By convenience sampling, one hundred and twenty (120) fecal samples were aseptically collected from diarrheic calves aged less than 1 year, by herd to herd visits of settled and migratory cattle herds in Ondo and Ekiti states, Nigeria between September 2014 and February 2015. Eighty samples (80) were from males while 40 were from females.

The fecal samples were immediately screened for the presence of group A rotavirus or transported to the laboratory on ice and stored at −20˚C until tested. The fecal samples were tested for Rotaviruses using the Certest EIA kit according to the manufacturer’s instructions and as previously reported [11].

For each of the 15 samples that were positive for rotaviruses, 10% (v/v) fecal suspensions were prepared in sterile Phosphate Buffered Saline, PBS (pH 7.2) after vigorous mixing and clarified by centrifugation at 3000 g for 20 min at 4˚C [12]. The resultant supernatants were harvested, divided into aliquots and stored at −80˚C until use for the downstream molecular assays for group A rotaviruses at the Department of Virology, University of Ibadan, Nigeria.

For each aliquot of the 15 rotavirus positive samples, rotavirus dsRNA genome was extracted from the 10% stool suspension using the Total RNA Purification kit (Jena Bioscience GmbH, Jena Germany) according to the manufacturer’ instructions.

First round One-Step Reverse Transcription and Amplification of the dsRNA genome targeting the full length (1062 bp) VP7 gene segment 9 encoding the VP7 Glycoprotein was performed using sBeg9 and RVG9 primers (Eurofins, Germany) with the invitrogen One-Step RT-PCR reagent kit (Invitrogen Life Technologies, Carlsbad USA) following the manufacturer’s instructions and a previous method with minor modifications at the cycling conditions in Table 1 [13]. Synthesized dsDNA amplicons of the target sequence were then utilized as template in the second round semi-nested PCR for genotyping the detected rotaviruses.

The VP7 G-Typing assays were conducted on each of the synthesized first round amplicons of the detected rotaviruses using a consensus anchor primer RVG9 along with type specific cocktail of forward primers (Eurofins, Germany) for G1 (aBT1), G2 (aCT2), G3 (aET3), G4 (aDT4), G5 (aFT5), G6 (aDT6), G8 (aAT8), G9 (aFT9), G10 (mG10), G11 (aBT11), G12 (G12) genotypes as previously described [13] [14] [15]. Semi-nested PCR reaction (Table 2) was conducted using the 5X Red Load Taq Master High Yield PCR Mix (master mix of thermostable DNA polymerase, dATP, dCTP, dGTP, dTTP, KCl, MgCl₂, red dye, gel loading buffer and stabilizers) (Jena Bioscience GmbH, Jena Germany) following the cycling conditions in Table 1.

First round One-Step Reverse Transcription and Amplification of the dsRNA genome targeting the VP4 gene segment (876 bp) was performed using Con3 and Con2 primers with the invitrogen One-Step RT-PCR reagent kit (Invitrogen Life Technologies, Carlsbad, USA) following the manufacturer’s instructions and a previous method with minor modifications, at the cycling conditions in Table 1 [16]. Synthesized dsDNA amplicons of the target sequence were then utilized as template in the second round semi-nested PCR for genotyping the detected rotaviruses.

The VP4 P-Typing assays were conducted on each of the synthesized first round amplicons of the detected rotaviruses using a consensus anchor primer Con3 along with type specific cocktail of primers (Eurofins, Germany) as previously described [15] [17]. Assays were performed for the P-genotype of each of the isolated first round amplicons, using a cocktail of the primers set (specific for genotypes P[4], P[6], P[8], P[9], P[10], P[11] and P[14]). Semi-nested PCR was conducted using the 5X Red Load Taq Master High Yield PCR Mix (master mix of thermostable DNA polymerase, dATP, dCTP, dGTP, dTTP, KCl, MgCl₂, red dye, gel loading buffer and stabilizers) (Jena Bioscience GmbH, Jena Germany) following the cycling conditions in Table 1.

