The distribution of Rotavirus G and P genotypes in children with acute gastroenteritis in Cukurova region, Turkey

Introduction Rotavirus is the most common cause of acute severe gastroenteritis in infants and young children worldwide. It is estimated that approximately 600,000 children die annually due to rotavirus gastroenteritis and more than 80% of deaths occur in developing countries. In developed countries, rotaviruses are associated with high morbidity, but low mortality and are the most frequently isolated pathogens in children hospitalized with acute gastroenteritis (1).


Introduction
Rotavirus is the most common cause of acute severe gastroenteritis in infants and young children worldwide.It is estimated that approximately 600,000 children die annually due to rotavirus gastroenteritis and more than 80% of deaths occur in developing countries.In developed countries, rotaviruses are associated with high morbidity, but low mortality and are the most frequently isolated pathogens in children hospitalized with acute gastroenteritis (1).
The genes encoding the outer capsid viral proteins VP7 and VP4, which are responsible for cell attachment and entry, allow the classification of group A rotaviruses into G and P genotypes, respectively.Up to now, at least 27 G types and 35 P types have been described for rotavirus group A, but it has been known that only 10 G (G1-G6, G8-G10, and G12) and 11 P (P1, P3-P6, P8-P11, P14 and P19) genotypes infect humans (4).Furthermore, epidemiological studies have shown that five of the G genotypes (G1, G2, G3, G4, and G9) and three of the P genotypes (P1A [8], P1B [4], and P2A [6]) are globally common in humans (1,3,5,6).Another issue that is of critical importance is the emergence of new reassortant virus strains in case of coinfection with different rotavirus strains.There is a large number of G and P genotype combinations circulating in the human population, however five major combinations of G and P genotypes worldwide are G1P [8],G2P [4], G3P [8], G4P [8], and G9P [8] (1,5).Considering the genetic diversity of rotaviruses, it is advisable to determine geographic variation of predominant strains for effective rotavirus vaccine development.At present, there are two rotavirus vaccines approved in many countries including Turkey, which are Rotarix (GlaxoSmithKline, Research Triangle Park, NC, USA), a live attenuated monovalent vaccine containing rotavirus P1A [8]G1 genotype and RotaTeq (Merck, Rahway, NJ, USA), a live attenuated pentavalent vaccine consisting of G1, G2, G3, G4, and P1A [8] types.However, rotavirus vaccines are not yet included in national immunization programme in Turkey.The efficacy of the Rotarix and RotaTeq against severe diarrhea was found to be 85% and 98%, respectively (3,7).While deciding the strategy for immunization against rotavirus, it is important to detect the regional incidence of rotavirus gastroenteritis and predominant circulating genotypes before and after immunization.The aim of this study was to determine the frequency of rotavirus infection and the distribution of rotavirus G and P genotypes among children under 5-years-old with acute gastroenteritis in Çukurova region, Turkey, between October 2009 and June 2010.The results of this study would provide epidemiological information before national implementation of rotavirus vaccines into routine childhood immunization schedule in Turkey.

Sample collection
A total of 846 stool specimens were collected from unvaccinated children with acute gastroenteritis, younger 5 years of age, admitted to Adana Obstetrics and Child Care Hospital from October 2009 to June 2010.The stool samples were stored at -80ºC until tested.Clinical symptoms and demographic data of the patients were obtained via a questionnaire filled out by a physician by face to face interview and after physical examination of the patients.

Rotavirus antigen detection
Stool samples were tested for group A rotavirus antigen using a solid phase sandwich type enzyme immunoassay (Ridascreen rotavirus ELISA test, R-Biopharm AG, Germany).Rotavirus antigen-positive samples were evaluated for rotavirus G and P genotyping by seminested multiplex RT-PCR test using the primers previously described by Gentsch et al.,(8) Gouvea et al., (9) and Iturriza-Gómara et al. (10).

RNA extraction from stool samples
Frozen stool samples were incubated on the bench to dissolve and then diluted 1:10 in phosphate-buffered saline.Viral RNA was extracted using the High Pure Viral RNA Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.

RT-PCR for rotavirus G and P typing
The G and P genotyping of rotavirus was performed by using two-step RT-PCR.For rotavirus G genotyping, the 1062 bp fragment of the VP7 gene was amplified with the forward primer Beg9 (5'-GGC TTT AAA AGA GAG AAT TTC CGT CTG G-3') and the reverse primer End9 (5'-GGT CAC ATC ATA CAA TTC TAA TCT AAG-3').For P genotyping, the consensus primers Con2 (5'-ATT TCG GAC CAT TTA TAA CC-3') and Con3 (5'-TGG CTT CGC CAT TTT ATA GAC A-3') were used to amplify VP4 gene fragment of the 876 bp (8)(9)(10).For G genotyping, the reaction was carried out with an initial reverse transcription step.Synthesis of cDNA was carried out in 20μl reaction volume by using 10U of reverse transcriptase enzyme (Roche Diagnostics, Mannheim, Germany), 25 pmol of each Beg9, and End9 primers, 20U RNasin (Roche, Diagnostics, Mannheim, Germany), 1mM dNTP mix, 4μl of 5X RT-buffer, deionized distilled water, and 5μl RNA extract, which were incubated at 55˚C for 30 minute, followed by heating at 85°C for 5 minute to inactivate the enzyme.The amplification reaction was performed with a 50μl reaction volume consisting of 10X PCR Taq Buffer, 1.5 mM MgCl2, 200 µM dNTP mix, 25 pmol of each Beg9 and End9 primers, 2 U Taq DNA polymerase (Fermentas Life Sciences), and 5 μl cDNA.The thermal cycling conditions were as follows: initial denaturation at 94°C for 3 minutes, followed by 35 cycles of 1 minute denaturation at 94°C, 2 minutes annealing at 42°C, and 1 minute extension at 72°C.The final extension step was carried out at 72°C for 10 minutes.PCR products were analyzed on an agarose gel stained with ethidium bromide and visualized in ultraviolet transilluminator.The same RT-PCR protocol was used for P genotyping of rotavirus with consensus primers Con2 and Con3 amplifying VP4 gene (876bp).

