Epstein-Barr virus genetics: talking about the BAC generation

Genetic mutant organisms pervade all areas of Biology. Early on, herpesviruses (HV) were found to be amenable to genetic analysis using homologous recombination techniques in eukaryotic cells. More recently, HV genomes cloned onto a bacterial artificial chromosome (BAC) have become available. HV BACs can be easily modified in E.coli and reintroduced in eukaryotic cells to produce infectious viruses. Mutants derived from HV BACs have been used both to understand the functions of all types of genetic elements present on the virus genome, but also to generate mutants with potentially medically relevant properties such as preventative vaccines. Here we retrace the development of the BAC technology applied to the Epstein-Barr virus (EBV) and review the strategies available for the construction of mutants. We expand on the appropriate controls required for proper use of the EBV BACs, and on the technical hurdles researchers face in working with these recombinants. We then discuss how further technological developments might successfully overcome these difficulties. Finally, we catalog the EBV BAC mutants that are currently available and illustrate their contributions to the field using a few representative examples

Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes

<p>Abstract</p> <p>Background</p> <p>Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-<it>ts</it>) attenuating (<it>att</it>) mutations primarily in the P/C gene, namely C^R84GHN^T553A(two point mutations used together as a set) and C^Δ170(a short deletion mutation), and two <it>ts att </it>mutations in the L gene, namely L^Y942A(a point mutation), and L^Δ1710–11(a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs).</p> <p>Results</p> <p>The rHPIV1 mutant bearing the novel L^Δ1710–11mutation was highly <it>ts </it>and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-C^R84G/Δ170HN^T553AL^Y942Aand rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11vaccine candidates were highly <it>ts</it>, with shut-off temperatures of 38°C and 35°C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-C^R84G/Δ170HN^T553AL^Y942Aprotected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host.</p> <p>Conclusion</p> <p>The rHPIV1-C^R84G/Δ170HN^T553AL^Y942Aand rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.</p

Human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium

Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology

Optimization of Naked DNA Delivery for Interferon Subtype Immunotherapy in Cytomegalovirus Infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200mg IFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMV gB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases

Efficacy and immunological mechanisms of type 1 interferon gene therapy in murine cytomegalovirus

This thesis presents a comparative analysis of the type I Interferon (IFN) subtypes and an evaluation of their potential as DNA vaccines in a model of murine cytomegalovirus (MCMV) infection and disease. MCMV induces acute and chronic phases of myocarditis, a heart disease characterised by an inflammatory cell infiltrate, in susceptible BALB/c mice. The type I IFNs comprise 14 IFN alpha genes in the human and >10 IFN alpha genes in the mouse with a single IFN beta gene in both species, however, the purpose of their multiplicity has remained unclear to date. An extensive panel of murine type I IFN subtype genes, including IFNA1, A2, A4, A5, A6, A9 and B, were sub-cloned into the mammalian expression vector pkCMVint (Vical, Inc.) for expression in BALB/c mice. These DNA constructs express biologically active IFN both in vitro and in vivo with systemic, low level expression persisting in the mouse for up to 4 weeks. The individual type I IFN subtypes differentially affect the immune response to MCMV challenge. IFNA6 proved most efficacious, reducing viral replication and inflammation in the acute and chronic phase of disease. Data suggests this occurs via induction of a Th1-like cytokine and antibody response. Furthermore, IFNA6 inoculation after the acute phase was shown to protect mice from the onset of chronic myocarditis. Characterisation of the immune cell response in IFN-treated, MCMV-infected mice demonstrated that type I IFN subtypes modulate the type of immune cells infiltrating the myocardium during myocarditis. Notably, reduced CD8+ and CD4+ T cells and B cell numbers in the heart was associated with reduced chronic myocarditis. Finally, coadministration of type I IFNs, in particular, IFNA6 and IFNB, synergistically improved immunotherapy against MCMV infection and myocarditis. The findings detailed here highlight the potential for the type I IFNs as DNA vaccines and most importantly, demonstrate that the type I IFNs have differential antiviral and immunomodulatory efficacies in the MCMV model of infection and myocarditis

The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection▿ †

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in children and the most common cause of viral croup. We performed a microarray-based analysis of gene expression kinetics to examine how wild-type (wt) HPIV1 infection altered gene expression in human respiratory epithelial cells and what role beta interferon played in this response. We similarly evaluated HPIV1-P(C−), a highly attenuated and apoptosis-inducing virus that does not express any of the four C proteins, and HPIV1-CF170S, a less attenuated mutant that contains a single point mutation in C and, like wt HPIV1, does not efficiently induce apoptosis, to examine the role of the C proteins in controlling host gene expression. We also used these data to investigate whether the phenotypic differences between the two C mutants could be explained at the transcriptional level. Mutation or deletion of the C proteins of HPIV1 permitted the activation of over 2,000 cellular genes that otherwise would be repressed by HPIV1 infection. Thus, the C proteins profoundly suppress the response of human respiratory cells to HPIV1 infection. Cellular pathways targeted by the HPIV1 C proteins were identified and their transcriptional control was analyzed using bioinformatics. Transcription factor binding sites for IRF and NF-κB were overrepresented in some of the C protein-targeted pathways, but other pathways were dominated by less-known factors, such as forkhead transcription factor FOXD1. Surprisingly, the host responses to the P(C−) and CF170S mutants were very similar, and only subtle differences in the expression kinetics of caspase 3 and TRAIL receptor 2 were observed. Thus, changes in host cell transcription did not reflect the striking phenotypic differences observed between these two viruses

Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes-0

<p><b>Copyright information:</b></p><p>Taken from "Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes"</p><p>http://www.virologyj.com/content/4/1/67</p><p>Virology Journal 2007;4():67-67.</p><p>Published online 2 Jul 2007</p><p>PMCID:PMC1939843.</p><p></p>edium was removed on days 0 (residual inoculum), 2 and 4–11 post-infection, frozen for later determination of virus titers, and replaced by fresh medium containing trypsin. The virus titers shown are the means of 3 replicate cultures