THE EVOLUTION OF HUMAN ROTAVIRUS STRAINS G4P[6] AND

G4P[8] CIRCULATING IN SOUTHERN AFRICA BETWEEN 1985 AND

2011

By

Mr. Fulufhelo Matshonyonge (201531645)

A mini dissertation in fulfilment of the requirements for the degree of

BSC HONOURS IN MEDICAL VIROLOGY

Submitted in the Department of Virology

School of Pathology and Pre-clinical Sciences, Faculty of Health

Sciences

Sefako Makghato Health Sciences University

Supervisor: Dr Martin Munene Nyaga (MSc, PhD)

Co-supervisor: Dr Mapaseka Luyanda Seheri (MSc, PhD)

ii

DECLARATION

I Fulufhelo Matshonyonge, hereby declare that the work presented in this mini-

dissertation is original and does not contain any work that has been submitted in

fulfilment of any degree or diploma at any other university. I carried it out under the

supervision of Dr M.M Nyaga and Dr L.M Seheri in the Department of Virology,

South Africa Medical Research Council Diarrhoeal Pathogens Research Unit

(DPRU), Sefako Makgatho Health Sciences University.

…………………………………...

Signature of candidate

.…08…. Day of .…01….. 2016

iii

DEDICATION

I dedicated this work to my sister Mrs Sarah Nedzingahe, my parents Mr Mackson

and Mrs Florah Matshonyonge and the entire Matshonyonge family, for their love,

support, prayers and encouragement.

iv

ACKNOWLEDGEMENTS

During the duration of this study I have received immense support and dependable

assistance from numerous peoples. The success of this research would not have

been possible without their contribution.

First my sincere appreciation goes to my supervisor Dr M.M. Nyaga and co-

supervisor Dr L.M. Seheri for their cooperation, guidance and expertise. They did not

only give me their undivided attention but encouraged me along the way.

My deepest appreciation goes to Prof R Burnett for her expertise, patience and

support. Her contribution throughout the study was highly appreciated.

Thank you to the staff of Department of Virology especially MRC-DPRU and to my

fellow student for their support.

I particularly want to thank Poliomyelitis Research Foundation (PRF) and National

Research foundation (NRF) for funding this study.

My sincere gratitude goes to my friends, brother and sisters, who constantly

encouraged me and had incomparable faith and belief in me.

Finally, I thank the almighty God Jesus Christ for the strength, courage and wisdom.

I give all the praise and honour to God.

v

ABSTRACT

Introduction: Group A rotaviruses are the leading cause of severe viral

gastroenteritis in infants with most deaths occurring in developing countries. The

major G- and P- type combinations circulating globally are G1P[8], G2P[4], G3P[8],

G4P[8] and G9P[8] which were detected during the 1980s and since remained as

common circulating strains globally. Recently, two G- and P- combinations, the

G9P[8] and G12P[8], re-emerged and emerged, respectively to become medically

important globally. During the same time when G9P[8] and G12P[8] became

increasingly common, genotype G4 declined in some geographical areas especially

in the African region. The temporal decline of G4 strains was accompanied by a

remarkable increase of G8, G9 and G12 strains in the African region during the

period of 1996–2007. In this study three G4 strains from South Africa, Zimbabwe and

Zambia associated with P[6] and P[8] genotypes collected between 1985 and 2011

were selected for complete genome analysis to determine if there is genetic

connection between the disappearing G4 strains and the strains that have emerged

within that time.

Methodology: A total of three stool samples were subjected to whole genome

sequencing. RNA was extracted from the samples using QIAamp® RNA extraction

Kit. Gene segments 4 and 9 were amplified using RT-PCR and genotyped using

multiplex primers to confirm the samples as genotype G4s. The whole genome

sequences of the three samples were compared to selected nucleotide sequences in

the GenBank using the NCBI-BLAST software. A dot conversation plot was

constructed using BioEdit comparing the study strains to the full genome sequences

of selected global published G4 strains. Maximum likelihood phylogenetic trees and

P-Distance Matrix of all the 11 gene segments of the three study strains were

constructed using MEGA software at 500 bootstraps.

Results: The disappearance of the G4 genotypes was found to be mainly due to

changes in antigenicity due to mutations at nucleotide level as well as genetic

reassortment. In this study we found that there is a direct evolutionary relationship

between the emerging RVA strains and the currently disappearing G4 genotypes.

The high genetic diversity observed within the MRC-DPRU20683 G4P[8] and the

MRC-DPRU1850 G4P[8] study strains on the VP7 encoding gene segment 9

substantiates the theory behind the disappearance of the G4s over the years. The

vi

G4P[8] study strain MRC-DPRU1850 clustered on lineage I In the VP7 tree forming

a distinct sub cluster, distantly related to the MRC-DPRU20683 study strain as well

as other reference African G4P[8] strains such as GR1107/86 and GR856/86 sharing

nucleotides identities ranging from 82.9% to 96.7%. The non-structural proteins of

the MRC-DPRU1752 study strain formed separate semi-cluster with other DS-1-like

African strains as expected. Whereas the MRC-DPRU20683 and MRC-DPRU1850

strains formed a single cluster with the Wa-like reference strains which substantiate

that the NSPs are generally conserved although minor variances were observed in

some genomes, they were not significant. The NSPs of the Wa-like and DS-1-like

study strains revealed 86-95% nucleotide similarities with 86.7% between the MRC-

DPRU20683 and MRC-DPRU1752.

Conclusion: The strains isolated between 1985 and 2000 have not undergone

significant evolution in the VP7 gene segment which is the major antigenic

determinant, a trend that was observed in the other 10 proteins. The study confirms

that genotype G4 of group A rotaviruses are in continuous evolution, primarily by

point mutation and in some cases, probably by reassortment mechanisms. Most of

the variable regions within all the 11 RVA genomes in the study strains were

considerably conserved with silent nucleotide mutations that did not change the

encoded proteins. The disappearance of the G4 genotypes could be mainly due to

changes in antigenicity as a result of mutations at nucleotide level giving rise to

either new strains or shifting to other genotypes. In conclusion, most of the variable

regions within all the 11 RVA genomes in the study strains were considerably

conserved with minor amino acid changes which substantiate that evolution resulting

in a genetic shift may take many more years which could be determined by

advanced bioinformatics studies such as Bayesian analyses.

vii

TABLE OF CONTENT DECLARATION .......................................................................................................... ii

DEDICATION ............................................................................................................. iii

ACKNOWLEDGEMENTS .......................................................................................... iv

ABSTRACT ............................................................................................................... v

TABLE OF CONTENT .............................................................................................. vii

CHAPTER 1: INTRODUCTION ................................................................................. 1

1.1. Introduction and rationale for the study ............................................................... 1

1.2. Problem statement .............................................................................................. 3

1.3. Purpose of the study ........................................................................................... 3

1.3.1 Research question ......................................................................................... 3

1.3.2 Aim of the study ............................................................................................. 3

1.3.3 Objectives ...................................................................................................... 4

1.4.5. Significance of the study ............................................................................... 4

CHAPTER 2: LITERATURE REVIEW ....................................................................... 5

2.1. Introduction ......................................................................................................... 5

2.2. Rotavirus proteins ............................................................................................... 6

2.2.1. Structural Proteins ........................................................................................ 7

2.2.2. Non-Structural proteins ............................................................................... 11

2.3. Rotavirus genotype distribution ......................................................................... 14

2.4. Life cycle and replication of rotavirus ................................................................ 16

2.4.1. Attachment and entry .................................................................................. 16

2.4.2. Transcription and Translation ..................................................................... 17

2.4.3. Replication of genomic RNA ....................................................................... 18

2.4.4. Genome packaging ..................................................................................... 19

2.4.5. Viral Assembly and release ........................................................................ 19

2.5. Pathogenesis and outcomes ............................................................................. 20

2.6. Detection, pevention and control ....................................................................... 21

2.6.1. Laboratory diagnosis .................................................................................. 21

2.6.2. Vaccination of rotavirus .............................................................................. 21

CHAPTER 3: MATERIALS AND METHOD ............................................................. 23

3.1. Ethical considerations ....................................................................................... 23

3.2. Study design ..................................................................................................... 23

3.2.1. Description of the study .............................................................................. 23

3.2.2. Study population and study sample ............................................................ 23

3.3. Laboratory methods .......................................................................................... 23

3.3.1 Enzyme immunoassay (EIA)........................................................................ 23

3.3.3. Reverse Transcription Polymerase Chain Reaction (RT-PCR) ................... 24

3.3.3.1. RNA Extraction ..................................................................................... 24

3.3.3.2. Reverse Transcription Polymerase Chain Reaction (RT-PCR) ............ 25

3.3.4. Gene segment 4 and 9 RT-PCR ................................................................. 26

3.3.6. Genotyping ................................................................................................. 26

3.3.6.1. G-typing ................................................................................................ 26

viii

3.3.6.2. P-typing ................................................................................................ 27

3.4. Data analysis ..................................................................................................... 27

3.4.1. Sequence analysis ...................................................................................... 27

3.4.2. Whole-genome phylogenetic analysis ........................................................ 27

CHAPTER 4: RESULTS .......................................................................................... 29

4.1. Confirmation of the G4P[6] and G4P[8] strains ................................................. 29

4.2. Genotyping of selected samples ....................................................................... 29

4.1.2. Nucleotide and amino acid pairwise analyses ............................................ 33

4.2. Phylogenetic analysis ........................................................................................ 33

4.2.1. Sequence analysis of VP7 .......................................................................... 34

4.2.2. Sequence analysis of VP4 ............................................................................. 38

4.2.3. Sequence analysis of VP6 .......................................................................... 39

4.2.4. Sequence Analyses of VP1–VP3 ................................................................ 43

4.2.5. Sequence analysis of Non-structural proteins (NSP1-NSP5) ..................... 46

CHAPTER 5: DISCUSSION AND CONCLUSSION ................................................ 51

5.1. EIA and PCR ..................................................................................................... 51

5.2. Phylogenetic Analysis ....................................................................................... 51

5.3. Full genome Sequence analysis of the G4P[6] and G4P[8] .............................. 53

5.4. Conclusion ........................................................................................................ 54

5.5. Recommendation .............................................................................................. 55

REFERENCES ......................................................................................................... 56

APPENDICES .......................................................................................................... 71

1. Preparation of 1% agarose gel and gel electrophoresis ....................................... 71

2. Preparation of 2% agarose gel and gel electrophoresis ....................................... 71

2. RT-PCR master mix. ............................................................................................ 72

3. PCR master mix. .................................................................................................. 72

4. Genotyping master mix. ....................................................................................... 73

5. Sequence identity matrix of aligned sequences ................................................... 73

1

CHAPTER 1: INTRODUCTION

1.1. Introduction and rationale for the study

Diarrhoeal infections are recognised in human and other mammals since ancient

times (Kapikian, 1996). In the early 1973 a virus particle was discovered in the

intestinal tissue of children admitted with diarrhoeal disease using an electron

microscope (Bishop et al., 1973). Morphologically, the virus resembled a wheel and

was subsequently named Rotavirus (rota, Latin for wheel) (Flewett et al., 1974;

Wyatt et al., 1978). Rotaviruses are recognised as the most common cause of

severe viral diarrhoeal infections in infants and young children around the world

(Kapikian and Hoshino, 2001).

Rotaviruses belong to the genus Rotavirus, in the family Roeviridae. They are

classified serologically into groups or serogroups (Hoshino and Kapikian, 2000).

Seven distinct groups (A to H) of rotaviruses have been described. Group A, B, C,

and H rotaviruses have been found both in humans and animals; groups D, E, and F

rotaviruses have been found only in animals (Krishnan et al., 1999; Hoshino and

Kapikian, 2000). Group A rotaviruses (RVA) are the major cause of virus induced

severe gastroenteritis in young children and infants below the age of five years

worldwide. The RVA differ geographically and have great socio-economic impact,

with most cases occurring in developing countries of Africa and Asia (Cook et al.,

1990; Tate et al., 2012). Recent rotavirus strains and serological surveillance studies

indicate that all young children are likely to have experienced at least one rotavirus

infection by the time they are five years of age (Kirkwood, 2010). RVA are estimated

to cause approximately 453,000 deaths in children less than 5 years of age annually,

of which more than 50% occur in African and Asian countries (Tate et al., 2012).

There is a great diversity in circulating RVA wild-type strains worldwide (Gentsch et

al., 2005; Dóró et al 2014; Seheri et al., 2014). The dominant circulating strains that

cause severe disease change from year to year and also differ by country. Rotavirus

diversity is mainly generated by several mechanisms such as accumulation of point

mutations or genetic drift that can lead to (a) antigenic changes; (b) reassortments

that may result in viruses with novel genetic and antigenic characteristics; (c) direct

transmission of animal strains to a human host; and (d) gene rearrangement such as

2

deletions, duplications and insertions into coding or non-coding regions (Palombo.,

2002; Parra et al., 2004; Gentsch et al., 2005; Ianiro et al., 2013; Zhou et al., 2015; ).

The Genus Rotavirus belongs to the family Reoviridae and within the Group III of the

Baltimore classification system. The rotavirus genome contains 11 of double-

stranded (ds) RNA segments, icosahedral in shape and non-enveloped. It is

surrounded by three-layered capsid proteins. The outer capsid protein is made up of

VP7 (glycoprotein, G-type) and VP4 viral spikes (protease sensitive, P-type) (Estes

and Kapikian, 2007). This way 37 G- and 27 P-types of rotavirus were reported as of

2008 (Matthijnssens et al., 2010; Matthijnssens et al., 2011; Trojnar et al., 2013). The

intermediate capsid layer consists of the VP6 (intermediate, I-type) which is the most

abundant rotavirus protein (Gonzalez et al., 1998; Matthijnssens et al., 2011; Trojnar

et al., 2013). The inner capsid layer consists of VP2 (core, C-type) encasing VP1

(RNA-dependent RNA polymerase, R-type) and VP3 (Methyltransferase, M-type),

and surrounds the genomic material (Estes and Kapikian, 2007). The RV segments

encode for six structural (VPs) or six non-structural proteins (NSPs) (Gonzalez et al.,

1998).

The major G- and P- type combinations circulating globally are G1P[8], G2P[4],

G3P[8], G4P[8] and G9P[8]. They constitute the majority of human rotavirus strains

worldwide that cause severe rotavirus disease in children in most countries for the

last 20 years (Gentsch et al., 2005; Kirkwood, 2010). The G1P[8] strains are most

prevalent, although large regional differences exist and strain types vary (Santos and

Hoshino, 2005; Page et al., 2010; Bányai et al., 2012; ; Dóró et al 2014; Seheri et al.,

2014).

Medically important G1P[8], G2P[4], G3P[8], and G4P[8] genotypes were first

detected during the 1980s and remained common during the 1990s and 2000s

(Bányai et al., 2012). During the 1990s and 2000s, two emerging strains, the G9s

and G12s, became medically important globally in addition to other historically well-

known important endemic RV strains (Matthijnssens et al., 2010). At the same time,

the prevalence of G4 common strains in Africa was declining over the years (Seheri

et al., 2014). In addition genotype G4P[6] prevalence was previously found to be less

common (0.2-2% prevalence range) compared to G4P[8] strain which was the most

commonly identified G4 combination globally (Bányai et al., 2012). The temporal

decline of G4 strains was accompanied by a remarkable increase of G8, G9 and

3

G12 strains in the African region between 1996 to 2007 (Seheri et al., 2010; Seheri

et al., 2014). The genetic mechanisms behind some of these changes were

investigated in this study.

1.2. Problem statement

Although genotype G4s were globally regarded as common strains, they are hardly

reported in southern Africa for the last decade (Dóró et al., 2014; Seheri et al., 2014).

No full-length whole genome data is available from G4s collected in Africa. It was

then Important to determine how the previously common genotype G4 from this

region disappeared from circulation, whereas at the same time, new genotypes (e.g.

the G9s and G12) emerged and are spreading rapidly across human populations

(Dóró et al., 2014; Seheri et al., 2014). It was necessary to determine if there is a

genetic connection between the disappearing G4 strains and the strains that have

emerged within that time through the genetic changes which have occurred over the

years.

