Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing.

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease's causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2.

Comparative analysis of cherry virus A genome sequences assembled from deep sequencing data.

Cherry virus A (CVA) is a ubiquitous graft-transmissible virus that mainly infects Prunus spp. Next-generation sequencing was applied to 39 tree fruit specimens infected with CVA, and 75 full and 16 partial-length CVA genome sequences were assembled. Phylogenetic analysis of these and 11 previously sequenced CVA genomes resulted in six major clusters with no observable relationship between the host and the assembled genome sequences.

Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics.

Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector.

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site.

Cloning and expression in E. coli of a functional Fab fragment obtained from single human lymphocyte against anthrax toxin.

Human lymphocytes derived from the blood of a donor immunized with anthrax vaccine were isolated and enriched for B-cells by Nycoprep density centrifugation. Individual anti-anthrax protective antigen (PA) B-cells were isolated by fluorescence activated cell sorting with fluorescence-labeled recombinant PA (rPA). The RNA from sorted single B-cells was extracted using plant total RNA as the carrier prior to purification by Nanoprep RNA isolation columns and then cDNA was prepared.

Detection of genetically modified coho salmon using polymerase chain reaction (PCR) amplification.

A PCR-based protocol for the identification of genetically modified salmon carrying a growth hormone transgene was developed. Several primer pairs were examined, and the primers that gave consistent results were selected to conduct routine testing. Comparison among several DNA extraction procedures, as well as different buffer compositions, led to the adoption of TriZol as the method of choice.