The Intestinal Eukaryotic Virome in Healthy and Diarrhoeic Neonatal Piglets.

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of European countries. The aim of the present study was to use viral metagenomics to examine a potential viral involvement in this diarrhoea and to describe the intestinal virome with focus on eukaryotic viruses. Samples from the distal jejunum of 50 diarrhoeic and 19 healthy piglets from 10 affected herds were analysed. The viral fraction of the samples was isolated and nucleic acids (RNA and DNA fractions) were subjected to sequence independent amplification. Samples from diarrhoeic piglets from the same herds were pooled whereas samples from healthy piglets were analysed individually. In total, 29 clinical samples, plus two negative controls and one positive control consisting of a mock metagenome were sequenced using the Ion Torrent platform. The resulting sequence data was subjected to taxonomic classification using Kraken, Diamond and HMMER. In the healthy specimens, eight different mammalian virus families were detected (Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picornaviridae, and Reoviridae) compared to four in the pooled diarrhoeic samples (Anelloviridae, Circoviridae, Picornaviridae, and Reoviridae). It was not possible to associate a particular virus family with the investigated diarrhoea. In conclusion, this study does not support the hypothesis that the investigated diarrhoea was caused by known mammalian viruses. The results do, however, indicate that known mammalian viruses were present in the intestine as early as 24-48 hours after birth, indicating immediate infection post-partum or possibly transplacental infection.

Molecular approaches to recognize relevant and emerging infectious diseases in animals.

Since the introduction of the first molecular tests, there has been a continuous effort to develop new and improved assays for rapid and efficient detection of infectious agents. This has been motivated by a need for improved sensitivity as well as results that can be easily communicated. The experiences and knowledge gained at the World Organisation for Animal Health (OIE) Collaborating Centre for Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine, Uppsala, Sweden, will here be used to provide an overview of the different molecular approaches that can be used to diagnose and identify relevant and emerging infectious diseases in animals.

Metagenomic approaches to disclose disease-associated pathogens: detection of viral pathogens in honeybees.

Metagenomic approaches have become invaluable for culture-independent and sequence-independent detection and characterization of disease-associated pathogens. Here, the sequential steps from sampling to verification of results are described for a metagenomic-based approach to detect potential pathogens in honeybees. The pre-sequencing steps are given in detail, but due to the rapid development of sequencing technologies, all platform-specific procedures, as well as subsequent bioinformatics analysis, are more generally described. It should also be noted that this approach could, with minor modifications, be adapted for other organisms and sample matrices.

Metagenomic detection of viral pathogens in Spanish honeybees: co-infection by Aphid Lethal Paralysis, Israel Acute Paralysis and Lake Sinai Viruses.

The situation in Europe concerning honeybees has in recent years become increasingly aggravated with steady decline in populations and/or catastrophic winter losses. This has largely been attributed to the occurrence of a variety of known and "unknown", emerging novel diseases. Previous studies have demonstrated that colonies often can harbour more than one pathogen, making identification of etiological agents with classical methods difficult. By employing an unbiased metagenomic approach, which allows the detection of both unexpected and previously unknown infectious agents, the detection of three viruses, Aphid Lethal Paralysis Virus (ALPV), Israel Acute Paralysis Virus (IAPV), and Lake Sinai Virus (LSV), in honeybees from Spain is reported in this article. The existence of a subgroup of ALPV with the ability to infect bees was only recently reported and this is the first identification of such a strain in Europe. Similarly, LSV appear to be a still unclassified group of viruses with unclear impact on colony health and these viruses have not previously been identified outside of the United States. Furthermore, our study also reveals that these bees carried a plant virus, Turnip Ringspot Virus (TuRSV), potentially serving as important vector organisms. Taken together, these results demonstrate the new possibilities opened up by high-throughput sequencing and metagenomic analysis to study emerging new diseases in domestic and wild animal populations, including honeybees.

New viruses in veterinary medicine, detected by metagenomic approaches.

In our world, which is faced today with exceptional environmental changes and dramatically intensifying globalisation, we are encountering challenges due to many new factors, including the emergence or re-emergence of novel, so far "unknown" infectious diseases. Although a broad arsenal of diagnostic methods is at our disposal, the majority of the conventional diagnostic tests is highly virus-specific or is targeted entirely towards a limited group of infectious agents. This specificity complicates or even hinders the detection of new or unexpected pathogens, such as new, emerging or re-emerging viruses or novel viral variants. The recently developed approaches of viral metagenomics provide an effective novel way to screen samples and detect viruses without previous knowledge of the infectious agent, thereby enabling a better diagnosis and disease control, in line with the "One World, One Health" principles (www.oneworldonehealth.org). Using metagenomic approaches, we have recently identified a broad variety of new viruses, such as novel bocaviruses, Torque Teno viruses, astroviruses, rotaviruses and kobuviruses in porcine disease syndromes, new virus variants in honeybee populations, as well as a range of other infectious agents in further host species. These findings indicate that the metagenomic detection of viral pathogens is becoming now a powerful, cultivation-independent, and useful novel diagnostic tool in veterinary diagnostic virology.