Development Of Gene Deleted Recombinant Pseudorabies Virus

Abstract

The glycoprotein E (gE) and thymidine kinase (TK) genes are virulence-associated genes of pseudorabies virus (PrV). The study conducted was to shut down the gE gene from an established local TK defective (TK¯) PrV strain (TK¯gE+PrV). The ultimate aim of the study was to develop a gene-deleted recombinant PrV with useful identification markers. A gE gene-deleted pseudorabies virus (TK¯gE¯PrV) was constructed by homologous recombinational techniques. The TK¯gE+PrV, regarded as the parental strain in the study, originated from a virulent local PrV isolate (TK+gE+PrV). Prior to the construction of the TK¯gE¯PrV, the gE of the parental strain was amplified, cloned and studied. Comparative sequence analysis showed that the gE sequence of TK¯gE+PrV was closely identical (98 %) to a Chinese Ea strain. The 10 nucleotide variations at nucleotide positions 237, 931, 1207, 1409, 1501, 1530, 1549, 1555, 1682 and 1842, led to six amino acids substitutions at amino acid residues 403 (A􀀙 P), 470 (V􀀙A), 501 (V􀀙I), 517 (P􀀙S), 519 (T􀀙A) and 561 (T􀀙N) in their open reading frames (ORFs) that code a 578 amino acid polypeptide. All 10 cystein clusters in the gE sequences of the PrV strains namely TK¯gE+ PrV, Ea strain and Rice strain were conserved. Despite the low overall level of amino acid sequence identity among the gE proteins (23 to 31%) of diverse animal species, the cystein clusters were relatively well conserved especially in the C-terminal of the protein. The 500 bp deletion introduced into TK¯gE+ PrV gE gene, was designed to remove three cystein residues and one potential N-glycosylation site at the C-domain of gE, while maintaining sufficient flanking regions within the gE gene to facilitate homologous recombination. The TK¯gE¯PrV constructed was identified by gene specific PCR assay, gE-PCR profiles and sequence analysis. Expression analysis by SDS-PAGE and immunoblots proved the absence of gE protein. The absence of gE-specific antibodies in the serum of TK¯gE¯PrV immunized murine models further substantiated the evidence. Besides, the protective nature of TK¯gE¯PrV resembled that of parental strain (TK¯gE+PrV). Based on the gE deletion site, TK¯gE¯PrV can be clearly differentiated from other PrV vaccine strains. Overall, the gE deletion was proven to be a functional genetic cum serologic marker. It is intriguing to know whether the virus is useful to deliver and express a foreign gene within the gE expression locus. Therefore, an E2 gene expression cassette, originally from classical swine fever (CSFV), was specially designed to be incorporated into the deleted gE gene as a foreign insert. It serves as a CSFV marker as well as for its immunogenic and protective properties against CSFV infection. An eukaryotic expression vector was constructed to express the CSFV E2 gene with specific functional domains. Following transfection of mammalian cells with the E2 encoded naked plasmid (pCDNA+E2), E2 protein was detected using immunoperoxidase staining, SDS-PAGE and immunoblot analyses. Before the gene was introduced into the gE gene of PrV, the ability of the expression plasmids to induce immune response in vivo was also evaluated in mice via gene gun and intramuscular injections. Both humoral and cell-mediated immunity were detected. Therefore, the CSFV E2 expression cassette developed was determined to be appropriate for a recombinant with TK¯gE¯ PrV. The recombinant PrV was successfully developed, primarily by genetically modifying the transfer plasmid. The pUC plasmid was manipulated and constructed to harbor the E2 expression cassette with flanking PrV gE nucleotide regions to facilitate homologous recombinant. The construct was transferred into TK¯gE¯ PrV genome by homologous crossovers DNA recombination. The expression of the E2 gene in a viral plaque indicated a successful integration of the gene in PrV genome. The formation of designated TK¯gE¯E2+PrV virus particles were verified by means of PCR and sequence analysis. Based on its characteristics, generally it is concluded that the gene deleted pseudorabies virus, TK¯gE¯PrV, is a good candidate for preparation of an attenuated vaccine as well as a viral vector.