Scientific publications

Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2
Wylezich et al. 2021

The detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples. A virus panel based on the principle of biotinylated RNA baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2-positive samples. Libraries generated from complex samples were sequenced via generic HTS (without enrichment) and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted. The VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector, or research question. The risk of cross-contamination needs to be taken into consideration. The application of the RNA-baits principle turned out to be user friendly, and even non-experts can easily use the VirBaits workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible as shown for SARS-CoV-2.

DOI:  https://doi.org/10.1186/s40168-020-00973-z  20 February 2021

Filling the gaps in the global prevalence map of clinical antimicrobial resistance
Oldenkamp et al. 2020

Surveillance is critical in containing globally increasing antimicrobial resistance (AMR). Affordable methodologies to prioritize AMR surveillance efforts are urgently needed, especially in low- and middle-income countries (LMICs), where resources are limited. While socioeconomic characteristics correlate with clinical AMR prevalence, this correlation has not yet been used to estimate AMR prevalence in countries lacking surveillance. We captured the statistical relationship between AMR prevalence and socioeconomic characteristics in a suite of beta-binomial principal component regression models for nine pathogens resistant to 19 (classes of) antibiotics. Prevalence data from ResistanceMap were combined with socioeconomic profiles constructed from 5,595 World Bank indicators. Cross-validated models were used to estimate clinical AMR prevalence and temporal trends for countries lacking data. Our approach provides robust estimates of clinical AMR prevalence in LMICs for most priority pathogens (cross-validated q2 > 0.78 for six out of nine pathogens).

DOI:   https://doi.org/10.1073/PNAS.2013515118   05 January 2021

Occurrence of Antibodies against SARS-CoV-2 in the Domestic Cat Population of Germany
Michelitsch et al. 2020

Domestic cats (Felis catus) are popular companion animals that live in close contact with their human owners. Therefore, the risk of a trans-species spreading event between domestic cats and humans is ever-present. Shortly after the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid spread around the world, the role of domestic cats in the transmission cycle was questioned. In the present study, the first large-scale survey of antibody occurrence in the domestic cat population in Germany was conducted, in order to assess the incidence of naturally occurring human to cat transmission of SARS-CoV-2. A total of 920 serum samples, which were collected from April to September of 2020, were screened by an indirect multispecies ELISA.

DOI:   https://doi.org/10.3390/vaccines8040772    17 December 2020

Novel NGS pipeline for virus discovery from a wide spectrum of hosts and sample types
Plyusnin et al. 2020

The study of the microbiome data holds great potential for elucidating the biological and metabolic functioning of living organisms and their role in the environment. Metagenomic analyses have shown that humans, along with for example, domestic animals, wildlife and arthropods, are colonized by an immense community of viruses. The current Coronavirus pandemic (COVID-19) heightens the need to rapidly detect previously unknown viruses in an unbiased way. The increasing availability of metagenomic data in this era of next-generation sequencing (NGS), along with increasingly affordable sequencing technologies, highlight the need for reliable and comprehensive methods to manage such data. In this article, we present a novel bioinformatics pipeline called LAZYPIPE for identifying both previously known and novel viruses in host associated or environmental samples and give examples of virus discovery based on it. LAZYPIPE is a Unix-based pipeline for automated assembling and taxonomic profiling of NGS libraries implemented as a collection of C++, Perl, and R scripts.

DOI:   https://dx.doi.org/10.1093%2Fve%2Fveaa091  02 December 2020

Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection
Freuling et al. 2020

Raccoon dogs might have been intermediate hosts for severe acute respiratory syndrome–associated coronavirus in 2002–2004. We demonstrated susceptibility of raccoon dogs to severe acute respiratory syndrome coronavirus 2 infection and transmission to in-contact animals. Infected animals had no signs of illness. Virus replication and tissue lesions occurred in the nasal conchae.

DOI:   10.3201/eid2612.203733   18 November 2020

Multi‐species ELISA for the detection of antibodies against SARS‐CoV‐2 in animals
Wernike et al. 2020

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease – referred to as COVID‐19 – unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS‐CoV‐2 infections in animals once test systems are evaluated for use in different species. Here, we developed an indirect multi‐species ELISA based on the receptor‐binding domain (RBD) of SARS‐CoV‐2.

