**5.2 Sample collection and processing**

Stool specimens were collected into clean universal containers. Supernatant obtained from stool suspension of 50% in 1 ml sterile phosphate buffered saline were stored at -20°C for RT-PCR analysis of norovirus.

### *5.2.1 RNA extraction*

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

#### *5.2.2 cDNA synthesis*

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 manufactures were followed for cDNA synthesis.

**Figure 1.** *Map of Southern Nigeria. States included in this study were Bayelsa, Delta and Edo.*

#### *5.2.3 Polymerase chain reaction*

The cDNA generated was then amplified by PCR in a 45 μL reaction mixture as described in a previous study [18]. Specific primers (G1SKRCAACCCARCCA TTRTACA) and G1FFN (GGAGATCGCAATCTCCTGCCC) were used for GI genotyping, while for genotyping GII noroviruses, primers GIIFBN (TGGGAGGGCGATCGCAATCT) and GIISKR (CCRCCNGCATRHCCRTTRTACAT), respectively, were used in an RT-PCR analysis. The products were visualized on UV illuminator and photographed using Polaroid camera [19]. The RT-PCR used is a very sensitive method, it can detect as few as 5 x 106 copies per gram of stool sample. U-TaQ DNA polymerase (SBS genetech, Beijing, China), a high-fidelity thermostable enzyme that can withstand prolonged incubation at high temperature up to 95°C without significant loss of activity was used for this RT-PCR protocol.

#### *5.2.4 Norovirus sequencing*

The amplicons from the partial gene regions of the viral capsid genes were purified using QIAquick PCR purification kit (Qiagen Inc., Valencia, CA). Nucleotide sequencing was done using Big Dye ® Terminator v 3.1 Cycle sequencing kit (Applied Biosystems, Carlsbad, CA) on 3130 DNA genetic analyzer (Applied Biosystems, Carlsbad, CA), Sequences were edited using sequencher® Version 5.4.6 DNA sequence analysis software (Gene codes Corporation, Ann Arbor, MI, USA). Norovirus genotypes were determined by comparison of corresponding sequences of norovirus strains using the online norovirus genotyping tool version 1.0. available at (www.rivm.nl/mpf/norovirus/typing tool).

#### *5.2.5 Phylogenetic analysis*

For confirmation of genotyping, nucleotide sequences obtained were aligned with reference sequences using MUSCLE [20]. The evolutionary history was inferred by using Maximum Likelihood method and Kimura 2-Parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale with the branch lengths measured in the number of substitutions per site. Accession Number KF307755, KF3077756, MN271357, MN271380 were used as outgroup.

### **6. Results**

Out of the 405 children enrolled, 45 (11.1%) were positive for norovirus using RT-PCR (**Table 1**). Only norovirus genogroup I and II were recovered in this study, norovirus genogroup IV were not detected. Norovirus genogroup II was the most prevalent (84.4%) among th**e** children (**Table 2**). GII noroviruses where also most commonly encountered among all centers included in this study (**Table 3**). Based on capsid gene sequences recovered from the 45 norovirus positive samples, two GI noroviruses genotype was detected in this study GI.3, 71.4% and genotype G1.5, 28.6% respectively. Seven genotypes of GII noroviruses were detected, genotypes GII.4 (63%), GII.12 (7.9%), GII.17 (7.9%), GII.6 (5.3%), GII.7 (5.3%), GII.14 (5.3%) and GII.2 (5.3%) (**Table 4**). GII.4 was found to be the most prevalent genotypes in all States studied, Delta (53.4%), Bayelsa (75.0%) and Edo State (46.2%) (**Table 5**).

