Molecular Characterization of Rotavirus Strains Circulating in Enugu Nigeria : 2011 to 2016

Rotavirus gastroenteritis is a major public health concern globally, estimated to cause 215,000 deaths among children < 5 years of age in 2013; with majority of mortality occurring in developing countries. In 2013, it was estimated that Nigeria was the second country with the highest number of rotavirus deaths. Monitoring of circulating rotavirus strains in Enugu, Nigeria is part of on-going rotavirus surveillance before the introduction of rotavirus vaccination. A total of 2694 stool samples were collected from enrolled under 5 years old children with diarrhoea between January 2011 and December 2016 and tested the virus using an antigen enzyme immunoassay. Randomly selected rotavirus positive samples were further characterized by rotavirus genotype methods to identify the G and P types circulating during the study period. Rotavirus was detected in 1242 (46%) of the 2694 samples collected over the six years period. Of these, 867 were randomly selected for genotyping. G and P types could be assigned for 832 samples (96%), while 31 (3.6%) could only #F. Akpali, N. Nwankwo, D. Ezenwaka, P. Nwachukwu, P. Oha, N. Anioke, A. Okonkwo, I. Onwusi, Past team members. How to cite this paper: Tagbo, B.N., Chukwubike, C., Mwenda, J.M., Seheri, M.L., Armah, G., Mphahlele, J.M., Ozumba, U.C., Benjamin-Puja, C., Azubuike, C., Okafor, H.U., Nnani, R.O., Okafor, V., Edelu, B.O., Eke, C.B., Udemba, O., Isiaka, A., Namadi, L., Umezinne, N., Njoku, R., Odume, C., Osaro, V., Ogude, N., Okwesili, M.U., Ezebilo, S.K., Yusuf, K.M. and Obidike, E.O., ICH UNTH Enugu Rotavirus Group (2019) Molecular Characterization of Rotavirus Strains Circulating in Enugu Nigeria: 2011 to 2016. World Journal of Vaccines, 9, 22-36. https://doi.org/10.4236/wjv.2019.91002 Received: November 28, 2018 Accepted: February 17, 2019 Published: February 20, 2019 DOI: 10.4236/wjv.2019.91002 Feb. 20, 2019 22 World Journal of Vaccines


Introduction
Rotavirus gastroenteritis is a major public health concern globally, estimated to cause 215, 000 (range, 197,000 -233,000) deaths among children <5 years of age in 2013.Four countries (India, Nigeria, Pakistan, and Democratic Republic of Congo) accounted for approximately half (49%) of all estimated rotavirus deaths in 2013 [1].In Africa, Nigeria has one of the highest under 5 years rotavirus disease prevalence of 56% [2] and mortality due to rotavirus accounted for an estimated 31,000 deaths in 2013 [3].Since improved sanitation, water supply and hygiene have little or no effect on rotavirus infections [2] [4] [5], vaccination is predicated to have great impact on rotavirus diarrhoea in Nigeria.Vaccines against rotavirus are available and have been shown to be effective in countries where they were employed [6] [7].Rotavirus surveillance is ongoing in Enugu State to determine the burden of rotavirus disease and circulating strains in anticipation of the introduction of rotavirus vaccine into the routine immunization schedule of Nigeria in 2019.

Methods
This surveillance was conducted as part of the WHO supported rotavirus diarrhoea disease in Enugu, South Eastern, Nigeria, the capital city of Enugu State, Nigeria.Many inhabitants of the state are Nigerians of Igbo ethnic nationality.
The main occupations of the inhabitants are public service, trading and agriculture-including animal rearing.
Between January 2011 and December 2016, stool samples were collected from children less than 5 years that were hospitalized with gastroenteritis at three main sites.These are; Children Emergency Room (CHER) of the University of Nigeria Teaching Hospital (UNTH) Ituku-Ozalla, Enugu (main site), Tender Care Specialist Hospital Enugu and Mother of Christ Specialist Hospital (MOCSH) Enugu.Inclusion and exclusion for rotavirus cases were based on the WHO generic protocol [18].The surveillance protocol was approved by the Health Research Ethics Committee of the University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu.Demographic and clinical information were collected following the generic protocol of the World Health Organization on surveillance for rotavirus gastroenteritis [18] after informed consent had been obtained from mothers or care givers of children.
Sampling: Stool specimens were obtained mostly within 48 hours after admission to exclude nosocomial infections.The stool samples were collected in clean and well labelled screw-capped containers and delivered to the laboratory within one hour or stored temporarily at +4˚C to +8˚C in hospitals before transporting under ice to the Virology Laboratory at University of Nigeria Teaching Hospital (UNTH) Ituku-Ozalla, Enugu.At the main laboratory, stool samples were stored at −60˚C till used.All demographic and clinical data were recorded on a standardized WHO Case Report Form and analyzed using SPSS DOI: 10.4236/wjv.2019.91002

Rotavirus Detection
Group A rotaviruses were detected by enzyme immunoassay (EIA) with the commercial rotavirus Prospect ® Rotavirus Kit, (Oxoid LTD, United Kingdom) provided by WHO/AFRO on 10% suspensions of faecal material, according to the manufacturer's instructions.

