Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV

High-Resolution Composition Analysis of an Inactivated Polyvalent Foot-and-Mouth Disease Vaccine

Appropriate vaccine selection is crucial in the control of foot-and-mouth disease (FMD). Vaccination can prevent clinical disease and reduces viral shedding, but there is a lack of cross-protection between the seven serotypes and their sublineages, making the selection of an adequately protective vaccine difficult. Since the exact composition of their vaccines is not consistently disclosed by all manufacturers, incompatibility of the strains used for vaccination with regionally circulating strains can cause vaccination campaigns to fail. Here, we present a deep sequencing approach for polyvalent inactivated FMD vaccines that can identify all component strains by their genome sequences. The genomes of all strains of a commercial pentavalent FMD vaccine were de novo assembled and the vaccine composition determined semi-quantitatively. The genome assembly required high stringency parameters to prevent misassemblies caused by conserved regions of the genome shared by related strains. In contrast, reference-guided assembly is only recommended in cases where the number of strains is previously known and appropriate reference sequences are available. The presented approach can be applied not only to any inactivated whole-virus FMD vaccine but also to vaccine quality testing in general and allows for better decision-making for vaccines with an unknown composition

A Novel Squirrel Respirovirus with Putative Zoonotic Potential

In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen

Borna disease outbreak with high mortality in an alpaca herd in a previously unreported endemic area in Germany

Borna disease virus 1 (BoDV-1) is the causative agent of Borna disease, an often fatal neurologic condition of domestic mammals, including New World camelids, in endemic areas in Central Europe. Recently, BoDV-1 gained further attention by the confirmation of fatal zoonotic infections in humans. Although Borna disease and BoDV-1 have been described already over the past decades, comprehensive reports of Borna disease outbreaks in domestic animals employing state-of-the-art diagnostic methods are missing. Here, we report a series of BoDV-1 infections in a herd of 27 alpacas (Vicugna pacos) in the federal state of Brandenburg, Germany, which resulted in eleven fatalities (41%) within ten months. Clinical courses ranged from sudden death without previous clinical signs to acute or chronic neurologic disease with death occurring after up to six months. All animals that underwent necropsy exhibited a non-suppurative encephalitis. In addition, six apparently healthy seropositive individuals were identified within the herd, suggesting subclinical BoDV-1 infections. In infected animals, BoDV-1 RNA and antigen were mainly restricted to the central nervous system and the eye, and sporadically detectable in large peripheral nerves and neuronal structures in other tissues. Pest control measures on the farm resulted in the collection of a BoDV-1-positive bicoloured white-toothed shrew (Crocidura leucodon), while all other trapped small mammals were negative. A phylogeographic analysis of BoDV-1 sequences from the alpacas, the shrew and BoDV-1-positive equine cases from the same region in Brandenburg revealed a previously unreported endemic area of BoDV-1 cluster 4 in North-Western Brandenburg. In conclusion, alpacas appear to be highly susceptible to BoDV-1 infection and display a highly variable clinical picture ranging from peracute death to subclinical forms. In addition to horses and sheep, they can serve as sensitive sentinels used for the identification of endemic areas

Ahead of Print - Novel Picornavirus in Lambs with Severe Encephalomyelitis - Volume 25, Number 5—May 2019 - Emerging Infectious Diseases journal - CDC

Using metagenomic analysis, we identified a novel picornavirus in young preweaned lambs with neurologic signs associated with severe nonsuppurative encephalitis and sensory ganglionitis in 2016 and 2017 in the United Kingdom. In situ hybridization demonstrated intralesional neuronotropism of this virus, which was also detected in archived samples of similarly affected lambs (1998-2014)