Subtyping analysis with the norovirus genotyping tool demonstrated that the majority of the norovirus GII.4 recovered in this study where homologous to the

**203**

**Table 4.**

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

PHC, Pessu/ Ugbuwangwe 68 15 (22.1) FMC Yenagoa 80 4 (5.0) Central Hospital, Benin 99 3 (3.0) Stella Obasanjo Hospital 75 10 (13.3)

*Norovirus infection detected by RT-PCR among children under 5 years from different health facilities in* 

**Norovirus genogroup Frequency (%)** GI 7 (15.6) GII 38 (84.4) **Total 45**

**Study Centre No. Tested Nov GI Nov GII** Central Hospital, Warri 13 2 (15.4) 11 (84.6) PHC, Pessu/Uguwangwe 15 2 (13.3) 13 (86.7) FMC, Yenagoa 4 1 (25.0) 3 (75.0) Central Hospital, Benin 3 1 (33.3) 2 (66.7) Stella Obasanjo CWH, Benin 10 1 (10.0) 9 (90.0)

*Frequency of occurrence of genogroup of norovirus in study population.*

*Distribution of norovirus genogroups among the health facilities in the study.*

*Distribution of genotypes of norovirus in study population.*

**Genotypes Frequency**

GI.3 5 (71.4%) GI. 5 2 (28.6%)

GII.2 2 (5.3%) GII.4 24 (63.0%) GII.6 2 (5.3%) GII.7 2 (5.3%) GII. 12 3 (7.9%) GII. 14 2 (5.3%) GII.17 3 (7.9%)

**Location No. Tested No. Infected (%) P value** Central Hospital, Warri 83 13 (15.7) 0.0032

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

*P < 0.05.*

**Table 1.**

**Table 2.**

**Table 3.**

GI

GII

*south–south region of Nigeria.*

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


#### **Table 1.**

*Norovirus infection detected by RT-PCR among children under 5 years from different health facilities in south–south region of Nigeria.*


#### **Table 2.**

*Frequency of occurrence of genogroup of norovirus in study population.*


#### **Table 3.**

*Distribution of norovirus genogroups among the health facilities in the study.*


#### **Table 4.**

*Distribution of genotypes of norovirus in study population.*


#### **Table 5.**

*Distribution of norovirus genotypes among study centers.*

#### **Figure 2.**

*Norovirus GII.4 variants among patients in this study.*

New Orleans 2009 variant 12 (50.0%), while 10 (41.7%) isolates showed homology to the Sydney 2012 strains. Two GII.4 sequences were unassigned. All sequences where further subjected to phylogenetic analysis, genotypic assignment of all sequences was based on bootstrap cut off values >70%. The result showed that norovirus GII.4 were more genetically diverse (**Figure 2**).

Three genetic clusters of Sydney 2009 variants where found to circulate among study participants, with varying sequence identity (range 74–100%). The two sequences that were unassigned using the genotyping tool were assigned following phylogenetic analysis. One GII.4 isolate (MN271364) did not cluster with either New Orleans or Sydney 2009 reference strains despite been assigned to be GII.4 Sydney strain by the genotyping tool, but had genetic sequence similarity to a norovirus strain isolated in 1993 in Bristol (X76716), which itself is a recombinant strain which was neither New Orleans nor Sydney 2009 strain (**Figure 3**). This strain is however deemed a putative novel recombinant.

**205**

**7. Discussion**

**Figure 3.**

South region of Nigeria.

worldwide [27].

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

A prevalence of 11.1% RT-PCR confirmed norovirus infection among children under 5 years of age was observed. This prevalence report is higher than the 6.7% in Ile –Ife, Osun State [21], but less than the 37.3% among children in Lagos, Nigeria [22]. This report supports the hypothesis that the prevalence of norovirus within Nigeria is not homogenous across communities and health centers. Hence, community and hospital-based surveillance are needed to provide an estimate of norovirus burden in Nigeria. The finding of this study might imply that norovirus may be one of the major etiologic agents of diarrhea among children less than 5 years in South–

*Phylogenetic tree of norovirus GII.4 identified in this study: There were a total of 1487 positions in the final dataset. Sequences in this study are represented with filled triangles, reference sequences are not marked.* 