Viral RNA Extraction and RT-PCR
Double-stranded RNA (dsRNA) was extracted from 10% faecal suspension of rotavirus EIA-positive and PAGE positive stool samples by the Phenol/Chloroform method as described previously [19] and purified with the RNaid ® Kit (Bio 101, Carlsbad, USA).The VP7 and VP4 genes of rotavirus were subjected to RT-PCR followed by multiplex PCR according to the methods described by Gouvea et al., [20] in a Gene AMP PCR Primus 25 System machine.Consensus primer sets that were published [21] [22] [23] were used to amplify the VP4 and VP7 gene segments of the rotavirus.Genotyping of the G and P were done with multiplex type specific primers [21] [22] [23] to detect G1 to G4, G8, G9, G10 and G12 and P [4], P [6], P [8], P[10] P [11] respectively.The PCR products were electrophoresed on 2.0% agarose gel containing ethidium bromide (0.5 ug/ml) (Sigma-Aldrich, Steinheim, Germany) and visualized under UV transilluminator.
Temporal year by year Rotavirus strain distribution in Enugu, Nigeria.
There were more males than females enrolled during the surveillance period.
Monthly distribution of rotavirus strains during the study period showed that 97% of strains were detected in the dry cool months of December to April (Figure 1).

Discussion
Rotavirus continues to be the leading cause of severe gastroenteritis among children below the age of 5 years globally [1].We report the detection and characterization of rotavirus strains in Enugu, Nigeria during 2011-2016.Our Surveillance has shown that rotavirus accounted for 46% of diarrheal episodes in children less than 5 years of age.This confirms previous reports in other African countries such as Cameroon, Ethiopia, Tanzania, Zimbabwe, Togo and Ghana where rotavirus prevalence was found to be between 23% -49% [24] [25] [26] [27].Rotavirus with G12 genotype specificity was the most commonly detected (27.6%) of genotyped samples.This finding is in conformance to previous studies in Ile-Ife, South West Nigeria [28] and Maiduguri, North East Nigeria [29].It is however, in sharp contrast to earlier reports from Nigeria where G1 genotype dominated [30] [31] [32].The detection of G12 genotype has also been reported from other African countries including Niger Republic, Ghana, Togo and Nepal in Asia [26] [27] [33] [34] [35].
Epidemiological studies demonstrated that G1P [8], G2P [4], G3P [8], G4P [8], G9P [8], and G12P [8] are responsible for 80% -90% of most rotavirus infections among children less than 5 years old worldwide [11] [12] [13].However, these globally common strains constituted only 41.5% of detected rotavirus strains in Enugu, Nigeria.G12P [8], G3P [6], G1P [8] and G1P [6] [38], the observed diversity of rotavirus strains in Nigeria should be monitored post vaccine introduction.One of the major finding of this surveillance report was the high proportion of mixed infections accompanied by the presence of multiple rotavirus P and G (strain) combinations, suggesting genome reassortment.However, other studies have also reported mixed infections [36] [39] [40].Non-typeable rotavirus G-types accounted for 3.2% (28/867) and the P-types accounted for 1.7% (15/867) in the 6 years study period (Table 1).This is in contrast to other studies carried out in the Regional Rotavirus Reference Laboratory in Ghana where higher proportions of non-typeable rotavirus strains had been documented [27] [41].The reduction in the proportion of non-typeable rotavirus strains was due to the use of expanded genotyping primer sets and sequence analysis (although more expensive) currently available in the two Rotavirus reference laboratories in South Africa and Ghana.
In our study area, rotavirus occurred mostly during December to April.This corresponded to the dry season in Nigeria and elsewhere in West Africa [41] [42] [43] [44] [45].Rotavirus infection showed a smooth rise in January and started declining in February through March and April to November.A rise was seen again during the cool dry month of December (Figure 1).The monthly distribution of rotavirus strain observed in this surveillance period is in accordance with findings reported previously [7] [42].The peak season for rotavirus infection was in January (52.2%; 435/867) followed by February (24%; 208/867), December (8%; 69/867) March (7.7%) and April (5%; 43/867).Rotavirus infection displays distinct seasonal pattern in temperate climates with epidemic peaks occurring in the cooler winter months of the year and absent or uncommon during the warmer months of the year [46] [47].In tropical areas rotavirus disease has been reported to occur all year round with seasonal peaks occurring during the cool dry months of the year [48] [49] [50].
More males were recruited into the study and were found to be infected with higher proportions of rotavirus strains (58%; 503/876) than the females (42%; 364/867) (P < 0.05).Rotavirus was detected in children of all age groups, but the highest detection rate was observed in children < 12 months (67.6%; 586/867).The rate of detection in children between the ages of 12 -23 months was 26.4% whilst those between 24 -59 months were 6%.It is interesting to note that G12P [8] was the most prevalent strain detected in all the age groups.Mixed genotypes were also seen mainly in children less than 12 months old.The proportion of mixed infection observed in the present study (15%) is higher than previous reports from Southern Nigeria [31] [50] and lower than the report by Aminu et al. [30] of Northern Nigeria.It is however, in accordance with findings reported previously elsewhere and some African countries [47] [48] [51] [52].The study has demonstrated that rotavirus infection as well as the most prevalent strains occurred in children below the age of 24 months.
Seasonal variations in the prevalence of some rotavirus strains were observed.The most common prevailing strain in the study from 2011-2013 was G12P [8], the sixth globally common circulating strain.However, the distribution pattern of this strain in Enugu, Nigeria changed during 2014-2016.The 2014-2015 year saw the emergence of G1P [8] the globally most common strain, replacing G12P [8] detected during the first three years of the study period.In 2016, G3P [6] an unusual strain, emerged as the dominant strain.G3P [6] was established as the second commonly detected strain in 2011, 2014 and 2015 whilst G10P [6] was detected as the second common strain in 2012 and 2013.G1P [6] an unusual strain was identified as the second dominant strain in 2016.G1P [8] the most common rotavirus strain detected globally, was virtually non-existent during 2012 and 2013.Some unusual rotavirus strains documented in this study (G1P [6], G3P [6], G12P [6] and G12P [6] occurred all year round (Figure 1, Table 2).

