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Group A Rotavirus is the most common cause of acute gastroenteritis worldwide. It causes more than 440.000 deaths among children less than 5 years annually, primarily in the developing countries. Rotavirus, which belongs to Reoviridae family and a member of Rotavirus genus, is a non-enveloped, wheel-like shaped virus that possesses 11 segmented double-stranded RNA (dsRNA) genome enclosed in a triple layered capsid. The triple layers consist of inner, middle, and outer capsid. The VP (viral protein) 1, VP2, and VP3 proteins, encoded by RNA segments 1-3, build the inner capsid. The middle capsid is VP6 protein, encoded by segment 6. The VP4 and VP7 proteins, that are encoded by segment 4 and 9 (or 7, or 8, depend on the strain), form the outer capsid. These two outer capsid proteins, VP4 and VP7, are rotavirus antigens to classify the virus into P and G type, where P stands for protease-sensitive protein and G for glycoprotein.
Rotavirus has been detected by numbers of different techniques such as direct detection, viral cultivation (in Ma104 cell culture), and immunoassay. Direct detection method is applicable for either viral particles (electron microscopy-EM) or viral genome (polyacrylamide gel electrophoresis-PAGE). The principle of immunoassay is the reactivity of viral antigen to neutralizing antibody, and the most common method used is enzyme-linked immunosorbent assay (ELISA/EIA). EM is a definitive method; nevertheless, it is considered impractical for routine use due to time-consuming and requires highly trained personnel to handle. ELISA is rapid and inexpensive for serotyping, but the application of monoclonal antibodies (Mab) holds the potency to cross-react with more than one serotype and limitation by the requirement of minimum amount of intact virus particles, which should present in specimens. As genomic RNA is more stable than protein in terms of laboratory diagnosis, a nucleic acid-based assay that correlates well with Mab-based serotyping of rotavirus VP7 gene was developed subsequently. The multiplex semi nested RT-PCR (Msn RT-PCR) converts the genomic RNA into cDNA and amplifies it using type-specific primers designed to hybridize to type-specific regions in the VP7 gene to classify Rotavirus into G genotypes. The high sensitivity and specificity offered by Msn RT-PCR for detection and genotyping should provide more reliable data for Rotavirus epidemiology studies.
Although Msn RT-PCR is the most widely method for Rotavirus identification in surveillance studies, however, the reagents and techniques that are used need to be regularly revised and updated. The accumulation of point mutations at the type-specific primer binding sites may lead to false negative results while the sharing of type-specific region among strains may lead to false multiple genotype identifications. The method itself retains a potential drawback that the primers may undergo interprimer competition or complementarity at 3'-ends. Primer competition may lead to incorrect genotype identification, while 3'-ends complementarity is the major cause of primer-dimers. The failure of the standard genotyping method to characterize rotavirus strains is also attributable to the emergence of novel genotypes for which type-specific primers may not be available.
Several cases of failure and false multiple genotype identification have been reported from different parts of the worlds. The mismatches at primer binding site are described in Bangladesh where the 9T1-1 primer failed to identify G1, as well as in Italy and United Kingdom where aFT9 primer can not hybridize to G9. The emergence of novel genotypes, such as G10 in India and G12 in Bangladesh, were previously thought as nontypeable. Some false multiple genotype identifications due to cross-priming include G3G8 in Guinea-Bissau, G3G10 in India, G3G9 and G4G9 in Brazil. Sequence analysis has successfully verifies the accurate VP7 genotypes of these misleading issues.
Toward ambiguous and / or false negative genotypes, the VP7 gene was sequenced to confirm the true G genotype by applying either random hexamers or a primer pair able to extend the full-length of VP7 gene. The 5'- and 3'-untranslated regions (UTR) flank the open reading frame (ORF) of VP7 gene. The regions are highly-conserved due to they play an important role in replication by initiating transcription, a function executed by a promoter at 3'-terminus and two enhancers at each 5' and 3' UTRs. The UTRs, therefore, provide suitable sequences for primer binding-site aimed to amplify and sequence the whole VP7 gene (1062 bp).
