Optimal Multiplicity Of Infection Research Articles

Stable lentiviral gene expression driven by constitutive promoters in mouse pluripotent stem cells

Stable lentiviral gene expression driven by constitutive promoters in mouse pluripotent stem cells

Rapid baculovirus titration assay based on viable cell side scatter (SSC)

The baculovirus expression system is one of the most powerful tools for the production of recombinant proteins on both laboratory and industrial scales. Multiplicity of infection (MOI) is the crucial parameter for efficient protein expression. To obtain an optimal MOI, it is important to determine titer of virus stock before protein production. Herein, we established a label-free, simple and rapid method for virus titration based on viable cell side scatter (SSC). Generally, the SSC of cells infected with a series of virus dilutions was measured by a flow cytometer at 48h post-infection, and the probability of infected cells at a given dilution was estimated. For each well with the infection probabilities between 0.20 and 0.80, the range of dilutions was chosen, and virus titer was determined with a statistical method. Log-scale comparison of the results between the SSC based method and a standard plaque assay showed a good correlation (R2=0.9853), suggesting the fine accuracy of this proposed method.

PHYSICO CHEMICAL CHARACTERIZATION OF A T5-LIKE SALMONELLA PHAGE ΦSP-3

Antimicrobial resistance is a problem faced by humanity for quite some time now. Search for an intelligent alterative to antibiotics led the scientific community to join hands with an age old foe of pathogenic bacteria - bacteriophages. The present study elaborates the potential of a previously isolated phage with biocontrol capability, to survive and adsorb in various physicochemical environments . ΦSP-3, a Salmonella specific lytic phage capable of infecting host in nutrient deprived states with an added advantage of high host specificity and absence of virulence genes as previously reported was chosen for the study. Salmonella Enteritidis was used as host. Basic growth parameters like time taken for phage adsorption, optimal multiplicity of infection (MOI) and one step growth curve, were determined, followed by physicochemical characterization. Various parameters studied include temperature, pH, salinity and presence of sugars and CaCl2. 25 minutes of exposure time was required for 100% adsorption and optimal MOI was calculated as one. The latent period and the rise period was 30 minutes each with a subsequent burst size of 60 phages per bacterium. ΦSP-3 could survive upto 70ºC. 1M NaCl was optimal for phage viability while 0.25M favored maximal adsorption. The optimum pH for ΦSP-3 viability and adsorption was 8. Among the sugars,Arabinose was most influential in inactivating ΦSP-3. 100% adsorption was achieved at both 37ºC and at 40ºC. 10mM of CaCl2 to be optimum for ΦSP-3. Thus the present study substantiates the candidature of ΦSP-3 as a sturdy biocontrol agent capable of thriving well in diverse physicochemical environments.

Isolation and characterization of a virulent bacteriophage SPW specific for Staphylococcus aureus isolated from bovine mastitis of lactating dairy cattle.

Mastitis in dairy cattle continues to be an economically important disease. However, control is complicated by a high prevalence of resistance to antibiotics. Phage therapy, therefore, is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a highly virulent phage SPW from wastewater of dairy farm, which possesses a strong lytic capability against mastitis-associated Staphylococcus aureus, the most important pathogen in bovine clinical and subclinical mastitis. The phage SPW produced large, round and clear plaques on bacterial culture plates. TEM showed phage SPW has an icosahedral head 62.5 nm in diameter and long tail of 106 nm, head and tail were held together by a connector of 18 ± 1.5 nm long and can be classified as a member of the Myoviridae family. Restriction analysis indicated that phage SPW was a dsDNA virus with an approximate genome size of 65-69 kb. One-step growth kinetics showed a short latency period of about 10-15 min and a rise period of 50 min and a relatively small burst size was 44 ± 3 phages particles/infected cell. Moreover, adsorption rates were not influenced by calcium ions and phage SPW was relatively stable in a wide range of temperature and pH values, and resistant to chloroform and isopropanol. The optimal multiplicity of infection (MOI) was 0.01. When phage SPW was used to infect five other clinically isolated pathogenic isolates, it showed relatively wide spectrum host range. Phage SPW was capable of eliciting efficient lysis of S. aureus, revealing it potentially as an effective approach to prophylaxis or treatment of S. aureus-associated mastitis in dairy cows.

T4-Like Phage Bp7, a Potential Antimicrobial Agent for Controlling Drug-Resistant Escherichia coli in Chickens

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens.

