Identification of Rotavirus Strains Causing Diarrhoea in Children under Five Years of Age in Yogyakarta, Indonesia

Background: Rotavirus is an important cause of severe diarrhoea in children. The aims of this study were to identify the rotavirus strains that cause diarrhoea in children in Yogyakarta and to determine the association between rotavirus positivity and its clinical manifestations. Methods: Clinical data and stool samples were collected from children hospitalised at Kodya Yogyakarta Hospital, Indonesia. Rotavirus was detected in stool samples using an enzyme immunoassay (EIA), which was followed by genotyping using reverse transcriptase polymerase chain reaction (RT-PCR). Electropherotyping was performed for the rotavirus-positive samples. Results: In total, 104 cases were included in the study, 57 (54.8%) of which were rotavirus-positive. Based on a multiple logistic regression analysis, age group, vomiting and stool mucous were associated with rotavirus positivity. Most of the 56 samples subjected to genotyping were classified as G1 (80.36%) and P[8] (69.64%) genotypes. The genotype combination G1P[8] was identified as the most prevalent strain (66.07%). Of the 19 samples subjected to electropherotyping, 17 G1 isolates and 1 G3 isolate had long patterns, and 1 G1 isolate had a short pattern. Conclusion: G1P[8] was the most dominant strain of rotavirus causing diarrhoea in children in Yogyakarta. Age group, vomiting and stool mucous were associated with rotavirus positivity.


Introduction
Rotavirus is an RNA virus that mainly infects the gastrointestinal tract. It is the most prevalent agent causing severe diarrhoea among infants and young children in both developed and developing countries, including Indonesia (1)(2)(3). It was recently estimated that rotavirus is responsible for 7,200,000-11,500,000 severe diarrhoea episodes and 133,000-284,000 deaths, most of which occur in developing countries (2). The incidence might be lower in countries that have introduced rotavirus vaccination (2).

Detection of rotavirus in stool samples
All stool samples were examined for the presence of group A rotavirus by enzyme immunoassay (EIA) using the IDEIA TM Rotavirus (DakoCytomation) kit according to the manufacturer's instructions. Other possible causes of diarrhoea were not examined.

RT-PCR genotyping
Rotavirus RNA was extracted from rotavirus-positive faecal specimens using Trizol (Invitrogen) according to the manufacturer's instructions. Rotavirus RNA was analysed to determine the VP7 (G-type) and VP4 (P-type) genotypes using reverse transcriptase polymerase chain reaction (RT-PCR) as previously described (13)(14)(15). For G typing, the primers 9con1L (13) and VP7R (15) were used during the first round of RT-PCR (20 cycles) to amplify the 905-bp of the VP7 gene segment. Then, 9con1L was used in the second round of PCR (20 cycles) with the type-specific primers 9T1 (G1), 9T-2 (G2), 9T-3P (G3), 9T4 (G4) and 9T-9B (G9) ( Table 1) (13). For P-typing, the primers con2 and con3 were used in the firstround of RT-PCR (20 cycles) to amplify the 877-Since rotavirus diarrhoea is prevalent among children in developed and developing countries, it is unlikely that improvements in sanitation and hygiene will be able to lower the incidence of the disease. Therefore, one of the best strategies for decreasing the global burden of disease is development and implementation of effective vaccines (1). The current strategy for developing rotavirus vaccines involves oral administration of live attenuated viruses that protection is expected to be similar to natural rotavirus infection (4,5).
Rotavirus has three essential antigenic specificities, determined by the viral proteins VP6, VP7 and VP4. Based on the VP6 protein, rotavirus is classified into seven groups (A to G), yet only groups A, B and C infect humans. VP7, a glycoprotein, determines G-genotype, while VP4, which is protease-sensitive, determines P-genotype. VP7 and VP4 form the outer layer of rotavirus and elicit neutralising antibody responses. Therefore, they are implicated in the dual classification of rotavirus group A as G-and P-type (6).
The effectiveness of rotavirus vaccine can be enhanced by identification of the patterns of circulating virus serotypes and the role of these serotypes in causing clinical illness (7). During vaccine implementation, strain surveillance is necessary to monitor antibody-escape variants and changes in the circulating rotavirus strains among humans (8).
Two rotavirus vaccines, Rotarix and RotaTeq, have been developed by GSK and Merck, respectively. RotaTeq is a live attenuated pentavalent vaccine containing G1, G2, G3, G4 and P [8] rotavirus derived from reassortants of human and bovine strains. Rotarix is a live attenuated monovalent vaccine derived from the most common human rotavirus strain, G1P [8]. These vaccines have been proven to be highly effective for the global prevention of severe diarrhoea (9,10). Before implementing the rotavirus vaccination programme in Yogyakarta, it is crucial to perform a study to ensure that the vaccines used in the programme match with the most prevalent and clinically relevant rotavirus strain in Yogyakarta.
The aims of this study were to identify the rotavirus strains currently circulating in Yogyakarta and to determine the association between rotavirus positivity and the illness's clinical manifestation. This study provides accurate information about the rotavirus strains circulating in Yogyakarta before and after the implementation of the vaccination programme.
were separated in 2% agarose gel and visualised under UV light after staining with ethidium bromide.