Of particular interest was that some RVA strains have emerged as a result of

interspecies reassortment which have resulted from insertions, deletions and

substitutions that have possibly and significantly altered the sequence arrangement

of genotype G4 (Gentsch et al., 2005; Bányai et al., 2014). Thus the evolutionary

mechanisms at the whole-genome level of this atypical RVA strain, was investigated

in order to obtain essential decisive data on the origin and evolution as well as to

gain insights into the overall genetic makeup.

1.3. Purpose of the study

1.3.1 Research question

How have human rotavirus strains G4P[6] and G4P[8] circulating in southern Africa

between 1985 to 2011 evolved?

1.3.2 Aim of the study

To investigate the evolutionary changes of human rotavirus strains G4P[6] and

G4P[8] circulating in southern Africa between 1985 and 2011.

4

1.3.3 Objectives

3.3.1. To analyse and compare the whole genome of human rotavirus genotypes

G4P[6] and G4P[8] in order to determine the genetic changes that could arise over

time in all 11 Gene segments of G4 rotavirus strains collected in southern Africa

compared to other parts of the world.

3.3.2. To determine the possible genetic variations and their implications in respect

to circulation of genotype G4 in southern Africa.

1.4.5. Significance of the study

The study provides substantial knowledge and mechanisms behind the

disappearance of previously dominant rotavirus strains and about strains that are

currently emerging in Africa, whether they are the result of reassortment of the

rotavirus genes and as well as the possible impact on the vaccine seroconvention.

The findings of this study were also significant to the on-going post-vaccine

surveillance and monitoring of the emerging rotavirus strains in southern Africa.

5

CHAPTER 2: LITERATURE REVIEW

2.1. Introduction

Rotaviruses were first isolated in tissue culture (Wyatt et al., 1980). However, the

isolation of human rotavirus was only successful after trypsin treatment. This

technique in combination with traditional viral culture methods provided more

information about the structure of rotavirus particles (Estes and Kapikian, 2007).

Rotaviruses are icosahedral in shape with three lipid capsid layers (Figure 2.1A).

Electron microscopy shows rotaviruses to be wheel shaped with a diameter of 80 nm

(Figure. 2.1B) (Yeager et al 1994; Prasad et al, 2001). The linear dsRNA genome of

rotaviruses consists of 11 dsRNA segments coding for 11 and sometimes 12

proteins (Ramig, 1997). The segment size ranges from 667 base pairs (bp) (gene

segment 11) to 3,302 bp (gene segment 1). The genome total size is approximately

18,550 bp depending on species. Gene segments 1-10 are monocistronic while gene

segment 11 codes for two proteins (NSP5 and NSP6) (Estes and Kapikian, 2007).

Figure 2.1: A; represent the Complete Rotavirus virion Cryo-EM structure filtered at 25-Å resolution. The segmentation of the structure is based on reconstructions of the complete virion and on published work of others (Yeager et al., 1994; Crawford et al., 2001; Grigorieff, 2007). B; Cryo-EM image of VP7-coated DLPs (Chen et al., 2009).

6

2.2. Rotavirus proteins

Rotavirus is a large and complex virus with a multi-layered capsid organization that

integrates the determinants of host specificity, cell entry, and the enzymatic functions

necessary for endogenous transcription of the genome that consists of 11 dsRNA

segments (Figure. 2.2A) encoding both structural and non-structural proteins

(Pesavento et al., 2006). The structural proteins are designated VP1 to VP4, VP6,

and VP7 (Figure. 2.2B) and the currently accepted nomenclature for the non-

structural proteins are NSP1 to NSP5. RNA viruses replicate in the cytoplasm of the

cell and they encode several non-structural proteins which aid in their replication and

morphogenesis inside the host cell (Pesavento et al., 2006; Estes and Cohen,

2007). However, structural studies resulting in successful expression of these genes

and the spontaneous formation of virus-like particles (VLP) have played an important

role to help understand the virus functions in the context of the three-dimensional

structures of the virus and virus-encoded individual proteins (Prasad et al., 1988;

Pesavento et al., 2006).

A clear understanding of the structure and function associations in rotaviruses has

been observed using electron cryomicroscopy (cryo- EM) techniques which had

paved the way for more elaborate structural characterization of this virus (Prasad

and Estes, 2000). X-ray crystallography has been successfully applied recently to

determine the atomic structures of several of the structural and non-structural

proteins of rotavirus (Deo et al., 2002; Dormitzer et al. 2004). Structural proteins are

named based on their molecular weights, with VP1, the largest at 125 kDa, and VP8,

one of the two proteolytic fragments of VP4, the smallest at 28 kDa (Graff et al.,

2002). The six structural proteins form the multi-layered capsid of the mature

rotavirus particle. The non-structural proteins are essential for virus replication

except for NSP1 which is an RNA-binding protein. NSP1 is an RNA-binding protein

that directly interacts with IRF-3 (Graff et al., 2002).

7

Figure 2.2: Rotavirus genome segments and their encoded proteins (Adapted from Prasad et al. 1996; Ruiz et al., 2009)

The mature infectious rotavirus particle is about 1000 Ǻ in diameter (including the

spikes), it is made of three concentric icosahedral protein layers commonly known as

TLPs that enclose the genome of 11 dsRNA segments (Shaw and Grenberg, 1999;

Pesavento et al., 2006). The capsid architecture is predominantly based on T=13

icosahedral symmetry (Parashar et al., 1998; Pesavento et al., 2006).

2.2.1. Structural Proteins

a). VP1

VP1 glycoprotein is encoded by the gene segment 1 and represents 2% of all the

virion proteins. It is an RNA polymerase enzyme and it forms the flower-shaped

VP1–VP3 transcription complex which is attached to the inside of the VP2 layer at

the five-fold icosahedral axes (Figure 2) (Lawton et al. 1997). VP1 sequences have

a long ORF encoding for the polypeptide of 1088 residues with a molecular weight of

125 kilo Daltons (Da) (Cohen et al., 1989; Arnoldi et al., 2007). VP1 contains

sequence motifs including NTP-binding activity whose analogue inhibits RNA

transcription (Mitchelle and Both., 1990). The viral polymerase (VP1) synthesizes a

capped mRNA containing 5’-methylated cap structures but lack poly-A tail from each

8

dsRNA segment (Harb et al., 2008; Estroz et al., 2012). This capped mRNA is

translocated to the cell cytoplasm where it is translated, thus the recombinant VP1

can direct template-dependent minus-strand synthesis in vitro in the presence of

VP2 (Valenzuela et al., 1991; Patton et al., 1997; Estes and Kapikian., 2007). VP1

sequences are highly conserved at the nucleotide and protein levels and the protein

is conserved in the region between amino acids 517 and 636 (Gorbalenya and

Koonin., 1988; Mitchelle and Both., 1990).

b). VP2

The innermost protein layer of the rotavirus structure VP2 is the product of rotavirus

RNA gene segment 2 which is responsible for stimulation of viral RNA replicase and

it is beneath the VP6, The VP2 encases VP1 and VP3, and the genome of 11

segments of double-stranded RNA (Conner et al., 1988; Zeng et al., 1997). The

particle structure at this level is referred to as the single-layer particle (SLP) which

encloses the dsRNA genome within a protein layer composed of 120 copies

organized into 12 decameric units of VP2 arranged in an unusual T=1 icosahedral

lattice with two molecules in the icosahedral asymmetric unit (Conner et al., 1988;

Lawton et al. 1997). VP2 5-fold hub is also important for interactions with VP1 and

VP1 is capable of recognizing viral plus-strand RNA. At the vertices of the core

protein VP2 is VP3 Guanylyl transferase mRNA capping coding enzyme. The flower-

shaped VP1–VP3 transcription complex is attached to the inside of the VP2 layer at

the five-fold icosahedral axis (Lawton et al., 1997).

c). VP3

VP3 is the glycoprotein encoded by Gene segment 3 and it is the minor core protein

component of the central core representing about one third the size of VP1. It is a

protein of 835 amino acids with a predicted molecular weight of 98 000 Da (Liu et al.,

1988). VP3 protein specifically binds to GTP and contains mRNA

guanylyltransferase and mRNA (guanine-N(7)-)-methyltransferase activities (Cheng

et al., 1999; Liu et al., 1992). It is a multifunctional enzyme involved in mRNA

capping (Cheng et al., 1999; Liu et al., 1992). VP3 also catalyses the formation of

the 5' cap structure on the viral plus-strand transcripts and drive the entry of the virus

into the cell (Patton, 2001). VP3 is also thought to impact on the transcription or

association with phosphatase or ATPase activity (Pizzaro et al., 1991). VP3

9

sequences also reveal high similarity with RNA polymerase from other viruses

suggesting that this protein is involved in RNA replication (Estes and Cohen., 1989).

d). VP4

VP4 is the product of gene segment 4 and it is the minor outer capsid which

constitutes only 2% of the viral mass and has a molecular weight of 88 000 Da (Liu

et al., 1988). The outer layer is decorated by 60 spikes, each of which is formed by a

dimer of VP4 resulting in 120 copies of VP4 in each rotavirus particle (Prasad et al.,

1990). The VP4 spike exhibits a distinct structure with two distal globular domains, a

central body, and an internal globular domain that is inserted inside the VP7 layer in

the peripentonal channel of the T=13 icosahedral lattice and it plays an important

role as the viral attachment protein and determinant of virulence (Shaw et al. 1993;

Mathieu et al., 2001).

The VP4 of RVA has a long ORF of 775 amino acids. The VP4 protein carries

neutralization-specific epitopes and it is post-translationally cleaved by trypsin at

conserved arginine residues at amino acid position 241 or 247, into two

polypeptides, VP5 (a protein of about 60 kDa) and VP8* (a protein of about 28 kDa)

which enhances the virion’s infectivity and cell penetration (Estes and Cohen., 1989;

Lopez et al., 1985; Greenberg et al., 1983). Additional cleavage sites have been

revealed at amino acid position 246 which has been suggested to be associated with

virulence. In addition to the conserved arginine residues mentioned above, other

conserved cysteine residues are found in positions 216, 318, 380 and 774 in all

rotavirus strains (Gorziglia et al., 1988) with most animal strains having another

additional cysteine residue at position 203 which has been suggested to stabilizes

the protein structure through disulphide bond (Mackow et al., 1988).

Variable regions were found close to the amino terminus in the region of VP8 at

amino acid 71-204 locating an antigenic site in the VP5 and five antigenic sites in

VP8 in amino acids 88/89, 114, 148/150 and 188 (Mackow et al., 1988). Three

epitopes have also been documented at amino acids 306, 393/440 and 433

(Mackow et al., 1988; Taniguchi et al., 1987).

e). VP6

VP6 glycoprotein is the product of the rotavirus RNA gene segment 6 and is the

major structural protein of the inner viral capsid constituting up to 50% of the virion

10

with a molecular weight of 44816 Da (Liu et al., 1988; Prasad et al. 1988). VP6

glycoprotein forms the intermediate layer and is in direct contact with the outer

surface VP7 glycoprotein. Particles carrying VP6 on the outside are called double

layered particles (DLPs) (Pesavento et al., 2006). The VP6 layer maintains the same

icosahedral symmetry as the VP7 layer with 780 copies of VP6 arranged as 260

trimerson a T=13 icosahedral lattice (Mathieu et al., 2001). These trimers are located

right below the VP7 trimers such that the channels in the VP7 and VP6 layers are in

correspondence. The DLP is the transcriptionally competent form of the virus during

the replication cycle (Prasad et al., 1988). VP6 is the major protein of the rotavirus

particle by weight and it plays a key role in the fundamentally organization of the

rotavirus architecture by interacting with the outer layer proteins, VP7 and VP4, and

the inner most layer protein VP2 (Shaw and Greenberg., 1999; Prasad et al. 1990). It

may also integrate two principal functions of the virus which are cell entry (outer

layer) and endogenous transcription (inner layer) (Kuhil et al., 1992; Prasad et al.

1996). VP6 is also the primary target antigen for routine serological diagnostic

techniques of RVA such as enzyme-linked immunosorbent assay (ELISA),

immunofluorescence, and immunochromatography (Greenberg et al., 1983; Estes

and Kapikian, 2007).

f). VP7

The major constituent of the outer layer is VP7 glycoprotein and it is the product of

RVA RNA gene segment 7. Seven hundred eighty copies of VP7 are grouped as 260

trimers at all the icosahedral and local three-fold axes of a T=13 icosahedral lattice

surrounding 132 channels (Prasad et al. 1996). The glycoprotein VP7 is glycosylated

and makes up to 30% of the viral mass (Liu et al., 1988).

The VP7 gene segment sequence analysis reveals two ORFs which encode

polypeptides of 326 and 286 amino acids (Gorziglia et al., 1986). The nucleotide

sequence predicts one major reading frame yielding a protein that includes two NH2

terminal hydrophobic regions and a single potential glycosylation site (Both et al.,

1983). Nearly all VP7 molecules are characterized by the present of glycosylation

site at position 69, whereas other glycosylation sites are located at amino acid 146,

238 and 318 (Desselberg and McCrave, 1994; Gorziglia et al., 1986). There are also

conserved cystei residues that can be observed in amino acid 82, 135, 165, 191,

11

207, 244 and 249. In addition there is also proline residues found at amino acid 58,

86, 112, 131, 167, 197, 254, 275 and 279 (Desselberg and McCrave, 1994).

2.2.2. Non-Structural proteins

a). NSP1

The product of RV gene segment 5 is the non-structural protein NSP1

(Desselberger, 2014). NSP1 is a 57-kDa protein whose length ranges from 486 to

496 amino acids. NSP1 is the least conserved gene segment among the RVA

proteome, with sequence variability highest in the C-terminal half (Mitchell and Both,

1990; Palombo and Bishop, 1994). Phylogenetic analysis from previous studies

reveals that NSP1 sequences cluster according to host species (Hua et al., 1993;

Kojima et al., 1996) indicating a possible role for NSP1 in host range restriction

(Ciarlet et al., 1998; Feng et al., 2011; 2013). An intact NSP1 protein is not essential

for RV to disseminate in permissive cell culture and a number of laboratory strains

contain a rearranged gene 5 that encodes a C-terminally condensed NSP1 (Hua and

Patton, 1994; Taniguchi et al., 1996). Phylogenetic and biochemical analyses have

identified key structural and functional domains on the NSP1 gene sequences

although significant sequence variability between NSP1 proteins exists (Patton et al.,

2001). NSP1 contains a cysteine-rich conserved zinc binding, putative C4-H-C3

RING domain (consensus sequence: CX2-C-X8-C-X2-C-X3-H-X-C-X2-C-X5-C) that

spans from residues 42–72 in most sequences (Brottier et al., 1992; Palombo and

Bishop, 1994).

Conservation occurs at amino acid positions between 37 to 81 containing one of the

zinc binding domains. NSP1 also have a specific affinity for all 11 rotavirus mRNAs

which mediate a specific interaction with the 50 termini of viral mRNAs, which may

prevent activation of cellular RNA sensors (Hua et al., 1994). NSP1 is not required

for rotavirus replication; however it clearly plays a role in the replication of the

rotaviruses (Tian et al., 1993).

b). NSP2

NSP2 is encoded by the Gene segment 8 and has a molecular weight of 37 000 Da

conforming to a polypeptide of 317 amino acids. The NSP2 carboxy-terminal half

contains highly conserved region of 37 amino acids at 205-241 that is extremely

basic and is linked to serve as the RNA-binding domain (Patton et al., 1993). NSP2

12

is a non-specific RNA binding protein in the infected cell where it is localized in

viroplasm and exhibits oligomeric NTPase activity and it is also involved in

packaging of viral mRNA (Ramig and Petrie, 1984; Estes and Kapikian, 2007). Thus

this suggests that NSP2 plays a major role in RNA replication or sera packaging into

sub-viral particles (Patton et al., 1995). It has also been shown that NSP2 has affinity

for RNA although the binding activity is not virus-specific (Kattoura et al., 1994).

