Rotavirus infection was diagnosed by commercial enzyme immunoassay from a bloody-mucoid stool sample of a 2-year-old boy treated for acute diarrhea overnight on 19–20 February 2005 at the emergency ward of the municipal hospital, in Zhanghua, Taiwan. Initial characterization of the VP7 and VP4 genes by nucleotide sequencing (Wu et al. 2009
) demonstrated that strain 04-94s51 belonged to G3P genotype, which is most closely related to canine rotaviruses. To further characterize this strain, we determined the sequence of the VP6 and NSP4 genes using primers and methods described by Wu et al. (2011
). Phylogenetic analyses were done by the maximum likelihood algorithm as implemented in MEGA5 (Tamura et al. 2011
) using an appropriate substitution model obtained for each gene by the AIC prediction method. Bootstrap analysis was performed using 500 psuedoreplicates.
In brief, the full-length open-reading frame (ORF) of the VP7 gene for strain 04-94s51 was determined and an 825
nt–long fragment was used to compare this strain to others by phylogenetic analysis. Strain 04-94s51 shared the highest nt similarity with canine RV52/96 and GC/KS05 G3 strains (98.5% and 97.6%
nt sequence identity) and with human PA260/97 strain (98.4%
nt sequence identity) but demonstrated moderate-to-low nt sequence identity with other G3 strains (91.2% or less). In the VP7 phylogeny, the 4 closely related strains comprised a single monophyletic group that was distantly related to other canine and canine-derived strains (e.g., feline Cat97 and human Ro1845 and HCR3A strains) (). In the VP4 gene sequence analysis, the variable region (822
nt in length) VP8* was compared to other related P strains (). The Taiwanese 04-94s51 strain showed the highest nt similarity to the canine RV198/95 strain (97.2%) and was closely related to other canine and canine-derived zoonotic human strains (range, 93.3–95.4%), but lower degrees of similarity were observed to ruminant and simian P rotavirus strains (range, 78.7–79.7%). The full ORF was determined for the VP6 gene of strain 04-94s51 and a 1225
nt–long fragment was used to determine relatedness to other strains (). The highest nt similarity was seen between 04-94s51 and canine strains RV52/96 (94.7%) and RV198/95 (94%), but the strain was only distantly related to other genotype I3 strains, such as the feline rotavirus-like human strain, AU-1 (85.7%), or the canine rotavirus-like human strains Ro1845 (85.5%) and HCR3A (85.1%). Phylogenetic analysis demonstrated that Taiwanese 04-94s51 strain comprises a single common cluster with the human strain T152, and the canine strains RV52/96 and RV198/95, representing a unique lineage within the I3 genotype of the VP6 gene that is divergent from other known feline, canine, and zoonotic human strains. The NSP4 gene of 04-94s51 shared the highest nt similarity along a 683
nt–long fragment to the canine RV52/96 strain (99.1%
nt identity) and the feline FRV348 strain (98.7%
nt identity) and was more closely related to some other genotype E3 feline strains (up to 91.5%) than to most of the known canine rotaviruses (86.8% or less). Phylogenetic analysis confirmed the distance-based estimation of relatedness among strains, demonstrating that the NSP4 gene of 04-94s51 forms a single common cluster with RV52/96 and FRV348 ().
Maximum-likelihood trees of rotavirus VP7, VP4, VP6, and NSP4 genes. Arrows indicate the G3P strain, 04-94s51, identified in Taiwan. Bootstrap values >60 are indicated.