Design up until this line

Norovirus (NoV):

• Major cause of gastroenteritis worldwide in humans

• Common cause of childhood diarrhoea

• Affects people of all ages

• Children, elderly and immunocompromised patients are at risk

of severe disease

• Transferred through faecal-oral route

• Associated with outbreaks in settings such as: hospitals, cruise

ships, nursing homes, schools, hotels and military facilities

• Symptoms include nausea, vomiting, non-bloody watery

diarrhoea and fever

Classification:

• Family – Caliciviridae

• Genus – Norovirus

• Genogroups – GI to GVII (based

on amino acid sequence of capsid)

• Genotypes – at least 38

• Affecting humans – GI, GII and GIV

• GII.4 is predominant

Structure:

• 27-35 nm virion

• Non-enveloped

• Icosahedral shape

• 3 open reading frames

(ORFs)

• Linear ssRNA (+)

• ~ 7.6 kb

Aim The aim of this project is to optimise NoV GI genotyping primers

and to apply the newly developed genotyping assay to study NoV GI

diversity in clinical specimens and the environment.

• The partial capsid region of NoV GI was amplified in 57% (20/35) of samples

• Viruses could be genotyped from 19 samples

• Viruses in sewage samples were difficult to genotype and required substantial

assay optimisation

• The most prevalent strain was GI.4, which circulated for 8 months and dominated

during 5 months

• GI.2, GI.3, GI.5 and GI.6 have all been previously detected in children with severe

NoV gastroenteritis in South Africa and are therefore clinically relevant

• The South African GI strains cluster with strains from 2002-2015 NoV outbreaks in

China, Russia, Sweden and Tanzania

• New genotyping primers were designed and characterisation of stool specimens is

in progress

• National Research Foundation for project funding and bursary

• Poliomyelitis Research Foundation for BSc (Hons) bursary

• Rand Water and Roney Hoffman Consultants for samples

Materials and Methods

Optimisation of genotyping and characterisation of norovirus genogroup I in

clinical specimens and sewage samples

Meno KD, Taylor MB, Mans J Department of Medical Virology, School of Medicine, University of Pretoria

Figure 2: Norovirus genome. Three open reading frames linked to

the viral protein genome (VPg) (solid black circle) at the 5′ end and

polyadenylated at the 3′ end. ORF1 encodes the 6 non-structural (NS)

proteins, ORF2 and ORF3 encode the structural components of the

virion, viral protein 1 (VP1) and VP2, respectively (Chan et al., 2015).

Figure 4: NoV GI diversity: GI.4 was the most

abundant strain and was detected in 63.2%

(12/19) of NoV GI positive samples. GI.2, GI.3 and

GI.5 were detected at similar levels of 10.5%

(2/19) whereas GI.6 was the least detected in 5%

(1/19) of samples.

Introduction

Results

Discussion and Conclusion

Five NoV GI genotypes circulated in Gauteng and the Free State between April 2015 to March 2016

Figure 3: Materials and Methods.

Samples were collected from the

Rotavirus Sentinel Surveillance

Programme (RSSP) and Randwater for

genotyping of NoV GI. Primers were

optimised for typing of Nov GI. NoV GI

samples were amplified, cloned and

sequenced to characterise genotypes.

Figure 3: Percentage of NoV GI positive sewage

samples from which the partial capsid region

could be amplified using semi-nested PCR.

TAN, M. & JIANG, X. 2005. The P domain of norovirus capsid protein forms a subviral particle that binds to

histo-blood group antigen receptors. Journal of virology, 79, 14017-14030.

CHAN, M. C et al. 2015. Rapid emergence and predominance of a broadly recognizing and fast-evolving

norovirus GII. 17 variant in late 2014. Nature communications, 6.

Figure 6: Neighbour-joining phylogenetic analysis of 35

capsid nucleotide sequences (323 bp, 5’-end) obtained from

sewage samples from Gauteng and the Free State. Reference

sequences, indicated by accession numbers, and the most

closely related sequences in GenBank are included. Bootstrap

values >70% are indicated.

Figure 1: Norovirus capsid. The capsid contains

180 proteins organised in an icosahedral shape.

Two major domains are the N-terminal shell (S) and

the C-terminal protruding (P) domains (Tan and

Jiang, 2005).

57% 43%

Amplification success rate(n=35)

Positive

Negative

0

1

2

3

4

5

May(n=2)

Jun(n=2)

Jul(n=1)

Aug(n=1)

Sep(n=2)

Oct(n=2)

Nov(n=2)

Dec(n=3)

Jan(n=2)

Feb(n=1)

Mar(n=1)

Nu

mb

er

of

No

V G

I cl

on

es

seq

ue

nce

d

Months

NoV GI genotypes circulating from May 2015 to March 2016

G1.6

G1.5

G1.4

G1.3

G1.2

Figure 5: NoV GI circulation: GI.4 circulated in 8/11 months and was the predominant strain

overall. GI.2 appeared in December 2015. Between one and three genotypes co-circulated per

month.

Sample selection

Sewage samples

(Apr 2015-March 2016)

Stool specimens

(<5 yrs with diarrhoea

from RSSP)

Recovery + easyMAG

nucleic acid extraction

Manual nucleic acid

extraction

RNA

Primer optimisation

based on NoV GI

sequences in GenBank

Genotyping

cDNA

Semi-nested RT-PCR

(partial capsid)

Sewage Stool specimens

Cloning Direct sequencing

Colony PCR

Sanger Sequencing (at

least 3 clones/sample)

Phylogenetic Analysis

Acknowledgements

10.5%

10.5%

63.2%

10.5%

5.3%

Genotype distibution (n=19)

GI.2

GI.3

GI.4

G1.5

GI.6