Studies on the virulence of pigeon paramyxovirus-1 (PMV-1). II. Mechanism of the virulence increase of pigeon PMV-1.
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Animals↗
Biomedical subjects
B Lomniczi
Publications and source records attributed to B Lomniczi.
Newcastle disease vaccine (La Sota) strain specific monoclonal antibody. Brief report.
Newcastle disease virus vaccine strain (La Sota) specific monoclonal antibody (La-1) was produced by immunizing mice with isolated glycoproteins of strain La Sota. This antibody was recognized only in the ELISA test in which it bound exclusively to La Sota strain out of a range of over 300 lentogenic, mesogenic and velogenic strains examined.
Animals↗
Multiple defects in the genome of pseudorabies virus can affect virulence without detectably affecting replication in cell culture.
Several independently isolated vaccine strains of pseudorabies virus were studied to identify the functions that play a role in the expression of virulence of this virus. All the strains that were studied grew well in three different cell types. No differences that could be correlated with avirulence could be detected either in the virus yield produced by the cells or in the length of the eclipse phases. All the attenuated strains, however, had lost their ability to replicate efficiently in the brains of day-old chickens. The defects leading to the decrease in the virulence for day-old chickens varied in the different vaccine strains. The Tatarov vaccine strain is defective in the thymidine kinase (TK) gene; restoration of a functional TK gene restores to this strain its virulence for day-old chickens and for pigs. Three out of four different, independently isolated avirulent strains were found to be defective in different loci, as determined by their ability to generate virulent recombinants. Two strains, Bartha and Buk Z300, however, yielded few virulent recombinants, indicating that they were defective in at least one closely linked function. Furthermore, all the virulent recombinants obtained from cells coinfected with different pairwise combinations of the vaccine strains had higher LD50 values than virulent wild-type virus, indicating that the recombinants had not acquired all the functions necessary for optimum expression of virulence. Partial virulence was also restored to Buk Z900 by marker rescue with sequences originating from three different regions of the wild-type pseudorabies virus genome. All three of these regions were different from those that had previously been shown to rescue virulence of the Bartha strain (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1987, J. Virol. 61, 796-801). Our results thus show that (1) defects in several different loci of the pseudorabies virus genome can affect virulence without detectably affecting growth in cell culture and (2) most vaccine strains have multiple defects contributing to their lack of virulence.
Animals↗
Role of a structural glycoprotein of pseudorabies in virus virulence.
The virulence of deletion mutants of pseudorabies virus defective in the expression of glycoprotein gI, gp63, or both was tested in 1-day-old chickens and young pigs. In the absence of expression of gI, the virulence of a fully virulent laboratory strain, PrV(Ka), for 1-day-old chickens was reduced approximately fourfold. Inactivation of glycoprotein gp63 appeared also to affect the virulence of PrV(Ka) only slightly, as did inactivation of both gI and gp63. The level of reduction in virulence, however, was considerably more marked in Bartha 43/25aB4, a less virulent virus strain. Inactivation of the expression of gI in Bartha 43/25aB4 reduced virulence for chickens at least 100-fold. The results obtained when the virulence of the mutants for pigs was determined were compatible with those obtained for chickens. These results indicate that gI plays a role in virulence, but that it does so in conjunction with at least one other viral function (a function that is defective in Bartha 43/25aB4).
Animals↗
Genome location and identification of functions defective in the Bartha vaccine strain of pseudorabies virus.
We have shown previously (Lomniczi et al., J. Virol. 52:198-205, 1984) that the Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome--a deletion that is related to the absence of virus virulence. This strain is, however, also defective in other genes involved in virulence. We show here that virulence can be restored by marker rescue of the Bartha strain to which an intact Us has been restored (but not to the parental Bartha strain) by sequences derived from approximate map units 0.460 and 0.505 of the wild-type virus genome. No difference in the ability to grow in cell culture was observed between parental Bartha, Bartha 43/25a (Bartha to which an intact Us has been restored), or the doubly rescued Bartha strains. However, only the doubly rescued Bartha strain was virulent for both chickens and pigs and replicated to high titers when inoculated directly into the brains of chickens. The sequences that could restore virulence to the Bartha 43/25a strain encode four genes, all of which are involved in processes leading to the assembly of nucleocapsids. Since these sequences rescue virulence, it appears that a function that plays a role in nucleocapsid assembly is defective in the Bartha strain and that this defect contributes to the lack of virulence of this virus.
Animals↗
Evolution of pseudorabies virions containing genomes with an invertible long component after repeated passage in chicken embryo fibroblasts.