Isolation and partial characterization of ΦSP‐1, a Salmonella specific lytic phage from intestinal content of broiler chicken

Salmonella enterica subsp. enterica serovar Enteritidis is a major causative agent of gastroenteritis with contaminated eggs and chicken meat being the major source of infection. Phages are seriously being considered as a safe and cheaper alternative to antibiotics. The intestinal content of chicken was used as source for isolating phages. Phage designated as ΦSP-1 was selected for the study. Transmission electron microscopy (TEM) of phage ΦSP-1 revealed that it belonged to family Podoviridae. The optimal multiplicity of infection (MOI) was 5 phages/cell. Latent and rise period were calculated to be 30 and 55 minutes respectively, while burst size was 44 phages/bacterial cell. The genome size of ΦSP-1 was estimated to be 86 kb from pulsed-field gel electrophoresis analysis (PFGE). The effect of different physical and chemical parameters like temperature, pH, salinity and CaCl₂ were analyzed to optimize the conditions for large scale production of phages and to check the viability of ΦSP-1 under different physiochemical conditions. A temperature of 40 °C, pH 8 and 0.25 M NaCl were found to be optimum for phage adsorption and it was able to survive up to a temperature of 50 °C for 3 min. Capability to survive under hostile environmental conditions, absence of virulence genes in genome and genus specificity suggest suitability of ΦSP-1 to be used as a biocontrol agent.

Response surface methodology to determine optimal measles-specific cytokine responses in human peripheral blood mononuclear cells

Limitations of assay variability, labor costs, and availability of cells can affect the conduct of large population-based studies. The ability to determine optimal conditions for laboratory assessment of immune outcomes, including measurement of cytokines, can reduce the number of peripheral blood mononuclear cells (PBMCs) needed, reduce the labor costs involved, and the variability in secreted cytokine response by pooling cytokines from the same cell culture supernatant. Previously, we used response surface methodology to predict optimal conditions for vaccinia virus-stimulated cytokine responses in recipients of smallpox vaccine. Here, we apply the same approach for a measles vaccine study.PBMCs were collected from vaccinated subjects, and seven cytokines (IFN-γ, IL-2, TNF-α, IL-10, IFN-α, IFN-λ1, and IL-6) involved in measles virus-specific cytokine immune responses were examined. PBMCs were stimulated with differing multiplicity of infection (MOI) and days in culture (incubation time). Response surface methodology was used to select the optimal MOI and incubation time for each secreted cytokine.Our results demonstrate that each cytokine's optimal conditions (MOI and incubation time) differ for each virus (measles vs. vaccinia) and each cytokine's optimal conditions for each virus can be predicted using response surface methodology. These conditions allow for cytokines with overlapping optimal conditions to be pooled from the same supernatant in culture to reduce the number of PBMCs used, the costs involved, and assay variability. Therefore, response surface methodology is an effective technique that can be used to optimize antigen-specific secreted cytokines prior to population-based studies.

Efficient Production of Porcine Circovirus Type 2 Capsid Protein using Baculovirus

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus associated with Postweaning multisystemic wasting syndrome (PMWS), which is considered to be an important infectious swine viral disease. PCV2 capsid protein encoded by ORF2 is a structural protein and expected as the high immunogenicity protein. In this study, we generated recombinant baculovirus containing ORF2 of PCV2 and analyzed the optimal conditions for the production of capsid protein in insect cell. Production and status of recombinant capsid protein in insect cell were confirmed by SDS-PAGE and Western blot analysis using His tag antibody and anti-PCV2 serum. The yield of recombinant capsid protein was high like as shown visible on SDS-PAGE. Optimal multiplicity of infection (MOI) and infection time of recombinant virus were determined as 5 MOI and 4 days, respectively. ORF2 is known to have N-linked glycosylation site, but we couldn't detect the glycosylation of recombinant protein in insect cells.

KEY PARAMETERS OF MEASLES VIRUS PRODUCTION FOR ONCOLYTIC VIROTHERAPY

Attenuated measles virus has revealed selective tum or cell killing and is currently tested in clinical trials for the therapy of cancer patients. The amount of i nfectious particles per dose needed for oncolytic therapy can be more than a million times higher compared to vaccination. This requires highly effectiv e production processes which guarantee the measles vi rus quantities needed for its use in cancer therapy . Referring to measles virus production itself, sever al factors are influencing process parameters and subsequent virus yields. These factors are medium o ptimization, feeding of nutrients, an optimal multiplicity of infection, localization of produced virus particles, temperature sensitivity and time of harvest. This review summarizes the available data concerning measles virus production in cell culture and factors influencing the virus yield.

