Variability in the genotypes of rotavirus detected in Côte d'Ivoire from 2010-2016

1 Département de Microbiologie, Université Félix Houphouët Boigny, UFR des Sciences Médicales, Côte d’Ivoire. 2 Laboratory of Sentinel Site Surveillance of Paediatric Bacterial Meningitis and Rotavirus Diarrhoea, CHU, Yopougon, Abidjan, Côte d’Ivoire. 3 Paediatric Service, CHU, Yopougon, Abidjan, Côte d’Ivoire. 4 Expanded Program on Immunization, Côte d'Ivoire. 5 NMIMR West African Regional Rotavirus Reference Laboratory, Accra, Ghana.


INTRODUCTION
Group A rotaviruses are the major viral agent of acute gastroenteritis and severe diarrhea in children <5 years old (Gasparinho et al., 2017).Globally, this virus is responsible for about 40% of cases of severe diarrhea with hospitalization and 5% of deaths in children under five years (Eteme et al., 2015).The number of *Correspondent author.E-mail: bonicatc@yahoo.fr.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License deaths is estimated to be 215,000/year in children <5 years with 80% cases in sub-Saharan Africa and South Asia (Tate et al., 2016).The envelope of rotavirus is made of VP7 and VP4 proteins which form the outer part of the capsid (15 genotypes G and 27 genotypes P) (Lorrot et al., 2012).The circulating genotypes of rotavirus are of different types all over the world; several studies in Africa have identified the G1P [8] genotype as the most common in pre-vaccination areas.The G1P [6], G8P [6], G6P [6], G8P [8], G12P [6] and mixed G and mixed P genotypes are also found in a lower rate in sub-Saharan Africa (Hokoro et al., 2014).The World Health Organization (WHO) recommends surveillance of circulating strains before and after introduction of vaccination programs in various countries (Damanka et al., 2016).However, the diversity of circulating strains in developing countries proves a real challenge to the vaccination programs (Todd et al. 2010).Currently, WHO recommends two types of oral vaccines: the Rotarix® vaccine (GlaxoSmithKline, Rixensart, Belgium) and Rotateq® vaccine (Merck & Co., Whitehouse Station, NJ).Rotateq® vaccine contains (RV5) five human-bovine reassortant viruses [W179-9 (G1P [5], SC-2 (G2P [5]), W178-8 (G3P [5]), ) and W179-4 (G6P [8]).Rotarix® vaccine (RV1) is a human G1P [8] virus RIX4414 derived from a serial passage in the cell culture of a virus recovered from the stool of an infected child (Agutu et al., 2017).Both recommended vaccines require multiple dose administration (two doses for Rotarix and three for RotaTeq); the first to be administered between 6 and 15 weeks of age to raise homo-and heterotypic immune response against RVA different strains.The two vaccines have been proven to be effective worldwide, but lower efficacy was observed in low-income countries from Africa and Southern Asia.Among the several hypotheses used to explain the differences in the immune response and consequent efficacy of these vaccines in low-versus high-income countries, RVA strains diversity, host genetic factors, malnutrition, host co-infection, deficient micronutrient ingestion, and interfering gut flora have been put forward (Gasparinho et al., 2017).Studies conducted in Côte d'Ivoire prior to the sentinel surveillance which started in 2010 showed a predominance of G1 followed by G2.A small number of G3, G8 and G9 variants were identified in this study (Akran et al., 2010).In view of the wide variety of circulating strains, it is therefore important to maintain continuous monitoring of the prevalence of rotavirus in order to understand the distribution of G and P genotypes in the country.Accurate information in respect of different types of circulating genotypes of rotavirus is essential to monitor the impact and effectiveness of the vaccine.
This study aimed to identify the different genotypes circulating in the country before the introduction of the Rotavirus vaccine.It proposes to describe the proportion of the different genotypes G and P circulating from 2010 to 2016 in the pre-vaccination area.Genotypic combinations were also determined.

Study population
Children with acute gastroenteritis aged 0 to 5 years, admitted in one of the sentinel surveillance sites were recruited.There are six collection sites altogether in five municipal localities in the city of Abidjan, the commercial capital of Côte d'Ivoire.Children either hospitalized or kept under observation for treatment of acute diarrhea (less than two weeks) were included in the study with stools that had no mucus or blood associated with fever from January 2010 to December 2016.