NSP2 have multiple activities which includes binding single-stranded RNA non-

specifically via Mg2+-dependent nucleoside triphosphate activity (NTP) in vitro with

purified recombinant and radiolabelled NTPs. NSP2 has helix-destabilizing activity

with purified bacterially expressed proteins and it is the component of replication

intermediates (Taraporewala and Patton, 2001; Taraporewala et al., 2006). This

indicates that NSP2 is capable of unwinding and also help package RNA and bind to

and work with VP1 in creating its functionality (Kattoura et al., 1992; Jayaram et al.,

2002). NSP2 appear to be responsible for the replication of virus RNA and for its

incorporation into new virions. NSP5 seems to facilitate NSP2 and VP2 to delay the

assembly of the outer capsid long enough to allow RNA replication to complete

(Berois et al., 2003; Taraporewala et al., 2006).

c). NSP3

NSP3 protein is encoded by Gene segment 7 and has a molecular weight of 36 000

Da conforming to a polypeptide of 315 amino acids. NSP3 has been found in large

amounts in infected cells with complexes containing replicas activity (Patton and

Gallegos, 1988). NSP3 is a slightly acidic protein that occurs in large quantities in the

infected cell and is associated with the viral cytoskeleton (Patton et al., 2001). NSP3

sequence analysis has revealed that it is predominantly helical in nature and

contains half of conserved hydrophobic heptad repeat (amino acids 181-236) region

within its COOH-terminus (Rao, 1995; Mattion et al., 1992).

NSP3 protein is responsible for helping to regulate the synthesis of mRNA from the

virus dsRNA genome (Patton et al., 2001). NSP3 has been found to interact with

eukaryotic initiation factor 4 gamma 1 (elFGI) complexes which regulates the

circularization of mRNA by binding simultaneously to the 3’ consensus sequence

and cap binding protein elF4G (Piron et al., 1998). Thus NSP3 is generally involved

in new viral mRNA translational regulation (Ogden et al., 2014)

13

d). NSP4

The NSP4 is a non-structural transmembrane protein of the rough ER-specific

glycoprotein. NSP4 is encoded by rotavirus Gene segment 10 and has a molecular

weight of 28 000 Da with a polypetide of 175 amino acid residues (Mattion et al.,

1994). NSP4 is the only non-structural protein of rotavirus that does not interact with

viral nucleic acid and it has been shown that it functions as an enterotoxin, viral

replication, and pathogenesis and is also essential for virus assembly (Estes, 1996;

Estes and Kapikian, 2007). NSP4 amino acid terminus is maintained in the luminal

side and carboxy terminus is extended into the cytoplasm (Chan et al., 1988).

Rotavirus NSP4 plays an important role in the morphogenesis in infected cells as it

act as intracellular receptor by mediating the conversion of double-layered particle in

the ER that transports sub-viral particles across the membrane of the ER (Chan et

al., 1988). NSP4 mutational changes have been associated with altered virus

virulence which plays a role in NSP4 pathogenesis (Zhang et al., 1998; Fredj et al.,

2012; Bertol et al., 2015). The other function of NSP4 lies in its ability to modulate

intracellular calcium levels and chloride secretion (Tian et al., 1994; Silvestri et al.,

2005). Previous studies showed that the enterotoxin activity of NSP4 may be

responsible for the severe diarrhoeal onset observed during initial rotavirus infections

of animals prior to the detection of histological changes in the intestine (Burns et al.,

1995; Ward et al., 1996; Zhang et al. 1998).

e). NSP5/6

NSP5 is the product of gene segment 11 with a molecular weight of 26 000. The

Gene segment 11 is the smallest genomic segment of rotavirus genome encoding

two proteins, NSP5 and NSP6. The NSP5 gene contains an ORF starting at

nucleotide 22 encoding polypeptide of 198 amino acids conforming to the molecular

weight of 21 500 Da and NSP6 protein of 92 amino acids conforming to molecular

weight of 11 000 Da; both of which accumulates in viroplasm (Petrie et al., 1984;

Mitchell and Both, 1988; Estes and Kapikian, 2007). NSP6 have been shown to

interact with NSP5 during the process of replication, although the NSP6 function

remains unclear (Torres-Vega et al., 200). NSP6 appears to change the three-

dimensional structure of NSP5 in order to allow the NSP5 to function more effectively

(Torres-Vega et al., 2000).

14

NSP5 is an O-glycosylated phosphoproten which have been shown to interact in a

sequence independent fashion with both double-stranded and single-stranded RNA

(Gonzalez and Burrone, 1991; Afrikanova et al., 1996; Vende et al., 2002). Some

rotaviruses do not have this ORF which may indicate that NSP6 is not essential for

virus replication (Mattion et al., 1991; Taraporewala and Patton, 2004).

2.3. Rotavirus genotype distribution

RVs are divided into eight distinct species or groups (RVA to RVH) based on either

electropherotype migration pattern or VP6 based antigenic properties (Estes and

Kapikian, 2007; Matthijnssens et al., 2012). A recently identified canine RV species

has been tentatively named Rotavirus I (RVI) and may represent the ninth RV group,

although further ratification by the International Committee on Taxonomy of Viruses

(ICTV) is warranted (Mihalov-Kovács et al., 2015). Rotaviruses are also further more

widely classified according to their outer capsid serotype and genotype as VP7

(designated G) and protease-sensitive VP4 (designated P) respectively (Hoshino et

al., 1985; Estes and Kapikian, 2000). As from 2008 studies have come up with a new

nucleotide-based complete genome classification system which assigns specific

genotypes to each of the 11 Gene segments according to the determined cut-off

values (Matthijnssens et al., 2008).

There is a high number of possible G/P genotype combinations circulating

worldwide, approximately 88% of RVA infections worldwide are instigated by the five

common human genotypes G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8] (Bányai et

al., 2012). The remaining of infections are instigated by strains that present

uncommon or rare genotypes or are untypable in either G or P genes, or are

associated with multiple RVA strains (Hoshino and Kapikian, 2000). The most

common circulating G- genotypes worldwide are G1–G4, G9 and three P- genotypes

(P[4], P[6], P[8]) (Matthijnssens et al., 2010). In African countries, RVA circulating in

the human population often exhibit genotypes markedly different from those

observed in the rest of the world (Armah et al., 2010). Genotypic distribution of

rotavirus strains shows temporal and geographical fluctuations. For example, in

Korea G1P[8] was the most frequent genotype in children while G4P[6] were the

most frequent genotype in neonates (Jae-Seok et al., 2013). Recent studies reports

that the molecular characterization of G4P[8] RVA strains circulating in Italy in 2013,

and the analyses conducted suggest that the co-circulation of different G4P[8]

15

variants occurs during the same season (Hoshino and Kapikian, 2000). Thus

different evolutionary lines of common G4P[8] rotaviruses were likely generated by

random point mutations as the study suggested, but part of intra-genotype

reassortment events played a major role in the diversity of this genotypes and thus

cannot be excluded, particularly for VP4, VP6 and NSP4 genes (Hoshino and

Kapikian, 2000).

Serotype analysis on the distribution of rotavirus G showed that 71% were serotype

G4, and there was no serotype G9s detected from 1977-1988 (Santos and Hoshino,

2005). The outcomes show that there has been a variation in the predominant strain

of RV from serotype G4 to serotype G9 and in some cases to serotype G12 in

children with diarrhoea over the last two decades and the frequency of individual

serotypes in a particular region can fluctuate annually, and within the same country

the RV genotype distribution can vary during the same year in different regions

(Santos and Hoshino, 2005). This G9 serotype transformation has implications for

vaccine development strategies and recommendations in which protection against

serotype G9 may be required for a successful vaccination program in a particular

country. Thus continuous surveillance is also important to detect any change in the

predominant strain over time (Hung et al., 2006).

This wide variation in the proportion of RV-associated diarrhoea could be due to

various factors such as differences in methodology of study, improvements in the

storage, transport and processing of stool specimens, an increase in the sensitivity

of the tests used to detect RV, or an actual increase in the proportion of RV-

associated diarrhoea (Hung et al., 2006). In temperate countries, seasonal variation

has been observed with a peak incidence occurring during the winter months.

Studies from some tropical countries where there are distinct cooler months have

also shown that the incidence is higher during the cooler months, while others have

shown no significant correlation (Hung et al., 2006; Chang et al., 2015). In tropical

countries where the main seasonal variation is the amount of rainfall, there was no

significant correlation observed between the incidence of RV-associated diarrhoea

and the amount of rainfall (Hung et al., 2006). Thus in many study periods over the

years; literature shows that there is no seasonality that can be significantly observed

in either site (Hung et al., 2006).

16

2.4. Life cycle and replication of rotavirus

Rotaviruses primarily infect the mature non-dividing enterocytes of the small

intestines as they have the necessary sialic acid receptors required for VP4 binding

and have been reported to cause extra intestinal infections as well (Crawford et al.,

2006; Ramig, 2004). Throughout the replication cycle, from entry to the release of

newly formed particles, the developing virions transit through different cellular

compartments characterized by a distinct [Ca2+] which is the determinant for the

replication process (Ruiz et al., 2009). Rotavirus entry into the host cell is a complex

multistep process in which different domains surface proteins interact with different

cell surface molecules, which act as receptors on the plasma membrane (Ruiz et al.,

2009).

Following endogenous transcription and release of the transcripts, the rotavirus

replication cycle may be viewed as having three subsequent major stages: (a)

translation and synthesis of the viral proteins; (b) replication, genome packaging, and

DLP assembly; (c) budding of the newly formed DLPs into the ER and assembly of

the outer layer to form mature TLPs (Estes, 2001). The positive-stranded RNA

transcripts encode the rotavirus proteins and function as templates for production of

negative strands to make the progeny dsRNA (Pesavento et al., 2006). Recent

studies with small interfering RNA (siRNA) have indicated that there are likely to be

two separate pools of mRNA for these distinct functions (Silvestri et al., 2004). The

non-structural protein NSP3 is implicated in the specific recognition of the rotavirus

mitochondrial RNA (mRNA)s and in facilitating their translation using the cellular

machinery (Vende et al., 2000).

2.4.1. Attachment and entry

Attachment of rotavirus to its receptor via the initial interaction of the outer capsid

proteins VP4 and VP7 stimulate the formation of an endocytic vesicle (Figure. 2.3)

thereby isolating the TLP within an intracellular compartment (Chemello et al., 2002;

Ruiz et al., 200). This is followed by a progressive decrease in Ca2+ concentration in

this vesicle from the extracellular to the intracellular by diffusion through activated

Ca2+ Channels or transport mechanisms (Gerasimenko et al., 1998). When the

endosomal Ca2+ concentration equals that of the cytoplasm and is below the crucial

levels for stability of the outer capsid, the virus sheds its outer proteins and this leads

17

to lysis of the vesicle membrane, permitting the DLP to pass into the cytoplasm (Ruiz

et al., 1997).

Figure 2.3: Replication cycle of rotavirus. The rotavirus triple layered particles (TLPs) first attach to sialo-glycans (or histo-blood group antigens) on the host cell surface, followed by interactions with other cellular receptors, including integrins and Hsc70. Virus is then internalized by receptor-mediated endocytosis. Removal of the outer layer, triggered by the low calcium of the endosome triggers the removal of the outer layer resulting in the release of transcriptionally active double-layered particles (DLPs) into the cytoplasm. The DLPs start rounds of mRNA transcription, and these mRNAs are used to translate viral proteins. The RNA genome is replicated and packaged into newly made DLPsin specialized structures called viroplasms once enough viral proteins are made, which interact with lipid droplets. The newly made DLPs bind to NSP4, which serves as an endoplasmic reticulum (ER) receptor,and bud into the ER. NSP4 also acts as a viroporin to release Ca2+ from intracellular stores. Transiently enveloped particles are seen in the ER. The transient membranes are removed as the outer capsid proteins VP4 and VP7 assemble, resulting in the maturation of the TLPs. The progeny virions are released through cell lysis. In polarized epithelial cells, particles are released by a non-classical vesicular transport mechanism (Desselberger,

2014).

2.4.2. Transcription and Translation

Transcription is activated when the DLP reaches the cytoplasm after the loss of the

outer capsid layer (Figure. 2.3). This transition may be linked to the low Ca2+ levels in

the cytoplasm (Charpilienne et al., 2002; Desselberger, 2014). The dsRNA segments

18

are transcribed within the structure of the DLP by VP1 which have RNA-Dependent

RNA polymerase activity and capped by VP3 which has both guanyltransferase and

methyltransferase activities (Cheng et al., 1999). The mRNAs that are synthesized

exit the DLP through 12 aqueous channels that pass through both the VP2 and VP6

protein layers (Pesavento et al., 2006).

Translation of the synthesized mRNAs takes place in the polysomes forming the

11/12 viral proteins (six structural and six/five non-structural proteins). NSP3 has

been shown to be involved in both rotavirus replication processes. The synthesized

mRNAs lacks poly (A) tail, however it has a consensus sequence at the 3’ ends that

binds NSP3 which serve as functional homologue of poly (A) binding protein (PABP)

(Chen et al., 1999). NSP3 has also been shown to interact with the cell-initiation

factor elF4GI and it has been shown that these interactions results in translation of

rotavirus mRNAs hence impairing the translation of cellular mRNAs at the same time

(Charpilienne et al., 2002).

2.4.3. Replication of genomic RNA

Replication of rotavirus dsRNA takes place in a conservative way whereby both

strands of the parental dsRNA remain within partially uncoated particles (Patton et

al., 2006). The dsRNA remains associated with the sub-viral particles after synthesis

which suggests that free dsRNA is not found in cells (Smith et al., 2005; Kapahnke et

al., 1986). The sub-viral particles in which dsRNA synthesis occurs consists of

structural proteins VP1, VP2 and VP6 and as well as non-structural proteins NSP1,

NSP2 and NSP3 (Helmberg-jones and Patton, 1986). Non-structural proteins are

involved in replication process due to their presence in replication complexes being

isolated from infected cells (Vende et al., 2002; Patton and Gallegos, 1988), their

nucleic acid-binding activity, their localization to viroplasms and their RNA negative

phenotype of the temperature sensitive mutants mapped to the genes encoding

these non-structural proteins (Mattion et al., 1992; Gonzalez et al., 1998; Chen et al.,

1990).

The Non-structural proteins NSP2, NSP5 and viral proteins VP1, VP2 and VP3 in the

viroplasm are the main constituent of the replication intermediates and are also

involved in genome replication and packaging (Gonzalez et al., 200). The role of the

non-structural protein NSP6 which is encoded by the Gene segment 11’s alternative

19

ORF and interacts with NSP5, in the replication process remains unclear, however it

has been suggested that NSP6 have regulatory role in the self-association of NSP5

(Torres-vegas et al., 200).

2.4.4. Genome packaging

Three models that describes how packaging occurs have been proposed and they

includes formation of precore replication intermediate precursor complexes

composed of viral mRNA, the viral RNA dependent RNA polymerase and the

capping enzyme that serves as a nucleation site for the binding of the VP2 core

protein (Pasavento et al., 2006). During encapsidation process the capsid assembly

begins with the association of 12 units consisting of pentamers of VP2 dimers in

complex with a transcription enzyme complex (VP1/VP3) and a genome, to form the

SLP and provide a temporary structure for the consecutive assembly of the VP6

trimers leading to the assembly of the DLP (Berois et al., 2003). The second model

is based on the ability of the rotavirus capsid proteins to self-assemble into empty

VLPs suggesting that empty cores are first made and that mRNAs would be inserted

into the cores (Crawford et al., 1994). The last model is based on the structural data

indicating that the core represents a collection of functionally separate pentameric

units, each containing its own RNA-dependent RNA polymerase activity and the

capping enzyme complex responsible for transcription of one of the Gene segments

(Lawton et al., 1997).

