Comparison of CRISPR-Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS.

The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR-Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR-Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR-Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.

Virus matrix interference on assessment of virucidal activity of high-touch surfaces designed to prevent hospital-acquired infections.

OBJECTIVES/PURPOSE: High-touch surfaces are a critical reservoir in the spread of nosocomial infections. Although disinfection and infection control protocols are well developed, they lack the ability to passively reduce the pathogenic load of high-touch surfaces. Copper and its alloys have been suggested as a surface that exhibit continuous biocidal effects. Antimicrobial studies on these surfaces are prevalent, while virucidal studies are not as well explored. The goal of this study was to first determine the virucidal activity of a copper-nickel-zinc alloy and to then examine the effect of soiling and virus preparation on virucidal activity. METHODS: A baculovirus vector was used as an easily quantifiable model of an infectious enveloped animal cell virus. Droplets containing virus were deposited on surfaces and allowed to stay wet using humidity control or were dried onto the surface. Virus was then recovered from the surface and assayed for infectivity. To examine how the composition of the droplet affected the survival of the virus, 3 different soiling conditions were tested. The first two were recommended by the United States Environmental Protection Agency and the third consisted of cell debris resulting from virus amplification. RESULTS: A copper-nickel-zinc alloy was shown to have strong virucidal effects for an enveloped virus. Copper, nickel, and zinc ions were all shown to leach from the alloy surface and are the likely cause of virucidal activity by this surface. Virucidal activity was achieved under moderate soiling but lost under high soiling generated by routine virus amplification procedures. The surface was able to repeatably inactivate dried virus droplets under moderate soiling conditions, but unable to do so for virus droplets kept wet using high humidity. CONCLUSION: Ion leaching was associated with virucidal activity in both wet and dried virus conditions. Soiling protected the virus by quenching metal ions, and not by inhibiting leaching. The composition of the solution containing virus plays a critical role in evaluating the virucidal activity of surfaces and surface coatings.

A critical evaluation of current protocols for self-sterilizing surfaces designed to reduce viral nosocomial infections.

BACKGROUND: Biocidal high-touch surfaces contact surfaces can be used to help reduce healthcare-acquired infections (HAIs). While the bactericidal protocols are well developed, there remains high variability in the methods used to evaluate the virucidal properties of such surfaces. This paper seeks to identify the most commonly used methods and critically evaluate the strengths of each method by comparing tests from standard testing organisations and related bactericidal protocols. METHODS: Three databases and grey literature were queried using a key-word search for relevant articles. Articles were selected if they met the criteria of virucidal properties of self-sterilizing surfaces designed to prevent HAIs. Of the resulting 177 articles, 38 met the inclusion criteria. RESULTS: The resulting papers varied greatly in their testing methods and recommendations. Further, no standard test adequately meets the needs for specifically testing virucidal properties of self-sterilizing surfaces. CONCLUSIONS: Studies have shown that temperature and humidity can affect the performance of virucidal touch-surfaces, but no standard protocols were found to test these factors.

Dissociation energies and potential energy functions for the ground X (1)Σ(+) and "avoided-crossing" A (1)Σ(+) states of NaH.

A direct-potential-fit analysis of all accessible data for the A (1)Σ(+) - X (1)Σ(+) system of NaH and NaD is used to determine analytic potential energy functions incorporating the correct theoretically predicted long-range behaviour. These potentials represent all of the data (on average) within the experimental uncertainties and yield an improved estimate for the ground-state NaH well depth of 𝔇e = 15797.4 (±4.3) cm(-1), which is ∼20 cm(-1) smaller than the best previous estimate. The present analysis also yields the first empirical determination of centrifugal (non-adiabatic) and potential-energy (adiabatic) Born-Oppenheimer breakdown correction functions for this system, with the latter showing that the A-state electronic isotope shift is -1.1(±0.6) cm(-1) going from NaH to NaD.