Biomedical subjects
Publications and source records attributed to G Siegl.
Hepatitis A virus (HAV) was highly purified from faeces. The genomic RNA was transcribed to cDNA and this DNA was then cloned into plasmid pBR 322 at the Pst I site, and clones were selected in presence of tetracycline. Most clones contained inserts which hybridized to HAV-specific RNA isolated from HAV-infected cell cultures derived from a human hepatocellular carcinoma. Two clones expressed low amounts of viral antigens.
The propagation of hepatitis A virus (HAV) in the cell line PLC/PRF/5 made possible the radiolabelling in vivo of mature, infectious hepatitis A virions and the determination of their physicochemical properties. In contrast to poliovirus type 2 (160S, 1.340 g/ml), HAV had a sedimentation coefficient of 156 +/- 2S and a buoyant density of 1.332 g/ml in CsCl. The genome of HAV consisted of linear single-stranded RNA which sedimented at 32.5S under non-denaturing conditions. Compared to the size and sedimentation behaviour of poliovirus RNA (2.6 X 10(6) mol. wt., 35S) this corresponds to a mol. wt. of 2.3 X 10(6). Electrophoresis under fully denaturing conditions, however, revealed a mol. wt. of 2.8 X 10(6) and indicates the existence of relatively extended regions with secondary structure. The purified virus genome, containing a poly(A) sequence, served as a messenger for the synthesis of virus antigen in PLC/PRF/5 cells. Finally, in accordance with previous observations, the capsid of the virion was found to be constructed of three major polypeptides (VP1, 31 X 10(3); VP2, 26 X 10(3); VP3, 21 X 10(3) mol. wt.) and of two less readily demonstrable components probably corresponding to VP4 (8 X 10(3) to 10 X 10(3) mol. wt.) and the precursor polypeptide VP0 (40 X 10(3) mol. wt.).
Hepatitis A virus was purified from fecal samples collected at various times in the incubation period of patients with naturally acquired hepatitis A. The proteins of particles banding at around 1.34 g/ml in CsCl and sedimenting at about 160S were radioiodinated in vitro and separated by electrophoresis on polyacrylamide gels in the presence of 0.1% sodium dodecyl sulfate and 8 M urea. Under these conditions, the capsid proteins resolved into four polypeptides with molecular weights of approximately 31,000, 24,500, 21,000, and 9,000, respectively. A fifth protein of about 40,000 daltons in size and assumed to be equivalent to the precursor polypeptide VP0 of the picornaviruses was present in particles sedimenting at only 150 to 155S and banding at around 1.33 g/ml in CsCl. The physicochemical characteristics of these particles are consistent with those of the provirion structures of picornaviruses. In several of the fecal samples, these particles represented a considerable fraction of all particles present. The significance of this finding with respect to the antigenicity of hepatitis A antigen extracted from stool specimens is discussed.
The genomes of canine parvovirus and mink enteritis virus were compared by restriction enzyme analysis of their replicative-form DNAs. Of 79 mapped sites, 68, or 86%, were found to be common for both types of DNA, indicating that canine parvovirus and mink enteritis virus are closely related viruses. Whether they evolved from a common precursor or whether canine parvovirus is derived from mink enteritis virus, however, cannot be deduced from our present data.
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Plaque formation with representative strains of feline panleukopenia virus (FPV) has been obtained using a permanent line of feline kidney cells under agarose overlay. FPV-infected cells appear as white plaques after neutral red staining. Plaque size is determined by the extent of cell division in the infected monolayer. FPV assay by the plaque procedure is rapid and gives infectivity titres which exceed those determined by the common inclusion body and immunofluorescent assays of FPV by a factor of about 100 and 10, respectively. Moreover, the plaque assay offers an effective means for the quantification of neutralizing antibodies in feline sera as well as for the detection of heat-stable substances in bovine sera which strongly interfere with replication of the virus.
