Molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in China

ALFRED, Niyokwishimira; LIU, Huan; LI, Mu Lan; HONG, Shao Feng; TANG, Hai Bo; WEI, Zu Zhang; CHEN, Ying; LI, Fa Kai; ZHONG, Yi Zhi; HUANG, Wei Jian

numbers KJ476832-KJ476856. Phylogenetic and genomic analysis: To identify strains of astroviruses circulating among cattle and water buffalo populations in Guangxi province, phylogenetic tree analysis was performed based on both partial and full-length ORF2 sequences (1). Phylogenetic analysis of partial ORF2 sequences showed that three isolates (BAstGX S7-7, BAstGX S12-7 and BAstGX S17-7) from Dairy Farm A clustered together with one isolate (BAstGX J25) from Dairy Farm B forming a subgroup, which had a high similarity with BAstV-B76-2/HK and BAstV-B18/ HK isolated from cattle in Hong Kong. Five of six isolates from water buffalo (Bu-fAstGX M52, BufAstGX M53, BufAstGX M54, BufAstGX M541 and BufAstGX M552) clustered together appeared to form a new independent subgroup. However, one water buffalo isolate (BufAstGX 141) formed a new subgroup and appeared to be very closely related to the CcAstV-1/ DNK/2010 isolate from a roe deer in Europe [17] , suggesting that the Guangxi isolates from water buffalo could be classified into two subgroups. Seven of eight isolates (BAstGX J7, BAstGX J8, BAstGX J22, BAstGX J23, BAstGX J27, BAstGX J251 and BAstGX 252) from Dairy Farm B formed a new subgroup, while one isolate (BAstGX G1) from Dairy Farm C formed a new subgroup on its own from other cattle isolates. Regardless of origin, the isolates from cattle were divided into three subgroups, while those from water buffalo were divided into two subgroups. However, five subgroups of the Guangxi cattle and water buffalo astrovirus isolates belonged to one genogroup previously composed of bovine and roe deer astroviruses (BAstv-B76-2/HK, BAstV-B18/ HK, CcAstV-1/DNK/2010 and CcAstV-2/DNK/2010) in the genus Mamastrovirus, genotype MAstV33 (Fig. 1a) . To confirm this classification, we next constructed another phylogenetic tree based on the seven predicted full-length ORF2 sequences deduced from amino acid sequences (five from cattle and two from water buffalo) obtained in this study together with other full-length ORF2 sequences of other mammalian astroviruses retrieved from the GenBank database. The phylogenetic relationships within the complete ORF2 sequences of different mammalian astroviruses were addressed in reference to the taxonomic proposals for astrovirus classification criteria as described by the Astroviridae Study Group in 2010, and each genotype was represented at least by one prototype strain. The obtained results confirmed the classification under the genus Mamastrovirus, genotype MAstV33, which, thus far, includes only cattle and roe deer astroviruses (Fig. 1b) . Both the partial and full length ORF2 sequences of water buffalo isolates constituted new subgroups in the clade of the genotype MAstV33. The above results classified water buffalo as new host species of the genus Mamastrovirus, genotype MAstV33 ( Fig. 1a and 1b) . Analysis of the ORF1b/ORF2 junction identified a highly conserved 17-nt sequence motif (5′-cgctccttgcctaaaat-3′) upstream of ORF2 within the ORF1b/ORF2 region (Fig. 2a) . The ORF1b/ORF2 overlapping region contained 56 nt that overlapped between the ORF2 start codon and the ORF1b stop codon, with the exception of BAstV NeuroS1 (a divergent bovine astrovirus). The most 3′-end of ORF2 of the bovine-related astroviruses was highly conserved by 42 nt (Fig. 2b) , while this region has been reported as conserved by only 19 nt in human astroviruses [12] .