




































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: harunaisamohammed@nsuk.edu.ng; 
 
 
 

Asian Journal of Immunology 
 
4(1): 135-145, 2021; Article no.AJI.77674 
 

 
 

 

 

Molecular Detection and Sequencing of Rotavirus 
VP4 among Children Aged 0-5 Years with 

Gastroenteritis in 2 Selected Healthcare  
Centres in Keffi, Nigeria 

 
I. Ibrahim1, R. U. Usman2, H. I. Mohammed3*, D. Ishaleku3  

and A. B. Shuaibu4 
 

1
Medical Microbiology Unit, Medical Laboratory Department, Federal Medical Centre, P.M.B. 004, 

Keffi, Nasarawa State, Nigeria. 
2
Science Department, Brightway International Academy, Keffi, Nasarawa State, Nigeria. 

3
Department of Microbiology, Nasarawa State University, P.M.B. 1022, Keffi, Nasarawa State, 

Nigeria. 
4
Department of Veterinary Medicine, Usman Danfodio University, P.M.B. 2346, Sokoto, Nigeria. 

 
Authors’ contributions  

 
This work was carried out in collaboration among all authors. Authors II and HIM designed the study, 

collected samples, performed laboratory and statistical analyses and wrote the first draft of the 
manuscript. Authors RUU, DI and ABS designed the study, managed literature searches, wrote the 

protocols and managed the analyses of the study. All authors read and approved the final manuscript. 
 

Article Information 
 

Editor(s): 
(1) Dr. Wagner Loyola, Brazilian Agricultural Research Corporation, Brazil. 

Reviewers: 
(1) Nirmal Kumar Mohakud, KIIT University, India. 

(2) Ahmed Samy El-Shafey, Tanta University, Egypt. 
Complete Peer review History, details of the editor(s), Reviewers and additional Reviewers are available here: 

https://www.sdiarticle5.com/review-history/77674 

 
 
 

Received 20 October 2021  
Accepted 27 December 2021 
Published 29 December 2021 

 
 

ABSTRACT 
 

Aims: This study was conducted to detect and sequence Rotavirus VP4 among children aged 0-5 
years with gastroenteritis in 2 selected healthcare centres in Keffi, Nigeria.  
Study Design: The study was a cross sectional study. 
Place and Duration of Study: Keffi, Nasarawa State, between March and June 2019. 
Methodology: Stool samples were collected from 303 (203 from FMC Keffi and 100 from PHC 
Angwan Waje, Keffi) children with gastroenteritis and information about them were obtained by 

Original Research Article 



 
 
 
 

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136 

 

structured questionnaires. All collected samples were screened for the presence of Rotavirus 
antigen using Aria Rotavirus antigen detection test kit (CTK Biotech, Inc, San Diego, USA). VP4 
was detected from Rotavirus positive samples by Reverse Transcriptase Polymerase Chain 
Reaction (RT-PCR) using specific primers. The sequences of the amplified VP4 genes were 
verified using MEGA software version 7 and Rotavirus strains were determined by pasting the 
FASTA (Text based format for representing nucleotide sequence) format into the Basic Local 
alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI). 
Data collected were analysed using Smith’s Statistical Package (version 2.8, California, USA) and 
P value of ≤ 0.05 was considered statistically significant.  
Results: Of the 303 children screened, 54(17.8%) were positive for Rotavirus infection. Highest 
prevalence of the viral infection was recorded among males (19.7%) aged 0-12 months (24.8%). 
Gender was found to be associated with rate of Rotavirus infection in this study (P<0.05). However, 
age was not significantly associated with the viral infection (P>0.05). Furthermore, based on the 
RT-PCR carried out, 3(5.6%) out of the 54 Rotavirus positive samples were positive for the VP4 
gene and sequences of this gene were all found to be of type P [11] Strain N115. 
Conclusion: This study reveals the presence of infection with type P [11] Strain N115 (5.6%) of 
Rotavirus in the study population. The detection of this rare rotavirus strain in this study is a cause 
for concern and hence there is an urgent need for the Nigerian health authorities to implement a 
nationwide surveillance system for monitoring rotavirus molecular epidemiology. 

 

 
Keywords: VP4; rotavirus; gastroenteritis; children; Keffi; Nigeria. 
 

