The objective of this study was to ascertain for the first time the P and G genotypes of circulating rotaviruses isolated from infected hospitalized children in Ireland. Genotyping is a well established and recognized epidemiological tool for examining strain diversity, and the correlation between genotype and serotype is well understood. The significance of determining serotypes of circulating rotaviruses has become increasingly recognized (21
), and RT-PCR has been shown to be the most sensitive assay for determining genotypes (9
). A rotavirus tetravalent vaccine that has recently been licensed specifically targets G1 to G4 serotypes, and it is essential to determine the predicted efficiency of this vaccine for each country before it is used.
The Republic of Ireland has a population of over 3.5 million and is served by over 50 medical microbiology laboratories. Sixteen of these routinely test pediatric patients (1 to 48 months) for rotavirus, and positive specimens were donated by over one half of these hospital laboratories. A total of 193 samples were collected from nine hospitals nationwide. Only four samples could not be assigned a G or a P type, which suggests that the incidence of unusual genotypes among Irish isolates is rare. The efficiency of the primers was determined by cloning and partially sequencing randomly chosen PCR products. As expected, in all cases the primers amplified the correct sequence (data not shown). After the P-typing assays it was discovered that our Irish results match those observed in studies from seven other countries involving 500 samples (6
), in that two main P types prevail, namely, P and P. It was also observed that their relative incidence over the 3 years varied. P types were detected almost exclusively for the first 2 years, whereas a notable shift occurred in year 3, when P became more prevalent (38%) (Table ). It is interesting that no P or P types were observed, even though the incidence of P in recent American (12
), Brazilian (18
), and Indian (11
) surveys seems to highlight this strain as a significant emerging genotype.
Of the samples tested, 165 were assigned a G type. As expected from similar surveys carried out worldwide, G1 was the most predominant type detected for each of the 3 years in Ireland, even though G2 emerged as a significant type in the third year (Table ). This seems to indicate a notable relative shift in the prevalence of circulating viruses, which should be monitored over the coming years. It is interesting that this bias away from G1 in December 1997 correlated with a simultaneous marked increase in the number of samples received by our laboratory (data not shown). This may suggest a greater susceptibility to emerging genotypes by Irish infants. Six mixed-type strains (all G1/G4) were recovered from patients in the last 2 years, and just two G3 isolates were detected during this survey. It is interesting that non-G1 to G4 genotypes are prevalent in other countries: G9 was the most predominant type reported in India (11
), and G5 was the third most reported G type in Brazil (6
Both G and P genotypes were assigned to 158 (81.8%) of collected viruses. Consistent with the findings of numerous reports, G1 P was by far the most common type, while G2 P and G4 P were less prevalent. However, in the third year of this study the number of G1 P isolates recovered was reduced by over 50% and an almost equal number of G1 P and G2 P viruses coexisted in Ireland (Fig. ). This would seem to indicate a significant genotypic shift, which will be of major importance for future studies carried out in Ireland. Although noteworthy, this is not an unusual phenomenon, as periodic changes in prevailing genotypes have previously been reported (8
). Even though a disparity exists between sample numbers and source, it appears that a correlation did not exist between the hospital source and a particular genotype. Each genotype appeared to be evenly distributed among the collection centers around the country (Table ).
In conclusion, genotyping by RT-PCR is an extremely sensitive technique (~50,000 times more so than enzyme immunoassays) (19
) and is adaptable to accommodating new and emerging types. However, the method is 10 times more expensive than other methods (9
), and the RNA used must be of a reputable quality. We found that the inclusion of random primers in the RT mix greatly reduced the appearance of nonspecific amplification during PCR and also halved the number of RT reactions required. This method was introduced in the last year of the study and, although more efficient, it was not able to detect previously untypeable samples. The detection of rotaviral genotypes for the first time in Irish children and the indication that the prevalance of certain genotypes may change over a rotavirus season is significant and mirrors observations from studies in other temperate climates (8
). If the tetravalent vaccine becomes available in Ireland, it appears that the coverage against infection would be significantly high, although monitoring of the genotypic changes among circulating viruses should be encouraged over the coming years.