David B. Boyle

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.

Capripoxviruses: An Emerging Worldwide Threat to Sheep, Goats and Cattle

Capripoxviruses are the cause of sheeppox, goatpox and lumpy skin disease (LSD) of cattle. These diseases are of great economic significance to farmers in regions in which they are endemic and are a major constraint to international trade in livestock and their products. Although the distribution of capripoxviruses is considerably reduced from what it was even 50 years ago, they are now expanding their territory, with recent outbreaks of sheeppox or goatpox in Vietnam, Mongolia and Greece, and outbreaks of LSD in Ethiopia, Egypt and Israel. Increased legal and illegal trade in live animals provides the potential for further spread, with, for instance, the possibility of LSD becoming firmly established in Asia. This review briefly summarizes what is known about capripoxviruses, including their impact on livestock production, their geographic range, host-specificity, clinical disease, transmission and genomics, and considers current developments in diagnostic tests and vaccines. Capripoxviruses have the potential to become emerging disease threats because of global climate change and changes in patterns of trade in animals and animal products. They also could be used as economic bioterrorism agents.

A dominant selectable marker for the construction of recombinant poxviruses

Mycophenolic acid has been shown to be a potent inhibitor of vaccinia virus growth. By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome. When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes. Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.

A general method for the construction of recombinant vaccinia viruses expressing multiple foreign genes

Plasmid vectors with multiple cloning sites adjacent to a vaccinia virus (VV) promoter were constructed and used to insert a protein coding sequence and a dominant selectable marker into a non-essential region of the VV genome. Recombinant viruses, selected on the basis of expression of the herpes simplex virus (HSV) thymidine kinase gene (tk), were shown to express in infected cells the model gene product, murine major histocompatibility complex (MHC) antigen H-2Kd, by cell-surface binding of antibody and by MHC-restricted recognition by cytotoxic T lymphocytes. Double recombinant VVs with insertions at two sites (in the VV tk gene and in the VV HindIII-F region) were constructed and shown to express influenza A/PR/8/34 haemagglutinin and H-2Kd antigen in addition to the HSV tk gene. The plasmids described allow the construction of recombinant VV expressing two genes of interest under the control of the same VV promoter. Such recombinant VVs can be used to study the interaction of immunologically important antigens simultaneously expressed.

Detection of Respiratory Viruses and Subtype Identification of Influenza A Viruses by GreeneChipResp Oligonucleotide Microarray

ABSTRACT Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.

A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene

Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.

Infectious bursal disease virus structural protein VP 2 expressed by a fowlpox virus recombinant confers protection against disease in chickens

SummaryTwo fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP 2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP 4-VP 3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP 2 and VP 3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP 2 contained only the VP 2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of theTK gene. The expression level of VP 2 from FPV-VP 2 was approximately 5 times higher than from FPV-VP 2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP 2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP 2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP 2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.

The Roles of Influenza Virus Haemagglutinin and Nueleoprotein in Protection: Analysis Using Vaccinia Virus Recombinants

Vaccinia virus recombinants expressing haemagglutinin (HA) or nucleoprotein (NP) from influenza virus A/PR/8/4 were used to investigate protective immunity in mice, with two protocols. Protection was assessed by mortality and morbidity rates and by lung virus titres after infection intranasally with A/PR/8/34. In the first protocol, mice immunized with vaccinia‐HA recombinaant virus and infected intranasally with A/PR/8/34 were almost totally protected, but mice immunized with vaccinia‐NP virus were very poorly protected. In the second protocol, the recombinant viruses were used to stimulate in vitro T cells that are specific for HA and NP; both populations of T cells, when transferred to A/PR/8/34‐infected mice, afforded good protection. The results indicate that an immune response specific for just HA provided protection that was almsot indistinguishable from that provided by whole A/PR/8/34. On the other hand, immunization with vaccinia‐NP provided poor protective immunity, despite the fact that transferred NP‐specific T cells were very effective and vaccinia‐NP immunization has previously been shown to stimulate cytotoxic T cells. These results demonstrate that a single viral antigen, delivered by live vaccinia virus, can provide effective protection, but that immunization for cross‐protection against heterologous influenza virus remains elusive.

Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants

Recombinant vaccinia viruses enable studies of immune recognition of antigens expressed from single viral genes. We have constructed recombinants expressing the haemagglutinin (HA) and nucleoprotein (NP) genes of the influenza virus A/PR/8/34 (H1N1). These recombinant viruses together with a recombinant expressing the HA from influenza virus A/JAP/305/57 (H2N2) have been used to examine the cytotoxic T lymphocyte (CTL) response to these influenza virus antigens. Both antigens are recognised by murine CTL and recognition of HA is influenza virus subtype-specific, whereas recognition of NP is crossreactive. In limiting dilution studies approximately 10% of the influenza CTL response is HA-specific, while approximately 30% of the response is NP-specific. Despite the ability of NP to stimulate a significant CTL response, mice immunised with the NP-vaccinia recombinant are not as well protected from subsequent lethal challenge with influenza virus, as mice immunised with the HA vaccinia recombinant. These studies demonstrate that viral antigens expressed from vaccine recombinants can provide protective immunity and that the influenza-poxvirus recombinants can provide data on protective immunity generated by individual viral proteins.