Virus Assay Methods: Accuracy and Validation

Darling, Allan J.; Boose, Jeri Anne; Spaltro, John

doi:10.1006/biol.1998.0134

Cited by 93 publications

(52 citation statements)

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“…Even though the cultured hyalinocytes and semigranulocytes were susceptible to PaV1, we could not develop a plaque assay for quantitative study of the virus, because lobster hemocytes do not undergo mitosis and no conXuent cell layer could be formed; i.e., the cells formed dispersed monolayers. However, a CPE assay using an estimate of the 50% tissue culture infectious dose (TCID 50 ) method provided an alternative to determine viral titer (see Darling et al, 1998). Such assays have been successfully applied to quantify the infectious titer of several other crustacean viruses including yellow head baculovirus (Assavalapsakul et al, 2003;Lu et al, 1995) and non-occluded baculo-like virus (Tapay et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Shields

2007

Journal of Invertebrate Pathology

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Section: Discussionmentioning

confidence: 99%

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Shields

2007

Journal of Invertebrate Pathology

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“…The resulting supernatant contained the extra-cellular virus was stored at 4°C. Virus titer was determined by end point dilution assay (EPDA) (Darling et al 1998).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk)

2010

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The Amsacta moorei entomopoxvirus (AMEV) genome has 279 open reading frames (ORFs) among which is the AMV197, composed of 900 nt and potentially encoding a protein of 299 amino acids. Sequence-derived amino acid analysis suggested it to be a serine/threonine protein kinase (PK) having conserved PK and serine/ threonine PK domains. For transcriptional analysis of the AMV197 pk gene, Ld652 cells were infected with AMEV and mRNA was isolated at different times thereafter. RT-PCR analysis indicated that the transcription of the AMV197 pk gene started at 4 h post infection (h p.i.) and continued to be expressed through 24 h p.i. Infection of Ld cells in the presence of Ara-C (inhibits DNA replication), followed by RT-PCR showed that AMV197 pk is transcribed as an early gene. Transcription was initiated at 54 nt upstream of the translation start site. The vaccinia virus early promoter element G was also found at the correct position (−21) in the AMV197 pk gene. Rapid amplification of the 3′ ends of the AMV197 pk transcript showed that there are two polyadenylation start points. They are located at 22 and 32 nucleotides downstream of translation stop site. Also, the translational stop site and poly (A) signal of AMV197 pk are overlapped. The termination signal TTTTTGT sequence of vaccinia virus early genes was found just upstream of the 3′ end of AMV197 pk gene. Conserved amino acid subdomains of the AMV197 PK were found by sequence comparisons with PK's from other organisms. Analysis of the protein sequence of AMV197 pk gene reveals close identity with PK genes of other organisms.

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“…Infectious particles (ip) in 50% tissue culture infective dose (TCID 50 ) were determined according to the statistical method of SpearmanÀKarber (Darling et al 1998).…”

Section: Methodsmentioning

confidence: 99%

Two Different Serum-free Media and Osmolality Effect Upon Human 293 Cell Growth and Adenovirus Production

Ferreira

Carrondo

et al. 2005

Biotechnol Lett

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Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

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Virus Assay Methods: Accuracy and Validation

Cited by 93 publications

References 0 publications

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk)

Two Different Serum-free Media and Osmolality Effect Upon Human 293 Cell Growth and Adenovirus Production

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