DOI:  https://doi-org.proxy.findit.dtu.dk/10.1111/tbed.13926   15 November 2020

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity
Cantuti-Castelvetri et al. 2020

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

DOI:   10.1126/science.abd2985    13 November 2020

Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans
Oude Munnink et al. 2020

Animal experiments have shown that nonhuman primates, cats, ferrets, hamsters, rabbits, and bats can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, SARS-CoV-2 RNA has been detected in felids, mink, and dogs in the field. Here, we describe an in-depth investigation using whole-genome sequencing of outbreaks on 16 mink farms and the humans living or working on these farms. We conclude that the virus was initially introduced by humans and has since evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period, several weeks before detection. Despite enhanced biosecurity, early warning surveillance, and immediate culling of animals in affected farms, transmission occurred between mink farms in three large transmission clusters with unknown modes of transmission. Of the tested mink farm residents, employees, and/or individuals with whom they had been in contact, 68% had evidence of SARS-CoV-2 infection. Individuals for which whole genomes were available were shown to have been infected with strains with an animal sequence signature, providing evidence of animal-to-human transmission of SARS-CoV-2 within mink farms.

DOI:   https://doi.org/10.1126/science.abe5901  10 November 2020

Phylogenetic Investigation of Norovirus Transmission between Humans and Animals
Villabruna et al. 2020

Norovirus infections are a leading cause of acute gastroenteritis worldwide, affecting people of all ages. There are 10 norovirus genogroups (GI-GX) that infect humans and animals in a host-specific manner. New variants and genotypes frequently emerge, and their origin is not well understood. One hypothesis is that new human infections may be seeded from an animal reservoir, as human noroviruses have occasionally been detected in animal species.

DOI:   https://doi.org/10.3390/v12111287  10 November 2020

Experimental Infection and Genetic Characterization of Two Different Capripox Virus Isolates in Small Ruminants
Wolff et al. 2020

Capripox viruses, with their members “lumpy skin disease virus (LSDV)”, “goatpox virus (GTPV)” and “sheeppox virus (SPPV)”, are described as the most serious pox diseases of production animals. A GTPV isolate and a SPPV isolate were sequenced in a combined approach using nanopore MinION sequencing to obtain long reads and Illumina high throughput sequencing for short precise reads to gain full-length high-quality genome sequences. Concomitantly, sheep and goats were inoculated with SPPV and GTPV strains, respectively. During the animal trial, varying infection routes were compared: a combined intravenous and subcutaneous infection, an only intranasal infection, and the contact infection between naïve and inoculated animals.

DOI:   https://doi.org/10.3390/v12101098   28 September 2020

The genetics of highly pathogenic avian influenza viruses of subtype H5 in Germany, 2006–2020
King et al. 2020b

The H5 A/Goose/Guangdong/1/1996 (gs/GD) lineage emerged in China in 1996. Rooted in the respective gs/GD lineage, the hemagglutinin (HA) gene of highly pathogenic avian influenza viruses (HPAIV) has genetically diversified into a plethora of clades and subclades and evolved into an assortment of sub‐ and genotypes. Some caused substantial losses in the poultry industry and had a major impact on wild bird populations alongside public health implications due to a zoonotic potential of certain clades. After the primary introduction of the HPAI H5N1 gs/GD lineage into Europe in autumn 2005 and winter 2005/2006, Germany has seen recurring incursions of four varying H5Nx subtypes (H5N1, H5N8, H5N5, H5N6) carrying multiple distinct reassortants, all descendants of the gs/GD virus.

DOI:   https://doi-org.proxy.findit.dtu.dk/10.1111/tbed.13843   22 September 2020

Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses
King et al. 2020a

Due to the frequent reassortment and zoonotic potential of influenza A viruses, rapid gain of sequence information is crucial. Alongside established next-generation sequencing protocols, the MinION sequencing device (Oxford Nanopore Technologies) has become a serious competitor for routine whole-genome sequencing. Here, we established a novel, rapid and high-throughput MinION multiplexing workflow based on a universal RT-PCR.

