Etti Harms

Correlation between RNA fragments of fowl plague virus and their corresponding gene functions

Abstract Fowl plague virus (FPV) RNA consists of eight fragments which, except for the two fragments with the highest molecular weight, can be clearly separated by polyacrylamide gel electrophoresis in the presence of 8 M urea. The base sequence homology between individual fragments of FPV RNA and the complementary RNA (cRNA) of various influenza A prototype strains has been established. Recombinants have been isolated by double infection of chick embryo cells with ts mutants of FPV and influenza A strains, which do not form plaques on this host. By hybridization of the cRNA of these recombinants with the 32P-labeled fragments of FPV RNA six of the eight RNA fragments can be correlated with gene functions of FPV.

Correlation of pathogenicity and gene constellation of an influenza a virus (fowl plague) I. Exchange of a single gene

Abstract Recombinants of fowl plague virus (FPV) with other influenza A prototype strains of human and animal origin in which only a single gene (RNA segment) is not derived from FPV were tested for their pathogenicity in chickens. Most of these recombinants had a lowered pathogenicity, while some were completely apathogenic. The apathogenic recombinants induced high titers of antibodies against the surface components of FPV in the infected chickens which were protected against a challenge infection with wild type FPV. Loss of pathogenicity depended on the gene which was exchanged as well as on the influenza A strain of which the foreign gene was derived, but no specific gene constellation could be detected for apathogenic recombinants. There is no clear correlation between growth rate and pathogenicity, indicating that other factors such as organ tropism might also play an important role in pathogenicity of influenza viruses.

A possible partial heterozygote of an influenza a virus

Abstract The gene constellation of recombinants between temperature-sensitive ( ts ) mutants of fowl plaque virus (FPV) and other prototype influenza A strains were analyzed by reciprocal molecular hybridization. Of 40 recombinants tested, only one (19/N) was found which carried RNA segments 3 (polymerase 2 gene) and 6 (neuraminidase gene) of FPV as well as those of virus N. Neuraminidase activity of FPV was fully expressed in cells infected with 19/N, whereas the enzyme activity of virus N was not detected. Virus N neuraminidase, however, was still synthesized as an antigen. During further plaque passages, this isolate finally segregated into stable recombinants carrying segment 3 either from the one or the other parent and segment 6 of FPV. Plaque isolates carrying segment 6 of both parents lost the gene which codes for the virus N neuraminidase in a stepwise manner during further passages through embryonated eggs. Using a specific antiserum against FPV neuraminidase, isolates were obtained which carried only the defective virus N neuraminidase gene. These isolates formed normal sized plaques and induced extremely low enzyme activities, although neuraminidase as an antigen was still produced. The data so far available make it unlikely that the original isolate consisted of a mixture of different recombinants and indicate that it represents an individual partial heterozygote.

Biochemical studies on influenza viruses I. Comparative analysis of equine 2 virus and virus N genes and gene products

Abstract Ribonucleic acid (RNA) from equine 2 and virus N was analyzed by polyacrylamidegel electrophoresis and separate RNA bands isolated. Base sequence homologies with various influenza prototype strains were established and gene functions assigned to particular RNA segments. Recombinant strains obtained by double infection of chick embryo cells with the fowl plague virus (FPV) mutant ts 113, temperature-sensitive in neuraminidase, and with equine 2 virus or virus N, respectively, carry the neuraminidase gene of the rescue virus and were characterized biochemically. Evidence is presented that equine 2-specific neuraminidase consists of subunits of different molecular weights.

Correlation between base sequence homology of RNA segment 4 and antigenicity of the hemagglutinin of influenza viruses

Abstract Base sequence homology between PR8 segment 4, the gene coding for the hemagglutinin, and the cRNAs of other influenza A prototype strains was determined. One hundred percent base sequence homology was found between PR8 (HO) and FMl (Hl), and 80% homology between PR8 and swine influenza virus (HSW1), although there is no cross-inhibition of hemagglutination. This is in clear contrast to other antigenically unrelated strains, which exhibit a base sequence homology between 20 and 40% with regard to segment 4. Fowl plague virus (FPV) and the Dutch strain, and equine 2 virus and the Hong Kong strain, which have antigenically related hemagglutinins, exhibit a base sequence homology of 100 and 80%, respectively. The available data suggest that swine, PR8, and FMl comprise one “subtype” genetically and are not derived by recombination, but differ by a number of point mutations.

