Figure 1

Figure 1. Distribution of rotavirus samples and their G/P genotypes in Hokkaido Prefecture (center map), Japan, 2014. The 4 locations from which the fecal samples were collected are shown on the map. Four...

During March–July 2014, we obtained rotavirus-positive fecal samples from 165 children in Hokkaido with acute gastroenteritis. The children were receiving care as inpatients or outpatients at 1 of 6 medical facilities (4 hospitals and 2 clinics) in the cities of Sapporo, Tomakomai, Muroran, and Urakawa (Figure 1).

For each fecal sample, we prepared a 10% fecal suspension, from which we extracted viral RNA. We performed reverse transcription PCR on the RNA by using the SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA); PrimeSTAR GXL DNA polymerase (Takara, Shiga, Japan); and previously described primers (3,4). We used the BigDye Terminator v.3.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA) to sequence PCR amplicons. For some of the rotavirus samples, next-generation sequencing was performed at the National Institute of Infectious Diseases in Tokyo, Japan, as described previously (5). Sequences of the rotaviruses used in this study were submitted to the DDBJ under accession numbers LC102884–LC103134 and LC105000–LC105532.

We successfully determined G and P genotypes for 148 of the 165 rotavirus samples by using the RotaC rotavirus genotyping tool (http://rotac.regatools.be/). The most common genotype was G8P[8], which was identified in 58 samples (39.2%), followed by G1P[8] (25.7%), G9P[8] (20.3%), and G2P[4] (12.8%) (Table 1; Technical Appendix).

We obtained clinical data for all 84 patients who sought care at the hospital or clinic in Tomakomai. Demographic and clinical characteristics (e.g., age, sex, history of rotavirus vaccination, duration of fever, and duration and frequency of diarrhea and vomiting) were not substantially different between 42 patients with G8P[8] rotavirus infection and 42 patients with non-G8P[8] rotavirus infection. The proportion of patients admitted to hospitals was also similar in the 2 groups (Table 1).

We selected 15 G8P[8] strains for whole-genome analysis. All strains had the same genotype constellation, G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, indicating a genomic backbone of the DS-1 genotype constellation. The genomes of these G8P[8] strains shared >99.6% nt identity with each other (Table 2; Technical Appendix). All 11 genome segments of strain To14-0 (the representative G8P[8] strain in this study) exhibited the highest nucleotide identity to human G8P[8] strains isolated in Southeast Asia in 2014 (represented by strain RVN1149 from Vietnam [>99.4% nt identity] and NP-130 from Thailand [>99.5% nt identity]) (6,7) (Table 2). This finding suggests that the strains share a common G8P[8] origin.

Figure 2

Figure 2. Phylogenetic analysis of the viral protein 7 gene of G8 rotavirus strains used in a study of the clinical and molecular features of a G8P[8] rotavirus outbreak strain, Hokkaido Prefecture, Japan,...

The VP7 gene of rotavirus strain To14-0 shared the highest nucleotide identity with the VP7 genes of human G8P[8] strains from Southeast Asia, including strains RVN1149 and NP-130 (99.4% and 99.7% identity, respectively), and it shared slightly lower identity to the VP7 gene of human strain 04-97s379 (97.8%) from Taiwan, which is speculated to be of bovine origin (8) (Figure 2). The VP7 genes of other G8 strains isolated in Japan were more distantly related to the To14-0 VP7 gene (e.g., human AU109 and bovine strains shared 89.5% and 81.9%–85.1%, respectively, with To14-0) (9). The VP7 genes of the human G8 strains prevailing in Africa were also distantly related (<90% nt identity) to the VP7 gene of To14-0.

Among the 11 To14-0 genome segments, 8 (VP2–VP4, VP6, nonstructural protein [NSP] 1–3, and NSP5) were highly similar to those of the human DS-1–like P[8] strains that have been isolated in Asia since 2012 (e.g., SKT-109, NT004, and LS-04) (10–12), including the strains isolated in this study (e.g., To14-41) (Table 2; Technical Appendix, panels B–H, J). In addition, the VP6 and NSP5 genes of the strains isolated in this study were also highly similar to those of human G2P[4] strains circulating in South Korea (strain CAU15-11) and Thailand (strain NP-M51) (12).

In contrast, the VP1 and NSP4 genes of To14-0 were only distantly related to those of the DS-1–like P[8] strains isolated in Asia (e.g., SKT-109, NT004, and LS-04), including the strains isolated in this study (e.g., To14-41) (Table 2; Technical Appendix Figure, panels A and I). The To14-0 VP1 gene shared high nucleotide identity with the VP1 genes of human G10P[14] strain PR457 from Italy (98.1%), which are probably the result of independent zoonotic transmissions (13). The To14-0 NSP4 gene shared high nucleotide identity with the NSP4 genes of human strains BSGH38 from India (96.7%) and the caprine G6P[1] strain GO34 from Bangladesh (96.0%) (14).

The clinical characteristics recorded for patients infected with G8P[8] rotaviruses and those infected with non-G8P[8] rotaviruses did not differ (Table 1). Our findings suggest that the severity of gastroenteritis caused by newly emerging G8P[8] rotaviruses could possibly be attenuated by 1) the existence of VP7/VP4 genotype cross-reactive (heterotypic) protective responses; 2) protective immunity associated with other segments, such as VP6 and NSP4 (3,15); or 3) both of these factors combined.

The VP7 genes of the human G8P[8] strains isolated in this study and in Southeast Asia appear to have a close relationship with bovine strains from Asia but not from Japan, and the VP7 gene of human G8 or bovine G8 strains previously isolated in Japan are distantly related to them. Therefore, the VP7 genes in the G8P[8] strains from this study may have originated from a bovine strain from Asia. As with the VP7 genes, the VP1 and NSP4 genes are also assumed to have been derived from artiodactyl strains.

Eight genome segments (VP2–VP4, VP6, NSP1–NSP3, and NSP5) of the human G8P[8] strains isolated in this study and from Southeast Asia are closely related to those of the DS-1–like P[8] strains that have emerged and spread in Japan and other countries of Asia since 2012 (Table 2). Therefore, these 8 genome segments of the G8P[8] strains from this study may be derived from the DS-1–like P[8] strains in Asia.

For the reasons we have stated, the G8P[8] strains isolated in this study were speculated to be formed outside of Japan by multiple reassortment events between the DS-1–like P[8] strains and bovine strains in Asia. The resulting strain was probably recently introduced into Japan.

The predominance of novel DS-1–like G8P[8] strains noted in this study indicates that these strains are sufficiently adapted to humans to sustain human-to-human transmission in an industrialized country. This finding suggests that these G8P[8] rotavirus strains could spread to other regions in the near future. Continuing surveillance is required to monitor the circulating wild-type strains, and rotavirus genotype constellations and clinical information must be analyzed to understand rotavirus virulence in humans.