Sample information
Between April 2019 and September 2019, we collected and analyzed 39 samples from five different farms with calf diarrhea in Hebei, China, including 30 diarrheal fecal samples, 5 blood samples and 4 tissue samples (lung, liver, kidney, heart). Samples were screened for BNoV by nested PCR.
Nucleotide extraction and PCR detection of BNoV
Total RNA was extracted by TRIzol reagent following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using the PrimeScriptTM1st strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China) with an oligo dT primer. Nested PCR primers (F, AGTTAYTTTTCCTTYTAYGGBGA; R, AGTGTCTCTGTCAGTCATCTTCAT (Smiley,Hoet,Traven,Tsunemitsu, & Saif, 2003); and nF, GTCGACGGYCTKGTSTTCCT; nR, CACAGCGACAAATCATGAAA (Park et al., 2006)) targeting the RdRp gene were used with the following reaction conditions: 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 54°C for 30 s and 72°C for 40 s, with a final extension at 72°C for 10 min. The reaction generated a 326 bp PCR product. In addition, three other diarrhea-related enteric viruses, i.e., bovine viral diarrhea virus, bovine rotavirus, and bovine coronavirus, were detected using previously reported molecular methods for the evaluation of possible co-infection with BNoV.