In vitro assay of mitogen stimulation of avian peripheral lymphocytes.
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Biomedical subjects
L F Lee
Publications and source records attributed to L F Lee.
Characterization of avian reovirus 24.
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Released and extracted virions of Marek's disease virus.
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An enzyme-linked immunosorbent assay for the detection of antibodies to Marek's disease virus.
A reproducible enzyme-linked immunosorbent assay (ELISA) using Marek's disease virus (MDV)-infected cells for the detection of antibodies to MDV is described. The optimum number of MDV-infected chicken embryo fibroblasts (CEF) was 5 X 10(4)/well, and test sera were positive at 1:400 dilutions. Compared with a purified virus preparation, MDV-infected CEF produced high specific and low nonspecific reactivities. Wells coated with whole cells could be stored at 4 C or -20 C for at least 3 months without loss of reactivity. With antibody-negative sera, the cutoff absorbency was 0.20 units. The ELISA was 20-to-40-fold more sensitive than indirect immunofluorescence. Homologous combinations of antisera in wells coated with CEF infected with different MDV serotypes were more reactive at higher dilutions than were heterologous combinations. The procedure described is specific and suitable for large-scale screening of both chicken and monoclonal antibodies against MDV.
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Kinetics of phytohemagglutinin response in chickens infected with various strains of Marek's disease virus.
Phytohemagglutinin (PHA) response of whole blood lymphocytes from white leghorn inbred line 7(2) chickens infected with various strains of Marek's disease virus (MDV) was monitored sequentially for 6 weeks postinfection. A significant difference between JM and GA strains was shown. A two-phase depression in the PHA response was observed in chickens infected with the JM strain. Early depression occurred 1 week postinfection and was followed by recovery a week later. The second depression occurred at 4 weeks postinfection and lasted until the end of the experiment. The GA strain-infected group, on the other hand, began to show depression 4 weeks postinfection, and most chickens died within a short time thereafter. PHA response of chickens infected with strain Md11/75C, attenuated in cell culture from highly virulent strain Md11, was almost the same as that of control chickens.
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Field trials to test the efficacy of polyvalent Marek's disease vaccines in broilers.
The efficacies of trivalent (Md11/75C + SB-1 + HVT), bivalent (SB-1 + HVT), and turkey herpesvirus (HVT) vaccines against Marek's disease (MD) were compared in commercial broiler flocks in four trials involving 11 farm locations and 486,300 chickens. In all four trials, chickens receiving polyvalent vaccines had lower leukosis (MD) condemnation rates than chickens vaccinated with HVT alone; when data were summarized for each vaccine type in each trial, condemnation rates for the bivalent- or trivalent-vaccinated groups were 56-96% (mean 78%) lower than those for HVT-vaccinated chickens. Polyvalent vaccination was clearly mor efficacious than HVT in 8 of 11 individual farms, although it did not always reduce leukosis condemnations to acceptable levels. Body weights of chickens vaccinated with polyvalent vaccines did not differ consistently from those vaccinated with HVT. Chickens inoculated with the trivalent vaccine had slightly lower overall leukosis condemnation rates (0.24%) than those inoculated with the bivalent vaccine (0.45%) in trials 1-3, where direct comparisons were made. Bivalent vaccines containing either 1,500 or 200 plaque-forming units of SB-1 virus were equally effective; thus, HVT may need to be supplemented with only small amounts of SB-1 to obtain the benefits of protective synergism. SB-1 virus did not appear to carry over from polyvalent-vaccinated flocks to subsequent HVT-vaccinated flocks in the same houses, even when old litter was used.
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Chicken lymphocyte stimulation by mitogens: a microassay with whole-blood cultures.
A micromethod is described that uses whole blood for studying mitogen stimulation of chicken lymphocytes. Procedures critical for obtaining a stimulatory response up to 200 times that of unstimulated control cultures include: 1) use of no more than 10 microliter of whole blood per culture; 2) incubation at 41 C for 72 hours in 0.2 ml of medium RPMI 1640 containing 100 microgram of stock phytohemagglutinin-P and 3) pulse labeling for 8 hours with 5-[125I] iodo-2'-deoxyruidine. The method is easy, rapid, and suitable for sequential studies of pathogenesis of disease in poultry.