Conclusion
We report the prevalence of rotavirus G and P genotypes circulating in infants and children in Enugu, Nigeria pre-rotavirus vaccine introduction.The predominant strains identified as part of routine rotavirus surveillance reflect the great diversity and unusual nature of rotavirus strains detected in Nigeria.This data will be useful in supporting evidence-based decisions on introduction of rotavirus vaccine into the national immunization programme in Nigeria.
diarrhoea surveillance was established in Nigeria in September 2010 as part of the African Rotavirus Surveillance Network with the support of the World Health Organization (WHO).The Institute of Child Health, University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu was chosen by the Ministry of Health to play a dual role of Paediatric hospital and National laboratory for rotavirus surveillance.Neighbouring hospitals including Tender Care Specialist Hospital, and Mother of Christ Specialist Hospital, both in Enugu are part of surveillance.
Rotavirus positive samples were subsequently sent to the WHO Regional Rotavirus Reference Laboratories (RRL) at South African Medical Research Council, Diarrhoeal Pathogens Research Unit, Sefako Makgatho Health Sciences University (SMU), Pretoria and Noguchi Memorial Institute of Medical Research, University of Ghana, Accra.Rotavirus positive samples were further subjected to semi nested reverse-transcriptase polymerase chain reaction (RT-PCR).

Table 1 .
Rotavirus G and P-genotypes detected in children < 5 years old hospitalized with acute watery diarrhoea.Numbers in parenthesis are percentage of each sample.Mix indicates at least 2 G or P found in one sample.NT means non-typable samples.

Table 2 .
Annual distribution of major rotavirus stains in children < 5 years hospitalized with acute watery diarrhoea in Nigeria: 2011-2016.
Mixed genotype indicates at least 2 G or P found in one sample.Partially typed indicates both the G or P is typed and the other is not.Only genotypes with at least 10 samples were represented, the rest were grouped as others.

Table 3 .
Major rotavirus strains distribution by age circulating among children < 5 years old with diarrhoea in Enugu Nigeria: 2011-2016.
Mixed genotype indicates at least 2 G or P found in one sample.Partially typed indicates both the G or P is typed and the other is not.Mix.Only genotypes with at least 10 samples were represented, the rest were grouped as others.DOI: 10.4236/wjv.2019.9100227 World Journal of Vaccines B. N. Tagbo et al.

Table 4 .
Distribution of major rotavirus strains by gender in Enugu Nigeria: 2011-2016.
[6]March (15%; 10/67), April (25.6%; 11/43) and December (37.7%; 26/69).G3P[6], the second most common strain detected Mixed genotype indicates at least 2 G or P found in one sample.Partially typed indicates both the G or P is typed and the other is not.Only genotypes with at least 10 samples were represented, the rest were grouped as others.