DOI:   https://doi-org.proxy.findit.dtu.dk/10.1186/s12879-020-05367-y   03 September 2020

Modelling West Nile Virus and Usutu Virus Pathogenicity in Human Neural Stem Cells
Riccetti et al. 2020

West Nile virus (WNV) and Usutu virus (USUV) are genetically related neurotropic mosquito-borne flaviviruses, which frequently co-circulate in nature. Despite USUV seeming to be less pathogenic for humans than WNV, the clinical manifestations induced by these two viruses often overlap and may evolve to produce severe neurological complications. The aim of this study was to investigate the effects of WNV and USUV infection on human induced pluripotent stem cell-derived neural stem cells (hNSCs), as a model of the neural progenitor cells in the developing fetal brain and in adult brain.

DOI:   https://doi.org/10.3390/v12080882    12 August 2020

Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands
Oude Munnink et al. 2020

In late December 2019, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands.

DOI:   https://doi.org/10.1038/s41591-020-0997-y   16 July 2020

COVID-19 in health-care workers in three hospitals in the south of the Netherlands: a cross-sectional study
Sikkema et al. 2020

10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers.

DOI:   https://doi.org/10.1016/S1473-3099(20)30527-2  02 July 2020

Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo’
Lavezzo et al. 2020

On 21 February 2020, a resident of the municipality of Vo’, a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo’ at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1–3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8–1.8%). Notably, 42.5% (95% CI: 31.5–54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9–9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon–Mann–Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.

DOI:   https://doi.org/10.1038/s41586-020-2488-1  30 June 2020

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model
Rockx et al. 2020

The current pandemic coronavirus, severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2), was recently identified in patients with an acute respiratory syndrome, coronavirus disease 2019 (COVID-19). To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or Middle East respiratory syndrome (MERS)–CoV and compared the pathology and virology with historical reports of SARS-CoV infections. In SARS-CoV-2–infected macaques, virus was excreted from nose and throat in the absence of clinical signs and detected in type I and II pneumocytes in foci of diffuse alveolar damage and in ciliated epithelial cells of nasal, bronchial, and bronchiolar mucosae.

DOI:  https://doi.org/10.1126/science.abb7314   29 May 2020

Human West Nile Virus Lineage 2 Infection: Epidemiological, Clinical, and Virological Findings
Pacenti et al. 2020

West Nile virus (WNV) lineage 2 is expanding and causing large outbreaks in Europe. In this study, we analyzed the epidemiological, clinical, and virological features of WNV lineage 2 infection during the large outbreak that occurred in northern Italy in 2018. The study population included 86 patients with neuroinvasive disease (WNND), 307 with fever (WNF), and 34 blood donors. Phylogenetic analysis of WNV full genome sequences from patients’ samples showed that the virus belonged to the widespread central/southern European clade of WNV lineage 2 and was circulating in the area at least since 2014. The incidence of WNND and WNF progressively increased with age and was higher in males than in females. Among WNND patients, the case fatality rate was 22%. About 70% of blood donors reported symptoms during follow-up. Within the first week after symptom onset, WNV RNA was detectable in the blood or urine of 80% of patients, while 20% and 40% of WNND and WNF patients, respectively, were WNV IgM-seronegative. In CSF samples of WNND patients, WNV RNA was typically detectable when WNV IgM antibodies were absent. Blunted or no WNV IgM response and high WNV IgG levels were observed in seven patients with previous flavivirus immunity.

DOI:  https://doi.org/10.3390/v12040458    18 April 2020

Novel HPAIV H5N8 Reassortant (Clade 2.3.4.4b) Detected in Germany
King et al. 2020

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) was detected in a greater white-fronted goose in January 2020 in Brandenburg, Germany, and, in February 2020, in domestic chickens belonging to a smallholding in Baden-Wuerttemberg, Germany. Full-genome sequencing was conducted on the MinION platform, enabling further phylogenetic analyses. The virus of clade 2.3.4.4b holds six segments from a Eurasian/Asian/African HPAIV H5N8 reassortant and two segments from low pathogenic avian influenza H3N8 subtype viruses recently detected in wild birds in Central Russia. These new entries continue to show the reassortment potential of the clade 2.3.4.4 H5Nx viruses, underlining the necessity for full-genome sequencing and continuous surveillance.

DOI:   https://doi.org/10.3390/v12030281    04 March 2020

 

https://www.veo-europe.eu/library/scientific-publications
18 APRIL 2021