2.4.5. Viral Assembly and release

The sub-viral particles of rotavirus bud through the membrane of the endoplasmic

reticulum and maturing particles are transitorily enveloped. The envelope acquired

during the process is lost as particles move towards the interior of the endoplasmic

reticulum and is replaced by a thin layer of protein that ultimately constitutes the

outer capsid of mature virions (Estes and Kapikian, 2007; Gonzalez et al., 2000).

NSP4 plays a major role in the assembly process and also serve as an enterotoxin

although it does not bind to RNA. NSP4 also contains binding sites for VP which

plays a role in removing transient envelops (Tian et al., 1996). The process of

rotavirus maturation depends on calcium but the budding process occurs in the

absence of calcium. However calcium must be present in cells for correct epitope

formation (Dormitzer and Greenberg, 1992).

20

Rotavirus infection cycle ends when the progeny virus exit the host cell following

host cell lysis from non-polarized cells (Jourdan et al., 1997). Drastic alteration in the

plasma membrane permeability of infected cells as well as extensive cytolysis during

infection results in the release of cellular and viral proteins (Dormitzer and

Greenberg, 1992). The VP4 protein interacts with the actin and lipid rafts and

remodels the filaments whereby the brush border membrane of polarized epithelial

cells may be destabilised to facilitate rotavirus exit from the host cells (Gardet et al.,

2006).

2.5. Pathogenesis and outcomes

The clinical presentation of RVA comprises of an extensive range of severity of

symptoms, ranging from asymptomatic infection to severe dehydrating

gastroenteritis. Pathological changes are limited to the small intestines (Aupiais et

al., 2009). The high mortality and morbidity are due to severe dehydrating diarrhoea

and vomiting. High costs to the health care system can also lead to death for

children who do not receive treatment in time and often occurs in developing

countries (Tate et al., 2012). There is little knowledge about the immunology of the

host or viral factors that contribute to severe disease (Bahl et al., 2005; Aupiais et al.,

2009). Rotavirus diarrhoea progression has been linked to several different

mechanisms, including malabsorption secondary to enterocyte destruction, a virus-

encoded toxin, stimulation of the enteric nervous system (ENS), and villus ischemia

(Ramig, 2004). Recent studies have investigated associations between infection with

rotavirus G types and P types and illness severity, but there are still no reliable

consistent patterns in terms of which genotypes are associated with particular severe

disease (Aupiais et al., 2009).

Iis thought that rotaviruses counteract many antiviral pathways in a strain-specific

manner, for example NSP1 protein induces the proteasome-dependent degradation

of IRF3, IRF5, and IRF7 to prevent their induction of IFN (Ramig, 2004). Rotavirus

can prevent STAT1 and STAT2 nuclear translocation. Host gene expression shut-off

by virus NSP3 protein evicts cytoplasmic poly-(A) binding protein (PABP) from

translation initiation complexes and thus shuts off the translation of cellular

polyadenylated mRNAs. Inhibition of host PABP by virus results in host translation

being shut-off in the intestine (Connar and Ramig, 1997; Aupiais et al., 2009)

21

2.6. Detection, prevention and control

2.6.1. Laboratory diagnosis

For direct detection of rotavirus, stool specimens should be collected during acute

infection preferably within the first 3-5 days of illness because virus shedding

coincides with the duration of the symptoms (Kapikian et al., 2005). Rapid test is

one of the most widely used methods of testing rotavirus specimens based on the

immunochromatography technique. This involves detection of viral antigens, viral

nucleic acids (dsRNA) or serological response. For rotavirus antigen detection

commercially available kits including Enzyme Immuno Assay (EIA) and Latex

Agglutination Test are available (Christensens, 1999). EM is another reliable method

used to detect rotaviruses based on their distinctive morphology. Examination EM of

a negatively stained stool sample by EM is a relatively rapid diagnostic method when

only a small numbers of specimens are tested. EM is widely used to detect groups A

and B rotaviruses as well as other enteric viruses and can be used as confirmatory

method when any discrepancies arise with other techniques (Christensens, 1999;

Estes and Kapikian, 2007). Faecal samples positive for RV antigen can be further

analysed through RT-PCR and genotyping to determine the specific genotypes

(Iturriza-G´omara et al., 1999).

2.6.2. Vaccination of rotavirus

World Health Organization (WHO) recommended that the rotavirus vaccine be

incorporated into routine national immunization programs in all countries particularly

in those countries with high diarrhoea-related mortality in children less than five

years (Danchin and Bines, 2009). There are two oral rotavirus vaccines currently

licenced for use worldwide, RotaTeq (Merck & Co., Whitehouse Station, NJ, USA)

and Rotarix (GlaxoSmithKline, Rixensart, Belgium). RotaTeq is a pentavalent

vaccine consisting human rotavirus A genotype genes (G1–4 and P[8]) into a bovine

WC3 strain to create five different reassortant strains. RotaTeq® is thus an oral

pentavalent human-bovine reassortant vaccine that is administered in a 3-dose

schedule (Kapikian et al., 2005). Rotarix is a monovalent vaccine composed of an

G1P[8] rotavirus strain that is designed to provide genotype-specific and heterotypic

protection against common rotavirus A genotypes. Rotarix® is an oral attenuated

human monovalent vaccine (G1P[8]) that is administered in a two-dose schedule.

22

These vaccines have proven to be safe and effective in clinical trials in different

countries.

South Africa was the first country in Africa to implement the use of rotavirus vaccine

Rotarix®, GSK Biologicals into the Expanded Program on Immunization (EPI) in

August 2009. It is administered at 6 and 14 weeks of age. RotaTeqTM is also in use

in the private sector EPI. Introduction of rotavirus vaccines has led to rapid decrease

of the burden of rotavirus disease in the world (Anderson, 2014)., However, rotavirus

infection in rotavirus-vaccinated children is still frequently noted in clinical cases but

with less severe disease. This observation could be due to the emergence of vaccine

escape mutations among the circulating genotypes (Rennels 1996; Bányai and

Gentsch, 2014).

23

CHAPTER 3: MATERIALS AND METHOD

3.1. Ethical considerations

The study was approved by the School of Pathology and Pre-clinical Sciences

Research Ethics Committee (SREC) and from the Sefako Makgatho Health Sciences

University Research Ethics Committee (SMU-REC) under the SMUREC number

(SMUREC/P/144/2015:PG). Only assigned laboratory numbers were used

throughout the study in order to delink sample information from the patients.

3.2. Study design

3.2.1. Description of the study

This was an exploratory study to characterize whole genome sequences of human

rotavirus genotype G4 with P[6] and P[8] combinations circulating in southern Africa

between 1985 and 2011.

3.2.2. Study population and study sample

The study population consisted of archival diarrhoeal stool specimens stored at -

200C at the South African Medical Research Council, Diarrhoeal Pathogens

Research Unit (MRC-DPRU). The samples were collected between 1985 and 2011

from children under 5 years of age presenting with rotavirus gastroenteritis in

hospital and sentinel sites in South Africa, Zambia and Zimbabwe. Three samples

that meet the selection criteria of being G4s were selected one was previously

genotyped as G4P[6] and the other two were G4P[8] strains.

3.3. Laboratory methods

3.3.1 Enzyme immunoassay (EIA)

Stool samples were confirmed for rotavirus antigen using a commercially available

rotavirus kit (ProSpecT™ Rotavirus Kit, Oxoid, Ltd. UK), according to the

manufacturer’s instructions. Briefly, five micro plate strips were removed from the kit

and placed into a microplate strip holder. The samples were diluted to a 10% dilution

by adding approximately 0.1g of faecal specimen into labelled tube containing 1ml of

sample diluent provided in the kit using a 1000μl pipette. The solution was mixed

thoroughly and 100μl of the 10% specimen was added into each microwell. In

addition, negative and positive controls provided in the kit were also added. Two

24

drops of conjugate was then added into each microwell and the plate was covered

and incubated at 300C for 60 minutes.

The washing process was done five times by completely filling each well with

working solution wash buffer, and then removing all fluid from the wells after each

wash. All traces of wash buffer were removed by thoroughly inverting the plate and

tapping it on absorbent paper towel in a bench top. Two drops of substrate were

added into each microwell. The five plates ware then covered and the microplates

were incubated at 300C for 10 minutes. Thereafter an acidic stop solution was

immediately added into each microwell and read spectrophotometrically at 450nm.

All positives specimens were stored at -200C and were later used in this study.

3.3.2. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

3.3.2.1. RNA Extraction

Extraction of rotavirus dsRNA was performed using the QIAamp® RNA extraction kit

(Qiagen, Netherlands) following the manufacturer’s instructions. The first step

involved the preparation of reagents, where the carrier RNA (Qiagen, Netherlands

was added to the AVL lysis buffer, and 100% ethanol was added to wash buffer

1(AW1) and 2 (AW2).

Thereafter 560μl AVL lysis buffer containing carrier RNA was pipetted into a labelled

1.5ml micro centrifuge tube. Each 100μl or 1 g of stool sample was diluted with 1ml

of distilled water and 140μl was added to the tube that contained lysis buffer, which

were then mixed by pulse-vortexing (Labsol Enterprise, India) for 15 seconds. The

tube was then incubated at room temperature for 10 minutes. The lysis buffer

inactivated RNases and ensured isolation of intact RNA, while carrier RNA improved

the binding of RNA to the QIAamp® membrane throughout the incubation period.

560μl of absolute ethanol (96%) was added to the tube and mixed by pulse-vortexing

for 15 seconds. Then 630μl of the solution was carefully transferred from the micro

centrifuge tube to a QIAamp mini spin column that was attached to a 2ml collection

tube (Qiagen, Netherlands). The column was then centrifuged (Eppendorf 5415R,

Eppendorf AG, Germany) at 8 000rpm for 1 minute. The collection tube containing

the filtrate was discarded and the QIAamp mini spin column was placed into a clean

2ml collection tube. An additional 630μl was transferred as described above and

centrifuged at 8 000rpm for 1 minute. The QIAamp mini spin column was placed in a

25

clean 2ml collection tube and the tube containing the filtrate was discarded. The

QIAamp mini spin column was opened cautiously avoiding contamination and 500μl

of wash buffer 1 (AW1) was added. The tube was then centrifuged at 8 000rpm for 1

minute. The tube containing the filtrate was discarded and the QIAamp mini spin

column was placed into a clean collection tube. Then 500μl of wash buffer 2 (AW2)

was added as above and centrifuged at full speed of 12 000rpm for 3 minutes. The

QIAamp mini spin column was centrifuged further after discarding the collection tube

with filtrate and replacing with another to ensure that all contaminants were

completely removed. The QIAamp mini spin column was then placed in clean 1.5ml

labelled micro centrifuge tube and 60μl RNase free elution buffer was added to the

column and incubated at room temperature for 5 minutes. RNA was eluted by

centrifuging at 8 000rpm for 1 minute. The column was then discarded and the 1.5

ml micro centrifuge tube containing total RNA was stored at -20°C. One Positive and

one negative control were included in this extraction for validity of the procedure.

3.3.2.2. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The RT-PCR involved denaturation of the extracted viral RNA, reverse transcribing

the dsRNA to cDNA and then amplifying cDNA though a PCR. An RT master mix

was prepared (Appendix 1.) in a sterile labelled 1.5ml micro centrifuge as shown in

Table 2.1 in a designated RT-PCR room. A PCR master mix containing all the

components required to amplify a DNA strand was also prepared in a labelled sterile

1.5ml eppendorf tube also in a designated PCR room.

Products were quantified using a SYBR green dsDNA detection assay (SYBR Green

I Nucleic Acid Gel Stain, Thermo Fisher Scientific, Waltham, MA, USA), and all 11

RT-PCR products for each genome were pooled in equimolar amounts. Pooled

amplicons were then sequenced using the Illumina sequencing platforms. Illumina

libraries were prepared using the Nextera DNA Sample Preparation Kit (Illumina,

Inc., San Diego, CA, USA) with half-reaction volumes and DNA amplicons were

tagmented at 55⁰C for 5 minutes. Tagmented DNA were cleaned with the ZR-96

DNA Clean & Concentrator Kit (Zymo Research Corporation, Irvine,CA,USA) (Nyaga

et al., 2014; 2015).

Illumina sequencing adapters and barcodes were added to tagmented DNA via PCR

amplification using 20µl tagmented DNA combined with 7.5µl Nextera PCR Master

26

Mix, 2.5µl Nextera PCR Primer Cocktail, and 2.5µl of each index primer (Integrated

DNA Technologies, Coralville, IA, USA) for a total volume of 35µl per reaction. Ion

Torrent libraries were prepared by shearing pooled RVA amplicons for 12 minutes,

and Ion Torrent compatible barcoded adapters were ligated to the sheared DNA

using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham,

MA, USA) to create libraries. Barcoded libraries were thereafter pooled in equal

volumes and cleaned with the Ampure XP Reagent (Beckman Coulter, Inc., Brae,

CA, USA).

3.3.3. Gene segment 4 and 9 RT-PCR

For each RT-PCR amplification reaction, a sterile 0.5ml eppendorf tube was labelled

including positive and negative amplification reaction controls. 1μl of each of the two

primers set sBeg9 and End9 for VP7 (Góuvea et al, 1990) and Con2 and Con3 for

VP4 were added separately to 8μl of the dsRNA template into the amplification

reaction tube and mixed by pipetting. The tubes were then incubated at 95°C for 5

minutes to allow the dsRNA to denature. Thereafter the tube was then removed from

the heating block (Dry block heaters, Thermo Fisher scientific Inc, USA) and placed

on ice and immediately after the tubes were removed from the heating block, 3.2μl of

the prepared RT master mix was added within 5 minutes. The tube was centrifuged

for 10 seconds at 12 000rpm and incubated at 42°C for 30 minutes. After incubation

the tube was centrifuged for 10 seconds at 12 000rpm and 40μl of the prepared PCR

master mix was added (Appendix 1). The cDNA was then placed in a thermocycler

(G-storm Thermal Cycler Systems, UK). The 30 cycles of PCR amplification

consisted of denaturation at 95°C for 2 minutes, annealing at 42°C for 1 minute and

elongation at 72°C for 7 minutes, and the reaction’s holding temperature was at 4°C

after the final cycle. Positive and negative controls were included in the reactions.

3.3.4. Genotyping

Genotyping master mix for G-typing and P-typing was prepared in a sterile labelled

1.5 ml micro centrifuge tube as shown in appendix 2.

3.3.4.1. G-typing

A sterile 0.5ml eppendorf tube was labelled for each amplification reaction including

a positive and negative control. 0.5μl of the gene segment 9 cDNA and 40μl of the

prepared genotyping master mix (Appendix 2) were added to the tube while working

27

on ice. The master mix contained a genomic segment 9 cocktail of primers

consisting RVG9 and aAT8v, aCT2, aDT4, mG3, mG9, mG10, G12, representing

G1-G4, G8-G10 and G12 (Góuvea et al., 1990; Iturriza-Gómara et al., 2004).

3.3.4.2. P-typing

A sterile 0.5 ml labelled eppendorf tube was prepared for each amplification reaction

which included positive and negative controls. At a volume of 0.5 μl the gene

segment 4 cDNA was added to the tube followed by 40 μl of the prepared

genotyping master mix while working on ice. The master mix contained a gene

segment 4 cocktail of primers: Con3 and 1T-1, 1T1-VN, 1T-1Wa, 2T-1, 3T-1, 4T-1

and 5T-1 representing P[6], P[4], P[8], P[11] and P[14] (Gentsch et al., 1992; Iturriza-

Gómara et al., 2004).

The cDNA for both G-typing and P-typing was amplified by PCR in a thermocycler.

The 30 cycles of PCR amplification consisted of denaturation at 95 °C for 2 min,

annealing at 42 °C for 1 min and elongation at 72 °C for 7 min, followed by

incubation on holding temperature at 4 °C after the final cycle (Gentsch et al., 1992).

