As a consequence, a total of 21 rotaviruses were available for further studies ( Fig. 1): these comprised genotypes G8P (MAL01, MAL02, MAL33, MAL47,
MAL55, MAL60, MAL70, and MAL81); see more G8P (MAL43); G12P (2 short pattern viruses MAL39 and MAL88, and 2 long RNA pattern viruses MAL12 and MAL40); G9P (MAL80 and MAL82); G1P (MAL23, MAL38, and MAL50); G1P (MAL63); G12P (MAL65); and G2P (MAL66). Since strains carrying G8P, G12P, G9P and G1P previously accounted for 89% of the strains identified among placebo recipients, single representative strains from each of these major genotype combinations (two in the case of G12P representing both short and long RNA patterns) were subjected to nucleotide sequencing of
the genome segment coding for VP7, VP4, VP6, and NSP4. These included MAL81 for G8P, MAL88 for G12P short RNA pattern, MAL12 for G12P long RNA pattern, MAL82 for G9P, and MAL23 for G1P. Nucleotide sequence analysis confirmed the G and P genotypes previously ascribed by RT-PCR, and assigned genotypes to VP6 and NSP4 (Table 1). All long RNA pattern viruses possessed VP6 and NSP4 genotypes of I1 and E1, respectively, whereas all short RNA pattern viruses had VP6 and NSP4 genotypes of I2 and E2, respectively. The results of RNA–RNA hybridization assays are shown in Fig. 2, Fig. 3 and Fig. 4. When the RIX4414 probe was allowed to hybridize with selleck screening library the panel of dsRNAs from representative Malawian strains carrying various genotype combinations, the probe produced 6–8 hybrid bands with genomic RNAs from MAL23 (G1P), MAL38 (G1P), MAL80 (G9P), and MAL12 (G12P), all of which had long RNA patterns. A caveat in interpretation of the result of liquid-phase RNA–RNA hybridization is that
there often occurs aberrant migration of a hybrid band in which a lesser degree of sequence homology exists between the probe segment and its Adenylyl cyclase corresponding minus strand of the genomic RNA because in such a case the hybrid band becomes much less compact than the homoduplex band, resulting in slower migration upon gel electrophoresis. However, judging from the level of aberrant migration of the hybrid bands on the autoradiograph, the homology of the RIX4414 probe appeared higher with the genomic RNAs from the prototype Wa strain compared with Malawian long RNA pattern viruses (Fig. 2). By sharp contrast, the probe produced almost no hybrid band with genomic RNAs from MAL88 (G12P), MAL43 (G8P), MAL60 (G8P), MAL70 (G8P) and MAL66 (G2P), all of which had short RNA patterns (Fig. 2). When the MAL60 (G8P) probe was allowed to hybridize with the panel of dsRNAs from representative Malawian strains carrying various genotype combinations, the probe produced 7 or more hybrid bands with genomic RNAs from MAL88 (G12P), MAL43 (G8P), MAL70 (G8P), MAL66 (G2P) and KUN (G2P).