A total of 378 fecal samples were collected from cattle and buffalo calves up to 3 months old that were suffering from diarrhea. These animals belonged to organized and unorganized dairy farms in different ecoregions of India including temperate western Himalaya (Tarai and foothills of Himalaya, Uttarakhand state), tropical, semi-tropical, and semi-arid regions (Haryana, Uttar Pradesh, and Madhya Pradesh) of India. A 10% fecal suspension (w/v) was prepared with phosphate buffered saline (0.01 M, pH 7.4; Sigma, USA). Samples were centrifuged at 2,000 × g for 10 min to remove coarse particulate matter and the upper aqueous layer was filtered through a 0.22-µm pore filter (MDI, India) into a fresh tube. The suspension was stored at -20℃ until further use. Reference NCDV (G6P[1]) and UK (G6P[5]) cell culture-adapted bovine RVA strains were obtained from the National Research Centre on Equine (India). RNA extracted from these samples was used to optimize reverse-transcription (RT)-PCR and genotyping conditions.

Total RNA was extracted from 500 µL of fecal suspension using an equal volume of TriReagent-LS (Sigma, USA) following the manufacturer's instructions. RNA was dissolved in nuclease-free water (NFW) (Life Technologies, USA) in a final volume of 25 µL. RNA quantity and quality was assessed using a Nanodrop Spectrophotometer (ND-1000; Thermo Scientific, USA) and stored at -20℃ until further use.

The extracted viral RNA was analyzed by PAGE performed as previously described by Laemmli [18] by using 5% stacking and 10% resolving gels. Briefly, extracted RNA (200~500 ng) was mixed with an equal amount of 2× RNA loading buffer (Thermo Fisher Scientific, USA) and loaded into the wells of the gels. Electrophoresis was performed at 120 V in 1× Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3). The gel was subsequently stained with silver nitrate as previously described by Svensson et al. [29]. All the samples were initially screened with PAGE.

RT of the viral RNA was performed using a random hexamer primer (0.2 µg/µL; Thermo Fisher Scientific, USA). Initially, 50~100 ng of viral RNA, 0.5 µL random hexamer primer, and 2 µL dimethyl sulphoxide (DMSO) were combined in a thin-walled 0.5-mL PCR tube (GenAxy, India). The reaction mixture was incubated at 95℃ for 5 min to denature the dsRNA strands and immediately snap chilled on ice. Next, 4 µL of 5× RT buffer (Promega, USA), 2 µL of 10 mM dNTPs (Thermo Fisher Scientific, USA), 40 U of RNase inhibitor (Life Technologies, USA), and 200 U of Moloney murine leukemia virus reverse transcriptase (Promega, USA) were added and the mixture was incubated at 37℃ in a thermocycler for 1 h (HR-PCR-96G; Haier, China). The reverse transcriptase was denatured at 80℃ for 5 min at the end of the incubation step.

The first-round of PCR amplification of the VP7 gene was performed with 3 µL cDNA, 5 µL 10× PCR buffer, 2 mM MgCl₂, 2 µL DMSO, 1 µL dNTPs, 50 pmol of forward and reverse primers (Table 1), and 1.25 U of Taq DNA polymerase (Thermo Fisher Scientific, USA). The total reaction volume was brought to 50 µL with NFW. The first-round of amplification of full-length VP7 (1062 bp) consisted of an initial denaturation at 94℃ for 4 min followed by 35 cycles of 1 min at 94℃, 2 min at 46℃, and 2 min at 72℃, and final incubation at 72℃ for 10 min. A similar PCR protocol was performed to amplify the partial-length VP4 gene (864 bp) for the first-round with the exception that the annealing temperature was set at 48℃. The PCR products were separated by conventional agarose gel electrophoresis (1%, w/v) at 100 V for 1 h in 1× TAE buffer with 0.5 µg/mL ethidium bromide, viewed with a UV transilluminator, and documented using Syngene gel documentation system (Syngene, UK).

Multiplex nested PCR genotype determination was performed as per the method and primers used by Gouvea et al. [12] for G3 (aET3) and G8 (aAT8), Gouvea et al. [13] for G5 and G6, and Iturriza-Gómara et al. [16] for G10. Primers used for the first-round of amplification and subsequent nested PCR for genotyping both the VP4 and VP7 genes are presented in Table 1. Genotyping was performed using 3 µL of the amplicons (1 : 100 dilution) from the first-round of PCR amplification and the typing primers (G3, 5, 6, 8, and 10) with the reverse primer used for generating the first-round VP7 gene product. Amplicons from the first-round of PCR (1 : 100 dilution) and the typing primers (P[1], [5], and [11]) with the forward primer (Bov4Com5) used for generating the first-round VP4 gene product were used for P typing. A diagrammatic sketch with specific amplicon size for the G and P types is presented in Fig. 1. PCR cycling conditions for the second-round were same as those of the first-round. The amplicons were analyzed as described above.

Selected bovine RVA isolates (n = 3) with the G3 genotype from cattle in different geographical areas were amplified as described above. The amplified PCR products were excised from the gel and purified with a High Pure PCR Product Purification kit (Roche Diagnostics, Germany) with final elution in 20 µL of elution buffer. The eluted PCR products were quantified in Nanodrop Spectrophotometer and sequenced with an automated sequencer (ABI 3130, USA) at the Foot and Mouth Disease Typing Facility Lab, Project Directorate on Foot and Mouth Disease, Indian Veterinary Research Institute, Mukteswar (India). Sequence data for these samples were entered into the NCBI GenBank database under the following accession numbers: B-46 (HM235510), BR-133 (JF 720877), and 0970 (HQ 199897).

