We initially screened 6 samples; 1 sample, Bat/KE4852/07, obtained from an E. helvum bat trapped in Vihiga, Kenya, was positive for rotavirus by VP6 RT-PCR. Using primers annealing to noncoding regions of each segment and internal primers, we then obtained full-length ORF sequences for VP2, VP6, VP7, NSP2, NSP3, NSP4, and NSP5, except for VP1 and VP4, for which partial-length gene sequences were obtained (for the remainder of this report, we will refer to each ORF, from ATG to stop codon, as a gene). None of the sequences reported in this study were inferred from primer sequences. Sequences were not obtained for VP3 and NSP1, despite repeated attempts to obtain amplicons by using panels of rotavirus A–specific primer pairs. Nucleotide sequences for VP1, VP2, VP4, VP6, VP7, NSP2, NSP3, NSP4, and NSP5 were deposited in GenBank under accession nos. GU983672–GU983680. Genetic analyses of Bat/KE4852/07 indicated that 7 genes were unique and 2 were similar to described rotavirus genotypes. Results are summarized below and in .
Figure 1 Percentage nucleotide and deduced amino acid homologies of A) viral protein 7 (VP7), B) VP4, C) VP6, D) VP1, E) VP2, F) nonstructural protein 2 (NSP2), G) NSP3, H) NSP4, and I) NSP5 gene segments of bat rotavirus strain Bat/KE4852/07 from Kenya compared (more ...)
The putative VP7 gene of strain Bat/KE4852/07 was 981 bp and encoded a 326 aa protein. The nucleotide sequence of Bat/KE4852/07 VP7 showed low levels of identity to the 24 established G genotypes (range 55.9%–67.4%) (, panel A). The VP7 gene of Bat/KE4852/07 was classified into a novel VP7 genotype, G25, by the RCWG (10
). When compared with other mammalian rotavirus VP7 protein sequences, amino acid dissimilarity was >17% overall (, panel A) and exceeded 20% in antigenic regions A, B, and C (26
An 829-bp region of the VP4 gene of Bat/KE4852/07 strain was amplified and sequenced by using standard VP4 RT-PCR primers (18
). The sequence corresponded to bases 24–851 of strain US1205 VP4 (GenBank accession no. AF079356.) BLAST searches indicated that the bat rotavirus VP4 sequence was closely related to human P strains from Africa (6809/ARN) and eastern Asia (CAU214) and showed 99% nt and 98% aa identities (, panel B). Relatively low sequence identities were found when compared with P sequences of animal origin (81.6% nt and 85.2% aa identities) (, panel B).
The complete VP6 gene (1,194 bp), with a protein of 397 aa, of Bat/KE4852/07 strain was determined. Overall nucleotide identity with reference genotypes I1-I13 strains ranged from 68.1% to 80.6% (, panel C). These nucleotide identity values fell below the VP6 genotype cutoff value of 85% (10
), indicating that Bat/KE4852/07 strain belongs to a novel VP6 genotype designated I15 by the RCWG. Bat/KE4852/07 VP6 amino acid sequence shared 69%–92.7% identity with other rotavirus VP6 sequences (, panel C).
A fragment of the VP1 gene was obtained for Bat/KE4852/07. The sequenced region was 1,198 nt, which was one third the expected full length of the VP1 gene and corresponded to bases 252–1451 of rotavirus strain S2 VP1 sequence (GenBank accession no. DQ870485). Comparison of the partial VP1 nucleotide and amino acid sequences of Bat/KE4852/07 with those of other mammalian and avian rotavirus strains showed low levels of identity similar to those of other VP1 genotypes (<59% and <38%, respectively; , panel D).