Semi-nested multiplex PCR for rotavirus specific G and P genotyping
Rotavirus specific G and P genotyping was performed using a semi-nested type specific multiplex RT-PCR test that detects seven G types and six P types (8)(9)(10).Briefly, in order to identify the specific G type, the amplicons obtained by consensus PCR, type specific primers (G1, G2, G3, G4, G8, G9, and G10) and consensus primer RVG9 for the VP7 gene were used in semi-nested type specific multiplex RT-PCR.For P genotyping, the amplicons obtained by consensus PCR, type specific primers (P4, P6, P8, P9, P10, and P11) and consensus primer Con3A for the VP4 gene were used (8)(9)(10).Both G and P genotyping protocols were identical and performed with a 50μl reaction volume consisting of 5 μl of 10X PCR Taq Buffer (100 mM Tris-HCl [pH 8.8], 500 mM KCl), 2 mM MgCl2, 200 µM dNTP mix, 20 pmol of each type specific primers and consensus primer, 2 U Taq DNA polymerase (Fermentas), and 2 μl product of first round PCR.The thermal cycling conditions were as follows: initial denaturation at 94°C for 3 minutes, followed by 35 cycles of amplification (1 minute at 94°C, 2 minutes at 42°C, 1 minute at 72°C for each cycles), and a final extension of 10 minutes at 72°C.All amplified products of G and P genotypes were examined by gel electrophoresis in 2% agarose gel.

Statistical analyzes
The dependent factor in our study was defined as "rotavirus positivity".The independent variables tested were sex, age, symptoms include diarrhea, duration of diarrhea, number of daily bowel movements, vomiting, daily number of vomiting, fever, dehydration, and hospitalization.Chi-square test (χ2) was used for statistical comparison using EpiInfo 6.0 software.The pvalue less than 0.05 were accepted as significant with 95% of confidence.

Discussion
The high incidence of rotavirus gastroenteritis in both developed and developing countries suggests that the improvement of personal and public hygiene is not enough to prevent the spread of the disease.Therefore, vaccination is inevitable for prevention against rotavirus infections.Currently, there are two available rotavirus vaccines targeting protection against severe diarrhea, for reducing emergency admission rates, mortality, morbidity, and economic burden due to rotavirus infections (1,6).Rotarix, monovalent vaccine against P1A [8]G1 genotype, is effective in protection against G1 type (96.4%), heterotypic G types G3 (93.7%),G4 (95.4%), and G9 (85%).13However, the efficacy of Rotarix vaccine against heterotypic G2P [4] has been reported to be from 41% in Latin America (11), 77% in Brasil (12) to 85.5% in Europe (13).
The rate of rotavirus infection in children with acute gastroenteritis has been previously reported in varying proportions of 13 (23)(24)(25)(26).The frequency of rotavirus infection in our study was 17% (144 cases).The rates of rotavirus gastroenteritis are comparable in both developed and developing countries, but mortality from diarrhea is greater in developing countries as a result of inadequate treatment (1,2).In our study, the most common rotavirus G genotype was G1 with the rate of 41.7%, followed by G2 (16.6%),G9 (11.1%), and G4 (1.4%).The genotype G3, which is also one of the most common types worldwide, was not identified during the study period.Interestingly, the predominant P genotype was P [4] (37.5%), but not P [8] (31.2%).There is limited number of studies conducted on the distribution of rotavirus genotypes in Turkey.Reports in different years from Ankara (24) and Izmir (25) regions in Turkey also showed that G1 was the dominant genotype with 86% and 75.1%, respectively, but being higher than our finding that of 41.7%.We found G2 genotype as the second most common (16.6%) type, was higher than the rates of 3.3% and 0.8% reported in Ankara and Izmir, respectively (24,25).Higher frequency of G1 genotype has also been consistent with previous results for rotavirus infection in France (61.7%) and Japan (72.7%) (27,19).Our finding of 41.7% rate for G1 type was similar to that reported from Italy in 2008 (G1 40.7%), however the second most common type observed in Italy was G9, but not G2 (28).The most prevalent genotype combination in our study was G1P [8] (22.2%) which was also identified as predominant genotype in the previous studies (14,(28)(29)(30).

Conclusion
In conclusion, although the predominant rotavirus genotypes circulating during the study period in our region are the targets of current rotavirus vaccines, uncommon, non-vaccine rotavirus genotype combinations such as G1P [4] (17.3%) and G9 [4] (6.3%) were also detected.The high rate of mixed rotavirus infections (29.2%) in the study population might increase emergence of new strains due to natural reassortment, which might decrease the effectiveness of current rotavirus vaccines against rotavirus infections.Therefore, continuous prospective monitoring of circulating strains of rotavirus is essential to evaluate the efficacy and convenience of rotavirus vaccines.

Table 1 .
Clinical and epidemiological features of children with and without rotavirus gastroenteritis.

Table 2 .
Distribution of rotavirus G and P types.
7 Figure 1.Monthly distribution of rotavirus positive and negative gastroenteritis cases.

Table 3 .
Distribution of rotavirus G-P genotype combinations of mixed infections.