*MN271364 had low bootstrap values, as such classified to be a putative novel recombinant.*

Only norovirus belonging to GI and GII genogroups were identified in this study, this concurs with findings from previous norovirus molecular epidemiologic studies [23, 24]. Norovirus genogroup II was the most commonly recovered norovirus in this study. This report concurs with findings from other parts of the World [25, 26]. This study further highlights the superior role of norovirus genogroup II in cases of norovirus induced gastroenteritis among children. In this study, GII.4 norovirus strains were most commonly detected. It has been well established that GII. 4 noroviruses are responsible for the majority of outbreaks

Phylogenetic analysis revealed two GII.4 sequence variants, two Sydney 2012 clusters and one cluster for the New Orleans 2009 strain and a putative recombinant GII.4 virus. It is established that new variants of GII.4 norovirus emerge every 1–2 years, due to genetic recombination and point mutations resulting in the generation of new genetic clusters, recombinants/genotypes allowing increasing genetic fitness and continuous spread in populations by evading host immune responses [28]. GII.4 Possess the largest number of intra-genotypic variants and recombinants [29]. It is also possible for two or more variants to co-circulate at the same time in a geographical location [29]. Notable GII.4 variants causing majority

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

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

#### **Figure 3.**

*Phylogenetic tree of norovirus GII.4 identified in this study: There were a total of 1487 positions in the final dataset. Sequences in this study are represented with filled triangles, reference sequences are not marked. MN271364 had low bootstrap values, as such classified to be a putative novel recombinant.*

#### **7. Discussion**

A prevalence of 11.1% RT-PCR confirmed norovirus infection among children under 5 years of age was observed. This prevalence report is higher than the 6.7% in Ile –Ife, Osun State [21], but less than the 37.3% among children in Lagos, Nigeria [22]. This report supports the hypothesis that the prevalence of norovirus within Nigeria is not homogenous across communities and health centers. Hence, community and hospital-based surveillance are needed to provide an estimate of norovirus burden in Nigeria. The finding of this study might imply that norovirus may be one of the major etiologic agents of diarrhea among children less than 5 years in South– South region of Nigeria.

Only norovirus belonging to GI and GII genogroups were identified in this study, this concurs with findings from previous norovirus molecular epidemiologic studies [23, 24]. Norovirus genogroup II was the most commonly recovered norovirus in this study. This report concurs with findings from other parts of the World [25, 26]. This study further highlights the superior role of norovirus genogroup II in cases of norovirus induced gastroenteritis among children. In this study, GII.4 norovirus strains were most commonly detected. It has been well established that GII. 4 noroviruses are responsible for the majority of outbreaks worldwide [27].

Phylogenetic analysis revealed two GII.4 sequence variants, two Sydney 2012 clusters and one cluster for the New Orleans 2009 strain and a putative recombinant GII.4 virus. It is established that new variants of GII.4 norovirus emerge every 1–2 years, due to genetic recombination and point mutations resulting in the generation of new genetic clusters, recombinants/genotypes allowing increasing genetic fitness and continuous spread in populations by evading host immune responses [28]. GII.4 Possess the largest number of intra-genotypic variants and recombinants [29]. It is also possible for two or more variants to co-circulate at the same time in a geographical location [29]. Notable GII.4 variants causing majority

of pandemic diseases are the Sydney 2012, New Orleans 2009, Farmington Hills 2002, US95/961995, Hunter 2004 and Den Haag 2009 [30]. Certain variants cause localized epidemics, Cairo 2007, Japan 2008 and Asia 2003 [30].

This study reports on the finding of norovirus GII.17 among children in Southern, Nigeria for the first time, this genotype is known to be very virulent [17]. First documented evidence of emergence of GII.17 occurred in the winter of 2014–2015 in Asia [31].

The finding of this study provides evidence of the existence of diverse genetic subtypes of norovirus in our locality. This data has illuminated the epidemiological profile of norovirus induced diarrhea/gastroenteritis in our locality.
