Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells.

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

In Vivo Chemical Genomics with Random Cyclized Peptides.

We have developed and applied a novel strategy that can best be described as in vivo chemical genomics, a concept where populations of any transformable organism may be screened for consequences of novel RNAs or peptides. We created a library of ~800,000 random DNA sequences biased only by third-position nucleotide substitutions that suppress the frequency of termination codons. The sequences may be shuttled to any plant, microbial, or animal expression vector with recombination cloning. We then generated large populations of Arabidopsis thaliana plants, each expressing a randomized DNA sequence, presumably giving rise to synthetic RNA species and/or the peptides they encode. These novel molecules are produced within the context of the cell and have been shown to affect plant biology with a relatively high frequency, as evidenced by diverse phenotypes. This chapter provides the protocols necessary to construct the libraries and isolate plants expressing randomized DNA sequences.

A synthetic peptide encoded by a random DNA sequence inhibits discrete red light responses.

We have identified a synthetic peptide that interrupts discrete aspects of seedling development under red light. Previous reports have demonstrated that plants transformed with random DNA sequences produce synthetic peptides that affect plant biology. In this report, one specific peptide is characterized that inhibits discrete aspects of red light-mediated photomorphogenic development in Arabidopsis thaliana . Seedlings expressing the PEP6-32 peptide presented longer hypocotyls and diminished cotyledon expansion when grown under red light. Other red light-mediated seedling processes such as induction of Lhcb (cab) transcripts or loss of vertical growth remained unaffected. Long-term responses to red light in PEP6-32 expressing plants, such as repression of flowering time, did not show defects in red light signaling or integration. A synthesized peptide applied exogenously induced the long-hypocotyl phenotype under red light in non-transformed seedlings. The results indicate that the PEP6-32 peptide causes discrete cell expansion abnormalities during early seedling development in red light that mimic weak phyB alleles, yet only in some aspects of seedling photomorphogenesis. The findings demonstrate that new chemistries derived from random peptide expression can modulate specific facets of plant growth and development.

Characterization of new transposable element sub-families from white clover (Trifolium repens) using PCR amplification.

Transposable elements (TEs) dominate the landscapes of most plant and animal genomes. Once considered junk DNA and genetic parasites, these interspersed, repetitive DNA elements are now known to play major roles in both genetic and epigenetic processes that sponsor genome variation and regulate gene expression. Knowledge of TE consensus sequences from elements in species whose genomes have not been sequenced is limited, and the individual TEs that are encountered in clones or short-reads rarely represent potentially canonical, let alone, functional representatives. In this study, we queried the Repbase database with eight BAC clones from white clover (Trifolium repens), identified a large number of candidate TEs, and used polymerase chain reaction and Sanger sequencing to create consensus sequences for three new TE families. The results show that TE family consensus sequences can be obtained experimentally in species for which just a single, full-length member of a TE family has been sequenced.