**2. Norovirus genome and genetic diversity**

Taxonomically, norovirus is a positive sense RNA, non-enveloped virus in the family caliciviridae [6]. The genus has one species, known as Norwalk virus [6]. Norovirus are highly genetically diverse, Phylogenetically, they can be classified into 10 different genogroups (GI-GX) and several genotypes in this order; 60 P-types (14GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII and 1 GX), with each of these genotypes having several genetic clusters and sub groups [7].

The genome is organized into three open reading frames (ORF) [8]. ORF 1 encoded six non-structural proteins (NS1-NS6) and the RNA dependent RNA Polymerase RdRP, while ORF 2 encodes the capsid proteins-the major structural proteins VP1 containing the shell (S) and protruding (P) domains. The S domain surrounds the viral RNA and the P domain, which consists of the P2 domain, is linked to the S domain through a flexible hinge [9]. ORF 3 encodes a minor structural protein [9]. In spite of the large genetic diversity of norovirus, it is noteworthy that norovirus of the genotype GII.4 are responsible for a majority of infections [10, 11].

## **3. Norovirus recombinants**

Recombination are mechanism in the evolution of RNA viruses, this creates changes in virus genomes by exchanging sequences, thereby generating genetic variation and producing new viruses [12]. RNA recombination is very common among RNA viruses belonging to the family *picornaviridae, coronaviridae, retroviridae and caliciviridae* [13]. Recombination commonly occurs at the ORF1-ORF2 junction [14] although other recombination sites have been reported. Recombination event is high among the GII.4 noroviruses [14]. Norovirus recombination has been recognized to be a major tool it uses to evade host immune recognition [15]. Monitoring the incidence of rate of generation of new norovirus recombinants is a vital tool in the understanding of norovirus evolution and continuous global spread. Norovirus recombination has been linked to increased rate of generation of new norovirus genotypes and subtypes [16], this has also hampered the possibility of a possible vaccine.

#### **4. Justification for this study**

The introduction of rotavirus vaccines throughout the world has made norovirus the most common aetiologic agent of gastroenteritis world over [17]. An update on the predominant norovirus genotype in a given population is needed for the development of effective vaccine. There are no data on the genetic diversity of norovirus among children in South–South, Nigeria. Also, there are no data on the prevalence of norovirus in Nigeria. Against this background, this study was conducted to determine the prevalent norovirus genotypes and existence of possible GII.4 recombinants among children under 5 years with diarrhea in South–South, Nigeria.

#### **5. Method**

#### **5.1 Study area and study population**

This cross-sectional study was conducted in the period, March, 2018 to February, 2019. 405 children with clinical symptoms of diarrhea/gastroenteritis

**201**

**Figure 1.**

*Sequencing of Norovirus in Southern, Nigeria: Prevalent Genotypes and Putative GII.4 Novel…*

from a pool of 2813, attending outpatient clinics of four secondary health facilities (Central Hospital, Warri, Central Hospital, Benin, Primary Health Centre Pessu and Federal Medical Centre, Yenagoa) in Delta, Edo and Bayelsa States, Niger-Delta region, Nigeria were randomly included. Inclusion criteria where at least 3 clinical episodes of diarrhea- with an onset of 1 to 7 days whose parents or guardians consented for their ward/children to participate were included in this study. One hundred (100) asymptomatic apparently healthy age and sex matched children who

Stool specimens were collected into clean universal containers. Supernatant obtained from stool suspension of 50% in 1 ml sterile phosphate buffered saline

RNA extraction was from thawed frozen samples were performed using AccuPrep® Viral RNA Extraction Kit (Bioneer, Daejon South Korea), following

cDNA synthesis was carried out on a 20 μl reverse transcription reaction of 1.0ug of extracted RNA on 0.2 ml tubes of Accupower Cycle script RT Premix (Bioneer Corporation, South Korea). Standard protocols as recommended by

*DOI: http://dx.doi.org/10.5772/intechopen.94389*

served as controls (**Figure 1**).

*5.2.1 RNA extraction*

*5.2.2 cDNA synthesis*

manufacturers instruction.

**5.2 Sample collection and processing**

were stored at -20°C for RT-PCR analysis of norovirus.

manufactures were followed for cDNA synthesis.

*Map of Southern Nigeria. States included in this study were Bayelsa, Delta and Edo.*

*Sequencing of Norovirus in Southern, Nigeria: Prevalent Genotypes and Putative GII.4 Novel… DOI: http://dx.doi.org/10.5772/intechopen.94389*

from a pool of 2813, attending outpatient clinics of four secondary health facilities (Central Hospital, Warri, Central Hospital, Benin, Primary Health Centre Pessu and Federal Medical Centre, Yenagoa) in Delta, Edo and Bayelsa States, Niger-Delta region, Nigeria were randomly included. Inclusion criteria where at least 3 clinical episodes of diarrhea- with an onset of 1 to 7 days whose parents or guardians consented for their ward/children to participate were included in this study. One hundred (100) asymptomatic apparently healthy age and sex matched children who served as controls (**Figure 1**).