Construction of recombinant lentivirus carrying mouse estrogen receptor α and identification in infected neurons

Objective To construct a recombinant lentivirus carrying C57BL/6 mouse estrogen receptor α(Erα) and to infect mouse neurons,so as to pave a way for further studying the relationship of Erα with some nervous system diseases.Methods Erα gene was inserted into the main virus vector LV-GFP-flag to construct recombinant lentiviral vector LV-Erα-flag,which was confirmed by agarose gel electrophoresis(AGE) and DNA sequencing.Recombinant lentivirus V-Erα-flag was produced by 293T cells following the co-transfection of LV-Erα-flag with the packaging plasmids pHelper 1.0 and pHelper 2.0,and the virus titer was examined.The neurons of C57BL/6 mouse were cultured using a serum-free culture medium,and then control lentivirus V-GFP-flag was used to infect the neurons.The infection efficiency and apoptosis rate were examined by flow cytometry to obtain optimal multiplicity of infection(MOI).GFP expression was detected daily under an inverted fluorescent microscope.After that,V-Erα-flag with the optimal MOI was used to infect neurons;the expression of Erα mRNA and protein in the neurons was detected by quantitative real-time PCR and Western blotting analysis.Results AGE and DNA sequencing confirmed that LV-Erα-flag was successfully constructed,and packaged into 293T cells with a titer of 2×10~8 TU/ml.Control lentivirus V-GFP-flag could infect neurons,with the infection efficiency being(89.8±4.03)% and the cell apoptosis rate being only(3.6±0.29)% when MOI=5.Neurons could survive in the culture for at least 8 weeks,during which the GFP was persistently expressed,indicating the lentivirus could efficiently and stably infect the neurons.The expression of Erα mRNA and protein was greatly increased in neurons infected with V-Erα-flag(MOI=5).Conclusion The recombinant lentivirus carrying Erα has been constructed successfully,which can infect neurons and lead to increase the expression of Erα mRNA and protein.

Bone Marrow-Derived Mensenchymal Stem Cells Modified by Mouse Interferon-γ Gene Reduce Fibrosis and Improve Function In a Mouse Model of Liver Fibrosis.

AbstractAbstract 3768 Objective:To investigate the effect of bone marrow-derived mensenchymal stem cells modified by mouse interferon-γ gene on experimental liver fibrosis in mice. Methods:(1) Mesenchymal stem cells (MSCs) derived from BM in BALB/c mice were cultured and identified by the flow cytometry. (2) The Ad-mIFN-γ, a recombinant adenoviral vector encoding mouse IFN-γ gene obtained by PCR from the vector pORF5-mIFN-γ, was created with the AdEasy Adenoviral Vector System. (3) MSCs were transfected with Ad-mIFN-γ. The transfection efficiency was detected by the flow cytometry at 24 hours after infection to determine the best multiplicity of infection (MOI). (4) The liver fibrosis of BALB/c mice were induced with concanavalin A (Con A) challenge weekly for six times. Then MSCs modified by the recombinant adenovirus encoding mouse IFN-γ gene were infused to BALB/c mice induced with Con A at the 24 hours after the first dose. The behavioral changes of mice were monitored closely. One week after the last dose of Con A, all mice were sacrificed and the fibrosis indexes were assessed by several histopathology methods systemicly. Moreover, hepatic hydroxyproline and serum parameters for liver fibrosis and liver function were evaluated and serum mIFN-γ concentrations were measured, respectively. Results:(1) The flow cytometry confirmed that the cells we obtained were MSCs. (2) The target gene mIFN-γ amplified by PCR from the vector pORF5-mIFN-γ, was identified by sequencing, which proved that the mIFN-γ gene was consisted of 468 nucleotides and was completely the same as the sequence published on GenBank. The target gene mIFN-γ was cut out by double endonucleases and then connected to the shuttle vector pAdTrack-CMV. Then the newly constructed vector was linearized by Pme I following transformed to the E.coli. BJ5183, which has the backbone vector of pAdEasy-1. The correct recombinant pAd-mIFN-γ was selected by endonucleases and by Kanamycin resistance and was transfected into AD-293 cells to obtain the adenoviral vector Ad-mIFN-γ. The Ad-mIFN-γ can be propagated in AD-293 cell line, the titre of which was 3.2×109 pfu/ml. (3) MSCs modified by recombinant adenovirus encoding mIFN-γ (IFN-γ/GFP-MSCs) at different multiplicity of infection (MOI) were detected by the flow cytometry. The optimal MOI for Ad-mIFN-γ was 400 with the transfection efficiency of 80.4%, and the optimal MOI for Ad-control was 200 with the transfection efficiency of 72.3%. (4) The changes of liver tissue and serum parameters for liver fibrosis showed liver fibrosis were reducd more obviously in the group treated with IFN-γ/GFP-MSCs (p<0.05), and liver functions were also improved more obviously in this group (p<0.05). The histopathology study using HE staining, masson trichrome staining and silver staining demonstrated that the structure of liver became much better in mice treated with IFN-γ/GFP-MSCs, with liver fibrosis improved significantly. Conclusion:Bone marrow-derived mensenchymal stem cells modified by mouse interferon-γ can reduce liver fibrosis and improve liver function on immuo-mediated liver fibrosis in mice induced by Con A. Disclosures:No relevant conflicts of interest to declare.

