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A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the 'spiked' PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples. [ABSTRACT FROM AUTHOR]

A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5′ or 3′ UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV. [ABSTRACT FROM AUTHOR]