1. Kgothatso (KD) Meno
BSc (Hons)
Department of Medical Virology
Supervisor: Co-Supervisor:
Dr J Mans Prof MB Taylor

2.  Noroviruses are a major cause of gastroenteritis
worldwide in humans and animals
 It affects people of all ages
 Children, elderly and immunocompromised
patients are at risk of severe disease
 Norovirus is a highly contagious virus, 18 viral
particles can initiate an infection

3.  Transmission by means of faecal-oral route
 The symptoms shown by an individual who is
infected by NoVs include nausea, vomiting, non-
bloody watery diarrhoea and fever
 Virus shedding for 3 weeks after infection
 Prolonged in immunocompromised individuals
 200 000 deaths of children younger than five
years old has been estimated in the developing
world annually

4. Norovirus (NoV) - A member of Caliciviridae family
Small (30 to 35 nm), non-enveloped, icosahedral
Linear, positive sense, single-stranded RNA genome

5. Norovirus Genome
 ORF1/2 polymerase-capsid junction used for detection and
genotyping NoVs
 NoVs are divided into six genogroups, seventh has been
proposed (Genogroups I, II and IV infect humans)
 GII.4 is the most prevalent of all genotypes
Structural Protein
(VP1) / Major Capsid
Protein
Non-structural proteins
including RNA-dependent
RNA polymerase

6.  Norovirus Capsid Model of VP1 dimer

7.  To date no commercial NoV vaccine exists
 Virus like particles (VLP) vaccines are in clinical
trials (Atmar et al., NEJM, 2011)
 Vaccine has been shown to prevent disease but
not infection
 Studies are ongoing to evaluate combinations of
genogroups and other aspects of the vaccine

8.  No specific treatment or antiviral therapy for
NoV infection
 Treatment: intravenous or oral hydration
 At least 40 genotypes within the 6 genogroups

9. GI
GII

10.  A large number of gastroenteric infections and
outbreaks caused by NoVs are reported but many
suspected cases are not laboratory confirmed
 The degree of genetic variability in NoVs makes
it difficult to design sensitive and specific
molecular diagnostic assays
 This increases the need and impact of primer
design that is optimised for molecular detection
and typing

11.  Real-time reverse transcriptase-polymerase
chain reaction (real-time RT-PCR) is considered
the “gold standard” for detection of NoVs
 Laboratory confirmation of NoV is important in
public health remediation and prevention as well
as enhancing the understanding of NoV
prevalence in the population

12.  Genogroup distribution in South Africa (2009-
2015)
 GI (17.7%)
 GII (78.5%)
 GI+GII (3.8%)
 Sizable proportion caused by GI
 GI detected but not possible to genotype,
there is diversity primers are not picking up

13.  Since GII is studied more globally, there is
lack of information on GI because research is
skewed to GII
 The number of complete genome sequences
of GI Vs. GII
 GI is circulating a lot in SA but complete
genomes are missing
 In HIV positive children, more severe disease
is associated with GI (Prof NA Page)
 GI might be a more relevant genogroup in SA

14.  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

15.  To genotype NoV GI detected from sewage samples (April
2015- March 2016) using standard GI primers targeting the
polymerase and capsid regions
 To construct and analyse multiple alignments of available
GI sequences from GenBank
 To design primers to optimise conventional RT-PCR
 To apply optimised methods to genotype NoV GI strains
from the Rotavirus Surveillance Sentinel Programme that
could not be typed previously (2011- 2013) and new strains
from 2015
 To amplify complete capsid gene from selected GI strains
for characterisation and phylogenetic analysis

16. Sample selecion
Sewage samples (Apr
2015-March 2016)
Stool specimens (<5 yrs with
diarrhoea from RSSP)
Recovery + extraction
(Victor Mabasa)
Manual nucleic acid
extraction
RNA
Primer optimisation
Genotyping
cDNA
Semi-nested RT-PCR
(partial capsid and RdRp)

17. Sewage
Cloning
Colony PCR
Stool Specimens
Direct sequencing
Sequencing
Phylogenetic Analysis

18. Rand Water Project 2015/2016
NoV GI Positive Samples – April 2015* to March 2016
4
31
9
Samples
Unscreened
Screened
Unscreened
Ct>37
n=44

19. 0
1
2
3
4
5
6
7
8
GIpositives
Months
Representation of number of norovirus GI positive samples
detected by real-time RT-qPCR and typed by Semi-nested
conventional PCR
Real-time detection
nested PCR typing

20. Round 1 Round 2
1kb+
marker
PCR &
CDNA
negative
Round 1 Round 2
Round 2Round 1
M
M
M M M

21. 1kb+
marker
M
M

22. Distribution of genotypes: all samples sequenced were
GI.4
25
6
4 Negative
Positive
Still to be
screened
n=35

23.  Collected all the complete genomes (33)
 Typed them using the norovirus genotyping
tool and aligned them using MAFFT software
in FASTA format
 Distribution of genotypes GI.1 22
GI.2 3
GI.3 2
G1.4 1
GI.6 3
GI.8 2

28.  GI Forward primer 1
TGG ACA GGA GAT CGC RAT CT
Tm:66°C
 GI Forward primer 2
ATG ATG ATG GCG TCT AAG GA
Tm:61°C
 GI Reverse primer
CCI ACC CAI CCA TTR TAC AT
Tm:61°C

29.  Problems encountered:
 Encountered problems with the Hot Start
DNA polymerase enzyme, a lot of sewage
samples were screened but there was no
amplification
 Although the enzyme worked on clinical
specimens
 The standard primer binding region not
completely conserved
 The denaturation time (30 sec) too short

30.  Alternative ways attempted:
 A different enzyme (Emerald) was used,
there was amplification
 High number of none specific amplification
 Emerald PCR products: Gel clean up

31.  Use my designed primers on the stool
specimens
 Go back and screen some of the sewage
samples with the new optimised primers and
the Hot start DNA polymerase
 Go back and screen the previously negative
sewage samples with Emerald enzyme
 Optimise the Emerald PCR
 Design genotype specific primer for capsid 3’
end
 Amplify and sequence the complete capsid
gene of selected strains

33. Part of a larger (ethically approved) project (Rotavirus
Sentinel Surveillance Programme), additional ethical
approval has been granted (127/2016)
Total: R71 850

34.  Prof NA Page together with the Rota network
 Randwater
 NRF

35. Any Questions?