1. INTRODUCTION 
 

Rotavirus is the leading cause of severe 
gastroenteritis particularly among infants and 
young children worldwide with over 70% of cases 
occurring in Nigeria and other endemic sub-
Saharan African countries [1-4]. It is believed that 
almost every child in the world is infected with 
rotavirus at least once by the age of five [5]. 
However, Immunity develops with each infection, 
so subsequent infections are less severe and 
hence adults are rarely affected [6].  
 

Rotavirus belongs to the virus family Reoviridae, 
it is a non-enveloped and has an icosahedral 
nueleocapsid structure, enclosing a double 
stranded (ds) RNA genome segmented into 11 
compartments. The genome codes for six 
structural proteins, (VP1 to VP4, VP6 and VP7) 
and five nonstructural proteins (NSP1 to NSP5) 
[7]. VP4 is on the surface of the virion that 
protrudes as a spike [8]. It binds to molecules on 
the surface of cells called receptors and drives 
the entry of the virus into the cell [9]. It has to be 
modified by the protease enzyme trypsin, which 
is found in the gut, into VP5 and VP8 before the 
virus is infectious [10]. It also determines 
how virulent the virus is and determines the P-
type of the virus [11].  
 

There are at least 10 distinct species/groups of 
Rotavirus (A- I, J), differentiated by their VP6 
antigenic properties [12]. Groups A, B and C are 
found to cause infection in both humans and 

animals, whereas groups D, E, F, G and I have 
been found only in animals [13]. There are 32 G 
(VP7) genotypes and 47 P (VP4) genotypes 
identified through molecular epidemiology           
[2]. 

 
Rotaviruses are transmitted by the fæcal-oral 
route, via contact with contaminated hands, 
surfaces and objects, and possibly by the 
respiratory route [3,4,14]. The viral infection is 
usually associated with fever, nausea and 
vomiting, followed by abdominal cramps, 
frequent watery diarrhea, which may last for 3-8 
days, cough and runny nose occurring mostly 
among infants [3,15]. 

 
Rotaviruses are stable in the environment and 
can survive for long periods of time on toys and 
ordinary surfaces found in most homes. They 
also are relatively resistant to most soap and 
commonly used disinfectants, hence preventing 
a child from exposure can be difficult [3]. 
Therefore, vaccination still remains the best hope 
for preventing infection with Rotavirus especially 
in resource constraint nations such as Nigerian 
[1,16]. Additionally, most hospitals and diagnostic 
centres in such nations do not routinely 
diagnosed Rotavirus infection probably due to it 
high cost and may be because it share similar 
signs and symptoms with other infantile 
gastroenteritis [14,15,17].This consequently 
usually leads to misdiagnosis and mistreatment 
of the viral infection.  



 
 
 
 

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137 

 

Thus the aim of this study was to detect and 
sequence Rotavirus VP4 among children aged 0-
5 years with gastroenteritis in 2 selected 
healthcare centres in Keffi, Nigeria. 
 

2. MATERIALS AND METHODS 
 

2.1 Study Area 
 

This study was conducted at Federal Medical 
Centre (FMC) and Primary Healthcare Centre 
(PHC), Angwan Waje in Keffi Local Government 
Area, Nasarawa State, Nigeria. Keffi town, where 
the 2 centres are located is approximately 68 km 
from Abuja, Nigeria’s Federal Capital Territory 
and 128 km from Lafia, the capital of Nasarawa 
State. It is located geographically between 
latitude 8°3’N of the equator and longitude 
7°50’E and situated on an altitude of 850 m 
above sea level [18]. 
 

2.2 Study Population 
 

The study participants were male and female 
children with gastroenteritis aged 0-5 years 
accessing health care in the 2 selected 
healthcare centres in Keffi, Nasarawa State, 
Nigeria. Their socio-demographic and other 
required information were obtained from their 
parents/guardians by the use of a designed 
questionnaire. 
 

