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bioRxiv2020

Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives

The COVID-19 pandemic has necessitated a rapid multi-faceted response by the scientific community, bringing researchers, health officials and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

microbiology↗

bioRxiv2016

DNA from dust: comparative genomics of large DNA viruses in field surveillance samples

Mareks disease (MD) is a lymphoproliferative disease of chickens caused by airborne gallid herpesvirus type 2 (GaHV-2, aka MDV-1). Mature virions are formed in the feather follicle epithelium cells of infected chickens from which the virus is shed as fine particles of skin and feather debris, or poultry dust. Poultry dust is the major source of virus transmission between birds in agricultural settings. Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last 40 years, we do not yet understand the genetic basis of MDV-1 pathogenicity. Our present knowledge on genome-wide variation in the MDV-1 genome comes exclusively from laboratory-grown isolates. MDV-1 isolates tend to lose virulence with increasing passage number in vitro, raising concerns about their ability to accurately reflect virus in the field. The ability to rapidly and directly sequence field isolates of MDV-1 is critical to understanding the genetic basis of rising virulence in circulating wild strains. Here we present the first complete genomes of uncultured, field-isolated MDV-1. These five consensus genomes were derived directly from poultry dust or single chicken feather follicles without passage in cell culture. These sources represent the shed material that is transmitted to new hosts, vs. the virus produced by a point source in one animal. We developed a new procedure to extract and enrich viral DNA, while reducing host and environmental contamination. DNA was sequenced using Illumina MiSeq high-throughput approaches and processed through a recently described bioinformatics workflow for de novo assembly and curation of herpesvirus genomes. We comprehensively compared these genomes to one another and also to previously described MDV-1 genomes. The field-isolated genomes had remarkably high DNA identity when compared to one another, with few variant proteins between them. In an analysis of genetic distance, the five new field genomes grouped separately from all previously described genomes. Each consensus genome was also assessed to determine the level of polymorphisms within each sample, which revealed that MDV-1 exists in the wild as a polymorphic population. By tracking a new polymorphic locus in ICP4 over time, we found that MDV-1 genomes can evolve in short period of time. Together these approaches advance our ability to assess MDV-1 variation within and between hosts, over time, and during adaptation to changing conditions.

Microbiology↗