First Report of Fludioxonil Resistance in Botrytis cinerea, the Causal Agent of Gray Mold, from Strawberry Fields in Maryland and South Carolina.

Botrytis cinerea Pers. is the causal agent of gray mold and one of the most economically important plant-pathogenic fungi affecting strawberry (Fragaria × ananassa). Control of gray mold mainly depends on the use of site-specific fungicides, including the phenylpyrrole fludioxonil. This fungicide is currently registered in combination with cyprodinil in form of Switch 62.5WG (Syngenta Crop Protection, Greensboro, NC) for gray mold control of small fruits in the United States. In June 2013, strawberries affected with symptoms resembling gray mold were observed despite the application of Switch in one field located in Federalsburg, MD, and one located near Chesnee, SC. Ten single-spore isolates, each from a different fruit, were obtained from each location and confirmed to be B. cinerea using cultural and molecular tools as described previously (3). In vitro sensitivity to fludioxonil (Scholar SC, 20.4% [v/v] active ingredient, Syngenta Crop Protection, Greensboro, NC) was determined using a conidial germination assay as previously described (4). Eight of the 20 isolates (six from Maryland and two from South Carolina) were moderately resistant to fludioxonil, i.e., they grew on medium amended with 0.1 µg/ml fludixonil and showed residual growth at 10 µg/ml (4). The in vitro assay was repeated obtaining the same results. To assess in vivo sensitivity on fungicide-treated fruit, commercially grown strawberries were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of conidia suspension (106 spores/ml) of either two sensitive or four resistant isolates (two isolates from Maryland and two isolates from South Carolina). Each isolate/treatment combination consisted of 24 mature but still firm strawberry fruit with three 8-fruit replicates. The fruit were kept at 22°C and lesion diameters were measured after 4 days of inoculation. The sensitive isolates developed gray mold symptoms on nontreated (2.5 cm lesion diameter) but not on Scholar SC-treated fruit. The resistant isolates developed gray mold on both, the water-treated control (2.3 cm lesion diameter), and the fungicide-treated fruit (1.8 cm lesion diameter). The experiment was performed twice. To our knowledge this is the first report of fludioxonil resistance in B. cinerea from strawberry fields in Maryland and South Carolina. Resistance to fludioxonil is still rare in the United States and has only been reported in B. cinerea isolates from a Virginia strawberry field (1). The increase in occurrence of resistance to fludioxonil may be a result of increased use of Switch following reports of resistance to other chemical classes in this pathogen in southern strawberry fields (2). References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 96:1198, 2012. (3) D. Fernández-Ortuño et al. Plant Dis. 95:1482, 2011. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

Herpes simplex virus-enhanced cationic lipid/DNA-mediated transfection.

Liposome plasmid DNA complexes (lipoplexes) are often inefficient in mediating gene transfer and expression because of DNA degradation in lysosomal vesicles. Because herpes simplex virus (HSV) enters cells by fusion of the virus envelope with the plasma membranes, thereby overriding the endosomal pathway, HSV/lipoplex mixtures could be useful for improving gene transfer particularly when the mixture uses highly defective HSV particles that fail to express cytotoxic viral gene products. To evaluate this possibility, lipoplexes composed of cationic liposomes and lacZ reporter plasmids were compared for their ability to transduce cells in culture in the presence and absence of infectious HSV particles. The results showed that HSV increased the efficiency of cell transduction by approximately 4-100-fold compared with lipoplex vector alone, depending on the cell type targeted for gene delivery. The increased efficiency of transduction was virus dose dependent and required virus entry.

Involvement of an ATP-dependent peptide chaperone in cross-presentation after DNA immunization.

Immunization with plasmid DNA holds promise as a vaccination strategy perhaps useful in situations that currently lack vaccines, since the major means of immune induction may differ from more conventional approach. In the present study, we demonstrate that exposure of macrophages to plasmid DNA encoding viral proteins or OVA generates Ag-specific material that, when presented in vitro by dendritic cells to naive T cells, induces primary CTL response or elicits IL-2 production from an OVA peptide-specific T-T hybridoma. The immunogenic material released was proteinaceous in nature, free of apoptotic bodies, and had an apparent m.w. much larger than a 9-11-aa CTL-recognizable peptide. The macrophage-released factor(s) specifically required a hydrolyzable ATP substrate and was inhibited by procedures that removed or hydrolyzed ATP; in addition, anti-heat-shock protein 70 antiserum abrogated the activity to a large extent. These results indicate the possible involvement of a heat-shock protein 70-linked peptide chaperone in a cross-priming method of immune induction by DNA vaccination. Such a cross-priming process may represent a principal mechanism by which plasmid DNA delivered to cells such as myocytes effectively shuttle Ag to DC or other APC to achieve CTL induction in vivo.