2.3 Sample Size Determination  
 

To determine the sample size, the formula by 
Naing et al. [19] for sample size calculation at 
0.05 level of precision was used; 
 

  
    

  
 

 

Where: 
 

n = required sample size  
Z = standard normal deviation at the required 
confidence interval (1.96) which corresponds to 
95% confidence interval. 
P = prevalence of Rotavirus infection from 
previous study (25.0%) (0.2) [20]. 
Q = 1 – p = 0.9 
d = degree of precision expected (0.05)  
  

   
                 

       
  

           

      
 

        

      
              

 

       
 
To ensure minimum error however, this was 
rounded up to 303 samples. 

2.4 Sample Collection and Storage 
 
A total of 303 stool samples (203 from FMC keffi 
and 100 from PHC Angwan Waje, Keffi) were 
collected from children with gastroenteritis aged 
0-5 years. The samples were collected into 
sterile wide mouth universal containers, labeled 
accordingly and stored at -20

o
C until ready for 

use [21]. 
 

2.5 Laboratory Analysis 
 
2.5.1 Detection of rotavirus antigen 
 
All collected stool samples were screened for the 
presence of Rotavirus antigen using Aria 
Rotavirus antigen detection test kit (CTK Biotech, 
Inc, San Diego, USA). The tests procedure and 
results interpretation were done according to the 
instructions of the manufacturer. 
 
2.5.2 Test procedure 
 
The specimen was brought to room temperature 
and the test device was removed from the pouch 
and placed on a clean flat surface and labeled 
accordingly. Two drops of the watery stool was 
added into the labeled sample dilution vial 
followed by vigorous shaking to mix. Thereafter, 
2 drops of the mixture was dispensed into the 
sample well of the test device and a timer was 
set up for 15 minutes after which the results were 
read and interpreted as positive, negative or 
invalid according to the manufacturer’s 
instructions.  
 
2.5.3 Molecular detection of rotavirus VP4   
 
The VP4 was detected by a Reverse 
Transcriptase Polymerase Chain Reaction (RT-
PCR) system previously described by Gentsch et 
al. [22] using specific primers (consensus 
primers VP4F/VP4R) adopted from the work of 
Simmonds et al. [23]. 
 

2.5.4 Rotavirus RNA extraction  
 
Viral RNA was extracted and purified from 10% 
faecal suspensions in phosphate-buffered saline 
using the TRIzol method (Gibco BRL, Invitrogen, 
Burlington, Canada). 
 

2.5.5 Rotavirus VP4 RT-PCR 
 
The VP4 gene was reversely transcribed and 
amplified using the following primers as adopted 
from the work of Simmonds et al. [23]: 



 
 
 
 

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138 

 

Con2 Forward (5′-ATTTCGGACCATTTATAACC-
3′)  
Con3 Reverse (5′-
TGGCTTCGCTCATTTATAGACA-3′) 
 
The complementary DNA was generated by 
reverse transcription at 45

o
C for 30 minutes with 

initial denaturation at 95
o
C for 5 minutes followed 

by 35 cycles of denaturation at 95
o
C for 1 

minute.  
 
2.5.6 Agarose gel electrophoresis 
 
The PCR products were analyzed by running a 
1% agarose gel stained with ethidium bromide. 
The sizes of PCR products were estimated in 
relation to the migration pattern of a 100bp to 
1000bp increments plus DNA molecular marker 
(BIONEER Daejeon, North Korea). 
 
2.5.7 Rotavirus VP4 gene sequencing 
 
The amplified VP4 genes obtained were sent to 
Inqaba Biotec, South Africa for sequencing. The 
method described by Sanger et al. [24] with 
modification was used.  Briefly: 10µL of the 
amplified VP4 genes were cleaned by mixing 
with 2.5µL EXO/SAP master mix and incubated 
at 37

o
C for 15 minutes. The reaction was 

stopped by heating the mixture at 80
o
C for 15 

minutes. Sequencing was then done on the 
mixture using Nimagen; Brilliant Dye™ 
terminator cycle sequencing kit version 3.1 BRD. 
The labelled products were then cleaned using 
ZR-96 DNA sequencing clean-up kit and 
analysed using Applied Biosystems ABI3500XL 
Genetic analyser yielding sequence 
chromatogram. Sequences were verified using 
MEGA software version 7. The strains were 
determined by pasting the FASTA (Text based 
format for representing nucleotide sequence) 
format into the Basic Local alignment Search 
Tool (BLAST) from the National Center for 
Biotechnology Information (NCBI). 
 