Stool samples
Stools samples were collected from each child after obtaining informed consent from the parents, in a sterile jar on the same day or the next day of admission.Participants were request to complete some forms containing details of the child's socio-demographics and clinical information.The samples were kept in refrigerator at a temperature of between 0 and 4°C, and sent to the laboratory at the Yopougon University Teaching Hospital, Bacteriology-Virology unit, where they were stored at 4°C for a maximum of 30 days until ELISA test was performed.ELISA positive samples were then stored at -20°C before being taken to one of the WHO rotavirus reference laboratories, namely the Limpopo Regional Laboratory in South Africa or the West Africa Regional Laboratory in Ghana for genotyping.

Detection of group A Rotavirus antigens
Samples were screened for the presence of rotavirus structural protein VP6 by the use of Rotaclone® a rapid EIA test kit following the manufacturers' instructions.Samples with optical density >0.25 at 450 nm wavelength were considered positive.

G-and P-genotyping assays
To determine the VP7 (G-) and VP4 (P-) genotypes, viral RNAs was extracted from the clarified supernatant of 20% stool suspensions using the QIAamp ® Viral RNA Mini kit (QIAGEN ® , Hilden, Germany) based on the manufacturer's instructions.Reverse transcription (RT)-PCR was performed using both forward and reverse consensus primers Beg9/End9 and Con3/Con2 to amplify a 1,069bp and 835bp fragments of the VP7 and VP4 genes respectively.Multiplex PCR was carried out for G-and P-typing with genotype specific primers as previously described (Gouvea, 1990;Gentsch, 1992;Iturriza-Gomara, 2004).PCR amplicons were electrophoresed on a 2% agarose gel in Trisborate-EDTA buffer together with a 100-bp DNA ladder.

Data analysis
All statistical analysis was performed with the EPI-Info version 3.5.4software (CDC Atlanta, USA).All categorical variables were summarized as proportions, and significance of their difference in distribution with the outcome was assessed using Pearson's Chisquare and Fisher test at 5% risk.