Electropherotyping
Rotavirus RNA was extracted using Minikit (Qiagen) according to the manufacturer's instructions.
RNA was subjected to polyacrylamide gel electrophoresis to separate the 11 segments of dsRNA, which were then visualised by silver staining.

Statistical analyses
The variables were described using frequencies and percentages. Chi-square tests and logistic regression analyses were used to determine the association between rotavirus infections and clinical manifestations using STATA 13. The variables that had P-values of less than 0.25 according to the chi-square tests were entered into a logistic regression analysis model. Age group and all the clinical symptoms analysed (vomiting, stool mucous, bloody stool and fever) had P-values of less than 0.25 (Table 3). Bloody bp fragment of the VP4 gene segment (14). Con3 was used in the second round of PCR (30 cycles) with the type-specific primers 1T-1D (P [8]), 2T-1 (P [4]), 3T-1(P [6]), 4T-1 (P [9]) and 5T-1 (P[10]) ( Table 2) (14).
A number of rotavirus-positive stool samples did not have sufficient volume for polyacrylamide gel electrophoresis (PAGE) analysis. Only 24 samples were subjected to PAGE analysis, and 19 isolates had good results. Eighteen samples were identified as G1, and stool was omitted because the chi-square test revealed cells with a count of zero. Therefore, age group, vomiting, stool mucous and fever were examined in the multiple logistic regression analysis. The fit of the model was checked using the Hosmer-Lemeshow test. The results were presented as crude and adjusted odds ratios with a 95% confidence interval. P-values of less than 0.05 were considered statistically significant.

Ethical approval
The research protocol was approved by the Ethical Committee of the Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Informed consent was provided by the parents or guardians of each child before the children were enrolled in the study.

Results
From February to August 2009, 104 children under five years of age who were hospitalised at Kodya Yogyakarta General Hospital, Indonesia due to acute diarrhoea were enrolled in the study. There were 70 (67.3%) male and 34 (32.7%) female patients (Table 3). Of the 104 samples collected, 57 (54.8%) were positive for rotavirus, as determined by EIA.
Rotavirus diarrhoea particularly affects young children; 70.2% of the cases involved children under two years of age. Most cases involved children aged 12-23 months, with the next largest group comprised of children aged 24-59 months (Table 3). Additionally, the chi-

First amplification of P-typing as described by Gentsch et al. (14)
Con  Our study found a high incidence of rotavirus infection in Yogyakarta, Indonesia, which is comparable to the findings of similar studies in surrounding countries, such as Malaysia (38%) (21), Thailand (43%) (22), Cambodia (56%) (23) and Myanmar (57%) (24). Our study provides evidence that rotavirus infection gives a substantial burden to the one sample was identified as G3. All of them showed the migration pattern of the genome of rotavirus group A: 4-2-3-2. The results of electropherotyping showed that 17 of 18 G1 isolates and 1 G3 isolate had long patterns, while 1 G1 isolate had a short pattern (Figure 1).

Discussion
This hospital-based study described a high incidence of rotavirus infection in Yogyakarta, Indonesia. Rotavirus infection was identified in 54.8% of 104 children hospitalised with acute diarrhoea. Compared to previous hospital-based studies performed in Indonesia in the late 1970s and 1990s, the incidence rate was similarly high. Bishop et al. (17) reported rotavirus infection in 38% of children with severe acute diarrhoea in Yogyakarta. Subekti et al. (18), who conducted a study in Jakarta from 1997-1999, showed that 170 of 236 (72%) children with diarrhoea were  ( Table 3). This phenomenon is probably due to the presence of anti-rotavirus immunoglobulin G (IgG) and IgA antibodies in breast milk (32). In their study of children in Yogyakarta, Chan et al. (33) found a high concentration of anti-rotavirus IgG and IgA in colostrum as well as in breast milk. Moreover, Clemens et al. (34) found that exclusive breastfeeding confers better protection against severe rotavirus infection than partial or no breastfeeding. However, further study is required to fully address this issue in our patient population.
The genetic diversity of circulating rotavirus strains may have a crucial role in the initiation of the vaccination programme and its clinical evaluation. Some studies have been conducted to identify the rotavirus strains circulating in Indonesia. Using samples collected in Yogyakarta from 1978-1979, Bishop et al. (17) identified G1 (2%); G2 (9%), G3 (53%) and G4 (36%) strains.
diarrhoeal diseases and it is critical to develop an effective approach to preventing infection.
Rotavirus infection in children clinically manifests with several symptoms, such as fever, vomiting and watery, bloodless diarrhoea (25,26). These clinical manifestations are more severe than those of other causes of viral gastroenteritis (27). In our study, vomiting was associated with rotavirus positivity (Table 4), which aligns with the findings of other studies (20,28). Those studies also reported that rotavirus-positive patients were less likely to have bloody diarrhoea (20,28), in agreement with our study.
Severe complications of rotavirus infection, such as bloody diarrhoea associated with necrotising enterocolitis (NEC) and bowel perforation, have been described (25,26,29). Even though they have not yet been fully elucidated, several factors were associated with the pathogenesis of the disease (30). However, an enterotoxin protein, NSP4, and the ability of rotavirus to trigger the apoptosis of intestinal cells might be the factors that most affect the manifestation of the disease (31).
Our study demonstrated that the percentage of children aged 0-11 months who suffered from rotavirus diarrhoea was lower than that of children aged 12-23 and 24-59 months  the vaccine on genotype distribution and the possible emergence of less common rotavirus strains circulating in certain geographical areas. It is also crucial to assess the effectiveness of the vaccination programme against rotavirus diarrhoea.
According to the migration of its 11 RNA gene segments, rotavirus can also be classified as either long or short RNA electropherotype using polyacrylamide gels. The short electropherotype occurs as a result of partial duplication in gene segment 11. This gene runs more slowly than gene segment 10; because of its standard size, gene segment 11 in long-electropherotype strains migrates faster than segment 10 (38). In this study, we found that most G1 and G3 isolates had a long electropherotype. Long electropherotypes usually belong to the Wa genogroup, whereas short electropherotypes typically belong to the DS-1 genogroup (36).

Conclusion
We found that rotavirus caused acute diarrhoea in 54.8% of children under five years of aged hospitalised at Kodya Yogyakarta General Hospital. The majority (66.07%) was G1P [8], the most prevalent strain identified globally. Age group, vomiting and stool mucous were associated with rotavirus positivity.