leukemic chicken of the lambda 11A1-1 (tdB77) group rapidly transformed yolk sac cells in vitro. Results from an infectivity interference assay and analysis of proviral DNA fragments generated with restriction endonucleases were consistent with the presence in leukemic cells of defective avian myeloblastosis virus and tdB77 as the helper virus.

Animals↗
PubMed1980

Avian myeloblastosis virus proteins in leukemic chicken myeloblasts.

We have analyzed the avian myeloblastosis virus proteins in two types of leukemic myeloblasts: established myeloblastic cell lines (DU 1765 and DU 11157) and leukemic myeloblasts obtained from the peripheral blood of a leukemic C/E Spafas chicken (no. 21957). Using monospecific antisera for immunoprecipitation and polyacrylamide gel electrophoresis, we have detected gag gene-related proteins in the myeloblasts. The DU 1765 and DU 11157 cells contained a p100 protein which possessed antigenic determinants of the viral proteins p27, p19, p15, and p12. The p100 was not found in leukemic myeloblasts from Spafas chickens, and pulse-chase experiments showed that the p100 was not a precursor for the viral proteins. However, the p100 is present in uninfected line 15 chicken embryos. A pr76-like protein was identified in DU 1765 cells but migrated slightly further into gels than the pr76 of Spafas-derived leukemic myeloblasts. The Spafas-derived myeloblasts produced a pr60, whereas the DU 1765 cells contained instead a related protein of 62,000 daltons. Using anti-avian myeloblastosis virus gp85 sera, a glycoprotein of 120,000 daltons (gp120) was detected in all the tested leukemic myeloblasts. The gp120 was also present, in low amounts, in uninfected embyonic spleen and yolk sac cells. The anti-gp85 sera also precipitated a 27,000-dalton protein (h27) in these same cells. Both the gp120 and h27 could not be detected in either uninfected or myeloblastosis-associated virus-infected fibroblasts. Limited peptide hydrolysis revealed that h27 is different from the viral structural protein p27. In conclusion, monospecific antisera for gag and env gene products of avian myeloblastosis virus did not precipitate any unique or aberrant avian myeloblastosis virus protein from leukemic myeloblasts.

Animals↗
PubMed1975

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.

Agar↗
PubMedDate not supplied

Comparison of Chinese field strains of avian leukosis subgroup J viruses with prototype strain HPRS-103 and United States strains.

Eight Chinese field strains of subgroup J avian leukosis viruses (ALV-J) were isolated from broilers or parent stocks during January 1999 to April 2001. One strain, SD9902, was an acute transforming virus, able to induce typical myelocytomatosis in 22-38 days after inoculation of 1-day-old meat-type chicks. The envelope protein and 3'-untranslated region (UTR) of the eight field strains were compared with the U.K. prototype HPRS-103 and several U.S. field strains isolated in 1993-97. All Chinese strains shared an almost identical deletion with the U.S. strain 4817 in the E element region of 3'-UTR when compared with the prototype HPRS-103, indicating that they have a very close phylogenic relationship. Every year, China has to import grandparent stocks of meat-type chickens, mainly from the United States. Chinese isolates should represent a part in the phylogenic tree of U.S. ALV-J evolution. Envelope protein gp85 amino acid sequence analysis demonstrated that, interestingly, all recent Chinese isolates were more closely related to HPRS-103 and the earliest U.S. isolates but not to the late U.S. isolates. The result implies that envelope gp85 may not have diverged from prototype and older strains. It is also possible that some recently imported birds could have been infected by the older viruses that were introduced in the late 1990s.

Abattoirs↗
PubMedDate not supplied

Protection of turkeys from hemorrhagic enteritis with a recombinant fowl poxvirus expressing the native hexon of hemorrhagic enteritis virus.