3.54. Data analysis

3.5.1. Sequence analysis

The sequencing reads from the Ion Torrent PGM and Illumina MiSeq v2 instrument

will be sorted by barcode, trimmed and de novo assembled using CLC Bio’s

clc_novo_assemble program (QIAGEN, Hilden, Germany). All sequence reads were

then mapped to the reference RVA segments which were selected using CLC Bio’s

clc_ref_assemble long program. At loci where both Ion Torrent and Illumina

sequence data agree on a variation (as compared to the reference sequence), the

reference sequences were updated to reflect the difference (Nyaga et al., 2014;

Nyaga et al., 2015).

3.5.2. Whole-genome phylogenetic analysis

Complete open reading frames were deduced for all 11 RVA segments. In addition,

reference genomes from the National Centre for Biotechnology Information (NCBI)

GenBank were selected for analyses in comparison with those obtained for the study

strains. Genotypes for all Gene segments were determined using the 2013 updated

version web-based tool, RotaC® 2.0 [http://rotac.regatools.be] (Maes et al., 2009).

28

Nucleotide sequence alignments for each Gene segment were constructed using the

MUSCLE algorithm implemented in MEGA 5.1 (Tamura et al., 2011).

29

CHAPTER 4: RESULTS

4.1. Confirmation of the G4P[6] and G4P[8] strains

Three G4 strains met the selection criteria and named as recommended by the

Rotavirus Classification Working Group (RCWG) as follows: Rotavirus group/species

of origin/country of identification/common name/year of identification/G- and P-type

(Matthijnssens et al., 2011) (Table 4.2). From this point onwards, study strains will be

referred to using their common name as MRC-DPRU1850; MRC-DPRU20683 and

MRC-DPRU1752.

Strains MRC-DPRU20683, MRC-DPRU1850 and MRC-DPRU1752 were confirmed

as rotavirus positives by Enzyme Linked Immunoassay (EIA) using a commercially

available rotavirus EIA kit (ProSpectT, Oxoid Ltd, UK). The Optical density (OD)

values for all the study strains are as in Table 4.1. The cut-off values were calculated

by adding 0.200 absorbance units to the negative control value as such, the cut-off

value was 0.262. The validity of the results from samples tested was confirmed using

a negative and positive control contained in the EIA kit (ProSpectT, Oxoid Ltd, UK).

For validity of the test results, the negative control must be less than 0.150

absorbance units (OD) and the positive control must be above 0.500 OD for the

standard valid EIA test. The level of positivity in the DPRU laboratory was

determined using the positive sign (+) and (-). OD cut-off value -<0.5=+/-; OD>0.5-

<1.0= +; OD>1.0-<2.0= ++; OD>2.0-<3.0= +++; OD>3.0= ++++.

Table 4.1: Confirmatory EIA results for the three study strains. The cut-off OD value was 0.262

Strain Optical Density value (Positivity)

MRC-DPRU Lab Interpretation

MRC-DPRU20683 0.852 + MRC-DPRU1850 0.904 + MRC-DPRU1752 0.788 +

Positive Control 1.310 ++

4.2. Genotyping of selected samples

The three study strains were subjected to the rotavirus RT-PCR methods as

described by Gomara et al., 2004 and Gentsch et al., 1992. Two of the three

30

selected strains were determined to be G4P[8] whereas one was confirmed as

G4P[6] (Figure 4.1). All of the study strains resulted in amplicons of 583 base pairs

(bp) which was the expected size of the G4 genotype. For the VP4, the amplicon

size for MRC-DPRU20683 and MRC-DPRU1850 strains was determined to be 345

bp which was the expected P[8] genotypes size (Figure 4.2), whereas strain MRC-

DPRU1752 was determined to be 267 bp amplification product, the expected P[6]

genotype size.

Figure 4.1: Rotavirus G4 strains confirmation by multiplex PCR using a set of VP7 (RVG) primers: Lane 1 and 7 shows the 1200 bp molecular weight marker with arrows indicating the 100 bp, 500 bp and 100 bp marks; Lane 2 (MRC-DPRU20683); Lane 3 (MRC-DPRU1850); Lane 4 (MRC-DPRU1752); Lane 5 (G1 Positive Control); Lane 6 (Negative control). All the study strains amplified at 305 bp and were confirmed as G4’s.

31

Figure 4.2: Rotavirus P[6] and P[8] strains confirmation by multiplex PCR using a set of VP4 (Con3) primers: Lane 1 and 7 shows the 1200 bp molecular weight marker with arrows indicating the 100 bp, 500 bp and 100 bp marks; Lane 2 (MRC-DPRU20683); Lane 3 (MRC-DPRU1850); Lane 4 (MRC-DPRU1752); Lane 5 (P[8] Positive Control); Lane 6 (Negative control). The study strains MRC-DPRU20683 and MRC-DPRU1850 amplified at 345 bp and were confirmed as P[8]’s whereas the MRC-DPRU1752 study strain amplified at 267 bp and were confirmed as P[6].

Genome typing using a 2013 updated version of an online rotavirus genotyping tool,

RotaC2.0 (Maes et al., 2009), revealed that study strains MRC-DPRU20683 and

MRC-DPRU1850 were G4-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 typical of the Wa-like

(genotype 1), whereas strain MRC-DPRU1752 exhibited a different genome

constellation G4-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 typical of the DS-1-like

(genotype 2) genogroup.

32

Table 4.2: Study and selected reference strains used in this study

RCWG Strain Name Genome constellation

S9 (VP7)

S4 (VP4)

S6 (VP6)

S1 (VP1)

S2 (VP2)

S3 (VP3)

S5 (NSP1)

S8 (NSP2)

S7 (NSP3)

S10 (NSP4)

S11 (NSP5)

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6] G4 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/ZAF/GR1106/86/1999/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/GR828/86/1999/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/GR856/86/1999/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ARG/Ros972/1997/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/GR/Ath144/2010/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/PRY/954SR/2005/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ARG/Res1717/1998/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/PRY /350/1999/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ARG/Res1730/1998/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/CHN/ E2484/2011/G4P[8] G4 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6] G1 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/MRC-DPRU457/2004G1P[6] G1 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/USA/DC4315/1988/G1P[8] G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ETH/MRC-DPRU906/xxxx/G1P[8] G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8] G1 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ARG /Mis864/1998/G4P[8] G4 P[8] I1 R1 C1 M1 A8 N1 T1 E1 H1 RVA/Human-wt/CHN/GX77/2013/G4P[6] G4 P[6] I1 R1 C1 M1 A8 N1 T1 E1 H1 RVA/Human-wt/HUN /BP1901/1991/G4P[6] G4 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Pig-tc/USA/LS00008/1975/G4P[6] G4 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/COD /KisB332/2008/G4P[6] G4 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Pig-tc/USA/LS00007_Gottfried/1975/G4P[6] G4 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/CHN/E931/2008/G4P[6] G4 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6] G2 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8] G2 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6] G2 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

The Wa-like and DS-1-like genogroups were assigned if at least seven Gene segments belong to the respective Wa-like or DS-1-like genotype (Mathheijnssens et al., 2008). A web based RotaC online classification tool (http://rotac.regatools.be/) for rotavirus genotypying of all 11 RVA gene segments was used according to the recommendations by the RCWG (Maes et al., 2009). The three study strains are highlighted in Tan background. Green Colour on genome constellation symbolizes the Wa-like, red the DS-1-like, Purple represent some typical animal strains, yellow AU-like strains, S; segment, VP; viral protein, NSP; non-structural protein, G; glycoprotein, P; protease sensitive, I; intermediate, R; RNA-dependent RNA polymerase, C; core, M; methyltransferase, N; NTPase, T; transferase, E; Enterotoxin and H; pHosphoprotein .

33

4.1.2. Nucleotide and amino acid pairwise analyses

Estimates of the evolutionary divergence between nucleotide and amino acid

sequences as obtained by a bootstrap procedure (500 replicates) conducted

using the P-Distance model with pairwise deletion in MEGA 6 showed all

positions contained gaps and those with missing data were eliminated

(Tamura et al., 2013). For MRC-DPRU20683, MRC-DPRU1850 and MRC-

DPRU1752, the nucleotide evolutionary divergence for VP7 and VP4 was

86.8–96.9% and 85.8–96.8%, respectively (Table 4.2-4.3). Among the P[8]

strains, nucleotide identities of the VP1, VP2, VP4, VP6, and NSP5 genes

were more than 95%, whereas lower identities were observed in the VP3,

NSP1, NSP2, and NSP3 genes (Appendix 6).

All the Gene segments of strain MRC-DPRU1752 were distantly related to

those of both MRC-DPRU20683 in Gene segment 8 (NSP2) and 10 (NSP4)

and MRC-DPRU1850 strains. By contrast, there was no evolutionary variance

estimated between all the Gene segments of the MRC-DPRU20683 and

MRC-DPRU1850 G4P[8] strains. Additionally, multiple sequence alignment of

all the Gene segments of the study strains revealed some nucleotide

variations that appeared not to affect the amino acid sequences of the

deduced proteins. Although the nucleotide sequences of the two G4P[8]

(MRC-DPRU20683 and MRC- DPRU1850) rotaviruses were remarkably

conserved in general, they did show minor nucleotide variations at certain

positions.

4.2. Phylogenetic analysis

Phylogenetic analyses were conducted using the complete open reading

frames (ORFs) of the consensus nucleotide sequence of all eleven Gene

segments. In addition to the three rotavirus study strains, reference genome

sequences attained in the NCBI GenBank were utilised to construct the

phylogeny in order to determine their possible origin and compare them with

other regional and global G4 strains. Multiple sequence alignments was

performed using MEGA6 (www.megasoftwares.com) and phylogenetic trees

were also constructed using MEGA6 applying the Maximum-Likelihood (ML)

method based on the Tamura-Nei model (Tamura and Nei, 1993).

34

4.2.1. Sequence analysis of VP7

A maximum likelihood phylogenetic tree of gene segment 9 (VP7) was

constructed. The nucleotide and deduced amino acid consensus of all the

Gene segments sequences of the study strains were determined. The VP7

gene sequences of the MRC-DPRU1752, MRC-DPRU20683 and MRC-

DPRU1850 strains collected from South Africa, Zimbabwe and Zambia were

sequenced and compared with other G4’s obtained from the GenBank. Strain

MRC-DPRU1850 clustered on lineage I together with strain GR/Ath14146

from Great Britain and Belgian strain BE1113, forming a distinct sub-cluster

that was distantly related to strain MRC-DPRU20683which also clustered in

lineage I and was closely related to the South African strains GR1160/86 and

GR833/86 (Figure. 4.4A). Interestingly, the MRC-DPRU1752 study strain

formed a distinct sub-cluster from that of other G4 DS-like strains such as

LS00008 from the USA and Chinese CHN/R1954, CHN/E2484 in lineage IV.

The MRC-DPRU1752 shared only 87.6% and 86.8% amino acid identity with

the MRC-DPRU20683 and MRC-DPRU1850 strains, respectively (Table 4.3).

Table 4.3: Nucleotide and Amino Acid similarities between the gene segment 9 (VP7) of the study strains and reference strains as determined by the p-distance algorithm in the MEGA 6 software. Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8. 2 96.9 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 87.6 86.8 ID

RVA/Human-wt/ZAF/GR1106/86/1999/G4P8 4 99.7 97 87.5 ID

RVA/Human-wt/ZAF/GR828/86/1999/G4P8 5 99.6 96.9 87.4 99.7 ID

RVA/Human-wt/ZAF/GR856/86/1999/G4P8 6 99.4 96.7 87.5 99.5 99.4 ID

RVA/Human-wt/ARG/Ros972/1997/G4P8 7 97.2 96 87.5 97.3 97.2 97 ID

RVA/Human-wt/GR/Ath144/2010/G4P8 8 96.7 99.4 86.8 96.8 96.7 96.5 95.8 ID

RVA/Human-wt/PRY/954SR/2005/G4P8(2) 9 96.7 95.3 87.9 96.6 96.5 96.5 99.3 95.1 ID

RVA/Pig-tc/USA/LS00007_Gottfried/1975/G4P6 10 87.6 88.1 86.9 87.5 87.6 87.4 87.9 88.3 87.9 ID

RVA/Pig-tc/USA/LS00008/1975/G4P6 11 87.6 88.1 86.9 87.5 87.6 87.4 87.9 88.3 87.9 100 ID

RVA/Human-wt/CHN/E931/2008/G4P6(2) 12 87.3 87 86.1 87.2 87.1 87.1 87.2 87 87.1 87.7 87.7 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 6 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

The Gene segment 9 (VP7) contains highly conserved sites in its nucleotide

sequences that are the major neutralizing sites targeted by the cytotoxic T-

Lymphocytes, leading to the production of neutralizing antibodies by B cells

35

(Dyall-Smith et al., 1986). There are at least nine known VP7 variable regions

(VR1-VR9) and six antigenic regions (ARs). The ARs are; A (aa 87–101), B

(aa 141–150), C (aa 208–224), D (aa 291), E (aa 189), and F (aa 235–245)

(Dyall-Smith et al., 1986; Ciarlet et al., 1997). Antigenic regions A, B, C, and F

correspond to VR-5, VR-7, VR-8, and VR- 9, respectively (Figure.2) (Dyall-

Smith et al., 1986; Green et al., 1989; Kirkwood et al., 1993; Ciarlet et al.,

1997).The first variable region [VR-1(9-200)] on the nucleotide sequences of

segment 9 (VP7) was similar between the study strains as well as the strains

obtained from GenBank.

Changes in nucleotide bases were observed in VR-3 (I40V and V43A) where

the base G was substituted with base A at nucleotide level for MRC-

DPRU1752 without altering the amino acid sequence. However, the same

VR-3 there was another change in base sequences where the base C was

substituted with Ton MRC-DPRU1850 and MRC-DPRU1752 resulting in a

change in amino acid sequence level from Leucine to Phenylalanine. In VR-4

the substitutions A66V and R72Q were reported. When study strains were

compared with other reference G4P[8]s collected before the year 2000, (i.e.

GR1106/86; GR828/86 and ARG/Ros972) the MRC-DPRU20683 revealed

great similarity in amino acid substitutions (99.4-99.7%). Comparison with

strains Isolated after the year 2000 revealed MRC-DPRU20683 strain was

slightly divergent.

36

VR-6 (119-132) VR-7/AR-B (141-150)

AR-E (189-190) VR-8/AR-C (208-224)

37

Figure 4.3: Gene segment 9 (VP7) antigenic mapping. A Dot conservation plot showing comparison of the Variable Regions (VR) and Antigenic Regions (AR) of Gene segment 9 (VP7) of the G4P[6] and G4P[8] study strains to the reference G4 strains from different regions worldwide obtained from the GenBank. The nine VRs (VR-1 to VR-9), identified in VP7 are boxed. VRs 5, 7, 8, and 9 includes the antigenic sites which defines serotypes, and correspond to ARs A, B, C, and F, respectively. Antigenic sites D and E occur at amino acid (aa) 291 and 189-190 respectively (Dyall-Smith et al., 1986). All study strains are also boxed.

Geographically, the MRC-DPRU20683 and MRC-DPRU1850 strains clustered

closely to South American G4P[8]s in the VP7 phylogenetic tree although they

showed a high level of divergence (Table 4.4). However in the VP7 tree, the

MRC-DPRU1752 study strain clustered with the MRC-DPRU309 and MRC-

DPRU1195 G12P[8] . This trend was also observed in most of the Gene

segments.

Comparison of the amino acid sequence from the VP7 of MRC-DPRU20683,

MRC-DPRU1850 and MRC-DPRU1752 study strains also reveals several

important substitutions at VR5/AR-A (S88T, P91Q, S95N and T97N), VR-6

(D131E), VR-7 (K144R and A146T) and VR-8 (T213V) where the MRC-

DPRU1752 gene sequence was altered at four positions and it was also

observed that amino acid substitutions on the MRC-DPRU1752 occurred in

38

the same position as that of MRC-DPRU1850 in certain positions .

