W. Rohde

On the origin of the human influenza virus subtypes H2N2 and H3N2

The RNA of the human influenza virus Singapore (H2N2) strain has been labeled in vivo by phosphorus-32 and separated by polyacrylamide gel electrophoresis into eight segments, which were correlated to the corresponding gene functions and/or proteins. The base sequence homology between the individual genes (segments) of the H2N2 virus and those of different influenza A strains has been determined by molecular hybridization. Segments 1, 5, 7, and 8 of the Singapore strain exhibit a base sequence homology of almost 100% as compared to the FM1 strain (HlNl), while the homology between the other segments is significantly lower (24–76%). For the Singapore and Hong Kong (H3N2) strains all segments except that coding for the hemagglutinin (HA, 24%) exhibit a homology close to 100%. The 32P-labeled segment 4 (HA-gene) of the avian influenza A strain duck Ukraine (Hav7Neg2) shows a homology of 92% to Hong Kong, while the homology of at least two other segments is significantly lower. These results are taken as an indication that the H2N2 subtype is derived from the HlN1 subtype by a recombination event retaining four H1N1 segments, while the other four segments are gained from another yet unknown strain. The H3N2 subtype is presumably derived from a H2N2 subtype, retaining seven segments of the H2N2 subtype, while the gene coding for the HA is obtained from the duck Ukraine or another highly related strain.

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.

Detection of complementary RNA intermediates of viroid replication by Northern blot hybridization

Molecular hybridization by the Northern blot technique in combination with 125I-labeled PSTV (+) RNA and 32P-labeled PSTV cDNA as probes has been applied to detect viroid-specific sequences in healthy and viroid(PSTV)-infected tomato plants. Conditions are described which allow differentiation of (+) and (-) viroid sequences on the basis of the different thermostabilities of the corresponding hybrid molecules. By this experimental approach, it is documented that no viroid-specific DNA sequences can be detected and that viroid replication proceeds via complementary RNA intermediates. Out of the seven (-) RNA species found, six are apparently larger than the circular viroid (+) RNA and one is about the same size as the linear (+) RNA molecule.

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.

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.

In vitro transcription of viroid RNA into full-length copies by RNA-dependent RNA polymerase from healthy tomato leaf tissue

RNA-dependent RNA polymerase from healthy tomato plant tissue accepts potato spindle tuber viroid (PSTV) RNA as a template for the in vitro synthesis of full-length RNA copies of the PSTV genome. Viroid transcription requires the presence of Mn2+ and/or Mg2+ ions and is not inhibited by concentrations of l0−5 M α-amanitin. This is the first report of a well-defined product synthesized in vitro by an RNA-dependent RNA polymerase from healthy plants.

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.

Viroid RNA is accepted as a template for in vitro transcription by DNA-dependent DNA polymerase I and RNA polymerase fromEscherichia coli

The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA polymerase, respectively, fromEscherichia coli . In vitro synthesis of complementary RNA produces distinct transcripts larger than unit length thus reflecting the in vivo mechanism of viroid replication. The influence of varying experimental conditions on the transcription process is studied; actinomycin D is found to drastically reduce complementary RNA synthesis from the PSTV RNA template by RNA polymerase.

On the origin of the gene coding for an influenza A virus nucleocapsid protein

SummaryThe gene coding for the nucleocapsid protein NP of the influenza A virus recombinant strain 413 1,1 was characterized biochemically by molecular hybridization and fingerprint analysis. The data presented suggest that this NP gene has evolved by intracistronic recombination between the NP genes of virus N and the fowl plague virus temperature-sensitive mutantts 19.