An epidemiology study of Rotavirus circulating in Indonesia between February 2004 and February 2005 reported G4G9 as the most common G genotype identified (37.42%). Generally, the prevalence of a certain type of multiple genotypes is much lower than the leading genotype that circulates in a population. This inconsistent fact initiated the idea to determine the VP7 gene sequences of Rotavirus isolates that had been identified as G4G9 to verify whether they represent the emergence of new predominant Rotavirus genotypes or the failure of the genotyping method.
MATERIALS AND METHODS
Fourteen Rotavirus RNA that had been identified as G4G9 in the previous study (Putnam et al., 2007) were selected randomly from the collection of Bacterial Diseases Program, United States Naval Medical Research Unit No.2 Jakarta.
DNA Synthesis and Amplification
Initially, C1 and C2 primer pair reverse-transcribed viral RNA and subsequently amplify its cDNA to generate the DNA copies of full-length VP7 gene (Taniguchi et al., 1992). The reaction used SuperScriptTM III One-Step RT-PCR System with PlatinumÂ® Taq DNA Polymerase kit [Invitrogen]. In short, a mixture of 23 Âµl of 5 Âµl RNA, 1 Âµl of 10 ÂµM of each C1 and C2 primer, and 16 Âµl of nuclease-free water was heated at 95o C for 5 minutes and directly chilled on ice. The mixture was added by 25 Âµl of 2X Reaction Mix and 2 Âµl of Platinum TaqÂ® DNA Polymerase, then subjected to cycle conditions as follow: 55o C of 30 minutes reverse transcription; 94o C of 2 minutes for initial denaturation; 40 cycles of 94o C for 15 second, 55o C for 30 second; 68o C for 1 minute; and then 68o C of 5 minutes for final extension. Three reverse transcription products were selected randomly as templates in Msn-PCR in order to obtain the sufficient DNA copies for type-specific fragments of G4 and G9 respectively. The Msn-PCR used AmpliTaq Gold DNA Polymerase System [Applied Biosystems] and three different primer cocktails (C1+S4)/ (C1+S9)/ (C1+S4+S9) (Taniguchi et al., 1992). The reaction mixture consisted of 2 Âµl of VP7 cDNA in 1:25 and 1:50 dilution, 5 Âµl of 10x Buffer II, 3 Âµl of 25 mM MgCl2, 0.5 Âµl of each 20 mM dNTP, 1.5 Âµl of 75% DMSO, 0.25 Âµl of 50 ÂµM C1 primer, 0.125 Âµl of each 50 ÂµM type-specific primer (S4/S9/S4+S9), 0.25 Âµl of AmpliTaq Gold DNA Polymerase, and adjustable volume of nuclease-free water to obtain a final volume of 50 Âµl. The cycle conditions were 94o C of 10 minutes for initial denaturation, 30 cycles of 94o C for 1 minute, 50o C for 2 minutes, and 72o C for 1 minute, then a 72o C hold of 7 minutes for final extension. The amplicons of type-specific fragments demonstrated the expected results that are sized 394 bp and 306 bp for G4 and G9, respectively. PCR products were detected in gel electrophoresis assay of 2% agarose gel stained with 0.05% (w/v) ethidium bromide.
The PCR products need to be purified to remove excessive salts and impurities. The purification of single-band RT-PCR products used the QIAquick PCR Purification Kit [Qiagen], while the purification of multiple bands Msn-PCR products used the QIAquick Gel Extraction Kit [Qiagen]. The Gel Extraction kit requires the PCR products to be initially electrophoresed on a 1.8% of low-melt point agarose gel and excised precisely at the target fragment under UV light illumination. Briefly, for the PCR Purification kit, one volume of PCR products was mixed with 5 volumes of buffer PBI, while for the Gel Extraction kit, one volume of gel-containing DNA was mixed with 3 volumes of buffer QG. DNA was isolated from contaminants through centrifugation steps. The DNA was washed with ethanol-containing buffer PE to remove any residual salts, and then eluted in 30 Âµl of buffer EB. Subsequently, the DNA was quantified per one Âµl volume in a 2% agarose gel electrophoresis together with 100 bp DNA ladder [Promega], which contained DNA fragments with recognized sizes and quantities. The quantity of DNA was estimated by visual comparison of the band intensity with that of the nearby-sized fragment in DNA ladder. For 200-500 bp and 1-2 kb of PCR products, 3-10 ng and 10-40 ng of DNA is required as an optimum amount of template in cycle sequencing. The DNA was sequenced in both directions with appropriate primers using the dideoxy chain terminator method of BigDye v3.1 Cycle Sequencing Kit [Applied Biosystems]. Cycle sequencing reaction is a 20 Âµl mixture consisted of 4 Âµl of 2.5X Ready Reaction mix, 2 Âµl of 5X BigDye v3.1/1.1 Sequencing Buffer, 1 Âµl of 5 ÂµM primer, and adjustable volume of DNA and nuclease-free water. The cycle conditions were 96o C of 1 minute for initial denaturation, and 25 cycles of 96o C for 10 second, 50o C for 5 second, and 60o C for 4 minutes. The cycle sequencing products were purified with BigDyeX TerminatorTM Purification Kit [Applied Biosystems] to remove unincorporated dye terminators and excessive salts. The purification reaction consisted of 10 Âµl of cycle sequencing products, 45 Âµl of SAM solution, and 10 Âµl of BigDyeX Terminator. Capillary electrophoresis was performed in a 3130xl genetic analyzer [Applied Biosystems]. During this step, high voltage was automatically applied to move the DNA molecules toward the cathode, but shortly before reaching it, the fluorescent dye-labeled DNA moved through the path of a laser beam such that the dye excited light at a particular wavelength. An optical device converted the fluorescence into digital data.
