Improvements in metagenomic virus detection by simple pretreatment methods

Highlights • Methods assessed for non-viral nucleic acid depletion from clinical samples.• SARS-CoV-2, human papillomavirus (HPV) and molluscum contagiosum virus (MCV) detection used as test of concept.• DNase I treatment followed by filtration dramatically improved virus detection efficiency.• Presenting a metagenomic workflow with Nanopore sequencing for prompt RNA and DNA virus detection.• The Delta variant of SARS-CoV-2, HPV-genotype and a co-infection of HPV and MCV-1 were correctly identified with this workflow. 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.

Experimental Infection of Pigs with Recent European Porcine Epidemic Diarrhea Viruses.

Porcine epidemic diarrhea virus (PEDV), belonging to the genus Alphacoronavirus, can cause serious disease in pigs of all ages, especially in suckling pigs. Differences in virulence have been observed between various strains of this virus. In this study, four pigs were inoculated with PEDV from Germany (intestine/intestinal content collected from pigs in 2016) and four pigs with PEDV from Italy (intestine/intestinal material collected from pigs in 2016). The pigs were re-inoculated with the same virus on multiple occasions to create a more robust infection and enhance the antibody responses. The clinical signs and pathological changes observed were generally mild. Two distinct peaks of virus excretion were seen in the group of pigs inoculated with the PEDV from Germany, while only one strong peak was seen for the group of pigs that received the virus from Italy. Seroconversion was seen by days 18 and 10 post-inoculation with PEDV in all surviving pigs from the groups that received the inoculums from Germany and Italy, respectively. Attempts to infect pigs with a swine enteric coronavirus (SeCoV) from Slovakia were unsuccessful, and no signs of infection were observed in the inoculated animals.

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.

Infection, recovery and re-infection of farmed mink with SARS-CoV-2.

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene as well as the A22920T mutation, resulting in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2.

In vitro Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms.

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.

Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark.

Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera-i.e., Pedacovirus, Nyctacovirus, and Myotacovirus-that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates-including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus-present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

Preliminary report of an outbreak of SARS-CoV-2 in mink and mink farmers associated with community spread, Denmark, June to November 2020.

In June-November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV-2-PCR-positive and approximately 27% (95% confidence interval (CI): 25-30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.

SARS-CoV-2 in Danish Mink Farms: Course of the Epidemic and a Descriptive Analysis of the Outbreaks in 2020.

SARS-CoV-2 infection is the cause of COVID-19 in humans. In April 2020, SARS-CoV-2 infection in farmed mink (Neovision vision) occurred in the Netherlands. The first outbreaks in Denmark were detected in June 2020 in three farms. A steep increase in the number of infected farms occurred from September and onwards. Here, we describe prevalence data collected from 215 infected mink farms to characterize spread and impact of disease in infected farms. In one third of the farms, no clinical signs were observed. In farms with clinical signs, decreased feed intake, increased mortality and respiratory symptoms were most frequently observed, during a limited time period (median of 11 days). In 65% and 69% of farms, virus and sero-conversion, respectively, were detected in 100% of sampled animals at the first sampling. SARS-CoV-2 was detected, at low levels, in air samples collected close to the mink, on mink fur, on flies, on the foot of a seagull, and in gutter water, but not in feed. Some dogs and cats from infected farms tested positive for the virus. Chickens, rabbits, and horses sampled on a few farms, and wildlife sampled in the vicinity of the infected farms did not test positive for SARS-CoV-2. Thus, mink are highly susceptible to infection by SARS-CoV-2, but routes of transmission between farms, other than by direct human contact, are unclear.

SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.

Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink (Neovison vison) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.

Animal Models for COVID-19: More to the Picture Than ACE2, Rodents, Ferrets, and Non-human Primates. A Case for Porcine Respiratory Coronavirus and the Obese Ossabaw Pig.

The ongoing COVID-19 pandemic caused by infection with SARS-CoV-2 has created an urgent need for animal models to enable study of basic infection and disease mechanisms and for development of vaccines, therapeutics, and diagnostics. Most research on animal models for COVID-19 has been directed toward rodents, transgenic rodents, and non-human primates. The primary focus has been on the angiotensin-converting enzyme 2 (ACE2), which is a host cell receptor for SARS-CoV-2. Among investigated species, irrespective of ACE2 spike protein binding, only mild (or no) disease has occurred following infection with SARS-CoV-2, suggesting that ACE2 may be necessary for infection but is not sufficient to determine the outcome of infection. The common trait of all species investigated as COVID models is their healthy status prior to virus challenge. In contrast, the vast majority of severe COVID-19 cases occur in people with chronic comorbidities such as diabetes, obesity, and/or cardiovascular disease. Healthy pigs express ACE2 protein that binds the viral spike protein but they are not susceptible to infection with SARS-CoV-2. However, certain pig breeds, such as the Ossabaw pig, can reproducibly be made obese and show most aspects of the metabolic syndrome, thus resembling the more than 80% of the critically ill COVID-19 patients admitted to hospitals. We urge considering infection with porcine respiratory coronavirus of metabolic syndrome pigs, such as the obese Ossabaw pig, as a highly relevant animal model of severe COVID-19.