Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus

Abstract

Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (106.42–107.01 copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded.

Development of a multiplex RT‐PCR for the detection of major diarrhoeal viruses in pig herds in China

“…Although viral metagenomics and nanopore sequencing technology (Theuns et al, ) are valuable assets for the diagnosis of enteric diseases in pigs and for the discovery and identification of novel porcine viral enteric disease complexes, there are some disadvantages, such as cost, long diagnosis times, and high requirements for instruments and analysers, limiting their usage in the field or clinical settings for diagnosis. Multiplex PCR/RT‐PCR is still widely used by most veterinary diagnostic laboratories due to simple procedures, rapid diagnostic results, high‐throughput methodologies, low cost and more advantages.…”

“…Zhao et al also reported the coinfection of PKV with PEDV, TGEV and PRV‐A in China (Zhao et al, ). Using next‐generation sequencing or nanopore sequencing technologies, Chen et al and Theuns et al reported that coinfections of swine enteric viruses were widely presented in pig farms in the United States and Belgium (Chen et al, ; Theuns et al, ).…”

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

“…In fact, MinION sequencing technology might end up being a useful and affordable diagnostic tool for swine veterinary medicine in general. This technology can provide a complete readout of RNA viruses and RNAs from the host or other pathogens present in a sample without the need for pre-existing knowledge of what might be present [84].…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…To summarize, small physical dimensions of MinION, along with other ONT sequencers, availability of DNA and RNA sequencing protocols, potentially high precision and high data processing rates create promising prospects for its clinical application [179]. So far, it has already been tested in veterinary science for detection of canine distemper virus (CDV) [203,204] and in biology for identification of numerous pathogens in plants [205] and fish [206]. Nevertheless, prior to extrapolating these positive results into the medical field, we need to develop and validate the workflows that would easily integrate into the everyday work of a clinical laboratory and not require any special bioinformatic training in the lab staff [207].…”