Restriction fragment length polymorphism analysis of rotavirus VP7-encoding gene from humans and animals of Northeast India: a relative study of Indian and global isolates

Abstract

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic relationship between 67 (29 Indian, 38 global) rotavirus isolates of human, bovine and porcine neonates. The assay involved direct digestion of RT-PCR amplified VP7 cDNAs with three restriction enzymes (VspI, HaeIII, NlaIV) independently. Forty-eight RFLP patterns were identified for all 67 strains, and of these 20 patterns were associated with Indian isolates. A correlation between the restriction patterns and G type was apparent through deduction of enzyme restriction sites from known sequences. Major G serotypes (G1, G2, G6, G8) with a few mixed types could be differentiated where there was a positive assortment of intrinsic serotypes from multiple host origin, and certain single or combined enzyme profiles were highly dominant in the population. Significant genetic variations were established between global and Indian isolates and none of the RFLP patterns were shared between them. These data suggest that the Indian wild-type rotavirus population is distinguishable based on the VP7 gene, and co-circulation of distinct strains in different hosts is foremost, indicating the possible likelihood of inter-species transmission.

Keywords: Diversity; inter-species transmission; rotavirus; serotypes.

Fig. 1.

VP7 gene enzyme profiles of 29 Indian isolates after digestion with three restriction enzymes, VspI, HaeIII, and NlaIV. All products were analysed by agarose gel (2%) electrophoresis and visualized by staining with ethidium bromide (0·1 mg/ml). (a, b) VspI digestion profiles; (c, d) HaeIII digestion profiles; (e, f) NlaIV digestion profiles. Isolates of similar host origin are grouped together over the lanes. Lane M, Molecular size marker, 100-bp ladder. (a, c, e) Lane 1, IA-07; lane 2, IA-56; lane 3, IA-12; lane 4, IA-15; lane 5, IA-139; lane 6, IA-92; lane 7, IA-122; lane 8, IA-21; lane 9, IA-102; lane 10, IA-18, lane 11, IA-71; lane 12, IA-68; lane 13, IA-132; lane 14, IA-88, lane 15, IA-109. (b, d, f) Lane 16, IA-219; lane 17, IA-222; lane 18, IA-224; lane 19, IA-228; lane 20, IA-231; lane 21, IA-17; lane 22, IA-128; lane 23, IA-171; lane 24, IA-178; lane 25, IA-98; lane 26, IA-209; lane 27, IA-172; lane 28, IA-212; lane 29, IA-110.

Fig. 2.

Combined restriction fragment length polymorphism (RFLP) patterns of 29 Indian isolates. The combination of three enzyme profiles produced 20 RFLP patterns. Most pattern correspond to the host species undergoing single, dual or multiple infection. The patterns V₄H₁N₃ and V₄H₁N₆ correspond to inter-species infection. The details are further discussed in text. The x axis corresponds to the characteristic combined RFLP patterns. The y axis corresponds to the number of host organisms revealing the particular RFLP pattern.