Gregory Duke

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells

BackgroundRecent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells.ResultsThe eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 106 to 107 plaque forming units per ml by 5 to 7 days post-transfection.ConclusionThis rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

BackgroundHuman cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks.ResultsA series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus.ConclusionThese data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

Cytomegalovirus vaccine strain towne-derived dense bodies induce broad cellular immune responses and neutralizing antibodies that prevent infection of fibroblasts and epithelial cells.

ABSTRACT Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.

Development of a high-throughput Alamar blue assay for the determination of influenza virus infectious dose, serum antivirus neutralization titer and virus ca/ts phenotype.

FluMist is an intranasal influenza live vaccine containing two Influenza A strains (currently H1N1 and H3N2) and one B strain (Yamagata or Victoria lineage). Characterization of the vaccine requires determination of the median tissue culture infectious dose (TCID(50)) titer, serum antivirus neutralization titer and vaccine cold adapted/temperature sensitive (ca/ts) phenotype. Visual cytopathic effect (CPE) readings are used widely in viral assays, but these are subjective and labor intensive. In response to the need for an efficient, inexpensive and high-throughput assay, a 96-well microplate assay was developed that uses Alamar blue dye staining as a replacement for CPE observation in the determination of influenza virus infectious dose, serum antivirus neutralization titer and virus ca/ts phenotype. Relative operating characteristic curves verified that there was a clear distinction between the fluorescence readings of the Alamar blue stained CPE positive and CPE negative wells. Virus titer was determined by use of both Alamar blue staining and CPE-based TCID(50) assays for wild-type and FluMist influenza vaccine strains as well as a plasmid-rescued influenza FluMist A strain containing a H5N1 derived hemmaglutinin and neuramidinase. Correlation of the two assays was measured by regression analysis and resulted in R(2) values of 0.814 (Influenza A), 0.983 (Influenza B) and 1.000 (H5N1), respectively. Serum microneutralization as well as virus ca/ts phenotype assays also showed a high concordance between readings based on CPE observation and Alamar blue staining. The Alamar blue dye assay is user friendly, environmentally safe and sensitive. Also, it is adaptable to automation, which could provide a high-throughput platform for analysis of pre-clinical and clinical samples.

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.