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Columbia, MD, United States

George S.K.,Advanced BioNutrition | George S.K.,Wake forest University | Dhar A.K.,Advanced BioNutrition | Dhar A.K.,Viracine Therapeutics Corporation
In Vitro Cellular and Developmental Biology - Animal | Year: 2010

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace's insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO2. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO2 supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies. © 2010 The Society for In Vitro Biology. Source


Bowers R.M.,University of Colorado at Boulder | Bowers R.M.,Advanced BioNutrition | Dhar A.K.,Viracine Therapeutics Corporation | Dhar A.K.,Advanced BioNutrition
Molecular and Cellular Probes | Year: 2011

The effect of different templates on generating standard curves that are needed for the absolute quantification of an RNA virus by real-time reverse transcriptase-PCR (RT-PCR) was evaluated. We used infectious pancreatic necrosis virus (IPNV), a major viral pathogen of wild and cultured salmon, as a RNA virus example for the study. A dilution series of four different templates representing the IPNV protease gene (two in vitro transcribed RNAs of 100 bases and 500 bases in length, a plasmid DNA and a DNA oligo) were used as template to produce standard curves to quantify IPNV load in rainbow trout. The slope, the goodness of fit (r 2), and the efficiency (e) of PCR were statistically equivalent irrespective of the nature of template used in the PCR. Using a factorial ANOVA, no significant difference in IPNV copy number was observed using the four different standard curves for absolute quantification of IPNV in experimentally-challenged rainbow trout. However, when IPNV transcript abundance was less than 100 copies per reaction and when the template size was bigger than the amplicon size amplification was more variable. The data suggests that the size of the template used to generate standard curve should be very similar to the size of the amplicon. A synthetic DNA oligo template would be optimal for this purpose as it can be custom made and only requires the sequence information for its synthesis. However, if the standard curve is generated with template copy number in excess of 100 copies per reaction, the nature of the template has no effect on the standard curve, and therefore, the cheaper template would be the preferred choice of template over the other more expensive options. © 2011 Elsevier Ltd. Source


Dhar A.K.,Viracine Therapeutics Corporation | Dhar A.K.,Advanced BioNutrition | Kaizer K.N.,Viracine Therapeutics Corporation | Kaizer K.N.,Origene Technologies, Inc. | And 4 more authors.
Virus Genes | Year: 2011

In silico analysis of three Penaeus stylirostris densovirus (PstDNV) promoters, designated P2, P11, and P61, revealed sequence motifs including the TATA box, downstream promoter element (DPE), GC- and A-rich regions, inverted repeat, activation sequence-1 like (ASL) box, and a conserved guanosine (G) at ?24. To delineate the regulatory role of these motifs on promoter activity, deletion constructs were made in a promoter assay vector, pGL3 Basic, that contains a luciferase reporter gene. Luciferase assay showed that P2 had the highest promoter activity followed by P11 and P61 in Sf9 cells. The deletions of inverted repeat, DPE, and GC-rich regions in P2 had the highest negative impact on this promoter. Deletions of DPE, G at the ?24, and ASL box in P11 had the highest negative impact on this promoter activity. In P61, DPE and G at ?24 are the two key regulators of transcriptional activity. Identification of the key transcriptional regulators is important in understanding the PstDNV pathogenesis in shrimp. This information is also valuable in constructing shrimp viral promoter-based vectors for protein expression in insect cell culture system as well as in shrimp. © Springer Science+Business Media, LLC 2011. Source


Patent
Viracine Therapeutics Corporation | Date: 2011-04-08

The invention relates to the fields of viruses, vaccines and compounds and methods for expression. In particular, the invention includes methods and agents capable of producing quantities of a vaccine to a positive sense single stranded RNA ((+)sense RNA)virus.

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