Biochemical studies on influenza viruses. II. Assignment of gene functions to RNA segments 5, 7, and 8 of fowl plague virus and virus N.

Abstract Two recombinant strains carrying all genes from fowl plague virus (FPV), with the exception of RNA segments 5 and 8 which are derived from virus N, have been characterized by comparative RNA and protein analysis. From tryptic peptide mapping it was ascertained that segment 5 codes for the nucleoprotein, segment 7 for the matrix protein, and segment 8 for the nonstructural protein. In connection with previously published results (Scholtissek et al., 1976; Rohde et al., 1977), these data complete the genetic maps for FPV and virus N.

Genetic Relationship between an Influenza A and a B Virus

The base sequence homology between all eight 32P-labelled RNA segment of fowl plague virus (FPV) and the complementary RNA (cRNA) of an influenza B virus (B-mass), and between segment 8 of virus N and the cRNA of the same influenza B strain has been determined. All segments of FPV and segment 8 of virus N show a significant base sequence homology, ranging from 18 to 50% suggesting that influenza A and B viruses have a common ancestor. The conserved regions in segments 4,6 and 8 of the influenza A strains are identical to corresponding regions of the influenza B virus tested.

Characterization of virus-like particles produced by an influenza A virus

SummaryThe influenza strain 413 1,1 segregated as a stable recombinant during passage of the isolate 19/N which was obtained after double infection of chick embryo fibroblasts by virus N and the fowl plague virus (FPV) mutantsts 19. Its gene constellation was determined by molecular hybridization. Upon infection of chick embryo cells by this recombinant strain, two particle populations of high (H) and low (L) buoyant densities were produced. By biological and biochemical parameters, the H-population (δ=1.22 g/cm3) cannot be distinguished from standard infectious influenza virus. In contrast, the noninfectious L-particles (δ=1.14 g/cm3) lack all virus-specific glycoproteins (HA, NA) as well as the matrix protein M and are visualized by electron microscopy as spikeless particles. Significant changes in the quantitative composition of the phospholipid bilayer are evident as compared to the H-particles. In addition to the previously characterized eight genes both populations contain a variety of smaller RNA fragments which hybridize with complementary RNA and presumably represent degradation products of full-length genes.

Effects of dexamethasone on the morphogenesis of two mutants of Rous sarcoma virus.

Japanese quail cells transformed by the replication-defective, Bryan high-titer strain of Rous sarcoma virus, BH RSV(-)Q, clone 3, revealed intracytoplasmic A-type particles after dexamethasone treatment. In the absence of dexamethasone, or when superinfected with a helper virus in the presence or absence of dexamethasone, no such particles were observed. Chick embryo cells (CEC) infected with a temperature-sensitive sarcoma virus mutant, LA 334, coordinately defective for transformation and viral replication, showed abnormal accumulation of viral core substances and aberrant budding at the non-permissive temperature (41 degrees). CEC infected with LA 334 and treated with dexamethasone at 41 degrees resulted in more extensive accumulation of abnormal budding without increased release of viral particles. Dexamethasone, however, did not lead to abnormal morphogenesis of virus under permissive conditions. The selective effects of dexamethasone on the morphogenesis of these two mutants of avian sarcoma virus are discussed.

Minor nucleic acids in influenza virus.

A nucleic acid fraction consisting of RNA and DNA sequences with an apparent mol. wt. of 1.4 to 1.5 x 10(6) is present in minor amounts in purified influenza virus. The RNA is virus-specific and in the case of fowl plague virus (FPV) contains sequences of genes 2 and 7 which code for one of the proteins constituting the polymerase complex and for the matrix protein respectively.