The Sequence Analysis v5.2 software [Applied Biosystems] basecalled the digital data into a chromatogram, namely a *.ab1 file of 4 different-color peaks that represented each DNA base. The shift and unresolved peak errors in raw chromatograms were edited by Sequencher v4.8 software [GeneCodes]. Following correction, a contig was assembled by aligning overlapped sequence reads with 85% of minimum match percentage and 20 nucleotides of minimum overlap parameters. The consensus sequence of a contig alignment was picked up as the final sequence. The final sequences were subjected to a web-based homology comparison with Basic Local Alignment Search Tool for nucleotide sequence (BLASTN) v.2.2.19 (Zhang et al., 2000). Subsequently, the full-length VP7 sequences were aligned against the G4 and G9 fragments in order to confirm whether the multiple genotype identification was due to cross-priming or co-infection of several Rotavirus genotypes. S4 and S9 primers were included in the alignment in order to understand their base-pairing nature to investigated VP7 sequences.
Generation of templates for DNA sequencing by PCR
Reverse transcription of Rotavirus dsRNA and subsequent amplification of the cDNA with C1 and C2 primers generated the expected full-length of VP7 gene with size of 1062 bp (results not shown). The results verified that C1 and C2 primer pair could amplify the whole population of VP7 gene in Rotavirus RNA identified as G4G9 in our study. The Msn-PCR conducted to three G4G9 isolates (GR 0003, GR 1067, and GR 1430), using three different primer sets (C1+S4+S9, C1+S4, and C1+S9), yielded three manners of result. The combination of C1+S4+S9 primers generated both fragments of G4 and G9 with the size of 394 bp and 306 bp, respectively (Fig 1A). The application of C1+S4 and C1+S9 in different reaction mixture resulted in type-specific fragments of G4 and G9 with the size of 394 bp and 306 bp respectively (Fig 1B and 1C). Fig 1 showed that S4 and S9 primers were able to anneal to the same DNA. In addition to that, Fig 1 indicated that G4 fragment had higher intensity than the G9 fragment; although when the primer combination C1+S9 and C1+S4 were employed separately the intensity of both fragments appeared similar. Even though it could be only an experimental error, however, this condition might also indicate that S4 outcompeted S9 primer to generate DNA copies in a reaction mixture that contained both primers. Template in 1:25 and 1:50 dilution were prepared to optimize Msn-PCR. However, the result showed that the dilution had no significant effect on amplification of target. All fragments, together with the 1062 bp fragment of VP7 gene, were sequenced in 5' and 3' directions using appropriate primers.