Killing of anaerobic pathogens by predatory bacteria

Recently, the predation of Bdellovibrio bacteriovorus on a periodontal pathogen has been described. The current study explores the potential antimicrobial activity of a range of predatory bacteria against key periodontal pathogens. A number of representatives from the Bdellovibrio, Bacteriovorax and Peredibacter lineages (called 'BALOs') were tested for their activity towards a group of key periodontal pathogens and an optimal multiplicity of infection was established. As the oral cavity contains a wide variety of bacteria that are not preyed upon, it was investigated if they can have an effect on the predation efficiency of BALOs. It was concluded that a number of important variables involved in bacterial predation are found to be compatible with the composition of the oral microbiota. This finding makes the case for continued study of the potential for BALOs to combat periodontal pathogens.

TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

Development of a suspension serum-free helper-dependent adenovirus production system and assessment of co-infection conditions

Helper-dependent adenovirus (HDAd), deleted in all viral protein-coding sequences has been designed to reduce immune response and favor long-term expression of therapeutic genes in clinical programs. Its production requires co-infection of E1-complementing cells with helper adenovirus (HAd). Significant progresses have been made in the molecular design of HDAd, but large scale production remains a challenge. In this work, a scalable system for HDAd production is designed and evaluated focusing on the co-infection step. A human embryo kidney 293 (293) derived cell line, the 293SF/FLPe was generated to produce efficiently HDAd while restricting the packaging of HAd. This cell line was adapted to grow in suspension and in serum-free medium. Multiplicity of infection (MOI) of HDAd ranging from 0.1 to 50 was evaluated in presence of HAd at a MOI of 5. Optimal MOIs for HDAd amplification were found in the range of 5–10. HAd contamination was only 1%. These results were validated in a 3 L bioreactor under controlled operating conditions where a higher HDAd yield of 2.6 × 10 9 viral particles (VP)/mL or 3.5 × 10 8 infectious units (IU)/mL of HDAd was obtained.

A Protein Kinase Cε-Anti-apoptotic Kinase Signaling Complex Protects Human Vascular Endothelial Cells against Apoptosis through Induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

Rat Adenocarcinoma Cell Line Infected With an Adenovirus Carrying a Novel Herpes-Simplex Virus-Thymidine Kinase Suicide Gene Construct Dies by Apoptosis Upon Treatment with Ganciclovir

Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5' untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5' untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing. Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit. Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir. Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.

Characterization of a Bipartite Recombinant Adeno-Associated Viral Vector for Site-Specific Integration

Adeno-associated virus type 2 (AAV2) is the only virus known to integrate into a specific locus in the human genome. The locus, AAVS1, is on the q arm of chromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site-specific integration, that is, the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector that does insert the transgene into AAVS1. One component, rAAVSVAV2, contains the rep gene, driven by the simian virus 40 early promoter rather than the P5 promoter. Thus, the integration enhancer element (IEE) within P5, which greatly enhances site-specific integration, has been deleted. The other component, rAAVP5UF11, contains the P5 IEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS1. We have not detected any clones that have rep inserted anywhere. With the optimal multiplicity of infection and ratio of rAAVSVAV2 and rAAVP5UF11, the transgene integrated specifically at AAVS1 with high efficiency (>60%). Most importantly, the cloned cell lines with the AAVS1 site-specific integrated green fluorescent protein (GFP) were healthy and stably expressed GFP for 35 passages. An AAV vector that would integrate at a specific site with high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site-specific integration gene therapy could provide a novel approach for diseases that need long-term gene expression.

