Rapid efficient production of baculovirus expression vectors.

Recombinant baculoviruses have been used to produce foreign proteins and have the potential to be safe, efficacious insecticides. Isolation of recombinant virus is usually by plaque phenotype. Typical recombination rates are less than 1%, thus requiring time consuming inspection of hundreds of individual plaques. We describe a method of generating recombinants which requires less time than current protocols and frequently produces recombinants at rates exceeding 30%. This protocol employs liposome-mediated transfection, reduced post-transfection incubation times, linearized parental virus which produces occlusion positive plaques in clones of the parental genotype, and colorimetric detection of recombinants. This protocol allows the initial, and frequently the final, isolation of recombinants in 7 days.     

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus

Summary.  The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. Accepted November 20, 1996 Received October 10, 1996     

Pathogenicity and vaccine efficacy of a thymidine kinase gene deleted infectious laryngotracheitis virus expressing the green fluorescent protein gene

Summary.  The deletion of the thymidine kinase (TK) gene of herpesviruses causes a reduction in their virulence. However, the effects of the TK gene in infectious laryngotracheitis virus (ILTV) have not been clearly elucidated. In the present study, we constructed a TK gene-deleted recombinant ILTV expressing the green fluorescent protein (GFP) gene as a marker. The GFP gene was inserted in place of the TK gene in both virulent and low virulence strains of ILTV. The GFP gene in the recombinants was expressed in chicken kidney cells, LMH cells and in the chorioallantoic membrane of embryonated chicken eggs. The recombinants produced cytopathic effects in chicken kidney cells and LMH cells and formed pocks in the chorioallantoic membrane of embryonated chicken eggs. The growth rate of the recombinant in chicken kidney cells was similar to that of wild type viruses. The recombinants showed a reduction of virulence compared to that of parental viruses and induced protection against virulent ILTV in specific pathogen free chickens. The recombinant expressing GFP gene may be a candidate for a genetically engineered vaccine and provide a means to study growth kinetics and mechanism of latent infection and reactivation of ILTV. In this study, we confirmed that the TK gene is directly related to virulence of ILTV. This is the first report to show the evidence that the TK gene is a major gene related to virulence of ILTV. Received May 1, 2001; accepted November 14, 2001     

A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.     

Construction of recombinant fowlpox viruses as vectors for poultry vaccines

Plasmid vectors have been constructed which allow the construction of infectious fowlpox virus (FPV) recombinants expressing foreign genes. The foreign genes were inserted within the thymidine kinase (TK) gene of FPV contained in these vectors. To facilitate the selection of recombinants the Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene was developed as a dominant selectable marker. This marker operates in a wide variety of cell types and obviates the need for TK⁻ cell lines for selection of TK⁻ recombinants when foreign genes have been inserted within the TK gene of FPV. The general approach adopted was to construct plasmid vectors in which the FPV TK was interrupted by the Ecogpt gene under the control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter. Selection of viruses expressing the Ecogpt gene simultaneously selects for recombinants carrying both the Ecogpt gene and the gene of interest. Using this approach a series of plasmid vectors was constructed in which the FPV TK gene was interrupted by the Ecogpt gene under the control of the P7.5 vaccinia virus promoter in tandem with the A/PR/8/34 haemagglutinin gene under the control of the PL11 vaccinia virus promoter. A recombinant FPV constructed using these plasmids had the expected genome arrangement, expressed influenza haemagglutinin, and induced haemagglutination-inhibiting antibodies when inoculated into chickens. These techniques should allow the construction of a variety of recombinant FPVs expressing poultry vaccine antigens. Such recombinants should be a very cost-effective means of delivering vaccines to poultry.     

Biosynthesis and interaction of glycoproteins E and I of canine herpesvirus.

In cells infected with canine herpesvirus (CHV), the mature form of glycoprotein E (gE) had a molecular weight of 94 kDa, and that of glycoprotein I (gI) had a broad range of molecular weights of 55-62 kDa. gE and gI formed a complex like gE and gI of other alphaherpesviruses. When cells were infected with the gI minus mutant of CHV (gI/Z), the mature form of the 94 kDa gE was not formed, but a 76 kDa gE polypeptide was found. Similarly, no mature gI was formed in cells infected with the gE minus mutant of CHV (gE/Z), but a 40 kDa gI polypeptide was formed. When cells were coinfected with gE/Z and gI/Z, the molecular masses of gE and gI were increased from 76 to 94 kDa and from 40 to 55-62 kDa, respectively. We constructed vaccinia virus recombinants which expressed CHV gE or CHV gI. Only when cells were coinfected with both the vaccinia recombinant which expressed gE and the vaccinia recombinant which expressed gI, gE and gI were processed into their mature forms. Our results suggest that the presence of both gE and gI is necessary for efficient processing of the precursors of gE and gI to their mature forms.     

In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Delta5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.     

