Robert M. Dougherty

Application of Immunohistochemistry to Study of Avian Leukosis Virus

Summary A modification of the peroxidase-labelled antibody technique was applied to study the distribution of virus antigens in chicken cells infected with two serotypes (A and B) of avian leukosis virus (ALV). Type-specific chicken antisera reacted only with cells infected with virus of the homologous envelope serotype. When unfixed cells were exposed to type-specific antivirus serum, only antigens located at the cell surface were stained, while cells exposed to type-specific antibodies after fixation revealed both surface and intracytoplasmic virus antigen. Cytoplasmic antigen was usually concentrated in discrete granules which often had a vesicular structure. Hamster antibodies against ALV group-specific (gs) antigen reacted with fixed infected cells regardless of envelope serotype, and the distribution of antigen was as shown with chicken type-specific antibodies. Intranuclear gs antigen may have been present in a few cells. Unfixed infected cells did not react with hamster gs antibody and confirmed the location of ALV gs antigen within the virus particle. Some chicken antisera with neutralizing antibodies against a single envelope serotype of ALV contained both type-specific and gs antibodies. These antisera reacted with fixed cells infected with virus of either the homologous or heterologous serotype and stained both surface and cytoplasmic antigens. With unfixed infected cells, these antisera combined only with the surface of cells infected with virus of the same serotype as the neutralizing antibodies in the serum. Thus, the reaction in heterologously infected fixed cells was with internal gs antigen. This confirms independently that chickens are not naturally tolerant to their homologous ‘C type’ gs antigens.

Detection of Avian Oncovirus Group-specific Antigens by the Enzyme-linked Immunosorbent Assay

A three-step sandwich enzyme-linked immunosorbent assay was developed for the detection of avian oncovirus group-specific (gs) antigens. The assay procedure was to coat the wells of microtitre plates with hamster anti-gs IgG, react with crude or purified antigen and finally with hamster anti-gs IgG linked to horseradish peroxidase. The sensitivity was 8 picograms (pg) of input avian myeloblastosis virus (AMV) protein, with negligible background. As the ELISA takes less than 2 h to perform, large-scale screening for infected birds is feasible. A blocking assay was also developed for detecting anti-gs antibodies by adding unlabelled antiserrum after the antigen step.

Multiple antigenic components of the group-specific antigen of the avian leukosis-sarcoma viruses.

Abstract Antigenic material was prepared from chick embryo fibroblast tissue cultures infected with avian leukosis-sarcoma viruses and from leukemic chicken plasma containing avian myeloblastosis virus. Four antigenic components were demonstrated by immunodiffusion and immunoelectrophoresis when these antigens were reacted with group-specific antibodies from tumor-bearing hamsters. At least two of the components were common not only to preparations from tissue cultures infected with each viral subgroup type, but also to the avian myeloblastosis virus preparation. Centrifugation experiments showed that the group-specific antigens were composed of both “soluble” and “insoluble” components. It was also found that sodium lauryl sulfate could alter the diffusion rate and electrical charge of these antigenic components.

Restriction endonuclease digestion eliminates product contamination in reverse transcribed polymerase chain reaction.

Restriction endonuclease digestion was used to eliminate false-positive signals caused by polymerase chain reaction (PCR) product DNA contamination in a reverse transcribed (RT) PCR for amplifying rubella virus (RV) RNA sequences. A restriction enzyme selected to cut the PCR product DNA between, but not within, the primer binding sites was used to digest reaction mixtures after reverse transcription but before PCR amplification. Because restriction enzymes generally react only with specific double-strand sequences, contaminating DNA was rendered inactive while reverse-transcribed single strand cDNA was amplified. Assays showed that restriction enzyme digestion reduced template activity of product DNA by a factor of 10(7), while leaving sensitivity of the RT-PCR unaffected.