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Comb lesions and mortality patterns in white leghorn layers affected by Marek's disease.
Marek's disease was diagnosed as the cause of prolonged high mortality in two commercial flocks of white leghorn layers by gross and microscopic examination of affected tissues and by immunofluorescent study of live tumor cells using anti-Marek's disease tumor-associated surface antigen, anti-immunoglobulin M, and monoclonal antibodies. The disease was characterized by swollen, necrotic, lymphomatous combs and mortality that rose above normal at 28 weeks of age, peaked at 35-36 weeks, and returned to normal by 45-46 weeks.
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Characteristics of CVI988/Rispens and R2/23, two prototype vaccine strains of serotype 1 Marek's disease virus.
Studies were focused on two attenuated serotype 1 Marek's disease (MD) vaccine viruses, CVI988/Rispens (passage 42) and R2/23 (passage 105). Both serotype 1 vaccine viruses provided much higher levels of protection than the prototype MD vaccine, turkey herpesvirus (HVT); the best protection was generally provided by CVI988/Rispens when compared with other vaccines. The efficacy of neither serotype 1 vaccine was improved by mixture with viruses of other serotypes (synergism). No differences between the two serotype 1 vaccines were revealed by cross-neutralization tests, thus excluding preferential in vivo neutralization by maternal antibodies as an explanation for differences in protective efficacy. Neither vaccine strain induced MD lesions or reduced growth rates in 8- or 18-week trials. Neither virus depressed humoral or cellular immune responses to antigenic challenge at 3 or 15 days after vaccination. Both virus strains exhibited altered characteristics during serial backpassage; R2/23 acquired increased oncogenic potential, and CVI988/Rispens acquired the potential for increased viremia titers, accompanied by an increased frequency of both histologic nerve lesions and gross thymic atrophy. During backpassage trials, contact spread was not observed for R2/23 and, surprisingly, seemed relatively limited for CVI988/Rispens. Studies on these two serotype 1 strains generally support the safety and efficacy of the serotype 1 class of MD vaccines.
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Characterization of a monoclonal antibody against a nuclear antigen associated with serotype-1 Marek's disease virus-infected and transformed cells.
A monoclonal antibody (MAb), 2BN90, was characterized on a panel of Marek's disease virus (MDV) isolates representing three antigenically related serotypes and lymphoblastoid cell lines established from Marek's disease (MD) tumors. It was reactive with all serotype 1 isolates, including CV1988, in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) test. The MAb immunoprecipitated and immunoblotted a 40-kilodalton protein, which was found to be phosphorylated by metabolic labeling with 32P inorganic phosphate, and localized to the cell nucleus using the IF test. It reacted with MDV-transformed lymphoblastoid cell lines, and the percentage of reactive cells was enhanced after treatment with iododeoxyuridine. MAb 2BN90 may be useful as a type-specific antibody of choice for serotyping MDV serotype 1 strains and as a probe for studying the role of pp40 in MDV infection and transformation.
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Protection and synergism by recombinant fowl pox vaccines expressing genes from Marek's disease virus.
Recombinant fowl poxviruses (rFPV) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B (gB1), C (gC), and D (gD) and tegument proteins UL47 and UL48, as well as genes from serotypes 2 and 3 MDV coding for glycoprotein B (gB2 and gB3). These rFPVs, alone and in various combinations, including combinations of fowl poxvirus (FPV)/gBs with turkey herpesvirus (HVT), were evaluated for ability to protect maternal antibody-positive (ab+) and -negative (ab-) chickens against challenge with highly virulent MDV isolates. The protective efficacy was also compared with that of prototype Marek's disease (MD) vaccines. No protection was induced in ab+ chickens by rFPV expressing gC, gD, UL47, or UL48. In contrast, the rFPV/gB1 construct protected about 23% of ab+ chickens against MDV challenge compared with 26% for cell-associated HVT. Levels of protection by rFPV/gBs of different MDV serotypes was highest for gB1, intermediate for gB2, and lowest for gB3. When rFPV/gB1 was combined with cell-associated HVT, protection was enhanced by an average of 138% compared with the best component monovalent vaccine, and the mean level of protection was 59% compared with 67% for the HVT+SB-1 bivalent vaccine. Relatively high protection (50%) and enhancement (200%) were also observed between rFPV/gB1 and cell-free HVT. These results suggest a specific synergistic interaction between rFPV/gB1 and HVT, possibly analogous to that previously described between serotypes 2 and 3 viruses. Levels of protection by rFPV/ gB1 alone or by bivalent rFPV/gB1+cell-associated HVT were similar to those of conventional cell-associated MD vaccines. However, the bivalent rFPV/gB1+cell-free HVT vaccine was clearly more protective than cell-free HVT alone and, thus, may be the most protective, entirely cell-free MD vaccine thus far described.