Enhanced isolation of low frequency herpes simplex virus recombinants using green-fluorescent protein and FACS

The generation of recombinant herpes simplex virus to study the effect of engineered mutations on viral biology relies on the isolation of recombinants from a mixed population of viruses following a marker transfer procedure. Currently, the E. coli lacZ or green-fluorescent protein (GFP) genes are most frequently used as markers for isolation and isolation of recombinants relies on visual screening of plaques. Alternatively, novel restriction site changes can be inserted into a gene followed by screening of individual plaques for the novel change. These methods are inefficient when the frequency of recombinants in the pool of viruses is low. Using GFP as a selection marker, a FACS procedure that results in a substantial enrichment of the frequency of recombinants is described. Cells were infected at a multiplicity of infection (MOI) of 1.0 in the presence of acyclovir and at 10h post-infection, either the GFP+ or GFP- cells were sorted by FACS, and the sorted cells were plated on fresh cells. After three rounds of selection, the frequency of GFP+ recombinants rose from 0.1 to 3-4%. A mutant virus with a GFP insertion in the US1 gene (alpha22 protein) was generated and then used to isolate a virus with a mutation, Y116C, in the alpha22 protein.     

Rana grylio virus as a vector for foreign gene expression in fish cells

In the present study, Rana grylio virus (RGV, an iridovirus) thymidine kinase (TK) gene and viral envelope protein 53R gene were chosen as targets for foreign gene insertion. ΔTK-RGV and Δ53R-RGV, two recombinant RGV, expressing enhanced green fluorescence protein (EGFP) were constructed and analyzed in Epithelioma papulosum cyprinid (EPC) cells. The EGFP gene which fused to the virus major capsid protein (MCP) promoter p50 was inserted into TK and 53R gene loci of RGV, respectively. Cells infected with these two recombinant viruses not only displayed plaques, but also emitted strong green fluorescence under fluorescence microscope, providing a simple method for selection and purification of recombinant viruses. ΔTK-RGV was purified by seven successive rounds of plaque isolation and could be stably propagated in EPC cells. All of the plaques produced by the purified recombinant virus emitted green fluorescence. However, Δ53R-RGV was hard to be purified even through twenty rounds of plaque isolation. The purified recombinant virus ΔTK-RGV was verified by PCR analysis and Western blotting. These results showed EGFP was expressed in ΔTK-RGV infected cells. Furthermore, one-step growth curves and electron microscopy revealed that infection with recombinant ΔTK-RGV and wild-type RGV are similar. Therefore, RGV was demonstrated could be as a viral vector for foreign gene expression in fish cells.     

Use of PCR and immunofluorescence to detect bovine herpesvirus 1 recombinants

Homologous recombination occurs frequently between strains of the same alphaherpesvirus species. Studies of this phenomenon require techniques that can differentiate parental strains from putative recombinant progeny viruses. Usually, progeny viruses generated by co-infection of two distinguishable parental strains are first cloned by selection of a single plaque and then characterised by PCR. An assay designed to investigate recombination between two bovine herpesvirus 1 (BHV-1) strains lacking either the glycoprotein gC or gE ORF is described. A PCR assay was developed in which a single step co-amplifies both BHV-1 glycoprotein-encoding sequences. Because the usual procedure for virus isolation, viral plaque picking, can lead to polyclonal virus preparations, a PCR protocol alone does not differentiate between samples containing recombinant viruses (gC+/gE+) and those containing a mixture of both single deleted parental strains (gC-/gE+ and gC+/gE-), and false positives resulting from recombination could occur. To reduce this possibility, double-label immunofluorescence staining of isolated plaques was developed, which coupled with PCR, allows straightforward discrimination between parental strains and progeny recombinant viruses. This assay will be useful for further studies of recombination, especially those evaluating the potential emergence of recombinants between BHV-1 marker vaccine and wildtype strains.     

Improved method for the generation and selection of homogeneous lumpy skin disease virus (SA-Neethling) recombinants

Lumpy skin disease virus (LSDV) is being developed as a vector for recombinant vaccines against diseases of veterinary importance. A strategy for generating viral thymidine kinase (TK) gene-disrupted recombinants which are stable and homogeneous using the South African Neethling vaccine strain of LSDV as vector has been developed. To assist with the selection process, the Escherichia coli beta-galactosidase (lacZ) visual marker gene was incorporated into the constructs. However, the use of lacZ has certain limitations. An improved strategy was then devised substituting lacZ with the enhanced green fluorescent protein (EGFP) under control of the vaccinia virus (VV) P11K late promoter. The EGFP marker was found to enhance the selection process, and with the inclusion of additional sonication and filtration steps the number of passages required to select recombinants to homogeneity has been reduced. In support of the improved method for generation and selection of recombinants described, three different LSDV recombinants expressing the glycoprotein genes of bovine ephemeral fever virus, Rift Valley fever virus and rabies virus were prepared and characterised.     

Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gene therapy of amyotrophic lateral sclerosis

Two-year experiments were performed to evaluate the neurotrophic effect of hypoxia-inducible factors (vascular endothelial growth factor and angiogenin) expressed in recombinant human adenoviruses in amyotrophic lateral sclerosis. Randomized placebo-controlled trial demonstrated safety and good tolerability of the recombinant antiviral drugs. The life span of patients under conditions of hypoxia increased after treatment with the test drug, which was probably related to improved resistance of motoneurons. The presence of virus-neutralizing antibodies decreases the effectiveness of adenoviral vectors, which necessitates differential approach to the selection of patients and continuous monitoring of gene therapy. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 4, pp. 467–470, April, 2008     

Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses

We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector. The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine. Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol. We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein.     

