Hepatitis B viral clearance studies using duck virus model.

We have shown data to suggest that the in vivo duck hepatitis B virus system represents an excellent animal model system for the study of hepatitis B virus. Because of the similarity of DHBV to human HBV (including comparable results in virus inactivation studies), the high level of sensitivity of the DHBV assay, and the rapidity, ease, and relative low cost of obtaining results, we propose that the in vivo DHBV titration system be used as a model for human HBV in process validation studies. Data generated in such validation studies have, in fact, been submitted by a number of blood products manufacturers to the U.S. F.D.A. in support of IND applications.

Quantitation of residual mouse DNA in monoclonal antibody based products.

The identification and characterization of cell substrates and testing of bulk and final products is an important issue which must be addressed by manufacturers. In view of the fact that hundreds of applications for Investigational New Drugs (IND) have been submitted over the past few years, there is an obvious need for testing of these products. Detection of DNA by molecular hybridization has been used for various applications including the quantitation and characterization of DNA in biological products. We have developed a precise assay based on hybridization for the detection and quantitation of residual genomic DNA. In order to reduce protein interference, a specific pretreatment method for isolation of DNA in monoclonal antibody based products was implemented. We have used the assay to evaluate levels of contaminating DNA in prepared lots of monoclonal antibodies. Validation experiments demonstrated a sensitivity below 10 pg DNA using nick-translated 32P-labelled genomic DNA probes. The assay allows accurate quantitation of residual DNA in biologics.

[Persistence of the Sindbis virus in cultures producing oncornavirus]. / Persistentsiia virusa Sindbis v kul'turakh, produtsiruiushchikh onkornavirus.

Continuous lines were obtained from primary cultures of BALB/C mouse embryo cells which were found by electron microscope and reverse transcriptase reaction to produce permanently oncoronavirus type C after exogenous infection with Rauscher leukemia virus (RLV). Sindbis virus (SV) was inoculated into virogenic cultures 398 days after infection with RLV. The system in characterized by rapid (3-21 days) disappearance of the infectious arbovirus from the medium and the cells, long-term (over 5 months) persistence on SV noninfectious antigen and signs of stimulation of oncornavirus activity. The level of reverse transcriptase activity in cultures in the presence of persisting arbovirus was 1.5-3.3-fold higher than in cultures infected with RLV alone. Two variants of the course of mixed chronic infection of the cultures with oncornavirus and arbovirus differing in the rate of transition of the arbovirus into the noninfectious form and inhibition or stimulation of oncornavirus functions are discussed.

Detection by immunofluorescence of avian myeloblastosis virus reverse transcriptase.

Peripheral blood cells of avian myeloblastosis virus (AMV-(infected chickens were examined at various intervals post infection by immunofluorescence. AMV revertase was identified in pro- and myeloblasts; it was localized mainly in the perinuclear zone or throughout the cytoplasm. No revertase was found in erythrocytes or granulocytes. Blood cells from uninfected chickens of man contained no revertase.

[Immunologic aspects of preparation of antiserum to the reverse transcriptase from avian myeloblastosis virus]. / Immunologicheskie aspekty polucheniia antisyvorotki k obratnoi transkriptaze (revertaze) virusa mieloblastoza ptits.

After immunization of rabbits the antiserum was prepared against purified reverse transcriptase (revertase) from avian myeloblastosis virus (AMV). The antiserum demonstrated enzymeneutralizing antibody activity that was associated with ummunoglobulin G fraction but not with IgM. The high antigenicity of AMV revertase for rabbits was shown. The active antiserum was obtained after 4 immunizations of rabbit with approximately 20 microgram of the enzyme. Non-specific revertase inhibitors were found in normal rabbit serum, which were absent in IgG fraction from this serum. The revertase activity of Rauscher leukemia virus (RLV) and Visna virus was not neutralized by antisera against AMV polymerase. This work was supported by the project "Revertase".

[Study of the reproduction conditions of the avian myeloblastosis virus for separation of the "reverse transcriptase" enzyme]. / Izuchenie uslovii razmnozheniia virusa ptich'ego mieloblastoza dlia vydeleniia fermenta "obratnaia transkriptaza"

Sensitivity of eight chick lines to the avian myeloblastosis virus as the main source for RNA-dependent DNA-polymerase recovery was studied in the course of the "revertase" project. The virus (0.1 ml) was inoculated intracardially or intraperitoneally to one-day chicks, and then the virus titer was determined according to the ATP-activity. C and D-lines of the Enya-cross were shown to be most sensitive (sensitivity 75%) among the lines studied.