Unbiased Virus Detection in a Danish Zoo Using a Portable Metagenomic Sequencing System.

Metagenomic next-generation sequencing (mNGS) is receiving increased attention for the detection of new viruses and infections occurring at the human-animal interface. The ability to actively transport and relocate this technology enables in situ virus identification, which could reduce response time and enhance disease management. In a previous study, we developed a straightforward mNGS procedure that greatly enhances the detection of RNA and DNA viruses in human clinical samples. In this study, we improved the mNGS protocol with transportable battery-driven equipment for the portable, non-targeted detection of RNA and DNA viruses in animals from a large zoological facility, to simulate a field setting for point-of-incidence virus detection. From the resulting metagenomic data, we detected 13 vertebrate viruses from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including avian leukosis virus in domestic chickens (Gallus gallus), enzootic nasal tumour virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA (CRESS DNA) viruses in several mammal species. More significantly, we demonstrate that the mNGS method is able to detect potentially lethal animal viruses, such as elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the newly described human-associated gemykibivirus 2, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for the first time.

Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

Improvements in metagenomic virus detection by simple pretreatment methods.

Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified; this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.

An alternative workflow for molecular detection of SARS-CoV-2 - escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020.

The World Health Organization has declared COVID-19 caused by the newly discovered SARS-CoV-2 a pandemic. Due to growing demand for reagents and/or kits to extract SARS-CoV-2 RNA for subsequent RT-qPCR diagnostics, there is a worldwide risk of shortages. With a detection sensitivity of 97.4% (95% CI: 86.2-99.9%), we describe a simple, fast, alternative workflow for molecular detection of SARS-CoV-2, where samples are simply heat-processed for 5 min at 98 °C before a commonly-used RT-qPCR procedure.