Attenuated marmoset herpesvirus isolated from recombinants of virulent marmoset herpesvirus and hybrid plasmids

Marmoset herpesvirus (MarHV) deletion mutants in the thymidine kinase (TK) gene were isolated after infection of OMK cells with TK+ MarHV DNA and the hybrid plasmid, pMAR401, which lacks a 2.6-kb KpnI-M fragment in the coding region of the MarHV TK gene. After plaque purification in TK- HeLa(BU25) cells, the DNA's from five recombinant araT-resistant MarHV clones were analyzed with restriction nucleases to verify that the 2.6-kb KpnI-M fragment (and a 0.9-kb BglII-Q fragment) were deleted from the viral DNA's. Molecular hybridization experiments using 32P-labeled pMAR4 probes and viral DNA fragments also showed that the recombinant viral DNA's lacked the KpnI-M and BglII-Q fragments, that BamHI-I of parental virus was shortened, and that three new HindIII fragments replaced the parental virus HindIII-G fragment. The recombinants did not induce TK activity in LM (TK-) cells. To study the relative virulence of the recombinants, 3-week-old Swiss mice were injected intracerebrally (Ic) or subcutaneously (Sc) in the sacro-lumbar region with either parental or recombinant viruses. The LD50 for the parental virus was 3 p.f.u. (Ic) and 7,600 p.f.u. (Sc). The recombinant viruses were significantly less virulent than TK+ MarHV after Ic inoculation (LD50 of 62,000 and 32,000 p.f.u., respectively, for viruses 5D-6B and 5D-4B) and gave no fatalities after Sc inoculation. Mice surviving TK- MarHV infections were protected from challenge with TK+ parental MarHV. Recombinant TK- MarHV's may be useful as vectors for the expression of foreign genes in animal cells and as the starting material for the design of vaccines.     

Biologically selected recombinants between Feline Leukemia Virus (FeLV) subgroup a and an endogenous FeLV element

In efforts to elucidate the proximal leukemogens that might be produced during a feline leukemia virus (FeLV) infection of cats, homologous recombinations between molecularly cloned exogenous and endogenous FeLV proviruses of known sequences were examined in cell cultures in vitro. A plasmid containing an infectious member of the most commonly occurring FeLV subgroup (FeLV subgroup A or FeLV-A) was coexpressed with noninfectious constructs containing the envelope (env) gene of an endogenously inherited FeLV-like feline genomic element in transfected feline fibroblasts. The viruses generated were selected for their ability to propagate in human cells which are resistant to infection by the parental ecotropic FeLV-A or the noninfectious endogenous constructs. An analysis of the recombinants thus derived identified a limited number of sites in the env gene which were preferentially utilized in the generation of recombinant FeLVs under the selection conditions used. These sites were clustered in the surface glycoprotein (SU) moiety of the env gene, and it appeared that most, but not all, of the SU gene product of FeLV-A, beginning from the N-terminus, can be replaced by sequences from an endogenous element, still allowing the virus to be biologically viable. In fact, these substitutions in the env gene expanded infectivity of the parental FeLV-A from ecotropic to polytropic cell tropism. Additionally, substitutions in the SU region yielded many recombinants in which a primary neutralizing pentapeptide epitope of FeLV-A was altered because of its variance in the endogenous element. In several of the recombinants, this sequence was also found to be frequently mutated. Consistent with the changes identified in this antibody-binding domain, the recombinant viruses were only weakly inhibited by a monoclonal antibody directed against this epitope, while FeLV-A was highly sensitive to neutralization.     

Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene.

A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.     

Expression of cauliflower mosaic virus gene I using a baculovirus vector based upon the p10 gene and a novel selection method

A new baculovirus expression vector based upon the p10 gene of Autographa californica nuclear polyhedrosis virus (AcNPV) and a novel system for the screening of p10 recombinants have been developed. The insertion of a cassette containing the lacZ gene under the control of a heat-shock promoter of Drosophila melanogaster downstream from the cloning site in p10 transfer vectors allows the convenient identification of putative recombinants by virtue of their expression of beta-galactosidase. Using this p10 transfer vector an AcNPV recombinant was engineered with a cDNA copy of gene I of cauliflower mosaic virus (CaMV) in place of the p10 coding sequence. This p10 recombinant expressed CaMV gene I at levels equivalent to those of p10 and polyhedrin, and was shown to be as effective in producing this protein as recombinants exploiting the polyhedrin promoter. CaMV gene I protein formed large numbers of hollow fiber-like structures in the cytoplasm of infected cells. Because the polyhedrin gene remains intact, these p10 expression vectors may be exploited for the expression of heterologous proteins in insects infected per os and for the enhancement of baculovirus pathogenicity for insect control.