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.