Interestingly, strain MRC-DPRU20683 collected in 1985 revealed great

variation in the amino acid sequence than MRC-DPRU1850 collected in 2011

in comparison with MRC-DPRU1752.

Table: 4.4: Gene segment 9 study strains and reference strains with amino acid substitutions and positions. MRC-DPRU20683 was the consensus for the amino acid sequences substitution comparison.

Common Names Amino Acid substitutions (VP7) MRC-DPRU20683 MRC-DPRU1850 L18F; I40V; RN72,73QD; D131E; A146T MRC-DPRU1752 L18F; IK27,28TR; RI36,37KV; V43A; I55M; A66V; TRN72-74QSD;

S87T; P91Q; S95N; T97N; V140I; K144R; T213V; I228V; V308I GR1106/86 I26M, I40V; R72Q GR828/86 I40V; R72Q GR856/86 I30V; I40V; R72Q ARG/Ros972 L18V; I40V;R72Q; D131E; A146T; R248Q GR/Ath144 I40V; RN72,73QD; D131E; A146T PRY/954 V43A; R72Q; P91S; I228V LS00007/Gottfried F11L; V19M; I27T; I28M; R72Q; S95N; N97T; Y135H; V140I; K144R LS00008 F11L; V19M; I27T; I28M; R72Q; S95N; N97T; Y135H; V140I; K144R CHN/E931 I27T; I33L; R72Q; S88T; P91O; N97T; I130V; V140I; K155R; I205V;

A214N; D221E; I228V; S233G

4.2.2. Sequence analysis of VP4

A maximum likelihood phylogenetic tree of gene segment 4 was constructed

(Figure 4.4B) based on the TN93+G Tamura-Nei model (Tamura et al., 2013)

to compare the study strains with other reference strains available on the

NCBI Gen Bank. The VP4 gene sequences of one MRC-DPRU1752 and the

two G4P[8] (MRC-DPRU20683 and MRC-DPRU1850) study strains were

compared with other G4’s obtained from the GenBank.

Distance matrix analysis based on nucleotide sequences revealed that the

two MRC-DPRU20683 and MRC-DPRU1850 study strains shared an identity

of 89.2% (Table 4.5). The MRC-DPRU20683 strain also shared 96.3% and

96.1% identity with the Belgian strain BEL/BE1014 and Russian strain Nov10-

N53, respectively. The Gene segment 4 of the MRC-DPRU20683 strains

revealed a significant increased discrepancy in the nucleotide sequences as

well as in amino acid sequences. However strains from the same region show

similarity. For example when study strains were compared to reference strains

Mis864 G4P[8]; Res1717 G4P[8]; Res1730 G4P[8] and PRY/350 G4P[8]

39

G4P[8] 99.9-100% similarities on the pairwise Identities in both nucleotide and

amino acid sequences was observed.

Table 4.5: Nucleotide and Amino Acid similarities between the gene segment 4 (VP4) of the study strains and reference strains as determined by the p-distance algorithm in the MEGA 6 software. Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8(2) 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8(2) 2 89.2 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 74.2 74.9 ID

RVA/Human-wt/CHN/E931/2008/G4P6(2) 4 74.7 75.6 90.9 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 74.7 75.6 86.7 87.2 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 74.7 75.6 90.9 100 87.2 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8(2) 7 89.3 99.1 75 75.6 75.7 75.6 ID

RVA/Human-wt/PRY/350/1999/G4P8(2) 8 89.3 99.1 75 75.7 75.7 75.7 99.9 ID

RVA/Human-wt/CHN/E2484/2011/G4P8(2) 9 88.7 96.6 75 75.3 75 75.3 96.7 96.7 ID

RVA/Human-wt/BEL/BE1014/2008/G4P8 10 96.3 88.6 74.5 74.7 74.9 74.7 88.8 88.8 88.1 ID

RVA/Human-wt/RUS/Nov10-N53/2010/G4P8 11 96.1 88.9 74.7 75 74.9 75 89.1 89.1 88.4 97.9 ID

RVA/Human-wt/Bethesda/DC2241/1977/G4P8 12 90.2 91.4 75.2 75.4 75.6 75.4 91.5 91.5 90.5 89.8 89.7 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 6 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

The same trend is also observed with North American strains which are

closely related to the South American, for example the DC2241 strain and the

ARG/Mis864 which exhibit 89.3 and 90.2% nucleotide. The MRC-DPRU1752

G4P[6] is closely related to the CHN/E931 and COD/KisB332 with 90.9% and

86.7% similarity on the pairwise Identity.

Sequence analysis of Segment 4 (VP4) also reveals that the MRC-

DPRU1850 study strain and MRC-DPRU1752 were significantly divergent at

85.4% nucleotide and 85.8% amino acid divergence. The MRC-DPRU20683

shows homology with the MRC-DPRU1818, Res1717 and Res 1730 with

100% nucleotide and amino acid homology on the P-distance algorithm.

Some G4P[8] such as PRY/350 are closely related to the G4P[6] (LS00008

and BP1901).

4.2.3. Sequence analysis of VP6

Gene segment 6 of study strains MRC-DPRU20863 and MRC-DPRU1850

G4P[8] were genotype I1, whereas MRC-DPRU1752 was genotype I2. Gene

segment 6 of the Wa-like study strains were closely related to other regional

strains and clustered with Ethiopian and South African strains MRC-

DPRU5010 and ZAF/3133WC, respectively. Gene segment 6 of the DS-1 Like

40

MRC-DPRU1752 study strain formed an entirely distinct cluster with Ugandan

MRC-DPRU3710, Paraguayan PRY/412 as well as the RotaTeg-BrB-9

vaccine strain Isolated from USA.

Gene segment 6 of the MRC-DPRU20683 and MRC-DPRU1850 shared

98.3% nucleotides and amino acid homology and they were closely related to

the Brazilian BRA/RJ124 and Russian Nov06-1255 G4P[8] with about 97-

100% nucleotide and amino acid similarities (Appendix 5). Furthermore, the

MRC-DPRU1752 strain showed significant divergence of 77.2% and 77.4%

nucleotide and amino acid variance with the MRC-DPRU20683 and MRC-

DPRU1850 study strains whereas it also reveals 96.8-97.5% similarity with

the Ros990 and PRY/54SR reference strains isolated from Russia and

Paraguay, respectively.

In addition the study strain MRC-DPRU20683 revealed a close relation with

the MRC-DPRU3506 and MRC-DPRU5171 reference strains since they both

clustered together in the VP6 tree.

41

A. Genome Segment 9 (VP7)

RVA/Human-wt/ARG/Res1717/1998/G4P[[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/CAN/RT038-09/2009/G4P8

RVA/Human-wt/BEL/BE1129/2009/G4P[8]

RVA/Human-wt/HUN/ERN5199/2012/G4P[8]

RVA/Human-wt/GR/Ath146/2010/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/BEL/BE1113/2009/G4P[8]

RVA/Human-wt/Bethesda/DC1285/1980/G4P[8]

RVA/Human-wt/ZAF/GR1107/86/1999/G4P8

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/ZAF/GR856/86/G4P[8]

RVA/Human-wt/ZAF/GR1106/86/1999/G4P[8]

RVA/Human-wt/ZAF/GR833/86/1999/G4P[8]

RVA/Human-wt/Bethesda/DC2241/1977/G4P[8]

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]

G4-Lineage I

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Human-wt/PRY/954SR/2005/G4P[8]

RVA/Human-wt/ARG/Ros972/1997/G4P[8]Lineage II

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

Lineage IV

Out Group94

100

81

72

96

99

100

90

99

88

90

0.05

Figure: 4.4: Phylogenetic analysis by Maximum Likelihood method (MLM) based on coding sequences; A.VP7 B, VP4 C, VP6 D, VP1 E, VP2 F, VP3 G, NSP1 H, NSP2 I, NSP3 J, NSP4 and K NSP5 genes. The evolutionary history was inferred by using the MLM with a bootstrap value of 500 based on the Tamura-Nei model (Tamura and Nei., 1993). Anticipated lineages and sub-lineages within a genotype (A–K) were assigned instinctively based on observation of clustering patterns. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved nucleotide sequences from the 3 study strains and selected reference strains attained from the NCBI GenBank. G8P[4] study strains are indicated by ▲ and G4P[6] by ■ for G4P[8]. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

42

B. Genome Segment 4 (VP4)

RVA/Human-wt/BEL/BE1014/2008/G4P[8]

RVA/Human-wt/RUS/Nov06-1486/2006/G1P[8]

RVA/Human-wt/ETH/MRC-DPRU842/2012/G9P[8]

RVA/Human-wt/BEL/BE0005/2008/G1P[8]

RVA/Human-wt/SEN/MRC-DPRU2051/2009/G9P[8]

RVA/Human-wt/BEL/BE1418/2009/G9P[8]

RVA/Human-wt/RUS/Nov10-N632/2010/G4P[8]

RVA/Human-wt/RUS/Nov10-N709/2010/G4P[8]

RVA/Human-wt/RUS/Nov10-N1107/2010/G4P[8]

RVA/Human-wt/RUS/Nov10-N53/2010/G4P[8]

RVA/Human-wt/RUS/Nov10-N735/2010/G4P[8]

RVA/Human-wt/RUS/Nov09-D187/2009/G1P[8]

RVA/Human-wt/RUS/Nov10-N205/2010/G4P[8]

RVA/Human-wt/TGO/MRC-DPRU5123/2010/G9P[8]

RVA/Human-wt/BEL/BE1183/2009/G3P[8]

RVA/Human-wt/BEL/BE1190/2009/G3P[8]

RVA/Human-wt/RUS/Nov08-3149/2008/G9P[8]

RVA/Human-wt/ZAF/MRC-DPRU2144/2003/G9P[8]

RVA/Human-wt/ZAF/MRC-DPRU2711/2008/G9P[8]

RVA/Human-wt/MWI/OP530/1999/G4P[8]

RVA/Human-wt/MWI/MW670/1999/G4P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

Lineage III

RVA/Human-wt/Bethesda/DC2241/1977/G4P[8]

RVA/Human-tc/USA/Wa/1974/G1P8[]Lineage I

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ZAF/MRC-DPRU135/2009/G1P8

RVA/Human-wt/USA2007744270/2007/G1P[8]

Lineage II

P[8]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/ZAF/MRC-DPRU774/2000/G9P[6]

RVA/Human-wt/ZAF/MRC-DPRU2130-05/2005/G12P[6]

RVA/Human-wt/ETH/MRC-DPRU1844-08/2008/G3P[6]

RVA/Human-wt/TGO/MRC-DPRU5164/2010/G3P[6]

RVA/Human-wt/ZAF/2371WC/2008/G9P[8]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1195/2009/G2P6P[8]

RVA/Human-wt/GMB/MRC-DPRU3190/2010/G2G12P[6]

RVA/Human-wt/CMR/MRC-DPRU1480/2009/G1P[6]

Lineage V

Out Group RVA/Human-wt/CHN/E931/2008/G4P[6]

P[6]94

99

96

59

99

85

100

41

100

10088

9820

99

100

57

31

99

10087

79

90

95

99

44

65

99

77

100

96

61

94

8369

67

99

0.1

C. Genome Segment 6 (VP6)

RVA/Human-wt/Bethesda/DC5751/1991/G3P[8]

RVA/Human-wt/USA/DC5411/1991/G1P[8]

RVA/Human-wt/USA/DC5411/1991/G1P[8]

RVA/Human-wt/ARG/Ros990/1998/G4P[8]

RVA/Human-wt/PRY/88/1998/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/USA/DC4312/1988/G1P[8]

RVA/Human-wt/USA/DC4089/1988/G1P[8]

RVA/Human-wt/Bethesda/DC1285/1980/G4P[8]

RVA/Human/JPN/Hosokawa/1983/G4P1A8

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-wt/BEL/B4633/2003/G12P[8]

RVA/Human-wt/BRA/RJ12419/2006/G12P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/RUS/Nov06-1255/2006/G4P[8]

RVAHuman-wt/RUS/Nov06-1255/2006/G4P[8]

RVA/HumaH-wtu/RUS/Nov07-2058/2007/G1P[8]

RVA/Human-wt/BEL/BE00025/2007/G1P[8]

RVA/Human-wt/BEL/BE00025/2007/G1P[8]

RVA/Human-wt/ETH/MRC-DPRU5010/2010/G12P[8]

RVA/Human-wt/ETH/MRC-DPRU1843/2009/G1P[8]

RVA/Human-wt/ETH/MRC-DPRU1843/2009/G1P[8]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

I1

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Human-wt/UGA/MRC-DPRU3710/2009/G2P[4]

RVA/Human-wt/IND/mcs72/2011/G8P[4]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/USA/VU06-07-36/2006/G2P[4]

RVA/Human-wt/SEN/MRC-DPRU2128/2009/G2P[6]

RVA/Human-wt/ZAF/MRC-DPRU228/2009/G1G2P[6]

RVA/Human-wt/SWZ/MRC-DPRU2995/2009/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU1061/2009/G2P[4]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

RVA/Human-wt/ZAF/MRC-DPRU1362/2007/G2P[4]

I2

Out Group Avian RV A PO-13

100

98

94

95

94

72

95

100

100

93

97

87

82

100

8594

100

88

100

99

87

0.2

43

D. Genome Segment 1 (VP1)

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ETH/MRC-DPRU906/XXXX/G1P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

R1

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]

R2

100

10098

100

100

100

100

100100

98

79

100

90

82

100

100

80

100

8598

97

100

100

0.05

4.2.4. Sequence Analyses of VP1–VP3

Based on the P-Distance matrices analysis, RotaC and phylogenetic

analyses, the Gene segment 1 of the MRC-DPRU20683 and MRC-

DPRU1850 study strains were genotypes R1, C1 and M1, whereas that of

the MRC-DPRU1752 genotype R2, C2 and M2 . Phylogenetic analysis of the

Wa-like study strains were closely related but clustered separately from other

published Wa-like reference strains used in this analysis (Figure 4.4D-F). The

Gene segment of the DS-1-like study strain formed separate semi-cluster with

other DS-1-like African strain.

The VP1 of the two Wa-like G4P[8] strains as well as the reference strains

contains the RNA-Dependent RNA Polymerase repetitive motifs at residues

512-527, 582-608, and 690-702, whereas substitution is observed on the

MRC-DPRU1752 DS-1-like study strain where S512A as a result of a sense

mutation at nucleotide level .

Gene segment 2 of the MRC-DPRU20683 and MRC-DPRU1752 study strains

shows a Wa-like origin and the MRC-DPRU1752 study strain is a DS-1-

44

like.The Gene segment 2 of the Wa-like study strains clustered together on

the same lineage with the G4P[8] (Res1730) and G1P[6] (MRC-DPRU4498

and MRC-DPRU457) reference strains whereas the MRC-DPRU1752 G4P[6]

study strain clustered distinctively with the G2s (MRC-DPRU2128 and MRC-

DPRU1280-05) as well as the USA G4P[4] strain VU06-07-36 as observed

also on the other Gene segments. The G4P[6] study strain is also closely

related to a Paraguayan mixed G1/G4 (PRY/412) strain and the Australian

mixed G1P[8]/[4] (CK20004) strain.

The MRC-DPRU1752 study strain has 15 amino acid shorter than the Wa-like

strains which is typical of the DS-1-like strains. The amino acid variations are

also observed from position 12-45 which included the RNA binding domain of

the VP2. There are also numerous differences on the nucleotide and in some

insistence amino acid sequences between the Wa-like and the DS-1-like

strains.

Gene segment 3 of the study strains also segregated into Wa-like (MRC-

DPRU20683 and MRC-DPRU1850) and DS-1-like (MRC-DPRU1752)

genotypes. The Gene segment 3 of the Wa-like and DS-1-like study strains

shows 86-95% nucleotide similarities (Appendix 5). The Wa-like study strains

shows 95-100% nucleotide similarities in comparison with other Wa-like

reference strains whereas in comparison with the DS-1-like reveals only 86%

similarities. The Gene segment 3 also showed the same trend as the other

genomes (VP1 and VP2) on the phylogenetic tree, The Wa-like genotypes of

the study strains clustered together whereas the DS-1-like clustered with the

USA vaccine strain (Rotateg-BrB-9) as well as the African G2s.