DNA sequencing and sequence analysis
The length of VP7 sequences that was obtained in this study ranged about 859 to 1001 bases out of the expected 1062 bases (Fig 2). Sequences that were shorter than the expected were due to truncation at the 5' and 3'-ends, i.e. the extension region following primer-annealing site. The average length of sequence reads is controlled by ddNTP and dNTP ratio. The higher ratio leads to shorter reads, while the presence of high dNTP concentration allows prolonged DNA synthesis until a ddNTP incorporate to the growing chains and terminate the synthesis (Ausubel & Albright, 1995). Cycle sequencing was performed with BigDye v3.1 Cycle Sequencing Kit [Applied Biosystems] which was designed to obtain long sequence reads such that it is unable to synthesize short sequence reads adjacent to the primer (Applied Biosystems, 2002a; Applied Biosystems, 2002b). The lost region near to 5' and 3'-ends varied between 28 and 202 nucleotides. BLAST results for VP7 sequences in this study presented 98-99% nucleotide similarity and E-value of 0.0 to Rotavirus VP7 G9. The statistical results that support an inference of similarity searching by BLASTN are percentage of identity and E-value. The E (expectation) value is the number of database hits expected to be found randomly. The higher the percentage of identity and the lower the E-value is, the more likely that the sequences are homologous. The results confirmed that there was only one VP7 genotype, G9, in RNA samples that had been identified as G4G9. Based on that, it suggested that cross priming of S4 occurred to its designed binding-site at VP7 gene (nucleotide 669-688). Sequencing to G4 and G9 fragments provided detailed explanation on this finding. The sequence of G4 fragments were 393 bp and 305 bp for G9 fragments, out of the expected 394 and 306 bp (Fig 3). BLASTN results for G4 fragments, produced from the amplifications by primer mix C1+S4+S9 and C1+S4, were 99% nucleotide identity similar and E-value of 0.0 to VP7 G9 sequences in database. BLAST results for G9 fragments, resulted from amplifications by primer mix C1+S4+S9 and C1+S9, were 98-99% nucleotide identity similar and E-value of 3e-152-7e-149 to VP7 G9 sequences in database. The results emphasized that the generation of G4 fragments in Msn-PCR was due to cross-priming of S4 to VP7 G9 and eventually identified as G4G9 by our genotyping method. Through alignment of S4 and S9 primers against all obtained fragments, it was known that with primer S4 which has annealing position 669-688 and size of 20 bp, all isolates presented five mismatches (75% homology at primer binding region) i.e., at the second, fourth, seventh, and nineteenth position of primer annealing site and with primer S9, that has annealing position 757-776; and the size of 20 bp, all isolates possessed three mismatches (85% homology at primer binding region) i.e., at the third, ninth, and seventeenth position of primer annealing site (Fig 4). It was surprising that S4 was able to amplify VP7 G9, even with higher efficiency than S9, although it is of lower homology than S9 and possess one mismatch at the second base at 3'-end. Theoretically, primers with 17-20 nucleotide long need three homologous bases at 3'-end for successful priming (Sommer & Tautz, 1989).
The globally striking increase of Rotavirus G9 since 1995-1996 has made it the fourth most predominate genotype following G1, G2, and G4 (Gentsch et al., 2005; Santos & Hoshino, 2005). However, recent G9 strains are believed not to originate from the same ancestor that gave rise to the prototype strain (WI61). From six phylogenetic lineages of G9 existing around the world, currently circulating G9 strains belong to lineage III, that emerged around 1993-1994, while the prototype strains, that emerged in the early 1980s, belong to lineage I (Laird et al., 2003; Phan et al., 2007; Martinez-Laso et al., 2009). Since the type-specific primers designed to differentiate VP7 genotypes were based on strains isolated in 1980s, the primers may no longer be appropriate for identification at the present time. Regarding G9 identification, misleading issues have appeared such as false negative results or false multiple genotype of G3G9 and G4G9 (Santos et al., 2003; Iturriza-Gómara et al., 2004a; Martella et al., 2004, Putnam et al., 2007).
In this study, we edited the identification of VP7 G4G9 that was obtained by Msn-PCR into G9 by sequence analysis of PCR products. Direct sequencing to PCR products is able to generate large data in a very limited time and sequence different templates that contain polymorphisms or mutations at once (Meltzer, 1993). Subsequently, the homology comparison against international sequence database by BLASTN enables immediate confirmation for unknown sequences (Bottu et al., 2003).
