Dataset Information

Development of a method for effective amplification of human adenovirus 40.

ABSTRACT: Human adenovirus 40 (Ad40) is an interesting candidate for vector construction because of its tropism for the gastrointestinal tract. Although effective preparation of the vector is necessary for its in vivo application, amplification of Ad40 has been very difficult. Ad40 E1 deletion mutants were detected by PCR in the viral DNA from Ad40 Dugan amplified by Ad5 E1-expressing human embryonic kidney (293) cells and in Ad40 Dugan plaques observed with Ad5 E1-expressing human retinoblastic cells. For the purpose of generating a single wild-type Ad40 clone, the entire Ad40 DNA was cloned into a plasmid by homologous recombination. A pure Ad40 was successfully generated by plasmid transfection and subsequently amplified with Ad5 E4orf6-inducible 293 (2V6.11) cells. 2V6.11 is an apposite cell line for effective Ad40 amplification and for future vector construction because Ad40 genetic integrity was maintained with this Ad5 E1 and E4orf6 trans-complementing cell line.

SUBMITTER: Yamasaki S

PROVIDER: S-EPMC4569093 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a method for effective amplification of human adenovirus 40.

Yamasaki Satoshi S Miura Yoshiaki Y Brown Eric E Davydova Julia J Yamamoto Masato M

Archives of virology 20100521 7

Human adenovirus 40 (Ad40) is an interesting candidate for vector construction because of its tropism for the gastrointestinal tract. Although effective preparation of the vector is necessary for its in vivo application, amplification of Ad40 has been very difficult. Ad40 E1 deletion mutants were detected by PCR in the viral DNA from Ad40 Dugan amplified by Ad5 E1-expressing human embryonic kidney (293) cells and in Ad40 Dugan plaques observed with Ad5 E1-expressing human retinoblastic cells. Fo ...[more]

PMID: 20490608

Similar Datasets

Project description:UnlabelledThis paper examines the recovery of the enteric adenovirus human adenovirus 40 (HAdV 40) by cross-flow ultrafiltration and interprets recovery values in terms of physicochemical interactions of virions during sample concentration. Prior to ultrafiltration, membranes were either blocked by exposure to calf serum (CS) or coated with a polyelectrolyte multilayer (PEM). HAdV 40 is a hydrophobic virus with a point of zero charge between pH 4.0 and pH 4.3. In accordance with predictions from the extended Derjaguin-Landau-Verwey-Overbeek theory, the preelution recovery of HAdV (rpre) from deionized water was higher with PEM-coated membranes (rpre (PEM) = 74.8% ± 9.7%) than with CS-blocked membranes (rpre (CS) = 54.1% ± 6.2%). With either membrane type, the total virion recovery after elution (rpost) was high for both deionized water (rpost (PEM) = 99.5% ± 6.6% and rpost (CS) = 98.8% ± 7.7%) and tap water (rpost (PEM) = 89% ± 15% and rpost (CS) = 93.7% ± 6.9%). The nearly 100% recoveries suggest that the polyanion (sodium polyphosphate) and surfactant (Tween 80) in the eluent disrupt electrostatic and hydrophobic interactions between the virion and the membrane. Addition of EDTA to the eluent greatly improved the elution efficacy (rpost (CS) = 88.6% ± 4.3% and rpost (PEM) = 87.0% ± 6.9%) with surface water, even when the organic carbon concentration in the water was high (9.4 ± 0.1 mg/liter). EDTA likely disrupts cation bridging between virions and particles in the feed water matrix or the fouling layer on the membrane surface. For complex water matrices, the eluent composition is the most important factor for achieving high virion recovery.ImportanceHerein we present the results of a comprehensive physicochemical characterization of HAdV 40, an important human pathogen. The data on HAdV 40 surface properties enabled rigorous modeling to gain an understanding of the energetics of virion-virion and virion-filter interactions. Cross-flow filtration for concentration and recovery of HAdV 40 was evaluated, with postelution recoveries from ultrapure water (99%), tap water (∼91%), and high-carbon-content surface water (∼84%) being demonstrated. These results are significant because of the very low adenovirus recoveries that have been reported, to date, for other methods. The recovery data were interpreted in terms of specific interactions, and the eluent composition was designed accordingly to maximize HAdV 40 recovery.

Dataset Information

Development of a method for effective amplification of human adenovirus 40.

Publications

Development of a method for effective amplification of human adenovirus 40.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets