Detection and sequencing of rotavirus among sudanese children

Abstract Introduction Diarrheal diseases are a big public health problem worldwide, particularly among developing countries. The current study was conducted to detect and characterize group A rotavirus among admitted children with gastroenteritis to the pediatric hospitals, Sudan. Methods A total of 755 stool samples were collected from Sudanese children with less than 5 years of age presenting with acute gastroenteritis during the period from April to September 2010. Enzyme-linked immunosorbent assay (ELISA) was used to Detection of Rotavirus antigens. Ribonucleic acid (RNAs) were extracted from rotavirus-positive stool samples using (QIAamp® Viral RNA Mini Kit). (Omniscript® Reverse Transcription kit) was used to convert RNA to complementary Deoxyribonucleic acid (cDNA). The cDNAs were used as template for detection of VP4-P (P for Protease-sensitive) and VP7-G (G for Glycoprotein) genotyping of Rotavirus using nested PCR and sequencing. Results Out of the 755 stool samples from children with acute gastroenteritis, 121 were positive for rotavirus A. Among 24 samples that were sequenced; the VP7 predominant G type was G1 (83.3%), followed by G9 (16.7%). Out of these samples, only one VP4 P[8] genotype was detected. Conclusion As a conclusion the VP7 predominant G type was G1, followed by G9 whereas only one VP4 genotype was detected and showed similarity to P[8] GenBank strain. It appears that the recently approved rotavirus vaccines in Sudan are well matched to the rotavirus genotypes identified in this study, though more studies are needed.


Introduction
Among children under five years old, rotavirus A is the most frequent cause of acute gastroenteritis in developing countries [1].
In Sudan rotavirus was the second causes of diarrhea among children [2]. Worldwide there is about 527,000 (range, 475,000-580,000) children die of rotavirus each year this translate into more than 1440 death daily, by other mean, by 5 years of age about 1 of 237 born children would die of rotavirus yearly [3]. There are 11 double-stranded RNA segments of the rotavirus genome that code for viral proteins (VPs) [4]. The outer capsid layer of rotavirus consists of two structural proteins and they were VP4 and VP7.
Based on the VP7 and VP4 gene sequences rotaviruses are classified into the (G) and (P) respectively. At present 27 G genotypes (G1-G27) and 35 P genotypes (P1-P35) genotypes have been discriminated so far [5]. Assessment of the potency of rotavirus vaccine against constantly changing strains will depend on continuous global strain surveillance [6]. The diversity of VP4 and VP7 rotavirus strains is a major challenge to the efficacy of the currently used vaccines [7]. Characterization of the common strains in Sudan will have an impact on the implementation of rotavirus vaccines in the country. There is scanty data about the genotypes of rotavirus in Sudan despite its potential impact in the implementation of vaccines. However, the monovalent rotavirus vaccine (Rotarix® GSK Biologicals, Rixensart) was introduced into the most populous state (Khartoum) in Sudan on July 2011 [8]. Therefore, in this study the VP4 and VP7 were selected for sequencing because of their impact upon vaccine implementation. This study was conducted to genetically characterize the VP4 and VP7 rotavirus strains among some Sudanese children with acute gastroenteritis.

Methods
It is a cross sectional hospital based study conducted in different hospitals from different states of Sudan during period from April to September 2010. A total of 755 stool specimens were collected from children (430 males and 325 females), those children were suffering from acute gastroenteritis and they under 5 years of age. A single stool sample was taken from each child on the first day of hospitalization. After obtaining written informed consent from the parent or guardians stool samples were collected along with demographic data and medical history, using a questionnaire. The nucleotide sequences obtained in this study were deposited in GenBank. The obtained data was entered in the Statistical Package for the Social Sciences software (SPSS version16). Chi-squire test was used to test for significant differences between the variables and a p value of less than 0.05 was considered as significant.

Results
Of the 755 fecal samples analyzed, 16% tested positive for rotavirus

Discussion
This study is the a cross sectional hospital based study of rotavirus genotypes in Sudan, and the results support the observation that the G1 and G9 genotype, particularly P[8]G1 and P [8]G9, is the more genotypes spreading throughout Africa [11]. This study described the rotavirus infection among patients presenting to pediatric hospitals in the period from April through September 2010.
One of the main aims of this study was to identify and sequencing of the VP7 and VP4 gene segments of the Sudanese For VP4 Only P [8] was detected and showing as example high similarly to wt/PHI/TGE12-045 strain deposit in GenBank accession number KP007144. This finding is similar to a report by Inairo GI et al [12].
Moreover in agreement with our result, recent literature worldwide showed that P [8] is the most prevalent genotype [13,14]. In this study, the detection rate of 83% VP7 genotype G1 in Sudan is relatively similar to the 82.4% from Egypt [15]. Furthermore, results from eight countries in the Middle East and North Africa showed the prevalence of VP7 genotype G1 to be more than rotavirus strains.
56% [14]. The VP7 G9 genotype has been documented since the early 1980s [16]. Throughout the 1980s and 1990s, G9 was considered to be a very rare strains then beginning to increase as a cause of gastroenteritis and later emerged as the most important strains in developed and developing countries [17]. In Africa the serotype G9 strains have been detected in various countries and sporadically were isolated in South Africa, Botswana, Kenya, and Cameroon and appeared to be circulating at population, however it appears to be the predominant serotype in some settings [18]. In the United States, the G9 genotype was detected through an outbreak in 1995-1996 [19]. Nevertheless, this may not reflect the current prevalence of the G9 genotype, since regional in some settings [20,21]. In the present study the 17% rate of G9 is similar to an Iranian [22] and Brazil rate [23]. Moreover, this rate is similar to report from Cameron [24]. On the other hand, our G9 prevalence rate is a little bit higher than the Kuwaiti 10.2% rate [25] and Iraq [26].
In this study the presence of G1P [8] as predominant strains is in agreement of introducing of the monovalent Rotarix ™ vaccine in Sudan. Moreover the G9 detected strain in the present study had a P [8] antigen which is included in the monovalent Rotarix ™ vaccine, so severe gastroenteritis due to G1P [8] and G9P [8] strain in Sudan is otherwise expected to wane by implementing the current vaccine.
As referred to other publication by Magzoub et al [9], a total of 755 fecal specimens were tested for rotavirus and 16% were positive, a finding similar to Elhag et al [27] and Parashar et al [3] moreover are slightly less than the previous findings in Marocco [28]. In comparison to the rotavirus infection rate between males and females, the study results showed a prevalence rate of rotavirus infections in males higher than females [9] this is in agreement to some worldwide studies which indicated that males are more susceptible to rotavirus infection and actually exhibited a higher rate of rotavirus in their faeces than females [29,30]. It has to be mentioned that this study dealt with strains isolated from hospitalized patients. Conducting community based surveillance can produce more accurate results in terms of sequencing and characterization of rotavirus strains in order to reflect the true prevalence and circulating genotypes in Sudan.

What this study adds
 Although it is hospital based study but it seems to be the first study in Sudan in term of rotavirus sequences;  It adds background and an idea of the circulated rotavirus strain in Sudan so, it will help to make a decision of distribute the vaccine in Sudan. Figure 1: Although it is hospital based study but it seems to be the first study in Sudan in term of rotavirus sequences