2.6 Data Analysis 
 

The data obtained were analyzed using Smith’s 
Statistical Package (version 2.8, California, 
USA). Chi-square test was conducted at 95% 
confidence interval and P values ≤ 0.05 were 
considered statistically significant. 
 

3. RESULTS AND DISCUSSION 
 
This present study was conducted to detect and 
sequence Rotavirus VP4 among children aged 0-

5 years with gastroenteritis in 2 selected 
healthcare centres in Keffi, Nigeria. A total of 303 
children (203 from FMC Keffi and 100 from PHC 
Angwan Waje, Keffi) majority of which were 
males (157/303) aged 0-12 months (153/303) 
were recruited and screened for Rotavirus 
antigen. Overall, 54 (17.8%) children tested 
positive for Rotavirus infection giving a total 
prevalence of 17.8% (Table 1). 
 
The 17.8% prevalence of Rotavirus infection 
recorded in this study was higher than the 6.0% 
reported among children in Kwara State [25], 
7.4% among children with diarrhoea in Kano 
State [Suleiman et al., 2020] and 12.5% among 
infants in Calabar [26]. It was However lower 
than the 24.8% reported among children with 
gastroenteritis in Akure [27], 25.0% among 
children less than five years of age in Abuja 
satellite towns [28] and 56.0% among children 
with diarrhea in Enugu State [29]. Interestingly, 
researchers from other parts of Africa and the 
world also reported varying rates of the viral 
infection. For instance, it was 14.5% in Kenya 
[30], 16% in Sudan [31], 18% in India [32], 39.2% 
in Benin Republic [33], 44.8% in Indonesia [34] 
and 56% in Portugal [35]. The differences 
observed in the prevalence rates from different 
studies were possibly due to differences in 
testing methods used, location of the studies, 
time and season of sampling and study 
population types with different associated risk 
factors [2]. 
 
This study did not record significant association 
between Rotavirus infection and age of the 
participants (P>0.05). However, Most of the 
children infected were between the age group 0-
12 months (24.8%), followed by 13-24 months 
(19.7%), 37-48 months (4.8%) and 25-36 months 
1(2.9%) while none was detected in age group 
49-60 months (Table 1). This observation agrees 
with the report of Theophilus et al. [20], 
Okebugwu et al. [27] and Dhital et al. [36] among 
children with gastroenteritis in Abuja, Akure and 
Eastern India respectively but disagrees with that 
of Mohammed et al. [37] and Aliyu et al. [38] who 
reported higher prevalence of the viral infection 
among Nigerian children within the age group 25-
50 months. The higher prevalence of the 
infection recorded among younger children in this 
present study may be attributed to the absence 
of well-developed immune system among them 
as older children are expected to acquire 
protective immunity during repeated exposures 
to the virus and therefore, subsequent infections 
are mild or asymptomatic [39]. 



 
 
 
 

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Table 1. Prevalence and distribution of rotavirus infection in relation to age and gender among 
children between ages 0-5 years with gastroenteritis in 2 selected healthcare centres in Keffi, 

Nigeria 
 

Parameter No. Examined No. Positive Prevalence (%)  p-value 

 (N=303) (N=54) (Overall=17.8)  

Age (months)      
0–12 153 38 24.8 0.7792 
13–24 71 14 19.7   
25–36 37 1 2.9   
37–48 21 1 4.8  
49–60  21 0 0.0  
Gender     
Male 157 31 19.7 0.0004* 
Female 146 23 15.8  

*Statistically significant 
 

 

 

Fig. 1. Agarose gel electrophoretogram of Rotavirus VP4 amplified gene. Samples 1, 6 and 9 
were positive for Rotavirus VP4 gene while samples 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, 15 and 16 
were negative. *L represents the molecular ladder, ‘ −ʼis the negative control while ‘ + ’ is the 

positive control 
 
There was significant association between 
gender and prevalence of Rotavirus infection in 
this study (P<0.05) as males were more infected 
(19.7%) compared to their female counterparts 
(15.8%) (Table 1). This is consistent with the 
reports of most other previous studies conducted 
in Nigeria [3, 15, 18, 26, 28, 29, 37] and other 
parts of the world [30, 31, 34, 40]. The higher 
prevalence of the infection among male subjects 
in most previous studies may be connected to 
the fact that females mount stronger humoral and 
cellular immune responses to infection or 
antigenic stimulation than the males [41].    
 
