Kgothatso (KD) Meno

BSc (Hons)

Department of Medical Virology

Supervisor: Co-Supervisor:

Dr J Mans Prof MB Taylor

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

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

Norovirus (NoV) - A member of Caliciviridae family

Small (30 to 35 nm), non-enveloped, icosahedral

Linear, positive sense, single-stranded RNA genome

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

Norovirus Capsid Model of VP1 dimer

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

No specific treatment or antiviral therapy for

NoV infection

Treatment: intravenous or oral hydration

At least 40 genotypes within the 6 genogroups

GI

GII

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

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

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

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

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

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

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)

Sewage

Cloning

Colony PCR

Stool Specimens

Direct sequencing

Sequencing

Phylogenetic Analysis

Rand Water Project 2015/2016

NoV GI Positive Samples – April 2015* to March 2016

4

31

9

Samples

Unscreened

Screened

UnscreenedCt>37

n=44

0

1

2

3

4

5

6

7

8

GI p

osi

tive

s

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

Round 1 Round 2

1kb+

marker

PCR &

CDNA

negative

Round 1 Round 2

Round 2Round 1

M

M

M M M

1kb+

marker

M

M

Distribution of genotypes: all samples sequenced were

GI.4

25

6

4 Negative

Positive

Still to bescreened

n=35

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

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

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

Alternative ways attempted:

A different enzyme (Emerald) was used,

there was amplification

High number of none specific amplification

Emerald PCR products: Gel clean up

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

Part of a larger (ethically approved) project (Rotavirus

Sentinel Surveillance Programme), additional ethical

approval has been granted (127/2016)

Total: R71 850

Prof NA Page together with the Rota network

Randwater

NRF

Any Questions?