Jon-Duri Tratschin

Cloning of infectious adeno-associated virus genomes in bacterial plasmids

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains.

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.

Cloning of the human parvovirus B19 genome and structural analysis of its palindromic termini

We describe the molecular cloning of the entire 5.6-kb single-stranded DNA genome of the human parvovirus B19 in bacterial plasmids. Stable amplification of the recombinant plasmid DNA was achieved in Escherichia coli JC8111 but not in HB101 cells. Sequence analysis of the cloned DNA shows that the terminal 383 nucleotides at each end of the genome are identical inverted repeats. The distal 365 nucleotides of the repeat represent an imperfect palindrome which presumably folds over to form a hairpin structure. The sequence of the hairpin occurs in two distinct configurations which are related in that one is the inverted complement of the other. Such alternative configurations of the terminal hairpins have been found for all parvoviruses analyzed so far and are referred to as flip and flop.

Replication of adeno-associated virus DNA: Complementation of naturally occurring rep− mutants by a wild-type genome or an fori− mutant and correction of terminal palindrome deletions

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.

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.

Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein.

We have mapped a minor spliced transcript with mRNA properties which is derived from the capsid gene promoter (P40) of the adeno-associated virus (AAV) genome. By S1 nuclease mapping as well as primer extension analysis this mRNA was found to occur by splicing at the same donor site (nucleotide, NT 1907) but at an alternative acceptor site when compared to the major, 2.3-kb spliced P40 transcript which encodes two of the three AAV capsid proteins, namely VP2 and VP3. This minor acceptor site is located at NT 2200, i.e., 27 NT upstream of the acceptor site used to generate the VP2/VP3 mRNA. We have also sequenced the AAV genome in this region and have found one important error in the sequence published by A. Srivastava, E. Lusby, and K. I. Berns (1983, J. Virol. 45, 555-564): residue at NT 2429 has to be deleted from the reported sequence. This correction of the sequence reveals that the open reading frame (ORF) which encodes all three capsid proteins extends from NT 2203 to NT 4321. Furthermore, this entire ORF is contained in the minor but not in the major spliced P40 transcript. We provide evidence that the largest AAV structural protein VP1 is translated from this hitherto undetected spliced transcript by initiation at its first AUG (NT 2203) and termination at NT 4321. The calculated molecular weight of this VP1 polypeptide is 78,247 Da.

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines

Abstract Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures oft he swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

The Physicochemical Properties of Infectious Hepatitis A Virions

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.).

Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-α/β and carry foreign genes.

Virus replicon particles (VRP) are genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRP fulfil the criteria of a safe vaccine and gene delivery system. With VRP derived from classical swine fever virus (CSF-VRP), a single intradermal vaccination protects from disease. Spreading of the challenge virus in the host is however not completely abolished. Parameters that are critical for immunogenicity of CSF-VRP are not well characterized. Considering the importance of type I interferon (IFN-α/β) to immune defence development, we generated IFN-α/β-inducing VRP to determine how this would influence vaccine efficacy. We also evaluated the effect of co-expressing granulocyte macrophage colony-stimulating factor (GM-CSF) in the vaccine context. The VRP were capable of long-term replication in cell culture despite the presence of IFN-α/β. In vivo, RNA replication was essential for the induction of an immune response. IFN-α/β-inducing and GM-CSF-expressing CSF-VRP were similar to unmodified VRP in terms of antibody and peripheral T-cell responses, and in reducing the blood levels of challenge virus RNA. Importantly, the IFN-α/β-inducing VRP did show increased efficacy over the unmodified VRP in terms of B-cell and T-cell responses, when tested with secondary immune responses by in vitro restimulation assay.

PORCINE CELLS PERSISTENTLY INFECTED WITH CLASSICAL SWINE FEVER VIRUS PROTECTED FROM PESTIVIRUS-INDUCED CYTOPATHIC EFFECT

Cytopathogenicity of classical swine fever virus (CSFV) depends on the presence of defective particles containing a subgenomic (sg) RNA with a defined deletion. In a previous report we described the spontaneous generation of this sg RNA and therefore of cytopathogenic (cp) CSFV in porcine kidney cell cultures persistently infected with CSFV. Frequently, some cells survived the CPE and could be further propagated. They remained positive for viral antigen and continued to shed complete virus and in most cases also defective virus particles. SK-6 cells that had survived the CPE (CPE(surv)cells) were used to investigate these findings further. In contrast to persistently infected cells that had not experienced a CPE, CPE(surv) cells were protected from the CPE when superinfected with cp CSFV or with cp bovine viral diarrhoea virus. Similarly, cells which were rescued and further propagated after acute infection with cp CSFV also proved to be protected from the CSFV-induced CPE. When either virus obtained from CPE(surv) cells that had spontaneously lost the sg RNA or virus from which defective particles had been removed was used to establish persistently infected cells, these cells were also protected from the CPE after superinfection with cp CSFV. These findings suggest that the virus contained in CPE(surv) cells confers on the host cell the ability to resist the CSFV-induced CPE. However, when naive cells were infected with supernatants from CPE(surv)cells that contained defective virus particles, the CPE reappeared within three to five virus passages, indicating that the sg RNA retained its cytopathogenic potential.