Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.

Generation of a recombinant chimeric Newcastle disease virus vaccine that allows serological differentiation between vaccinated and infected animals.

Using a recently developed reverse genetics system, we have generated a recombinant Newcastle disease virus (NDV) vaccine in which the gene encoding the hemagglutinin-neuraminidase (HN) has been replaced by a hybrid HN gene consisting of the cytoplasmic domain, transmembrane region, and stalk region of HN of NDV, and the immunogenic globular domain of HN of avian paramyxovirus type 4 (APMV4). The objective was to generate a chimeric live vaccine that induces a protective immune response against NDV by eliciting neutralizing antibodies against the fusion (F) protein, but which can be differentiated from wild-type NDV on the basis of different antibodies elicited by their HN proteins. Pathogenicity tests in day-old chickens showed that the recombinant was non-virulent (intracerebral pathogenicity index [ICPI]=0.00). A vaccination-challenge experiment in 4-week-old specific pathogen free chickens demonstrated that the recombinant was completely safe and was able to protect chickens from challenge with a lethal dose of virulent NDV. By using a secreted form of HN produced in Pichia pastoris, a test was developed that allowed serological differentiation between animals vaccinated with the recombinant vaccine and animals infected with NDV. These results demonstrate that genetically modified marker vaccines can be generated from small RNA viruses that lack non-essential genes.

Genome replication of Newcastle disease virus: involvement of the rule-of-six.

We examined replication of Newcastle disease virus (NDV) by using minigenomes consisting of the 3' leader and 5' trailer regions of NDV flanking a reporter gene encoding secreted placental alkaline phosphatase (SEAP). Negative-sense minigenome RNA was generated from transfected plasmid DNA by means of in vivo transcription. Subsequent replication of minigenome RNA was determined either after infection with NDV helpervirus or after contransfection with helperplasmids that expressed the essential viral replication proteins NP, P, and L. In both systems, efficient replication of minigenome RNA was observed only if the genome size was a multiple of six nucleotides. Hence, in these systems, replication of NDV minigenome RNA's is strictly dependent on the rule-of-six. When the supernatant from helpervirus-infected, transfected cells was used to infect fresh monolayers, efficient transfer of SEAP activity by virus-like particles was observed only if the size of the minigenome RNA obeyed the rule-of-six. However, after several serial passages, we also observed efficient transfer of SEAP activity by virus-like particles derived from minigenome RNA's that did not obey the rule-of-six. Evidence was obtained which indicated that successful replication of these minigenomes was not due to a change in genome size.

Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence.

A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR downward arrowL) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR downward arrowF). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.

Molecular analysis of a flagellar core protein gene of Serpulina (Treponema) hyodysenteriae.

The flaB2 gene encoding a protein located in the core of the periplasmic flagella of Serpulina hyodysenteriae was cloned and sequenced. The FlaB2 protein consists of 285 amino acids and has a calculated molecular mass of 31.1 kDa. Southern blot analysis indicated that at least one, and possibly two genes related to flaB2 are present in the genome of S. hyodysenteriae. Comparison of the amino acid sequence of FlaB2 to sequences present in data banks showed significant similarity with the core flagellins of other spirochaetes, in particular with a FlaB2 protein from Treponema phagedenis.

Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein.

A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S. hyodysenteriae periplasmic flagella. A single immunoreactive plaque was chosen for further analysis. The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella. DNA sequence analysis showed that the gene encodes a protein of 320 amino acids. The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins. We designated the cloned S. hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA. The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm. Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein. Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified. Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.