496. AAV-Mediated, Somatic Gene Transfer to RPE Cells in Order To Study the Pathogenesis of Macular Degeneration: Proteomic and Genomic Analysis

Top of pageAbstract The use of AAV-mediated gene therapy appears to be an exciting approach for the treatment of inherited retinal diseases. However, there is at present, minimal information regarding the effect on the genome and proteome of the target cell following rAAV infection. Additional studies are critically needed to determine which genes and proteins are differentially expressed as the result of rAAV infection and as a result of the inclusion of a transgene. Using ARPE-19, a retinal pigment epithelial (RPE) cell line and recombinant AAV2/5 viruses, AAV2/5.EGFP, AAV2/5.EGFP-RO (reverse orientation), AAV2/5.EFEMP1wt and EFEMP1mut, as well as uninfected cells, we have examined the proteomes by 2D differential in-gel electrophoresis (DIGE) and the genomes by RNA transcript profiling. EGFP encodes the enhanced green flourescent protein and infection of RPE cells with this virus allowed the determination of the optimal multiplicity of infection (MOI) which was 1 |[times]| 106. The AAV2/5.EFEMP1wt and AAV2/5 EFEMP1mut encode epidermal growth factor containing fibrillin-like extracellular matrix protein wild-type and mutant R345W, where an arginine codon is substituted for a tryptophan codon. In patients with Malattia Leventinese (ML), an autosomal dominant macular degenerative disease, the EFEMP1 protein has been shown to accumulate within RPE cells and between the RPE and drusen. In vitro studies have shown that the EFEMP1wt protein is secreted while the EFEMP1mut protein is misfolded, poorly secreted and retained within cells. These results suggest that a misfolding and aberrant accumulation of EFEMP protein may underlie drusen formation and other abnormalities in the retinas of patients with ML and AMD. Initial analysis of the DIGE data shows the presence of both downregulated and upregulated proteins. Analysis of the microarray data shows the downregulation of a number of cell cycle regulator, DNA replication, kinase, kinesin-like (motor), and survivin [baculoviral IAP (apoptosis inhibitor protein) repeat-containing 5 and survivin-beta] genes for rAAV infected cells compared with uninfected RPE cells. Interestingly, there appears to be very little or no change in immune response, inflammatory response or signal transduction genes. Additional proteomic studies using DIGE and SILAC (stable isotope labeling with amino acids in cell culture) as well as continuing microarray studies are in progress to provide further understanding of the effect on the proteome and genome of these important gene therapy tools for the treatment of inherited retinal diseases.

A rapid method for estimation of baculovirus titer based on viable cell size

Baculovirus protein expression system is a powerful tool for producing recombinant proteins. To optimize conditions for efficient recombinant protein expression, it is important to determine titer of virus stock for arriving at an optimal multiplicity of infection (MOI) that maximizes recombinant protein expression. Traditionally plaque assays have been used for titer determination. Other methods such as endpoint dilution, quantitative real-time polymerase chain reaction and flow cytometry have been developed to aid the determination of virus titers. However, most of these methods are time-consuming and labor intensive. In this regard, a simple and rapid method for determination of virus titers based on the cytopathic effects that lead to viable cell size increase following virus infection is presented in this paper. In this study, the Vi-CELL (Beckman Coulter) was used to measure cell-diameter over a range of virus dilutions, following infection. Applying statistical modeling techniques, the viable cell-diameter data was used to estimate the virus titer. The results indicated that the viable cell-diameter based titer estimation to be reliable and comparable to titers determined by the traditional plaque assay.

Receptors for Theiler's murine encephalomyelitis virus: characterization by using rabbit antiviral antiserum.

An immunological assay was developed to characterize the binding of Theiler's murine encephalomyelitis virus to BHK-21 cell receptors. After absorption of the virus and formaldehyde fixation, rabbit antibodies and Staphylococcus aureus protein A labeled with 125I formed a specific complex on the surfaces of the cells. The optimal multiplicity of infection in this system was 10 PFU per cell. The virus was internalized at 33 and 37 degrees C, but internalization did not take place at 25 or 4 degrees C. The binding was proportional to the number of cells and was significant within 30 s. Cell surface receptors were still active after fixation, and only intact viruses were bound, as demonstrated by the lack of binding of the purified, isolated virion proteins VP1, VP2, and VP3.