The genome of pseudorabies virus consists of two components, short (S) and long (L). Only the S component is bracketed by inverted repeats, and only the S component inverts itself relative to the L component, giving rise to two isomeric forms of the genome. An attenuated vaccine strain of pseudorabies virus (Norden), however, has a genome which is found in four isomeric forms (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). To determine the basis for the atypical structure of the genome of the Norden strain, we examined more than 40 field isolates of pseudorabies virus; all contained genomes in which the L component was fixed in only one orientation relative to the S component. Several independently generated vaccine strains which have been passaged extensively in chicken embryos and chicken embryo fibroblast (CEF) cell cultures were also analyzed; they possessed an invertible L component. Furthermore, emergence of pseudorabies virus variants with an invertible L component was observed after passage of the virus in CEF, but not in rabbit kidney or pig kidney, cells. The invertibility of the L component was associated consistently with a translocation of sequences from the left end of the genome to a position next to the inverted repeat sequence of the S component. Three observations indicate that genomes with an invertible L component (and the translocation) have a selective growth advantage over standard pseudorabies virus when grown in CEF. The proportion of virions with such genomes does not increase linearly as would be expected if the translocation events occurred repeatedly, most genomes eventually experiencing the translocation. Instead, after a lag, the proportion of such virions in the population increases relatively rapidly. The genome structures that are generated upon independent passage in CEF of each virion population were relatively homogeneous. Some heterogeneity was observed at relatively early stages of the emergence of the genomes carrying the translocation; at later stages, virions with genomes with a specific size translocation predominated in the virus population. Parallel passages in CEF of the same pseudorabies virus strain resulted in the emergence of populations of virions with genomes with different size translocations. However, in each of the passaged populations of virions the majority of virions had genomes with the same size translocation. The most likely interpretation of these results is that virions with genomes carrying the translocations that emerge upon passage of the virus in CEF have a selective advantage when grown in these cells.
Animals↗
Role of glycoproteins of pseudorabies virus in eliciting neutralizing antibodies.
The experiments described in this paper were designed to assess the role of the various virus glycoproteins of pseudorabies virus (PrV) in eliciting the production of neutralizing antibodies during the normal course of infection of swine. They also address the question of the degree of antigenic variation within each glycoprotein between different virus isolates. The results show the following: Antigenic variation between strains of PrV isolated from different geographic areas are readily detectable; antigenic differences between strains isolated from the same geographic area are less common. No antigenic drift in glycoprotein gII was observed. Glycoprotein gIII and, to some extent, also glycoprotein gI showed a high level of antigenic drift. The neutralizing activity of pooled convalescent sera of swine is not directed against glycoprotein gI. A large part of the neutralizing activity of pooled convalescent sera of swine is directed against glycoprotein gIII.
Animals↗
Differentiation of Newcastle disease virus strains by one-dimensional peptide mapping.
One-dimensional peptide mapping was used for the differentiation of Newcastle disease virus (NDV) strains. Virions were purified in one step, and digested with Staphylococcus aureus V8 protease or chymotrypsin without prior separation of their proteins. Peptides were separated by polyacrylamide gel electrophoresis and stained with Coomassie blue. This method proved to be a simple, economic and reproducible means of differentiating NDV strains.
Electrophoresis, Polyacrylamide Gel↗
Deletions in the genomes of pseudorabies virus vaccine strains and existence of four isomers of the genomes.
As part of a study designed to identify the genes responsible for the virulence of pseudorabies virus, we have mapped the genomes of two independently derived vaccine strains (Bartha and Norden) by restriction enzyme analysis. The structures of these genomes have been compared with that of the genome of a laboratory strain previously mapped, of restriction fragments which had been cloned. The genome of the Bartha strain was found to be very similar to that of other pseudorabies virus strains, except that a deletion of approximately 2.7 X 10(6) daltons was found in the unique short (US) region. This deletion was also observed in the genome of the Norden vaccine strain but was not observed in the genomes of any other pseudorabies virus strains that have been studied (more than 20). The genome of the Norden strain differs from that of other pseudorabies virus strains in several other respects as well. The most important difference is that in contrast to all other pseudorabies virus strains analyzed to date, which contain a type 2 herpesvirus DNA molecule (in which the US region only inverts itself relative to the unique long [UL] region), the genome of the Norden strain is a type 3 molecule in which both the US and the UL regions of the genome invert themselves, giving rise to four isomeric forms of the genome. The ability of the UL region to invert itself is probably related to the fact that a sequence normally present in all other pseudorabies virus strains at the end of the UL only is found also in inverted form at the junction of the UL and the internal inverted repeat in the Norden strain.
Chromosome Deletion↗
Genetic basis of the neurovirulence of pseudorabies virus.
Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (US) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessary for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-]) revealed the following. None of the recombinant plaque isolates that were either TK- or which had a deletion in the US was virulent. Not all recombinant plaque isolates which were both TK+ and had an intact US were virulent. These results indicate that both thymidine kinase activity and an intact US were necessary but not sufficient for the expression of virulence. Marker rescue experiments involving cotransfection of the Bartha strain DNA and a restriction fragment spanning the region of the genome that was missing from the Bartha strain resulted in the isolation of virions to which an intact US had been restored. These virions were not virulent but had an improved ability to replicate in the brains of chicks compared with that of the parental nonrescued Bartha strain. Our results show that genes in the US region, which are missing from the Bartha strain, were necessary for virulence but that this strain was also defective in other genes required for the expression of virulence. Thus, the virulence of pseudorabies virus, as measured by intracerebral inoculation into chicks, appears to be controlled multigenically.
Animals↗
Oligonucleotide fingerprinting of the RNA of different strains of infectious bronchitis virus.
11 distinct oligonucleotide fingerprints were obtained in studies of the RNA of 13 isolates of IBV. Different serotypes had distinct fingerprints, but so did varieties within a serotype; allowing a greater degree of strain differentiation than was previously possible. Some conclusions can be drawn from the fingerprints concerning theories of origin and spread of IBV.
Coronaviridae↗
Polypeptides of infectious bronchitis virus. I. Polypeptides of the virion.
Infectious bronchitis virus (IBV), strain Beaudette, grown in cultured cells contained five structural proteins with apparent mol. wt. of 170 000 (p170), 94 000 (gp94), 50 000 (pp50) 30 000 (gp30) and 26 000 (p26). Both gp94 and gp30 are glycopeptides since they were labelled with [3H]glucosamine. The only phosphorylated polypeptide was pp50, and both it and gp94 were occasionally resolved into two bands. Two other polypeptides with mol. wt. of 28 000 (p28) and 14 000 (p14) were sometimes associated with the virus. In egg-grown virus two additional proteins were found with mol. wt. of 110 000 (p110) and 75 000 (gp75). The cell protein, actin, was also found in highly purified IBV virions. Different serotypes of either tissue culture-grown or egg-grown virus showed one of the two distinct polypeptide patterns of IBV described by Nagy & Lomniczi (1979) and Collins & Alexander (1980 a, b). Strain Beaudette gave a pattern characteristic of the M type, while strain Connecticut gave a pattern characteristic of the C type. The polypeptides present in Connecticut virus were p170, gp98, pp50, gp28 and p26. Thus, the differences between the two pattern involve the mobility of both the large (gp94/gp98) and small (gp30/gp28) glycopeptides..
Actins↗
Oligonucleotide fingerprinting of ribonucleic acids of infectious bronchitis virus strains.
A total of 11 distinct oligonucleotide fingerprints were obtained in studies of the ribonucleic acids of 13 isolates of infectious bronchitis virus. Different serotypes had distinct fingerprints, but so did varieties within a serotype, allowing a greater degree of strain differentiation than was previously possible. Some conclusions can be drawn from the fingerprints concerning theories of origin and spread of infectious bronchitis virus.
Coronaviridae↗
Polypeptide patterns of infectious bronchitis virus serotypes fall into two categories. Brief report.
Molecular weights of six major polypeptides of infectious bronchitis virus (IBV) are: 1. 75,000; 2. 50,000; 3. 45,000; 4. 35,000; 5. 28,000 or 24,000, and 6. 22,000 dalton. According to the mobility of protein 5 the polypeptide patterns of IBV serotypes fall into two main categories.
Coronaviridae↗
Transcription of the influenza virus genome.
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Cell Line↗
Influenza virus infection of a cell-cycle mutant of Chinese hamster ovary cells.
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Animals↗
Biological properties of avian coronavirus RNA.
RNA with a sedimentation coefficient of 64S was isolated from infectious bronchitis virus, an avian coronavirus. The SNA contained a polyadenylic acid tract and was found to be infectious.
Coronaviridae↗
Genome of infectious bronchitis virus.
Techniques are described for the growth and rapid purification of the avian coronavirus infectious bronchitis virus (IBV). Purified IBV has a sedimentation coefficient of 320S and a buoyant density of 1.22 g/ml in sucrose-deuterium oxide equilibrium gradients. IBV RNA extracted by proteinase K in the presence of sodium dodecyl sulfate and further purified by phenol extraction and gradient centrifugation is single stranded and has a sedimentation coefficient of 64S, as determined by isokinetic gradient centrifugation. Analysis on sucrose gradients under both aqueous and denaturing conditions together with agarose gel electrophoresis in the presence of the chaotropic agent methylmercuric hydroxide gave a value of 8 X 10(6) for the moleclar weight of IBV RNA. This value was confirmed by RNase T1 fingerprinting, which also indicated that IBV RNA is haploid. No evidence was found of subunit structure in IBV RNA. From these results together with the recently reported observation that IBV RNA is infectious and contains a tract of polyadenylic acid (Lomniczi, J. Gen. Virol., in press), we conclude that the genome of the coronaviruses is a single continuous chain of about 23,000 mononucleotides that is of messenger polarity.
Base Sequence↗