45

E. Genome Segment 2 (VP2)

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]

C2

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ETH/MRC-DPRU906/2009/G1P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

C1

100

100

9485

71

100

100

100

96

100

7690

70

70

100

79100

100

85

99

0.05

F. Genome Segment 3 (VP3)

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/CMR/MA88/2011/G12P[8]

RVA/Human-wt/ZAF/MRC-DPRU822/2005/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/MLI/Mali-042/2008/G1P[8]

RVA/Human-wt/SWZ/MRC-DPRU4550/2010/G1P[8]

RVA/Human-wt/BEL/BE00034/2008/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU1370/2004/G12P[6]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/CMR/MRC-DPRU3043/2009/G12P[8]

RVA/Human-wt/ETH/MRC-DPRU906/2009/G1P[8]

RVA/Human-wt/BGD/Dhaka25/2002/G12P[8]

RVA/Human-wt/PRY/473/2000/G9P[8]

RVA/Human-wt/PRY/7SR/2002/G9G4P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

M1

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]

RVA/ST3xUK reassortant (UKg9ST3)

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

M2

Out Group

99100

100

100

100

100

100

100

77100

98

93

98

100

87

83

7187

99

100

99

99

100

78

97

96

79

99

0.05

46

G. Genome Segment 5 (NSP1)

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

A2

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

A8

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/ETH/MRC-DPRU906/XXXX/G1P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

A1

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]A3

100

100

100

99

97

90100

96 75

99

97

100

7699

98

93

0.05

4.2.5. Sequence analysis of Non-structural proteins (NSP1-NSP5)

The Gene segment 5 of the study strains were Wa-like (MRC-DPRU20683

and MRC-DPRU1850) and DS-1-like (MRC-DPRU17520) origin. The

phylogenetic analysis indicates that the Gene segment 5 of the Wa-like study

strain (MRC-DPRU1850) were closely related with the G1P[6] strains (MRC-

DPRU4498,MRC-DPRU457) and G1P[8] strain (MRC-DPRU539), whereas

that of MRC-DPRU20683 study strain is closely related with the G1P[8]

(DC4315) collected in 1988. The Gene segment of the DS-1-like study strain

formed a separate cluster with the DS-1-like strains isolated from South

Africa, Ghana, Paraguay and Australia.

47

H. Genome Segment 8 (NSP2)

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ETH/MRC-DPRU906/XXXX/G1P[8]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Human-wt/PRY/412/1999/G1G4P[4][

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P8

N1

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

N2

Out Group9770

100

100

99

97

98

92

80

89

91

9599

8793

71

97

100

1

The conserved cysteine-rich motif C-X2-C-X8-C-X2-C-X3-H-X-C-X2-C-X5-C

was present in all the NSP1 of the study strains spanning from amino acid 42-

72 and is believed to be a zinc- and virus-specific RNA-Binding domain

protein (Huan et al., 1994). However it was located from amino acid 36-66

(Figure 4.5). Amino acids Q64E and G/D70S that separate depending on the

Wa- and DS-1-like Genotypes (Heiman et al., 2008) were conserved within

the cysteine rich motifs, however they were at position 58 and 64 respectively.

All the Wa-like study strains were having similar amino acid sequences at

these motifs whereas the DS-1-like study strains have variations within these

cysteine-rich motifs at the amino acid position R59Q, T52L and M53I.

Gene segment 8 (NSP2) of the study strains were also of Wa-like (MRC-

DPRU20683 and MRC-DPRU1850) and DS-1-like (MRC-DPRU1752) origins.

Phylogenetic analysis revealed a close relationship between Gene segment 8

of the Wa-like study strains as in Gene segment 5, which were related to the

G1P[8] and G1P[6] N1 genotype strains. The Gene segment 8 of the DS-1-

48

like study strains clustered with the other southern African reference strains

and also related to the other G4P[8] study strain indicating similarity.

Gene segment 7 (NSP3) of the study strains was also of Wa-like (MRC-

DPRU20683 and MRC-DPRU1850) and DS-1-like (MRC-DPRU1752). The

phylogenetic analysis of the Gene segment 7 revealed close relationship

between MRC-DPRU1850 and the DC4315 G1P[8] reference strain from the

USA which was collected in 1988, whereas the study strain was collected in

2011. The analysis also reveals the close relationship between the MRC-

DPRU20683 study strain and the recently isolated emerging G12s (i.e VU08-

09-40) and the G9s (i.e MRC-DPRU4595). There is also evidence of a direct

divergence of the G12s (MRC-DPRU906) and G9s (MRC-DPRU4515) from

the G4s (MRC-DPRU20683) The MRC-DPRU1752 study strain shows 99%

similarity with the MRC-DPRU1280-05 G2P[8] reference strain, both were

collected between 2005 and 2009 (Appendix 5).

Figure 4.5: Gene Segment 5 (NSP1). A Dot conservation plot showing the conserved cysteine-rich motif C-X2-C-X8-C-X2-C-X3-H-X-C-X2-C-X5-C of Gene segment 5 (NSP1) of the G4P[6] and G4P[8] study strains to the reference G4 strains from different regions worldwide obtained from the GeneBank. The cysteine rich motif (36-66), identified in NSP1 are boxed. (Huan et al., 1994). All study strains are also boxed.

49

The Gene segment 10 (NSP4) of the strains MRC-DPRU20683 and MRC-

DPRU1850 were genotype E1, whereas the MRC-DPRU1752 G4P[6] strain

was of the genotype E2. The Gene segment 11 (NSP5/6) of the MRC-

DPRU20683 and MRC-DPRU1850 study strains also revealed a stable Wa-

like genetic backbone, whereas the MRC-DPRU1752 study strain was of the

DS-1-like backbone as in the Gene segment 10. The NSP4 and NSP5 gene

of both study strains and reference strains were the most conserved among

the 11 Gene segment regardless of the genotype or year of collection.

I. Genome Segment 7 (NSP3)

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/ETH/MRC-DPRU906/2009/G1P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/HUN/BP1901/1991/G4P6

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

T1

RVA/Human-wt/AUS/CK20004/2000/G1P[8]P[4]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

T2b

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]T2a

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]T6

99

100

9684

94

10099

89

98

100

76

96

95

86

94

97

0.05

50

J. Genome Segment 10 (NSP4)

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/ETH/MRC-DPRU906/XXXX/G1P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/CHN/R1954/2013/G4P[6]

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]

E1

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

E2.

E2 RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

E 2 RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

93

97

8199

98

79

99

100

98

96

99

100

0.5

K. Genome Segment 11 (NSP5)

RVA/Human-wt/ARG/Res1730/1998/G4P[8]

RVA/Human-wt/PRY/350/1999/G4P[8]

RVA/Human-wt/ARG/Res1717/1998/G4P[8]

RVA/Human-wt/ARG/Mis864/1998/G4P[8]

RVA/Human-wt/ZAF/MRC-DPRU4498/2002/G1P[6]

RVA/Human-wt/ZAF/MRC-DPRU539/2003/G1P[8]

RVA/Human-wt/ZAF/MRC-DPRU457/2004/G1P[6]

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P[8]

RVA/Human-wt/ETH/MRC-DPRU906/XXXX/G1P[8]

RVA/Human-wt/UGA/MRC-DPRU4595/2011/G9P[8]

RVA/Human-wt/TGO/MRC-DPRU5171/2010/G12P[8]

RVA/Human-wt/USA/VU08-09-40/2008/G12P[8]

RVA/Human-wt/CHN/E2484/2011/G4P[8]

RVA/Human-wt/ZMB/MRC-DPRU3506/2009/G12P[6]

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P[8]

RVA/Human-wt/COD/KisB332/2008/G4P[6]

RVA/Pig-tc/USA/LS00008/1975/G4P[6]

RVA/Human-wt/HUN/BP1901/1991/G4P[6]

RVA/Human-wt/CHN/E931/2008/G4P[6]

RVA/Human-wt/USA/DC4315/1988/G1P[8]

H1

RVA/Human-wt/CHN/GX77/2013/G4P[6]

RVA/Human-wt/AUS/CK20004/2000/G1P8P[4]

RVA/Human-wt/PRY/412/1999/G1G4P[4]

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P[6]

RVA/Human-wt/ZAF/MRC-DPRU1280-05/2005/G2P[8]

RVA/Human-wt/ZAF/MRC-DPRU1815/1999/G2P[6]

RVA/Human-wt/GHA/MRC-DPRU1818/1999/G2P[6]

H2

RVA/Vaccine/USA/RotaTeq-BrB-9/1996/G4P7[5]

RVA/Bovine-tc/USA/ ST3xUk/1986/G4P[x]H3

RVA/Human-wt/CHN/R1954/2013/G4P[6]91

86

8699

92

72

99

73

8998

73

0.2

51

CHAPTER 5: DISCUSSION AND CONCLUSSION

5.1. EIA and PCR

The selected study strains were confirmed as rotavirus positives using EIA.

The optical values were ranging from 0.7-0.9 OD which was within positive

values range. The three study strains subjected to RVA RT-PCR and

genotyping were G4P[8] and a G4P[6]. Agarose gel electrophoresis of the

genotyped cDNA amplicons of the VP4 and VP7 resulted to the expected size

of 345 bp and 583 bp, respectively for the study strains with G4P[8]s . The

VP4 cDNA amplicon size for the study strain with P[6] exhibited the expected

267 bp amplification product when checked against a 100bp MW marker

(Bioline HyperLadder™ 100bp, UK).

5.2. Phylogenetic Analysis

The whole genome classification system for RVA proposed by the RCWG

(Matthijnssens et al., 2011), revealed genetic relationships among rotaviruses

from different host species. It was determined that the human Wa-like

genogroup have a common origin with porcine rotavirus strains, whereas the

human DS-1-like with bovine RV strains (Matthijnssens et al., 2011). These

could be due to several mechanisms such as genetic reassortment and

zoonosis which generate great diversity of rotavirus and strains which show

considerable geographic and temporal variations (Bányai et al., 2012; Dóró et

al., 2015).

The G4s in combination with P[6]s were previously found to be less common

as compared to G4s in combination with P[8]s, which are among the most

commonly identified G4 constellations globally (Bányai et al., 2012; Wang et

al., 2010). A high genetic heterogeneity was observed for these genotypes

most commonly generated by point mutations and reassortment in the VP7

encoding gene (Matthijnssens et al., 2009). Nucleotide sequence analyses of

the three human rotaviruses in this study indicated the presence of several

genetically distinct, co-circulating clades of G4P[6] and G4P[8] rotavirus

strains, which contained minor but significant differences in their encoded

52

proteins (Esona et al., 2013; Nyaga et al., 2013). Although the nucleotide

sequences of the MRC-DPRU20683 and MRC-DPRU1850 rotaviruses were

remarkably conserved in general, they exhibited minor nucleotide variations at

certain positions in the VP7 gene. NSP1 was found to be the least conserved

member of the RVA proteome with higher sequence variability in the C-

terminal half (Mitchell and Both, 1990).

Human RVAs having the G4 genotype have been rarely detected lately in

surveillance studies in most African countries although they were the most

prevalent genotypes. It was thought that the disappearance of these

genotypes is mainly due to changes in antigenicity due to mutations at

nucleotide level as well as genetic reassortment (Gentsch et al., 2005). In this

study we found that there is a direct evolutionary relationship between the

G12 emerging RVA strains and the currently disappearing G4 genotypes. The

high genetic diversity observed within the MRC-DPRU20683 G4P[8] and the

MRC-DPRU1850 G4P[8] study strains on the VP7 encoding gene segment 9

substantiates the theory behind the disappearance of the G4s over the years.

This observation is also supported by the fact that the G4P[8] study strain

MRC-DPRU1850 clustered on lineage I In the VP7 tree forming a distinct sub

cluster, distantly related to the MRC-DPRU20683 study strain as well as other

African G4P[8] strains such as GR1107/86 and GR856/86.

Gene segment 6 (VP6) of the MRC-DPRU-20683 and MRC-DPRU1850

study strains were closely related to Southern African strains and also

clustered with other African regional strains. Whereas the Gene segment 6 of

the MRC-DPRU1752 study strain formed an entirely distinct cluster with other

global DS-1-like strains. This was also observed in the non-structural proteins.

For instance the MRC-DPRU-20683 and MRC-DPRU1850 strains formed a

single cluster together with reference strains which substantiate that the non-

structural proteins are generally conserved although minor variances were

observed in some genomes, they were not significant. The phylogenetic

analysis of the Gene segments of the non-structural proteins also revealed

that they were closely related to other non G4P[8] Wa-like strains like G1P[6]s

and G1P[8]s suggesting that the G4s may have probably mutated into other

genotypes.

53

The MRC-DPRU20683 collected in 1985 did not show great divergence in

amino acid substitutions (99.4-99.7%) which could be an indication that

strains isolated between 1985 and 2009/2011 did not evolve significantly. To

further illustrate this the distance matrix analysis of the VP4 gene segment

based on nucleotide sequences revealed that the two G4P[8] strains (MRC-

DPRU20683 and MRC-DPRU1850) shared an identity of 89.2% which is

significantly divergent. Gene segment 6 of the MRC-DPRU20683 and MRC-

DPRU1850 shared 98.3%(98.1%) nucleotides and amino acid homology

which suggests that the declining of the G4’s could be due to possible genetic

shift as a results of mutations leading to the emergence of either G12’s or

other emerging genotypes.

5.3. Full genome Sequence analysis of the G4P[6] and G4P[8]

The nucleotide sequences of the MRC-DPRU20683 and MRC-DPRU1850

rotaviruses were generally conserved but minor nucleotide variations were

noted at certain positions. Multiple sequence alignment of all the Gene

segments of study strains revealed some nucleotide variations that appeared

not to affect the amino acid sequences of the deduced proteins. Gradually

over a number of years, the variations accumulated and probably affected the

amino acid sequence leading to a shift of circulating genotype which could

have resulted to disappearance of G4s. These could be as a result of

nucleotide substitutions observed on the MRC-DPRU1850 study strain

isolated in 2009 which appears to have accumulated base substitutions which

could resulted in amino acid changes when compared toMRC-DPRU20683

G4P[8] strain collected in 1985.

In addition, comparison of the amino acid sequences from the VP7 of the

G4P[8] and G4P[6] study strains also revealed several important substitutions

at certain positions where the G4P[6] study strain gene sequence was altered

at four positions and it was also observed that amino acid substitution

positions on the G4P[6] were quite similar with those of G4P[8] in certain

variable positions such as in VR-1 as well as VR- 4 (L18F, RN72-73QD). The

first variable region [VR-1(9-20)] on the nucleotide sequences of gene

segment 9 (VP7) is completely similar between the MRC-DPRU1850 G4P[8]

and MRC-DPRU1752 G4P[6] study strains whereas the MRC-DPRU20683

54

G4P[8] revealed a slightly different nucleotide sequence with a substitution in

position L18F. This indicated that the G4s have acquired slightly evolutionary

changes in some cases with significant impact on the genotype constellation.

The VR-9 of all the study strains was mostly conserved. The VP4 of the MRC-

DPRU20683 strains revealed a significant divergence in the nucleotide

sequences as well as in amino acid sequences ranging from 75.6% to 89.2%

which is slightly above the cut-off value set at 75% by the RCWG.

5.4. Conclusion

This is the first study to investigate the full genomes of the G4 RVA strains in

Africa and among the first globally (Ghosh et al 2012. This study was

conducted to investigate the evolution of the G4 RVA strains from southern

Africa at the molecular level in order to understand and identify the evolution

of the G4P[6] and G4P[8] strains by comparing them with the full genome

reference strains already available in NCBI GenBank and also to understand

their mechanisms of genetic diversity over time. The strains isolated between

1985 and 2011 did not significantly diverge in the VP7 gene segment which is

the major antigenic determinant, a trend that was observed in the other 10

proteins. Although generally the study confirms that rotaviruses are in

continuous evolution, primarily by point mutation, and in some cases by

reassortment mechanisms (Bányai and Gentsch, 2014). The disappearance

of the G4 genotypes is mainly due to changes in antigenicity as results of

mutations at nucleotide level giving rise to either new strain or shifting to other

genotype (Gentsch et al., 2005).