The sequence chromatogram was visualized with sequencing analysis software (ABI, USA). MegaBLAST (NCBI, USA) was used to analyze the predicted sequence within the non-redundant nucleotide database and confirm the presence of the gene specific for RVs. RotaC web-based RV classification software (Katholieke Universiteit Leuven, Belgium) was also used for sequence confirmation. RVA sequences from animals in different geographical locations within India and the rest of the world were downloaded from the NCBI nucleotide database. Sequence analysis was performed using the LaserGene software package (DNASTAR, USA). Percent identity and divergence of the nucleotide and predicted amino acid sequences were determined to calculate the percentage homology with other G3 RV sequences from different geographical locations. A phylogenetic tree was constructed using Mega 4.0 software [30].

PAGE analyses detected the RV genome in 33 out 378 diarrheic fecal samples from bovine calves that were up to 3 months old. All PAGE-positive RV samples showed a typical 4 : 2 : 3 : 2 "long" electropherogram migration pattern, characteristic of RVA. In the majority of the samples, genome segments 7, 8, and 9 co-migrated as a triplet. PAGE-positive samples were also confirmed by using RT-PCR targeting the group A-specific VP6 gene.

First-round amplification with generic primers of VP7 and VP4 genes produced full-length 1062-bp amplicons for the VP7 gene and partial-length 864-bp products for the VP4 gene in all 33 samples. G typing produced amplicons 373, 581, 884, and 396 bps in size, indicating the presence of G3, G6, G8, and G10 genotypes, respectively (Fig. 2A). None of the samples were found to have the G5 or G6 type RV. P typing of the samples produced amplicons 334, 453, and 661 bp in size, corresponding to P[11], P[1], and P[5] types, respectively (Fig. 2B). However, P[5] was not detected in any of the field isolates (Fig. 2B). A number of samples were confirmed to possess dual G types, either G3G8 or G3G10. The distribution of different combination of genotypes found throughout the study is summarized in Table 2. Typing primers (G&P) also confirmed the reference bovine RVA as G6P[1] for the NCDV strain and G6P[5] for the UK strain (Figs. 2A and B). G3 was found to be the main G type circulating among the ecoregions of India we examined between 2007 ~ 2010 with both single and/or dual infections (G3G8 or G3G10) observed (Fig. 2A). Overall, 13 of the 33 typed samples (39.4%) were identified as G3, and 20 (60.6%) were identified as mixed (G3G8 or G3G10) G types (Table 2). Additionally, 31 (93.9%) of the 33 P typed samples had the P[11] genotype and two (6.1%) exhibited dual P[1]P[11] types (Table 2). The majority of the mixed G types were G3G10 (55%, 11/20) following G3G8 (45%, 9/20) types.

The distribution of the G types showed that 52.9% were G3 and 47% were mixed G types. Among these, 35.3% were G3G8 (6/17) and 11.8% G3G10 (2/17) in the temperate region of western Himalaya while 25% of G3 and 75% mixed G types were found in the tropical, subtropical, and semi-arid regions. Among the mixed types, the most commonly detected G combination was G3G10 (55%). P types found in the temperate region of western Himalaya were mostly P[11] (94.1%) while 5.9% were the dual P type (P[1] P[11]). In the tropical, sub-tropical, and semi-arid regions (Table 2), 93.8% (15/16) were P[11] type and 11.1% (1/16) were the dual P type (P[1] P[11]).

The multiplex nested PCR typing results were further confirmed with sequencing, BLAST, and automated genotyping RotaC software analysis of selected isolates. As shown in Fig. 3, VP7 coding region sequence analysis of the bovine G3 isolates (B-46, 0970 and BR-133) showed that the predicted amino acid sequence identities with other bovine G3 RVA strains were between 90.5% (Bo/UK/1984 strain UKg9P GQ225779) and 98.8% (Bo/Ind/2006/RUBV3 EF200549). Identity was lower with bovine strains from the UK (90.5% to 92%) while two other bovine G3 strains isolates previously obtained from two different parts of India showed higher identities of 96.3% (J63 strain from central India) to 100% (RUBV3 strain from eastern India). Sequence identities of these three G3 isolates were between 89.9% and 95.1% with feline and canine G3 isolates, 93.3% and 94.5% with simian isolates, and 91.7% and 93.1% with equine isolates from Greece, Japan, and Germany. Equine G3 isolates from India had higher identities of 93.9% to 98.8%. All the G3 bovine isolates showed the lowest identity with porcine strains (82.8% to 91.4%). Sequence identities of these bovine strains with human G3 isolates from different parts of the world were between 89% and 94.5% (Fig. 3).

UK bovine G3 RVs showed a maximum of 183 variations at the nucleotide level corresponding to changes in the amino acid sequence at 26 positions compared to B-46, 0970, and BR-133 G3 strains. The RUBV3 strain from eastern India was more conserved as only one change was noticed at nucleotide level while there was no change at amino acid level. The J-63 G3 strain from central India showed 20 changes at the nucleotide level and eight changes at amino acid level (positions 22, 75, 146, 147, 211, 213, 221, and 242). Compared to the J-63 strain, the B-46 G3 strain isolated from the same location showed only 10 variations at the nucleotide level and four changes (59, 71, 76, and 317) at amino acid level.

Phylogenetic analysis of the B-46, 0970, and BR-133 G3 strains demonstrated that these three bovine strains clustered with RUBV3 and J-63 (bovine) and Erv-80 (equine). This finding indicated the genetic proximity of Indian bovine G3 strains to equine RVs (Fig. 3). On the other hand, other bovine RVA G3 strains from the UK (CP-1, PP-1, and UKg9P) clustered with human, canine, feline, and porcine G3 strains (Fig. 3).