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Retroviral insertional mutagenesis of a herpesvirus: a Marek's disease virus mutant attenuated for oncogenicity but not for immunosuppression or in vivo replication.
Our earlier studies have shown that retrovirus insertion into herpesvirus is an efficient process that engenders recombinant herpesviruses with altered biological properties. The RM1 clone is derived from the JM strain of Marek's disease virus (MDV) through retrovirus insertional mutagenesis and contains sequences of reticuloendotheliosis virus inserted at the junction of the internal repeat and unique short regions of the genome. In previous studies, the RM1 clone appeared attenuated for oncogenicity but caused marked atrophy of the thymic lobes. The present studies represent a detailed analysis of the biological characteristics of the RM1 clone in order to better understand mechanisms of oncogenicity and gene function of MDV. RM1 was almost fully attenuated for oncogenicity but retained other in vivo properties of virulent viruses such as thymic and bursal atrophy, early immunosuppression, early cytolytic infection followed by efficient replication, and contact spread--all normally absent in attenuated strains. This suggests that, for serotype 1 MDV, oncogenicity is not tightly linked with immunodepression or viral replication and that these properties may be controlled by different genes or mechanisms. The mutation was stable through serial passage of the virus in chickens as determined by molecular analysis. None of the mutant viruses demonstrated expansion of the 132-bp repeat region of the genome, indicating that such expansion is not required for attenuation. Chickens vaccinated with RM1 clones were protected against challenge with virulent MDV, and levels of protection exceeded those of other attenuated serotype 1 vaccine viruses. Thus, attenuation by selective mutation may be an advantageous strategy for development of serotype 1 Marek's disease vaccines.
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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.
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Serological characteristics of a membrane glycoprotein gp82 of Marek's disease virus.
Marek's disease virus (MDV) is a cell-associated herpesvirus of gallinaceous fowl. Glycoproteins of herpesviruses have been reported to be important in stimulating humoral and cell-mediated immune responses against viral infection. At least eight glycoproteins of MDV have been identified, and glycoprotein B (gB) has been reported to produce protective immunity. In addition to glycoproteins, other cell membrane proteins may also be important for engendering immunity. In an attempt to determine whether the protein is involved in immune responses, we developed three monoclonal antibodies (Mabs) and rabbit polyclonal antibodies specific for MDV membrane protein gp82. Three Mabs (Mab2.1, 3.7, and 5.7) were produced by using TrpEgp82 fusion protein as the immunogen that was expressed by recombinant pATH expression vector pATHgp82 in Escherichia coli. Indirect immunofluorescence assay (IFA), immunoprecipitation, and Western blot analysis were used to determine the characteristics of gp82. All of the Mabs reacted with gp82 protein irrespective of its conformation. The gp82 protein was predominantly anchored on the surface membrane of a chicken embryo fibroblast (CEF) infected with serotype 1 MDV (MDV-1). There was no detectable immunofluorescence staining in the cytoplasm or nucleus in the MDV-1-infected CEFs. These results indicate that gp82 is a membrane protein. The distribution pattern of immunofluorescence staining of gp82 varies between CEFs infected with the low passage MDV-1 isolate Md11, and those infected with a high passage derivative, Md11/75c. In SB-1 (Serotype 2 MDV [MDV-2]) and Turkey herpesvirus (HVT) (serotype 3 MDV [MDV-3]) infected CEFs, gp82 was not detected by immunoprecipitation, Western blot, or IFA using both monoclonal and polyclonal antibodies. These results suggest that gp82 in serotype 2 or 3 has a modified epitope, or that the gene is permanently repressed.
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