The extension of C1 and C2 primers generated the full-length VP7 gene sequences. Due to technical problems, we could not obtain the complete 1062 bp of VP7 gene. Yet, the sequences obtained were informative enough to describe the investigated VP7 gene as G9 by homology comparison. Sequence analysis to the G4 and G9 type-specific fragments originated from amplification with combination of C1+S4+S9, C1+S4, and C1+S9 primers showed that homology comparison for the two forms of type-specific fragment strengthened the initial confirmation from VP7 gene sequences that the RNA specimens typed as G4G9 were in fact G9.
Alignment of S4 primer to all sequences obtained in this study showed 5 consistent nucleotide mismatches (75% similarity) at its corresponding binding site to VP7 gene (nt 669-688). The mismatches (5'à3 ) were at nucleotide 670 (CàA); 671 (AàT); 672 (AàT); 675 (GàC); and 687 (TàG). Compared to 3 mismatches (5 à3 ) of S9 to its binding site (nt 757-776; 85% similarity; at 759 (AàT); 765 (AàG); and 773 (CàA)), it is unclear why the priming process allow annealing of a primer over another regardless of lower similarity to primer binding site and the position of nucleotide mismatches. An identical circumstance has been observed in Brazil where the investigated G4G9 isolates belonged to G9 lineage III (Santos et al., 2003). The study also revealed that even though the prototype strain (lineage I) and S4 primer shared 80 % similarity at nucleotide 669-688, it was not typed as G4G9. Meanwhile, the factors that favor primer-template annealing are strict base-pairing nature of primer to template, optimum Mg concentration, higher annealing temperature, shorter annealing time, lower primer and polymerase concentration (Eeles & Stamps, 1993). One of the altered conditions might have enabled S4 to prime the amplification of G4 fragment based on our VP7 G9 template; therefore further study is required.
Point mutation, together with genome reassortment and rearrangement, has contributed to the genetic diversity of Rotavirus strains either in individual or in combination of genes. Due to the absence of 3'à5 exonuclease activity and post-replication error correction of RNA polymerase, Rotavirus possesses a high mutation rate of ~5 X 10-5/ site/ replication (Domingo et al., 1996; Ramig, 1997). The segmented nature of Rotavirus genome enables random reassortment following mixed infection of co-circulating strains. Although viable progeny is limited by selection for fitness of all possible reassortants, this mechanism has led to the emergence of epidemiologically significant G9P in the United Kingdom at 1996 by reassortment of VP7 gene from G9P strain with VP4 gene from the common G1P, G2P, G3P, and G4P strains (Iturriza-Gómara et al., 2001). Rearrangement occurred when a certain sequence is introduced into its homologous RNA segments to produce a new gene containing genetic information from more than one source (Worobey & Holmes, 1999). A characterized recombinant strain of G9 is CIT-254 that has recombination break points at positions 380-520 where it is 92% similar with G1 strain 87H140 and only 78% similar with G9 strain R136 (Phan et al., 2007).
Since Msn RT-PCR aims to identify Rotavirus genome, genetic diversity naturally occurred by the three mentioned mechanisms strongly reasoned the failure or reduced accuracy of genotyping process. Inaccurate prevalence data of Rotavirus genotypes circulating in a locality during a defined timeline and emerging strains with new antigenic specificity, such as antibody-escape mutants, may complicate the efforts to develop vaccines that are able to give optimal protection against strains of globally / regionally epidemiologic importance or provoke the potency of a large outbreak in a population with naÃ¯ve immunity. Evaluation of this genotyping method, therefore, needs to be performed on a regular basis.
In this study, by using sequence analysis, Rotavirus RNA that was initially genotyped as G4G9 by Msn RT-PCR, was subsequently proved in fact G9. The occurrence of multiple genotype identification was due to cross priming between S4 primer, which was aimed to identify G4, and G9 at the right position for its designed binding site (nucleotide 669-688) in VP7 gene. Since this is not the first finding of false identification of G4G9, cautions must be addressed to the simultaneous usage of S4 and S9 primers to genotype field isolates of Rotavirus.
LIMITATIONS OF THE STUDY
Due to truncation at the 5' and 3' ends of sequences obtained in this study, we were not able to explore further about Indonesian Rotavirus G9, includes the credible conserved region for designing type-specific primer that is more appropriate for Indonesian isolates or internal primers targeted to sequence the complete VP7 gene. Further study on complete VP7 sequences will be able to reveal the most probable origin of Indonesian Rotavirus G9 strains and / or suggest their potency to form distinguished lineage from the globally circulating strains.