Propagation and assay of hepatitis A virus in vitro.

Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37 degrees C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.

Stability of hepatitis A virus.

The stabilities of hepatitis A virus (HAV) and of poliovirus type 2 were compared under strictly controlled, identical conditions of pH value, temperature, and salt concentration. Although the resistance of the viruses proved to be the same from pH 3 to 11, the temperature at which 50% of poliovirus particles became disintegrated during heating at pH 7.0 for 10 min (T50,10 = 43 degrees) differed significantly from that characteristic for HAV (T50,10 = 61 degrees). In the presence of 1 M MgCl2, the T50,10 for poliovirus and for HAV shifted to 61 degrees and 81 degrees, respectively. Destabilization of the physical integrity of HAV by heating resulted in the release of viral RNA and, simultaneously, in the generation of several 'empty' capsid structures or dissociation products thereof. Empty capsids and further dissociation products were still serologically reactive with anti-HAV IgG contained in human convalescent sera. With further heating (greater than T50,10 = 67 degrees or greater than T50,1 = 87 degrees), however, they irreversibly lost this reactivity.

Canine parvovirus: relationship to wild-type and vaccine strains of feline panleukopenia virus and mink enteritis virus.

Canine parvovirus (CPV), feline panleukopenia virus (FPLV) and mink enteritis virus (MEV) were compared serologically, by determination of their host range in cell cultures, as well as by restriction enzyme analysis. Maps of the virus genomes were established using seven different restriction enzymes cutting at a total of 56 sites. MEV and FPLV gave maps which were identical except for one restriction site. The map of CPV is closely related to those of FPLV/MEV since their DNAs share about 80% of the restriction sites tested. However, CPV is clearly distinct from FPLV/MEV since either eight (German isolate) or nine (Belgian, Swiss and American isolates) restriction sites are different. The DNAs of six vaccine strains of FPLV and MEV were also analysed. They gave maps which closely resembled those of the respective wild-type strains. CPV and FPLV/MEV also differed with respect to antigenicity, as well as to host range in cell cultures.

A plaque assay for feline panleukopenia virus.

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.

Multiplication of parvovirus LuIII in a synchronized culture system. IV. Association of viral structural polypeptides with the host cell chromatin.

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.