The second round multiplex PCR products were resolved by gel electrophoresis at 120 V for 60 mins in 1.5% Agarose gels containing 5 µl of 0.5 µg Ethidium bromide/ml along with a 100 bp DNA Molecular weight Standard (Jena Bioscience GmbH, Jena Germany) using freshly prepared 1X Tris Boric Ethylene Diamine

Tetra Acetic Acid (TBE) for agarose preparation and running buffer, in Owl® Gel Tank powered by a BIORAD® PowerPac. The resultant bands were visualized under ultraviolet light with the BIORAD® GelDoc system and documented. The specific G and P-genotypes were determined by the electrophoretic mobility bands and sizes (bp) of the amplicons in comparison to the molecular weight DNA Ladder [15].

Representative amplicons of the identified genotypes comprising the G1, G3, G9, G10, G12, and VP4 P[6] strains were randomly selected for nucleotide sequencing and investigation of their evolutionary relationships to those of other strains circulating in different regions of the world. The full length VP7 genes (1062 bp) and the partial VP4 genes (876 bp) of strains that could not be typed by multiplex semi-nested PCR were also selected for nucleotide sequencing. The amplicons were shipped to Macrogen Inc, Seoul, South Korea, for purification and sequencing in the forward direction with the respective specific genotyping primers stated above. The generated query sequences were extracted using the fasta format and edited using MEGA 6 software. Each query sequence was queried to the GeneBank Database by BLAST to retrieve reference sequences of the rotavirus genotype and aligned using the CLUSTALW program in MEGA 6 software. Pairwise distances of the sequences were estimated. The confidence values of the internal nodes in the phylogenetic trees were calculated by performing 1000 bootstrap replicates of the sequence alignment datasets, and the phylograms were constructed using the neighbor-joining method with MEGA version 6 [18].

Fifteen fecal samples tested positive for rotavirus antigens from the 120 samples that were screened. This represents a total prevalence of 12.5%. Representative results of viral assay of the fecal samples are presented in Figure 1(a) and Figure 1(b). Appearance of red band and green band at the test and control portions of the result window indicated positive test, while the green band alone indicated absence of rotavirus antigen. Invalid result was indicated by absence of green control band or both the control band and the test band. Intensity of the red band is directly proportional to the virus concentration. Rotavirus was recovered from male calves (10/80: 12.5%) at similar rates in the female calves (5/40: 12.5%) and no statistically significant association (χ² = 0.00, p = 1) existed between rotavirus infection and gender of the calves (Table 3).

From the 10 rotavirus positive stool samples that were submitted to genotyping assays, 14 isolates comprising 6 strains were identified (Table 4). The gel documentation of some of the electrophoretic assays for the VP7 genotyping are presented in Figure 2 and Figure 3 respectively. The full length VP7 genes (1062 bp) of strains that could not be typed by multiplex semi-nested PCR were determined by nucleotide sequencing. G-genotypes comprising G1, G5, G9, G10 and G12 were identified of which the most prevalent types were G1. The only identified P genotypes was P[6]. Multiple genotypes co-infection by G1G5 was observed in sample EK21, and G1G10 in samples OD44 and OD72 (Figure 3). The Genotype

BoRVA = Bovine group A Rotavirus. Chi square equals 0.00 with 1 degree of freedom. The p-value equals 1. No statistically significant association existed between rotavirus infection and gender of the calves.

identities of the rotavirus isolates from the positive stool samples are presented in Table 4. By genotype combinations, strain G1P[6] was the predominant strain identified, while G5P[6], G9P[6], and G10P[6] occurred at the same frequency.

The bovine RVA G1 isolates showed close identity to a Nigerian human isolate detected in year 2014 while the other G1G10 mixed genotype was similar to an India isolate. The G10 genotype had 85% nucleotide similarity to a human G10 isolate detected in Honduras in year 2011 to which it clustered. The G12 isolate in this study is closely related to G12 strains identified in India in year 2009 (Figure 4).