Hemorrhagic enteritis (HE) is an economically important disease of turkeys caused by a type II aviadenovirus, hemorrhagic enteritis virus (HEV). The vaccines currently available to the commercial poultry producer are highly effective in preventing disease outbreaks; however, they are immunosuppressive. A recombinant fowl poxvirus (rFPV) expressing the native hexon of HEV has been shown to induce an anti-HEV humoral immune response in turkeys. In this study, the rFPV expressing the native hexon of HEV was compared with a commercial HEV vaccine (vxHEV) for its ability to protect turkeys from virulent HEV challenge. Complete protection from the enteritis of HE was achieved in experimental groups vaccinated with either the rFPV or the vxHEV. Lymphocyte stimulation was measured in turkeys inoculated with rFPV, vxHEV, or a sublethal dose of HEV or not inoculated. No statistically significant immunodepression was observed in turkeys receiving the rFPV.

Adenoviridae Infections↗
PubMedDate not supplied

Tissue-specific restriction of latent turkey HVT transcription.

Using in situ hybridization, latent turkey herpesvirus (HVT) transcription was examined in lymphoid and/or nonlymphoid tissues. Blood samples were taken for virus isolation from chickens at 7 and 240 days post infection (PI) representing time points for productive and latent turkey herpesvirus infections, respectively. Spleen, thymus, sciatic and brachial nerves from infected chickens were analyzed for latent HVT transcription and HVT glycoprotein B (gB) expression at 240 days PI. Using indirect immunofluorescence, HVT gB expression was not detected in any tissues examined at 240 days PI. HVT genomic fragments from a HVT BamHI library were used as probes in in situ hybridization assays. In the spleen, thymus, sciatic and brachial nerves, latent HVT transcription occurred from the repeat regions flanking the unique long region (TRL and IRL). However, fine-mapping of this region revealed a difference in latent HVT transcriptional pattern. A SmaI map of the HVT BamHI-F fragment was made to further fine-map latent HVT transcription. A 1.6 kbp SmaI subfragment hybridized to cells infected with latent HVT in the spleen and thymus. However, the 1.6 kbp SmaI subfragment did not hybridize to cells of the brachial or sciatic nerves. In addition, a 2.0 kbp SmaI subfragment hybridized to cells in the thymus but not in the spleen, sciatic or brachial nerves. The above results suggest that latent turkey herpesvirus exhibits tissue-specific transcription.

Animals↗
PubMedDate not supplied

MDV glycoprotein D is expressed in the feather follicle epithelium of infected chickens.

Glycoprotein D (gD) and its homologues are essential for many alphaherpesvirus life cycles. A gene encoding a homologue of gD was recently found in the Marek's disease virus (MDV) genome. Interestingly, gD-negative MDV mutants apparently replicate unimpaired in both cell culture and chickens. In this study, we show the expression of the gD homologue of MDV in feather follicle epithelium in infected birds. The gD homologue was detected in a few feather follicles even when most of the follicles were expressing pp38 or gB, other MDV-specific proteins. These observations indicate that the MDV gD homologue is expressed in a very limited set of cells and may be differently regulated. Since feather follicle epithelium of infected birds are the only place where the infectious cell-free MDV virions can be recovered, analysis of the transcriptional regulation of gD may lead to the understanding of the cell-associated nature of MDV.

Animals↗
PubMedDate not supplied

Differentiation of pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDVs) by the polymerase chain reaction amplification of the tandem direct repeats within the MDV genome.

There are no simple, direct methods to reliably distinguish oncogenic serotype 1 Marek's disease viruses (MDVs) from their attenuated variants. The present study was an attempt to apply polymerase chain reaction (PCR) to develop a rapid and sensitive assay for the presence of the MDV genome. PCR oligos were chosen to flank the 132-base-pair tandem direct repeats in the serotype 1 MDV genome. The PCR reaction was specific for serotype 1 MDVs, amplifying fragments corresponding to one to three copies of the tandem repeats present in Md11/8, JM/102W, and GA viruses. A high-molecular-weight DNA smear was observed when the DNA from an attenuated Md11/100 was PCR-amplified. Use of the PCR technique allowed the detection of two copies of the 132-base-pair repeat in the DNA extracted from MDV-induced lymphomas removed from two chickens. No DNA was amplified from the DNA extracted from lymphomas induced by either an avian leukosis virus (RAV-1) or reticuloendotheliosis virus (chick syncytial virus).