The presence of avian leukosis virus group-specific antibodies in chicken sera☆

Abstract Experiments designed to investigate by complement-fixation inhibition tests the group-specific antibody response to an injected dose of either subgroup A or subgroup B strains of avian leukosis virus (ALV) were carried out in chickens 2 1 2 , 6, and 23–25 weeks of age. Many 2 1 2 and 6-week-old birds responded with group-specific (gs) antibody whereas the antibody response of birds injected at 23–25 weeks was very poor. Birds congenitally infected with an ALV of subgroup A were immunologically tolerant to the gs antigen and did not respond with gs antibody whether the challenge virus was subgroup A or B. A comparison between neutralizing and gs antibody activity in 70 immune chicken sera showed: (1) the greatest number of sera containing gs antibody were those with titers of neutralizing antibody > 1 1000 , (2) all gs-positive sera had titers of neutralizing antibody > 1 200 , and (3) only 56% of the sera with high neutralizing activity contained gs antibody. Fractionation of two chicken sera containing gs antibody was carried out to separate the IgM and IgG fractions. The gs antibody activity, as well as most of the neutralizing activity, was found in the IgG fractions.

Transformation of rodent cells by RFV, the human papovavirus with dual genome

Abstract Hamster, rat, and guinea pig cells were transformed by a variant of BK virus, RFV, whose genome consists of two complementary defective molecules, one, R1, with a deletion corresponding to 20–30% of the coding region for capsid proteins VP2 and VP3 and the other, R2, with a deletion corresponding approximately to 50% of the coding region for large T-antigen of BKV. Blot-hybridization of cellular DNA reveals that all three transformed cell types contain viral DNA integrated into high-molecular-weight DNA. Moreover, all the cell lines contain the viral DNA species which has the intact early region. None of the cell lines contains both DNA species. We examined the genome organization of the virus used in these transformation experiments to confirm our previous observations (Pater et al., J. Virol. 36 , 480–487, 1980a) on the dual genome state of RFV. We found a very similar but not identical map for the plaque-purified RFV DNA used in this study when compared to that for plaque-purified RFV DNA examined previously.

Transformation of African green monkey kidney cells with the RF strain of human papovarivus BKVB

Abstract Infection of semipermissive African green monkey kidney (AGMK) cells with human papovavirus RFV prodced a chronic infection with slow cytopathic effects. Prolonged maintenance of cell cultures in the presence of virus-neutralizing antibody resulted in the emergence of a stable cell line presently at the 100th generation intissue culture. The cell line MRF-325 has many of the properties of a tumor cell in culture, including increaed saturation density in monolayers, growth in soft agar and in suspension, and a decreased serum requirement for growth. Papovavirus T antigen and U antigen were detected in 100% of the MRF-325 nuclei by immunoperoxidase staining. No virion antigens were detected, and no infectious virions were isolated by cocultivation or fusion of MRF-325 with permissive human embryo kiney cells. Susceptibility of MRF-325 cells to SV40 infection was decreased compared to untransformed AGMK cells. MRF-325 cells supported high levels of replication of human adenovirus type 5 compared with nontransformed AGMK cells. Thus, RFV apparently provides a helper effect for adenovirus replication in AGMK cells similar to that observed for SV40.

PREPARATION AND USE OF SOLUBLE FERRITIN-ANTIFERRITIN COMPLEXES AS A SPECIFIC MARKER FOR IMMUNOELECTRON MICROSCOPY

Soluble immune complexes of ferritin and chicken antiferritin have been prepared. This has been used with unlabeled chicken antibody to avian leukosis virus and rabbit antichicken globulin to demonstrate the presence of specific viral antigens in tissue culture by electron microscopy.

Cytological observations of “nonproducer” Rous sarcoma cells☆

Abstract “Nonproducer” cells, obtained by infection of cultivated chick embryo fibroblasts with Rous sarcoma virus (Bryan strain), were compared and contrasted with similarly infected cells that were producing virus in large quantities and with uninfected control cells. Both types of infected cells differed markedly from uninfected cells but, despite quantitative differences between them, the morphology of the two types of infected cells was essentially the same. Inasmuch as the “nonproducer” cells did not involve infection with the “helper” Rous-associated virus, the alterations observed reflected only infection with Rous sarcoma virus.