In conclusion, most of the variable regions within all the 11 RVA genomes in

the study strains were considerably conserved with minor amino acid changes

which substantiate that evolution resulting in a genetic shift may take many

more years which could be determined by advanced bioinformatics studies

such as Bayesian analyses.

55

5.5. Recommendation

Based on the sample size of this study,. A larger sample size may give a

much better understanding of the mechanisms of genetic diversity and

evolution of G4 rotaviruses. Continuous monitoring of co-circulating RVA

strains with both common and uncommon genotypes could help envision the

possible spread of emerging rotaviruses in human and animal populations

especially in Africa and other developing parts of the world. A detailed

genomic and amino acid exploration of the G4 strains may help detect the

occurrence of changes that might eventually influence the diversity as well as

host pathogen relationships in African countries.

56

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APPENDICES

1. Preparation of 1% agarose gel and gel electrophoresis

A gram (1 g) of agarose powder was weighed and dissolved in 100 ml of 0.5

X Tris Borate EDTA buffer (TBE), heated in a microwave for 3 min and cooled

down. A volume of 10 μl of ethidium bromide was added to the solution which

was then poured into gel plates to solidify. The RT-PCR products together

with a 100 base pair (bp) molecular weight marker (Bioline reagents Ltd, UK)

were ran on a 1% TBE agarose gel stained with ethidium bromide at 100 volts

for 1 hour (hr) until the marker separated. The gels were visualised under

Ultra Violet (UV) gel doc (Vacutec, Syngene, UK) and positives were defined

as bands of 1062 bp and 876 bp for gene segments 9 and 4, respectively.

2. Preparation of 2% agarose gel and gel electrophoresis

In a 250 ml beaker 2 g of agarose powder was dissolved in 100 ml of 0.5 X

TBE buffer. The agarose powder was dissolved by heating the beaker in a

microwave and cooled down. A volume of 10 μl ethidium bromide was added

to the solution which was then poured into gel plates to solidify. The

genotyping products together with a 100 bp molecular weight marker were run

on a 2% TBE agarose gel stained with ethidium bromide at 100 volts for 1 hr

until the marker separated. The gels were visualized under UV and positives

were defined as the presence of bands with molecular weight of 559 bp for

G12’s and 345 bp (P[8]), 483 bp (P[4]), 267 bp (P[6]), 543 bp (P[14]) and 312

bp (P[11]).

72

3. RT-PCR master mix.

Table 2.1: RT master mix reagents, volume and concentration

Reagents

Manufacturer Concentration for 1 reaction

Volume per 1 reaction

Avian Myeloblastosis Virus (AMV)Reverse transcriptase enzyme

Thermo Fisher scientific, USA

2.4 U 0.2 μl

AMV buffer

Thermo Fisher scientific, USA

5X 2 μl

Deoxynucleotide triphosphates (dNTPS)

Bioline reagents Ltd, UK 10 mM for each dATP, dGTP, dCTP and Dttp

1 μl for each dATP, dGTP, dCTP and dTTP

Total 3.2 μl

4. PCR master mix.

Table 3.1: PCR master mix reagents, volume and concentration Reagents

Manufacturer Concentration per 1 reaction

Volume per 1 reaction

Water 26 μl Magnesium chloride (MgCl2)

Bioline reagents Ltd, UK 50 mM 1.2 μl

dNTPs

Bioline reagents Ltd, UK 10 mM for each dATP, dGTP, dCTP and dTTP

1 μl for each dATP, dGTP, dCTP and dTTP

Taq buffer

Bioline reagents Ltd, UK 5 μl 10 X

Taq polymerase

Bioline reagents Ltd, UK 0.3 μl 5 U

Primer 1

Integrated DNA technology Inc, USA

1 μl 10 pmol

Primer 2

Integrated DNA technology Inc, USA

1 μl 10 pmol

Total 40 μl

73

5. Genotyping master mix.

Table 3: Genotyping master mix reagentsents, volume and concentration Reagents

Manufacturer Concentration per 1 reaction

Volume per 1 reaction

Water 26 μl MgCl2

Bioline reagents Ltd, UK

50 mM 1.2 μl

dNTPs

Bioline reagents Ltd, UK

10 mM for each dATP, dGTP, dCTP and dTTP

1 μl for each dATP, dGTP, dCTP and dTTP

Taq buffer

Bioline reagents Ltd, UK

5 μl 10 X

Taq polymerase

Bioline reagents Ltd, UK

0.3 μl 5 U

Cocktail of primers

Integrated DNA technology Inc, USA

1 μl for each primer 10 pmol

Total 40 μl

6. Sequence identity matrix of aligned sequences for the whole genome segments of the study strains.

Table 5.1: VP6 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 98.1 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 76.8 77.1 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 4 88.3 87.1 77.1 ID

RVA/Human-wt/ARG/Ros990/1998/G4P8 5 97.9 96.8 76.6 88.2 ID

RVA/Human-wt/Bethesda/DC1285/1980/G4P8 6 97.9 96.6 77.1 88.9 97.6 ID

RVAHuman-wt/RUS/Nov06-1255/2006/G4P8 7 98.4 99.6 76.8 87.4 96.8 96.8 ID

RVA/Human-wt/GR/Ath146/2010/G4P8 8 98.3 99.5 77.1 87.6 96.7 96.7 99.7 ID

RVA/Human-wt/PRY/54SR/2002/G4P8 9 97.5 96.9 76.8 88.4 98.9 97.5 96.9 97 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/XXXX/G4P6 10 76.8 77.1 100 77.1 76.6 77.1 76.8 77.1 76.8 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 11 90.6 90.1 78 89.2 90.2 90.6 90.1 90 90.2 78 ID

RVA/Human-wt/CHN/E931/2008/G4P6 12 90.5 89.9 77.6 89.6 90.2 90.6 90.1 89.8 90.2 77.6 92.5 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 6 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

74

Table 5.2: VP1 Strain common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 94.7 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 73.5 73 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 86.3 86.6 71.9 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 82.9 83.7 70.6 82.6 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 88.2 87.3 71.8 86.1 83.3 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 95.2 98.3 73.3 87 83.5 87.9 ID

RVA/Human-wt/PRY/350/1999/G4P8 8 95.2 98.1 73.2 87.1 83.4 87.9 99.8 ID

RVA/Human-wt/CHN/E2484/2011/G4P8 9 96.3 93.9 73.3 86.3 83 88.1 94.3 94.1 ID

RVA/Human-wt/USA/Bethesda/DC1285/1980/G4P8 10 98 95.1 73.8 86.8 83.3 88.5 95.4 95.3 97.6 ID

RVA/Human-wt/ARG/Ros990/1998/G4P8 11 97.3 94.1 73.7 86.3 82.8 88.1 94.6 94.6 95.5 97.3 ID

RVA/Human-wt/ARG/Tuc1650/1998/G4P8 12 96.6 94.2 73.3 86.3 83.2 88.4 94.5 94.4 98 98 96.1 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 1 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

Table 5.3: VP2 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 95.6 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 31.6 31.7 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 33.1 33 30.4 ID

RVA/Human-wt/PRY/1809SR/2009/G4P6 5 32.7 32.6 30.3 92.2 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 33.2 32.9 30.4 92.6 92.1 ID

RVA/Human-wt/ARG/Res1717/1998/G4P8 7 95.8 99.3 31.6 33 32.5 32.9 ID

RVA/Human-wt/ARG/Res1730/1998/G4P8 8 33.1 33.2 32.2 31.8 32.2 31.6 33.4 ID

RVA/Human-wt/USA/Bethesda/DC1208/1980/G4P8 9 33.1 32.9 30.8 93.3 92.3 93.6 32.9 32.2 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 10 95.7 99.2 31.6 33 32.6 32.9 99.8 33.4 33 ID

RVA/Human-wt/PRY/350/1999/G4P8 11 95.7 99.3 31.6 33 32.5 32.8 99.9 33.5 33 99.8 ID

RVA/Human-wt/HUN/ERN5232/2012/G4P8 12 33.2 33.2 32.2 31.7 32.2 31.5 33.3 99 32.2 33.3 33.4 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 2 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

Table 5.4: VP3 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 12 13

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 90.4 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 74.6 74.2 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 87.7 88.6 73.4 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 86.9 87.5 73.1 90.6 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 83.8 83.2 73.4 83 82.6 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 90.7 98.8 74.4 88.8 87.6 83.2 ID

RVA/Human-wt/PRY/350/1999/G4P8 8 90.6 98.7 74.5 88.9 87.7 83.3 99.8 ID

RVA/Human-wt/ARG/Ush2037/1999/G4P8 9 90.6 98.7 74.3 88.8 87.6 83.1 99.8 99.7 ID

RVA/Human-wt/PRY/186/1998/G4P8 10 90.7 98.8 74.3 88.9 87.7 83.2 100 99.8 99.8 ID

RVA/Human-wt/HUN/ERN5121/2012/G4P8 11 91.5 97.8 74.5 88.3 87.3 83.4 97.7 97.6 97.5 97.7 ID

RVA/Human-wt/HUN/ERN5232/2012/G4P8 12 97.9 90.2 74.2 87.2 86.5 83.6 90.4 90.2 90.2 90.3 91.5 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 3 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

75

Table 5.5:NSP1 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 82.4 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 71.7 70.3 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 78.5 78.6 71.9 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 83.7 83 70.9 79.6 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 75.6 76.3 70.1 76.2 74.4 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 82.6 98.4 70.5 79 83.5 76.3 ID

RVA/Human-wt/ARG/Res1717/1998/G4P8 8 82.6 98.4 70.5 79 83.5 76.3 100 ID

RVA/Human-wt/ARG/Res1730/1998/G4P8 9 82.6 98.2 70.5 79.1 83.4 76.2 99.8 99.8 ID

RVA/Human-wt/PRY/350/1999/G4P8 10 82.6 98 70.7 79 83.4 76.2 99.7 99.7 99.4 ID

RVA/Human-wt/CHN/E2484/2011/G4P8 11 93.4 81.8 72.2 78 83.1 75.6 81.7 81.7 81.7 81.7 ID

RVA/Human-wt/HUN/ERN5199/2012/G4P8 12 82.4 99.2 70.6 79 83.1 76.2 98.6 98.6 98.4 98.2 81.5 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 5 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

Table 5.6: NPS2 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 87.6 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 83.6 82.5 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 93.2 87.2 83.7 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 29.8 29.5 28.5 29.1 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 92.6 88.1 83.3 92.1 28.6 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 97 88.9 83.6 92.3 29.7 92.1 ID

RVA/Human-wt/ARG/Res1717/1998/G4P8 8 97 88.9 83.6 92.3 29.7 92.1 100 ID

RVA/Human-wt/ARG/Res1730/1998/G4P8 9 97 88.9 83.6 92.3 29.7 92.1 100 100 ID

RVA/Human-wt/PRY/350/1999/G4P8 10 97 89.1 83.8 92.5 29.8 92.1 99.8 99.8 99.8 ID

RVA/Human-wt/CHN/E2484/2011/G4P8 11 89.5 93.7 82.7 88.3 29.3 89.4 89.5 89.5 89.5 89.8 ID

RVA/Human-wt/HUN/ERN5232/2012/G4P8 12 87.9 99.6 82.8 87.6 29.6 88.3 89.1 89.1 89.1 89.3 94.1 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 8 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

Table 5.7: NSP3 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 97.4 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 78.7 78.5 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 82.9 83.4 77.5 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 32.1 31.7 31.9 33.3 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 90.7 89.8 79.1 83.3 32.1 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 98 99 78.3 83 31.6 89.9 ID

RVA/Human-wt/PRY/350/1999/G4P8 8 98.1 99.1 78.4 83.1 31.7 90 99.9 ID

RVA/Human-wt/ARG/Res1730/1998/G4P8 9 97.2 96 78.2 82.6 31.7 90.1 96.5 96.6 ID

RVA/Human-wt/USA/Bethesda/DC1285/1980/G4P8 10 98.4 97.8 78.3 83.1 32.4 90.6 98.3 98.4 97.1 ID

RVA/Human-wt/ARG/Ros990/1998/G4P8 11 98.3 97.4 78.4 82.9 32.2 90 98 98.1 97 98.1 ID

RVA/Human-wt/HUN/ERN5232/2012/G4P8 12 97.4 99.6 78.9 83.6 31.7 89.8 99 99.1 96 97.8 97.4 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 7 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

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Tabale 5.8:NSP4 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 93.9 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 80.3 78.6 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 89.4 90.5 79.5 ID

RVA/Human-wt/CHN/E931/2008/G4P6 5 92.4 93.2 80.7 91.5 ID

RotaA/Human-wt/TUN/1125/2007/G4P6 6 27.7 28.1 25.6 27.7 27.9 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 94.7 97.3 79.9 90.9 93.9 27.7 ID

RVA/Human-wt/PRY/350/1999/G4P8 8 94.5 97.2 80.1 90.7 93.8 27.7 99.8 ID

RVAhuman/Bethesda/DC1285/1980/G4P8 9 98.3 93.6 80.3 89.4 92.8 27.5 94.3 94.1 ID

RVA/Human-wt/ZAF/GR833/86/1999/G4P8 10 99.8 94.1 80.5 89.6 92.6 27.7 94.9 94.7 98.5 ID

RVA/Human-wt/ARG/Tuc1650/1998/G4P8 11 97.7 94.1 80.5 89 92.4 28.3 94.5 94.3 97.5 97.9 ID

RotaA/Human-wt/TUN/6970/2006/G4P8 12 28.5 29 25.4 27.5 28.3 97.5 28.5 28.5 28.3 28.5 28.8 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 10 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).

Table 5.9: NSP5 Strain Common Name Number 1 2 3 4 5 6 7 8 9 10 11 12

RVA/Human-wt/ZAF/MRC-DPRU20683/1985/G4P8 1 ID

RVA/Human-wt/ZWE/MRC-DPRU1850/2011/G4P8 2 98.5 ID

RVA/Human-wt/ZMB/MRC-DPRU1752/2009/G4P6 3 67 66.8 ID

RVA/Human-wt/HUN/BP1901/1991/G4P6 4 97.3 97.4 66.5 ID

RVA/Human-wt/COD/KisB332/2008/G4P6 5 97.3 97.1 67 97.3 ID

RVA/Human-wt/CHN/E931/2008/G4P6 6 95.9 95.7 67 95.9 94.8 ID

RVA/Human-wt/ARG/Mis864/1998/G4P8 7 98.8 99 67 97.4 97.4 95.7 ID

RVA/Human-wt/ARG/Res1717/1998/G4P8 8 99 99.2 67.2 97.6 97.6 95.9 99.8 ID

RVA/Human-wt/ARG/Res1730/1998/G4P8 9 99 99.2 67.2 97.6 97.6 95.9 99.8 100 ID

RVA/Human-wt/PRY/350/1999/G4P8 10 99 99.2 67.2 97.6 97.6 95.9 99.8 100 100 ID

RVA/Human-wt/CHN/E2484/2011/G4P8 11 97.6 97.4 67.2 96.9 95.9 94.8 97.8 98 98 98 ID

RVA/Human-wt/HUN/ERN5232/2012/G4P8 12 98.6 99.2 67.9 97.3 96.9 95.9 99.2 99.3 99.3 99.3 98 ID Evolutionary percent divergence between the nucleotide sequences of Gene segment 11 of the study strains and other sequences obtained from the NCBI GenBank, sharing highest identity to the study strains. The results are based on sequence identity matrix of aligned sequence on Mega 6 sequence alignment editor (Tamura et al., 2013).