Animals↗
PubMedDate not supplied

Restriction endonuclease analysis of Marek's disease virus DNA: differentiation of viral strains and determination of passage history.

The restriction endonuclease (RE) patterns of DNA from serotype 1 Marek's disease viruses (MDVs) are unique to serotype 1 viruses and can also be used to differentiate between low and high cell-culture-passaged viruses. We compared the RE patterns of DNA from seven serotype 2 and 3 MDVs before and after serial in vitro passage. Passage of four serotype 2 strains resulted in a variety of changes in the RE pattern. Individual strains within a serotype exhibited unique restriction patterns that allowed individual isolates to be differentiated. In a similar manner, the serotype 3 virus strains displayed RE pattern variations that were unique to each strain, as well as differences between low and high cell-culture passage. Our findings, together with earlier reports, suggest that the RE patterns of MDV DNA provide a simple and accurate method to: 1) differentiate between the three MDV serotypes, 2) differentiate between virus strains within a serotype, and 3) determine whether the viruses have been passaged extensively in cell culture.

Animals↗
PubMedDate not supplied

New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: selected biological and molecular characteristics.

Two new Marek's disease vaccine viruses, Md11/75C/R2 (serotype 1) and 301B/1 (serotype 2), were evaluated in chickens with maternal antibodies (ab+) or without maternal antibodies (ab-). Strain Md11/75C/R2 was mildly pathogenic in ab--chickens, but this pathogenicity was markedly reduced in ab+ chickens. Md11/75C/R2 spread less by contact and replicated better, both in vivo and in vitro, than CVI988/C, another serotype 1 vaccine virus. Strain 301B/1 was similar to SB-1, another serotype 2 vaccine virus: both were nonpathogenic for ab--chickens, spread readily by contact, and replicated well in vivo. In vitro, 301B/1 grew more rapidly and produced larger plaques than SB-1. Notable characteristics of strain CVI988/C included absence of pathogenicity, poor replicative ability, and the absence of one epitope detected by a common serotype-1-specific monoclonal antibody. All four viruses could be distinguished from each other by restriction enzyme analysis of viral DNA. We conclude that Md11/75C/R2, although exceptionally protective, may require further attenuation. On the other hand, 301B/1, which in other studies induced higher levels of protection than SB-1, is nonpathogenic and may be considered for use as a commercial vaccine.

Animals↗
PubMedDate not supplied

Properties of producer and non-producer clones of a Marek's disease turkey lymphoblastoid cell line.

The MDTC-RP30 lymphoblastoid cell line established from Marek's disease (MD) tumors in turkeys consisted of a heterogeneous population of cells 10 to 25 micron in diameter. Large-cell fractions obtained from a bovine fetal serum gradient had a higher titer of cell-associated MD virus (MDV) than the small-cell fractions. Seven single-cell clones were established from MDTC-RP30 cell line: two consisted of large cells, and the other clones consisted of small cells. Infectious MDV was rescued from large-cell clones in chicken embryo fibroblast cultures but not from small-cell clones. All clones contained MDV DNA sequences when hybridized against cloned MDV DNA. All clones were positive for a Marek's-disease-tumor-associated surface antigen and surface immunoglobulins. All but two small-cell clones caused MD in susceptible chickens. The two large-cell-type clones were uniformly tetraploid, whereas one small-cell clone was diploid and the four others were a mixture of diploid and tetraploid, with an occasional triploid cell. Evidence of translocation involving the male (Z) chromosome and the chromosome #3 was seen in one clone. These results suggest that MDV transforms different subpopulations of lymphocytes.

Animals↗
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Characterization of monoclonal antibodies to avian leukosis viruses.