Hepatitis A virus (HAV) was isolated directly from human feces and propagated serially in an HBsAg producing human hepatoma cell line. No cytopathic effect was observed in the tissue culture and no detectable amounts of HAV were present in the tissue culture supernatant fluid. However, increasing amounts of hepatitis A antigen (HAAg) were detected by radioimmunoassay in the cell extracts obtained by freezing and thawing of cells. Specificity of the HAAg determination was shown by neutralization with convalescent sera of marmosets experimentally infected with the MS-1 strain of hepatitis A and by the absence of this neutralization with preinoculation sera. HAAg was first detected after four weeks in the cell extract of infected cultures after inoculation of 10(2)--10(4) tissue culture infectious doses of HAV from second passage.
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Virus-like particles were purified from stools of patients in an epidemic of hepatitis A in Germany. When reference MS-1 chimpanzee pre-inoculation and convalescent sera were used, the close serological relationship of the purified particles to well-known isolates of hepatitis A could be established. On the other hand, the physicochemical characteristics of the particles were determined in parallel to the characteristics of a marker parvovirus (LuIII) and a marker picornavirus (poliovirus type 2). It could be shown that the majority of the hepatitis A-associated particles band at 1.34 g/ml in CsCl and, like poliovirus, sediment at about 160S. In addition, a distinct hepatitis A antigen was observed, which banded at 1.305 g/ml and sedimented between 50 and 90S. A further component accumulated in the density range of between 1.38 and 1.44 g/ml. However, it seemed to be rather labile. Upon reisolation from CsCl and sedimentation in sucrose, it resolved into a 160S, a 90 to 100S, and a 50S form. The size of the 160S particles (27 to 29 nm) could be readily distinguished from that of the parvovirus (22 to 24 nm). It is concluded, therefore, that hepatitis A-associated virus particles are more likely to be classified with the picornaviruses than with the parvoviruses.
Virus particles banding at 1.34 g/ml in CsCl and sedimenting at 160S in sucrose gradients were isolated from fecal specimens of patients suffering from hepatitis. In the presence of 4 M urea and about 90% formamide, these particles released linear nucleic acid molecules of the kinked appearance characteristic of single-stranded RNA or single-stranded DNA. They could be distinguished from the nucleic acid of phage lambda added to the preparation as a marker for double-stranded configuration. Experiments in which the virus particles under investigation were incubated at pH 12.9 at 50 degrees C for 30 min revealed that their nucleic acid molecules were hydrolyzed as readily as the RNA genome of poliovirus type 2 analyzed in parallel. Both the single-stranded DNA of phage phiX174 and that of parvovirus LuIII, however, proved unaffected by this treatment, and the double-stranded DNA of phage lambda was denatured to single-stranded molecules. It was concluded, therefore, that the virus of human hepatitis A contains a linear genome of single-stranded RNA and has to be classified with the picornaviruses.
The LuIII parvovirus was adapted to the newborn hamster and produced a systemic infection with massive intestinal hemorrhage. Inoculation of pregnant hamsters lead to transplacental infection of the fetuses and abortion. Most fetal deaths were observed in animals inoculated on days 8 and 10 of gestation. Virus was recovered from dead fetuses, placentas, and viable fetuses. Histological lesions were found in the heart, liver, kidney and CNS of infected fetuses.
The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, double-stranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967).
Newly synthesized structural polypeptides of parvovirus LuIII, VP1 (62,000 daltons) and VP2 (74,000 daltons), were detected in nuclei of synchronized, infected HeLa cells at 11 to 12 h postinfection, i.e., after cells had passed through the S phase of the cell cycle. At this time, most of intranuclear viral polypeptides were associated with the chromatin acidic proteins. However, 13 to 14 h postinfection, about one-third of intranuclear VP1 and VP2 also could be extracted in the fraction containing nuclear sap proteins. According to pulse-chase experiments, VP1 and VP2 accumulated in the chromatin with a time lag of 20 to 30 min. About 90% of these chromatin-associated viral polypeptides represented empty viral capsids. In addition, chromatin prepared at 14 h postinfection contained 90 to 95% of the total intranuclear viral 16S replicative-form DNA. Since viral replicative-form DNA and empty viral capsids seem to be associated specifically with cellular chromatin, we assume that this subnuclear structure is the site of the synthesis of progeny viral DNA and the formation of complete virions.
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