Modulation of mucosal and systemic immunity by enteric administration of nonreplicating herpes simplex virus expressing cytokines.

In this report the ability of enteric immunization with recombinant replication deficient (ICP4-/-) HSV expressing IFN gamma to generate protection and modulate mucosal and systemic immunity was evaluated. ICP4-/-HSV, ICP4-/-HSV expressing IL4, live replicating, and uv HSV were used as controls. Following enteric administration of live HSV, a Th1 cytokine response was induced in the spleen, while both Th1 and notable Th2 cytokine production were detected at mucosal sites. Modulation of mucosal and systemic immune response was achieved when nonreplicating recombinant HSV viruses expressing cytokines were used. Compared to the control replication defective viruses, decreased frequency of Th2 cytokine producing cells in Peyer's patches was observed following enteric administration of nonreplicating HSV expressing IFN gamma. When IFN gamma expressing virus was given enterically, modulation was observed at the systemic level, measured by ELISPOT for cytokine producing cells, ELISA from the in vitro restimulated splenic cell cultures, and by the increase of the IgG2a/IgG1 ratio in the serum. This report provides evidence that replication defective viruses expressing cytokine genes in contrast to uv HSV, are immunogenic when administered enterically and can generate significant immunomodulatory effects at the mucosal and systemic levels.

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications.

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFN gamma). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate--early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of longterm transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

Enhancement of immune response to naked DNA vaccine by immunization with transfected dendritic cells.

Immunization with plasmid DNA encoding various proteins promises to be a valuable vaccine approach especially if its immunogenicity could be optimized. In this study we show that the intramuscular delivery in dendritic cells (DC) of naked plasmid DNA encoding two proteins of herpes simplex virus (HSV) leads to the induction of significantly enhanced levels of resistance to viral challenge. Whereas DC transfected in vitro with DNA induced enhanced immunity, similarly transfected macrophage (M phi) populations lacked immunogenicity even though plasmid expression occurred in vitro. The enhanced immunity induced by DC-delivered DNA appeared to be associated mainly with an increased Th1 CD4+ T cell response. Our results add evidence that DC are the essential antigen-presenting cell types involved in immune responses to intramuscularly administered DNA vaccines.

Rapid method for construction of recombinant HSV gene transfer vectors.

Herpes simplex virus type 1 (HSV-1) is a neurotrophic human pathogen that naturally persists in neurons in a latent state and carries a large number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications. In this report we describe a two-step method for insertion/deletion mutagenesis of HSV genes and the efficient insertion of transgenes into these locations in the viral genome. The first step is the insertion of a reporter gene (lacZ) cassette flanked by Pacl restriction enzyme sites not otherwise found in the viral genome, using standard marker transfer procedures to interrupt a portion of the target HSV gene. The second step is substitution of the reporter gene with other foreign cDNAs by digestion of the vector DNA with Pacl to remove the lacZ gene and subsequent repair of the vector genome by homologous recombination with a transgene expression plasmid. Potential recombinants identified by a 'clear plaque' phenotype after X-gal staining arose at high frequency (80-100%). Of these recombinants containing the transgene in place of the lacZ gene ranged from 19-65%. Insertion of the transgene expression construct into the viral genome eliminates the Pacl sites, allowing this method to be used repeatedly for the sequential deletion of multiple HSV genes while inserting multiple transgenes. This procedure was repeated in succession to produce a vector carrying two independent expression cassettes at distinct viral loci.

Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes.

Plasmid DNA encoding proteins represent a convenient novel approach to vaccination. We have investigated this "genetic immunization" approach as a means to protect against herpes simplex virus (HSV) infection using a mouse zosteriform model that mimics several aspects of reactivated HSV infection of humans. After i.m. immunization with plasmid DNA-encoding glycoprotein B (gB), (pc-gB), 80% of BALB/c mice were completely protected and lesions were delayed in the remaining animals. Upon pc-gB vaccination, the animals developed both gB- and HSV-specific IgG Ab response and the isotype examination revealed a predominance of IgG2a. These mice also have low levels (1/16) of HSV-neutralizing Abs. Immune splenocytes obtained from pc-gB-immunized mice, when restimulated in vitro with HSV resulted in production of type 1 cytokines. Evidence for CD(8+)-mediated cytotoxic T lymphocyte response was equivocal. Protection could be adoptively transferred to nude mice recipients by CD4+ T cells from pc-gB-immunized mice but not by CD8+ T cells. Our results demonstrate that genetic immunization is a potent means of inducing protection against HSV and that the mechanism of immunity responsible for clearing virus from cutaneous sites is principally by CD4+ T cells. It is likely that these cells are Th1 cells because type 1 cytokines were the major cytokines detected upon in vitro Ag stimulation.

