Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing

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Abstract

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

Original languageEnglish
Pages (from-to)141-150
Number of pages10
JournalFood and Environmental Virology
Volume10
Issue number2
DOIs
Publication statusPublished - Jun 2018

Fingerprint

Norovirus
Waste Water
Italy
wastewater
Genotype
genetic variation
genotype
Sewage
sewage
Polymerase Chain Reaction
reverse transcriptase polymerase chain reaction
High-Throughput Nucleotide Sequencing
sampling
molecular epidemiology
capsid
Molecular Epidemiology
monitoring
Capsid
gastroenteritis
Gastroenteritis

Keywords

  • Caliciviridae Infections/epidemiology
  • Gastroenteritis/epidemiology
  • Genetic Variation
  • Genotype
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Molecular Epidemiology
  • Norovirus/genetics
  • Sequence Analysis, DNA
  • Sewage/virology
  • Waste Water/virology

Cite this

@article{8368a6ae7b6b4d2a975ad64035b48640,
title = "Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing",
abstract = "Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.",
keywords = "Caliciviridae Infections/epidemiology, Gastroenteritis/epidemiology, Genetic Variation, Genotype, High-Throughput Nucleotide Sequencing, Humans, Molecular Epidemiology, Norovirus/genetics, Sequence Analysis, DNA, Sewage/virology, Waste Water/virology",
author = "E Suffredini and M Iaconelli and M Equestre and B Valdazo-Gonz{\'a}lez and Ciccaglione, {A R} and C Marcantonio and {Della Libera}, S and F Bignami and {La Rosa}, G",
year = "2018",
month = "6",
doi = "10.1007/s12560-017-9328-y",
language = "English",
volume = "10",
pages = "141--150",
journal = "Food and Environmental Virology",
issn = "1867-0334",
publisher = "Springer New York",
number = "2",

}

TY - JOUR

T1 - Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing

AU - Suffredini, E

AU - Iaconelli, M

AU - Equestre, M

AU - Valdazo-González, B

AU - Ciccaglione, A R

AU - Marcantonio, C

AU - Della Libera, S

AU - Bignami, F

AU - La Rosa, G

PY - 2018/6

Y1 - 2018/6

N2 - Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

AB - Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

KW - Caliciviridae Infections/epidemiology

KW - Gastroenteritis/epidemiology

KW - Genetic Variation

KW - Genotype

KW - High-Throughput Nucleotide Sequencing

KW - Humans

KW - Molecular Epidemiology

KW - Norovirus/genetics

KW - Sequence Analysis, DNA

KW - Sewage/virology

KW - Waste Water/virology

U2 - 10.1007/s12560-017-9328-y

DO - 10.1007/s12560-017-9328-y

M3 - Article

VL - 10

SP - 141

EP - 150

JO - Food and Environmental Virology

JF - Food and Environmental Virology

SN - 1867-0334

IS - 2

ER -