Establishing a workflow for gastroenteritis virus surveillance, and norovirus whole genome characterisation by next-generation sequencing technologies

Abstract

Acute gastroenteritis infection is a common illness seen around the world, with most cases being caused by viral pathogens such as rotavirus, norovirus, adenovirus, sapovirus, and astrovirus. Infection causes inflammation of the intestine and stomach, which results in nausea, vomiting, diarrhoea, and dehydration. In most cases, the acute diarrheal disease can resolve itself. However, some infections may lead to significant morbidity and, in extreme cases, death. According to the Centers for Disease Control, viral gastroenteritis infections account for over 200000 deaths per year globally. It is believed that children are potential reservoirs for new emerging viral strains due to their vulnerable immune systems. Thus, it is imperative to do routine gastroenteritis surveillance to allow researchers and vaccine developers to stay up to date with the genetic diversity currently circulating. The norovirus GII.4 genotype has been the predominant strain circulating the globe for the past 20 years, with a new GII.4 variant emerging every few years, allowing it to replace the pre-existing circulating strain. Other genotypes such as GI.5, GI.7, GII.3, GII.6, GII.2, and GII.17 have also been regularly detected in children ( 5 years of age). The lack of complete genome sequence data of non-GII.4 and new emerging GII.4 strains available on the GenBank database illustrates the gap in our knowledge of norovirus diversity and subsequently its epidemiology.

In this study, a workflow was established for gastroenteritis virus surveillance in children under the age of five years, and norovirus whole genome characterisation (WGC) by next-generation sequencing technologies.

Between January 2022 and July 2023, diarrhoeal stool specimens from hospitalised children < 5 years (115/362) and hospitalised individuals > 5 years (17/362) were screened for five gastroenteritis viruses using the Seegene Allplex™ GI Virus plus assay, which allowed for the detection of adenovirus, sapovirus, rotavirus, astrovirus, and norovirus. From the screening results, adenovirus was the most common virus detected (59%), followed by rotavirus (21%) and norovirus (12%). However, the screening kit used to detect the gastroenteritis viruses screened for all types of adenovirus. Thus, to differentiate gastroenteritis infections from others, a second screening assay targeting adenovirus types F40/F41 needs to be done. In addition, 58 norovirus-positive stool specimens were obtained from a private sector diagnostic laboratory screened for co-infections with the multiplex screening assay.

Norovirus GI and GII strains detected were subsequently genotyped based on Sanger sequencing of a ~570 bp region spanning the 3’ end of the RNA-dependent RNA polymerase gene and the 5’ end of the capsid gene. Noroviruses genotypes belonging to genogroup (G)II was more common than genotypes of GI. Genotype GII.4 predominated, being detected in 29.9% of genotyped specimens, followed by GII.17, detected in 16.4%, and GII.1 in 9.1%. In addition to detecting the classic GII.4[P31] Sydney 2012 strain, the recently reported novel GII.4 San Francisco variant was also identified. The GII.4 San Francisco strain was detected more frequently (18.2%) than the GII.4[P31] Sydney 2012 strain (7.6%). Only two GI strains were detected, namely GI.3[P13] and GI.7[P7].

Near complete genome sequencing of selected norovirus strains was achieved by amplifying the genome in 12 overlapping fragments. Both Illumina NextSeq sequencing and Oxford Nanopore singleplex and multiplex sequencing were used. Overall, Illumina sequencing was the most cost-effective method for whole genome sequencing and produced more data than Oxford Nanopore. However, Illumina sequence data contained multiple gaps throughout the sequence. Oxford Nanopore eliminated this limitation which allowed for more certainty of any indels or mutations occurring within the sequence data and better identification. When comparing Oxford Nanopore singleplex and multiplexing sequencing methods, singleplex sequencing is significantly more expensive, however it produces more data than multiplexing. Nevertheless, both Oxford nanopore sequencing methods produce the same quality data.

Comprehensively, this study’s results illustrate that norovirus is among the top three causes of viral gastroenteritis infections. The two most common variants detected were GII.4 and GII.17, which had a high percentage identity with other norovirus isolates circulating the globe, including the novel GII.4 San Francisco strain. Viral gastroenteritis surveillance also revealed which viruses were most prominent in children under the age of five years in the Pretoria, Gauteng area of South Africa. The age groups which were most vulnerable to infection, differences between the public and private sector and seasonality.

By achieving near-complete norovirus genome sequencings allowed for more accurate identification and will facilitate the management of laboratory diagnostics, identification of structural variants throughout the genome, and contribute knowledge of current stains to norovirus vaccine development, prevention and disease management strategies.