RESULTS
A total of 1472 <5 year old children were recruited in this study and 1472 stool samples were collected.Of the recruited children, the male sex was predominant about 57.4 %( 845/627) .The sex ratio was 1.36.ELISA positive stool specimens were 31.8%(468/1472) (Table 1).RT-PCR and genotyping were performed on 85.2% (399/468) of the positive samples.93.7% (376/399) of the tested samples were positive to VP7 with 6.3% (25/399) non-typable strains (Table 2) and 94.2% (376/399) were positive to VP4 and 5.8% (23/399) non-typable strains (Table 2).Regarding the VP 7 genotype, the G1 was predominant with 39.6% (158/399) followed by G12 with 27% (108/399) and Mix G 8% (32/399) (Table 2).Concerning the VP4, the predominant genotype was P [8] which was found in 50.4% (108/399) followed by P [6] 31.6% (32/399) (Table 2).Throughout the study period, rotavirus genotype G12 was most prevalent in 2012, 2013 and 2014, except the year 2010 when G9 was the most predominant genotype (Figure 1a).Genotypes G1, G2 and G3 were detected throughout the study period at varying frequencies.On the other hand, there was no change in the predominant P-type as P [8] remained dominant over the study period (Figure 1b).  is high, particularly in the developing countries (Boula et al., 2014).Children under 12 months are the most affected by this virus (Todd et al., 2010).The peak of rotavirus gastroenteritis was found in some countries such as Kenya between 6 and 24 months (Agutu et al., 2017).In Cameroon, a prevalence of 44.7% was found in children under 24 months (Ndze et al., 2013).Hospitalization rates of 50.6% were observed in children aged 6-8 months in Ghana (Damaka et al., 2016).In this study, higher detection rates were observed than those found in previous studies in Côte d'Ivoire (Akoua-Koffi et al., 2007, 2014;Akran et al., 2010).The administration of the vaccine is therefore recommended in early infancy in sub-Saharan Africa as the infection is acquired early there than in Western countries (WHO, 2009).WHO recommends that the first dose of either RotaTeq or Rotarix be administered at age 6-15 weeks; the maximum age for administering the last dose of either vaccine should be 32 weeks (WHO, 2009).A predominance of male gender was observed in our study.This has also been observed in other studies (Selvarajan et al., 2017) but there was no statistical difference.Studies showed male susceptibility to rotavirus infection (Junaid et al., 2011).However, several studies have suggested the absence of gender-related occurrence (Saluja et al., 2014).Rotavirus is the primary cause of diarrhea in children under five years of age with a prevalence rate of 41% (Selvarajan et al., 2017) in area where vaccination is not yet introduced.The rate of 45% found in our study is similar to that found in other African countries before the introduction of the vaccine (Bwogi et al., 2016).However, in neighboring countries to Ivory Coast, where the vaccine has been introduced, rotavirus remains the leading viral cause of diarrhoea in children under five years of age, with high prevalence.In Burkina Faso, Ouedraogo found Rotavirus (63.5%), adenovirus (31.2%) and genogroup II norovirus (18.2%) in a study conducted (Ouedraogo et al., 2016).In Ghana, similar results have been found, but with lower prevalence rate of rotaviruses (27.9%), astroviruses (7.5%), noroviruses (6.8%) and adenoviruses (5.4%) (Akkufo et al., 2017).The outer layer protein, VP4 and VP7 of the group A rotavirus induce the production of neutralizing antibodies.The attachment protein VP 4 determines the type P.This has a more conservative specificity than type G determined by the glycoprotein VP 7.Ten G genotypes and 8 P genotypes have been detected in humans (Wylie et al., 2015).Two rotavirus vaccines are currently licensed by WHO.RotaTeq® (RV5) (Merck & Co) consists of a mixture of 5 bovine viruses that contain VP7 and VP4 genes from human G1, G2, G3, and G4 and P viruses.Rotarix® (RV1) (GlaxoSmithKline Biologicals) consists of an attenuated virus derived from a human G1P [8] strain (Wylie et al., 2015).genotypes have been identified with similar rates in previous studies (Damanka et al., 2016;Kirkwood et al., 2014) with a predominance of P (Boula et al., 2014).
G12 genotype is a non-common strain with resurgence at 27%.This strain is not included in the target strains of the two vaccines recommended by WHO.Its emergence has been observed globally in several studies.In Thailand, Maneekarn et al. (2014) observed the predominance of the G12 from 2007 to 2009 only.In Australia, Kirkwood et al. (2014) reported incidence of G12P [8] (23%) in 2012, and Wylie (2014Wylie ( , 2015) ) reported an emergence of this strain in the same year in the Saint Louis, United States.This emergence was also observed in Africa, particularly in Cameroon in the same period (Ngum Ndze et al., 2012).Its significance is yet to be evaluated in the post vaccine era.Concerning genotypic combinations, the G1P [8], G12P [8], G1P [6] and G12P [6] genotypes are predominant in our study.The association of the genotype G12 with the different genotypes P suggests a high capacity of adaptation of this unusual strain.In this study, a lower rate was observed for non-typable and mixed strain.

Conclusion
Continuous monitoring of circulating strains is important as vaccine pressure may lead to the emergence of new epidemic strains in the post vaccine era.The determination of the different genotypes of rotavirus Boni-Cisse et al. 1667 strains before the introduction of vaccination is fundamental to better understand the mechanisms leading to the emergence of new strains.Countries are encouraged to monitor strains in circulation before and after the introduction of the vaccine to determine the impact of the vaccine on circulating strains which might potentially escape protection covered by the currently recommended vaccines.

Figure 1 .
Figure 1.Temporal rotavirus genotype distribution in Côte d'Ivoire.a).Rotavirus G-type distribution, January 2010 to December 2016; b).Rotavirus P-type distribution, January 2010 to December 2013.MIX: multiple genotypes detected for either G, P or both; NT: either G, P or both were nontypable.

Table 2 .
Rotavirus strain distribution between the period January 2010 and December 2016.
GMix/P[Mix]: Multiple genotypes detected for either G, P or both; GNT/P[NT]: either G, P or both were nontypable