Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing

Abstract

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

Keywords: Pretoria; South Africa; Tshwane; next-generation sequencing; norovirus; recombinants; wastewater.

Figure 1

Distribution and prevalence of norovirus GI and GII in wastewater samples. Norovirus GI and GII detected in raw sewage (A) and final effluent (B) samples collected between June 2018 and August 2020. The numbers in parentheses indicate the number of samples collected in each month.

Figure 2

Norovirus GI molecular typing. Distribution and abundance of norovirus GI strains identified from (A) raw sewage and final effluent (B) samples between June 2018 and August 2020. The amplicons from different sampling sites were pooled equimolar based on wastewater sample type and month of collection. The numbers in parentheses indicate the number of merged reads for each month. The key shows known RdRp-capsid combinations, putative novel recombinants (?) and Sanger-sequencing confirmed novel recombinants (*).

Figure 3

Norovirus GII molecular typing. Distribution and abundance of norovirus GII strains identified from (A) raw sewage and final effluent (B) samples between June 2018 and August 2020. The amplicons from different sampling sites were pooled equimolar based on wastewater sample type and month of collection. The numbers in parentheses indicate the number of merged reads for each month. The key shows known RdRp-capsid combinations and putative novel recombinants (?).

Figure 4

Phylogenetic analysis of the 265 bp at 3′-end of norovirus GI ORF1. Representative sequences identified in this study are indicated by black circles (●). All strain sequence names contain specific ID, genotype and P-type. Reference strains and the most closely related strains were downloaded from GenBank. The Maximum Likelihood phylogenetic trees were constructed using MEGA X software [46] and 1000 bootstrap replicates based on the Kimura 2-parameter model. The sequences were labelled according to month and year of detection, wastewater type, P-type and genotype, e.g., 618RGI.2[P2] = GI.2[P2] virus detected in raw sewage collected in June 2018.

Figure 5

Phylogenetic analysis of the 314 bp at 5′-end of norovirus GI ORF2. Representative sequences identified in this study are indicated by black circles (●). All strain sequence names contain specific ID, genotype and P-type. Reference strains and the most closely related strains were downloaded from GenBank. The Maximum Likelihood phylogenetic trees were constructed using MEGA X software [46] and 1000 bootstrap replicates based on the Kimura 2-parameter model. The sequences were labelled according to month and year of detection, wastewater type, P-type and genotype, e.g., 618RGI.2[P2] = GI.2[P2] virus detected in raw sewage collected in June 2018.

Figure 6

Phylogenetic analysis of the 265 bp at 3′-end of norovirus GII ORF1. Representative sequences identified in this study are indicated by black circles (●). All strain sequence names contain specific ID, genotype and P-type. Reference strains and the most closely related strains were downloaded from GenBank. The Maximum Likelihood phylogenetic trees were constructed using MEGA X software [46] and 1000 bootstrap replicates based on the Kimura 2-parameter model. The sequences were labelled according to month and year of detection, wastewater type, P-type and genotype, e.g., 618RGII.2[P2] = GII.2[P2] virus detected in raw sewage collected in June 2018.

Figure 7

Phylogenetic analysis of the 305 bp at 5′-end of norovirus GII ORF2. Representative sequences identified in this study are indicated by black circles (●). All strain sequence names contain specific ID, genotype and P-type. Reference strains and the most closely related strains were downloaded from GenBank. The Maximum Likelihood phylogenetic trees were constructed using MEGA X software [46] and 1000 bootstrap replicates based on the Kimura 2-parameter model. The sequences were labelled according to month and year of detection, wastewater type, P-type and genotype, e.g., 618RGII.2[P2] = GII.2[P2] virus detected in raw sewage collected in June 2018.

Figure 8

Phylogenetic analysis of the 302 bp at 5′-end of norovirus GII.2, GII.4 and GII.17 ORF2. Representative sequences identified in this study are indicated by black circles (●). All strain sequence names contain specific ID, genotype and P-type. Reference strains and the most closely related strains were downloaded from GenBank. The Maximum Likelihood phylogenetic tree was constructed using MEGA X software [46] and 1000 bootstrap replicates based on the Kimura 2-parameter model. The sequences were labelled according to month and year of detection, wastewater type, P-type and genotype, e.g., 1018RGII.2[P2] = GII.2[P2] virus detected in raw sewage collected in October 2018.