Furthermore, based on the RT-PCR carried out 
in this study, 3(5.6%) out of the 54 samples that 

tested positive with the lateral flow immunoassay 
kit were positive for the VP4 gene (Fig. 1). The 
low rate of rotavirus VP4 gene detected by RT-
PCR in this present study could possibly be as a 
result of PCR inhibitors in the faeces that were 
carried over into the RNA extracts [42]. It could 
also be attributed to prolonged storage of the 
stool samples, method of RNA                      
extraction and effects of freezing and thawing 
[43]. 
 
The 3 amplified VP4 genes were sequenced and 
were found to be of type P [11] Strain N115 (Figs 
2A and 2B). This strain showed 88.08% 
nucleotide identity to human rotavirus A strain 
G12 P [11] with accession number MH559158.1 



 
 
 
 

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140 

 

in the GenBank. The nucleotide sequence of the 
N155 strain detected in this study has been 

deposited in the GeneBank under the accession 
number: EU200796.1 (Figs 3 and 4). 
 

 

 

Fig. 2A. Chromatograph of the Sequenced RotavirusVP4 Genes (Forward Primer) 
 

 

 
Fig. 2B. Chromatograph of the Sequenced Rotavirus VP4 Gene (Reverse Primer) 



 
 
 
 

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141 

 

 

 
Fig. 3. Image of BLAST of sample showing sequence with significant alignment with that in the 

Gen Bank 
 

 

 
Fig. 4. Phylogeny of rotavirus VP4 gene 

 
This strain of Rotavirus detected in this present 
study has not been reported in Nigeria. In 
contrast however, other genotype combinations 
have been reported in Nigeria and other African 
countries. For instance, genotype G12 was 
detected in Asaba, Delta State [44], Ibadan, Oyo 
State, Nigeria [45],  Nairobi, Kenya [46], Dakar, 

Senegal [47], Democratic Republic of Congo [48] 
and in Tunisia [49].   
 
Furthermore, in India, Giri et al. [50] identified 
genotype G12 P [11] while Ray et al. [51] and 
Ramani et al. [52] reported the detection of strain 
N155 with a difference in the G-type (G10) 



 
 
 
 

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among neonates with gastroenteritis in India. 
Additionally, Libonati et al. [53] also reported the 
detection of G10 P [11] type in India. These 
strains of group A rotavirus are believed to be 
from bovine origin as a result of bovine-human 
gene re-assortment [50]. 
 

4. CONCLUSION 
 

This study recorded 17.8% prevalence of 
infection with type P [11] Strain N115 (5.6%) of 
Rotavirus among children aged 0-5 years with 
gastroenteritis in 2 selected healthcare centres in 
Keffi, Nigeria. The detection of this rare rotavirus 
strain in this study is a cause for concern and 
hence there is an urgent need for the Nigerian 
health authorities to implement a nationwide 
surveillance system for monitoring rotavirus 
molecular epidemiology, before considering 
introduction of rotavirus vaccination into the 
expanded program on immunization (EPI). This 
will help to give necessary information on current 
genotypes and novel introductions as well as 
evolution of mutant strains to help augment 
current rotavirus prevention and control. 
 

CONSENT 
 

All parents/guardians of the children included in 
this study completed and signed an informed 
consent form.  
 

ETHICAL APPROVAL 
 
Formal ethical approval to conduct this study      
was obtained from the Research Ethics 
Committee of Federal Medical Centre, Keffi 
(FMC/KF/HREC/207/17). Permission was also 
obtained from the management of Primary 
Healthcare Center Angwan Waje, Keffi. 
 

ACKNOWLEDGEMENT 
 
The study team would like to thank the 
management of Federal Medical Centre and 
Primary Healthcare Centre, Angwan Waje, Keffi, 
Nigeria for their kind permission to conduct the 
research work. We are also grateful to all 
parents/guardians of the children who       
voluntarily participated in the study. However, 
this research did not receive any form of grant 
from governmental or non-governmental 
organizations. 
 

COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 

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