Role of macrophages and dendritic cells in primary cytotoxic T lymphocyte responses.

The successful induction of class I restricted cytotoxic T lymphocytes (CTL) responses with soluble non-replicating antigens relies upon vehicles which deliver antigen in vivo appropriately to antigen presenting cells (APC), which for CTL may be dendritic cells (DC). In this study, we have followed the distribution of liposomes and their incorporated antigen and compared the efficacy of splenic macrophages (Mo) and DC at inducing primary CTL responses in vitro. Our results show that whereas liposomes locate predominantly in the splenic red pulp and marginal zone locations, some of their soluble antigen contents redistribute to the central white pulp, comprising mainly DC and T cells. Such antigen redistribution was most apparent following administration of pH-sensitive liposomes. In comparisons of the APC activity of Mo and DC taken at various times post-injection, DC were always superior to Mo. However, if Mo were depleted prior to antigen exposure, DC were ineffective APC for CTL induction. Furthermore, addition of supernatant from OVA-liposome treated Mo to naive DC-T cell cultures in vitro resulted in OVA-specific T cell responses. These studies indicate a role for Mo in enhancing the antigen presenting function of DC.

Induction of protective immunity against herpes simplex virus with DNA encoding the immediate early protein ICP 27.

Using a mouse zosteriform model that mimics human herpes simplex virus (HSV) infection in several aspects, the effectiveness of plasmid DNA encoding the immediate early protein ICP 27 was evaluated as a vaccine. Animals were immunized intramuscularly twice with DNA, then either challenged with virus or killed, and the nature of the immune response induced was measured. After intramuscular injection with plasmid DNA encoding ICP 27 (pc-ICP 27), solid protection was evident in 70-80% of mice and the lesions were delayed in the remaining animals. Immune splenocytes obtained from pc-ICP 27 immune mice showed HSV-specific lymphoproliferation, MHC-class I restricted cytotoxic T-lymphocyte (CTL) activity, and type 1 cytokine production. These animals also exhibited delayed-type hypersensitivity (DTH) reactions. Adoptive transfer studies conducted on syngeneic nude mice revealed that those recipients of immune CD4+ T cells, but not CD8+ T cells, were protected from subsequent HSV-1 (strain 17) challenge. Thus pc-ICP 27 DNA immunization protected the mice principally by CD4+ T cells and it is likely that these cells were Th-1 type because only type 1 cytokines were detectable after in vitro antigen stimulation. Our results indicate the potential value of DNA encoding nonstructural viral proteins as vaccines against HSV.

Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simplex virus proteins.

Vaccines which successfully protect against virus infections usually need to induce a broadly reactive immune response which includes the induction of cytotoxic T lymphocytes (CTL). In this study, we have used a convenient in vitro approach to investigate if plasmid DNAs encoding proteins of herpes simplex virus (HSV) are capable of inducing primary CD8+ CTL. Dendritic cells or macrophages were transfected with either plasmid DNA encoding glycoprotein B or DNA encoding the immediate-early protein ICP27. These antigen-presenting cells (APC) were then used to stimulate enriched populations of naive T cells in microcultures for 5 days in vitro. Antigen-specific CD8+ CTL which reacted both with specific protein-expressing targets and with syngeneic targets infected with HSV could be demonstrated. Dendritic cells, as APC, generated the maximal responses, but such cells needed to be transfected with DNA in the presence of a cationic lipid. However, macrophages could act as APC when they were exposed to purified DNA. HSV-primed splenocytes were also shown to generate specific CTL responses when they were stimulated with purified DNA encoding ICP27. The novel approach described in this paper promises to be extremely useful, since defining immunogenicity profiles and identifying epitopes on viral proteins should be easier and more convenient when working with DNA and investigating variables in vitro. This is particularly the case with complex viruses such as HSV, most of whose encoded proteins have yet to be isolated in sufficient quantity or purity to perform in vivo immunological studies.