Hybridoma cell lines secreting monoclonal antibody (MCA) to avian leukosis virus (ALV) structural proteins p27 and p19 have been established. In an indirect enzyme-linked immunosorbent assay (ELISA), MCA 6AL20 (IgG1 isotype) reacted with RPL-40 (ALV subgroup A), avian myeloblastosis virus (AMV) (a mixture of subgroups A and B), Rous-associated virus (RAV)-2 (subgroup B), and Carr-Zilber strain of Rous sarcoma virus (CZ-RSV) (subgroup D) but not with Prague strain of RSV (PrC-RSV) (subgroup C) or the endogenous virus RAV-0 (subgroup E). MCA 6AL22 reacted as above and also reacted marginally with PrC-RSV. Both MCAs immunoprecipitated p19 from 35S-methionine-labeled chicken embryo fibroblasts (CEFs) infected with RPL-40 or RAV-1, but not from CEFs infected with RAV-0, thus identifying the viral structural protein p19 as a polypeptide with subgroup-specific epitopes. Both MCAs can be used to differentiate RPL-40 from RAV-0 infection either in an indirect antibody ELISA or by immunoprecipitation. A third MCA, 6AL42 (IgG2a isotype), reacted with the above viruses of subgroups A, B, C, and D at an antibody titer up to 1000-fold higher than with subgroup E RAV-0 virus in indirect ELISAs. MCA 6AL42 immunoprecipitated p27 from cells infected with RPL-40, RAV-1, or RAV-0. These MCAs are potentially useful in developing immunological tests for differentiation of ALV strains.

Animals↗
PubMedDate not supplied

Identification and localization of glycoprotein B expression in lymphoid tissues of chickens infected with turkey herpesvirus.

One-day-old chickens were inoculated with turkey herpesvirus (HVT). Using an indirect immunofluorescence assay with a monoclonal antibody against HVT glycoprotein B (gB), we determined the course of productive HVT infection in peripheral blood mononuclear cells (PBMCs), spleen, thymus, and bursa. PBMCs were examined from days 4 through 35 postinfection (PI). The spleen, thymus, and bursa were examined from 21 through 70 days PI. Although productive infection in PBMCs was detected at 4 to 12 days PI, it ended by 14 days PI. Splenic cells expressed gB at 21, 28, 35, and 70 days PI, whereas the thymus was positive for gB expression at 21 and 35 days PI. The bursa was never positive for gB expression. At 21, 28, 35, and 70 days PI, plaque formation after co-cultivation of PBMCs with chicken embryo fibroblasts indicated the presence of HVT in infected chickens by co-cultivation assays. On the basis of indirect immunofluorescence assay, gB expression in the spleen and thymus indicates a productive HVT infection in chickens.

Animals↗
PubMedDate not supplied

A simple immunoperoxidase plaque assay to detect and quantitate Marek's disease virus plaques.

We report an immunoperoxidase-based staining technique that can be used to rapidly and accurately detect and quantitate Marek's disease virus (MDV) plaques. Monolayer cultures were fixed and incubated with a monoclonal antibody specific for MDV. After washing, a second antibody of horseradish peroxidase-conjugated goat anti-mouse IgG was applied, incubated for 1 hr, and washed with phosphate-buffered saline. After the cultures were incubated with diaminobenzidine, CoCl2, and H2O2, the plaques appeared as black spots and were easily seen and counted. Significantly more immunoperoxidase-stained serotype 1 MDV plaques could be counted at 4 days postinoculation than were seen in unstained cultures. With serotype 2 MDV-infected cells, the difference in plaque counts was less dramatic. Nevertheless, at 3 days postinoculation, significantly more stained serotype 2 plaques were seen than unstained plaques. Immunoperoxidase staining of turkey herpesvirus plaques did not increase the sensitivity of viewing plaques. Similar numbers of stained and unstained plaques were seen at 2 days postinoculation. We also demonstrated that we could count serotype-specific MDV plaques in a mixed infection that contained all three serotypes.

Animals↗