Despite the uniqueness of the bat rotavirus VP1 sequence, BLAST search and alignment results with amino acid sequence displayed the highest degree of identity with rotavirus A VP1 sequences (48% conservation of similar amino acid residues) (). The Bat/KE4852/07 VP1 gene partial sequence includes the region encoding the 19 residue polymerase F motif described (27
), but within this domain, the percent amino acid identity with other group A rotaviruses is <37% (53% similarity), and none of the 3 arginine residues (RR452, R457, R460) of a predicted functional role in rotaviruses are present in Bat/KE4852/07 VP1 (). The partial gene nucleotide sequence identity was well below the cutoff value of 83% that has been used to classify VP1 genotypes. The amino acid sequence divergence (>62%) exceeds that observed when group A and C rotavirus VP1 proteins are compared (>53%) (27
). Although the minimal sequence length set by the RCWG for new candidate genotypes does not permit official designation of a new genotype derived from the partial gene sequence of Bat/KE4852/07, we showed that this bat rotavirus contains a highly divergent VP1 gene provisionally designated genotype R8.
Figure 2 Alignment of viral protein 1 (VP1) amino acid sequence of bat rotavirus strain KE4852/07 from Kenya with cognate VP1 sequence of reference rotavirus A strain S2. The consensus line shows conserved amino acid residues and similar residues (indicated by (more ...)
The VP2 gene of strain Bat/KE4852/07 was 2,712 bp and encoded a deduced protein of 903 aa. The sequence of the VP2 gene segment of Bat/KE4852/07 strain was longer than those of most rotavirus strains because of several nucleotide insertions within the ORF near the 5′ end of the gene. As a result of these insertions, the predicted VP2 protein was 21 aa longer than that of most mammalian and avian rotaviruses. Nucleotide sequence comparisons indicated that the VP2 sequence of Bat/KE4852/07 was distantly related to all 6 established VP2 genotypes; identities ranged from 68.3% to 80.6% (, panel E). These values were below the cutoff value of 84% nt sequence identity that has been used to classify VP2 genotypes (10
). Thus, Bat/KE4852/07 was assigned into novel genotype C8 by the RCWG. VP2 amino acid identities between Bat/KE4852/07 and other rotaviruses were <94% (, panel E).
The NSP2 gene of strain Bat/KE4852/07 was 954 bp and encoded a polypeptide of 317 aa. Bat/KE4852/07 NSP2 gene sequence shared <78% identity with other rotavirus NSP2 strains (, panel F). This value was below the cutoff value of 85% nt sequence identity that has been used to classify NSP2 genotypes (10
). The RCWG assigned novel genotype N8 to the NSP2 gene of strain Bat/KE4852/07.
The NSP3 gene of strain Bat/KE4852/07 was 936 bp and encoded a deduced protein of 311 aa. Bat/KE4852/07 strain NSP3 nucleotide and amino acid sequences exhibited <74% identity with other NSP3 genotypes (, panel G). Because this value is below the cutoff value of 85% nt identity used to differentiate T genotypes (10
), this gene was assigned to a novel NSP3 genotype (T11) by the RCWG.
The putative NSP4 gene was 528 bp and encoded a polypeptide of 175 aa. Analyses of nucleotide and deduced amino acid sequences indicated that the Bat/KE4852/07 NSP4 gene shared >99% nt and >98% aa identities with human (I321, B1711 and DS-1), ovine (OVR762), simian (PTRV), and bovine (WC3) rotavirus strains of the NSP4 genotype E2 (, panel H).
The NSP5 gene of strain Bat/KE4852/07 was 630 bp and encoded a 209-aa polypeptide. This gene was longer than NSP5 genes of most rotavirus strains because of several nucleotide insertions within the ORF. Thus, the putative NSP5 is 11 aa longer than that of most mammalian and avian rotaviruses. The NSP5 gene of strain Bat/KE4852/07 shared <85% nt identity with other NSP5 genotypes (10
) (, panel I) and was assigned to a novel NSP5 genotype (H10). Within the NSP5 gene of strain Bat/KE4852/07, there was a second ORF at nucleotide positions 80–346 (+1 reading frame) which corresponded to a putative NSP6 gene.
Phylogenetic Relationships of Bat/KE/4852/07 Genes to other Rotavirus Strains
Bayesian phylogenetic analyses of Bat/KE4852/07 and other rotavirus strain sequences indicated that 2 genes (VP4 and NSP4) were closely related to described mammalian rotaviruses; that 6 bat rotavirus genes (VP2, VP6, VP7, NSP2, NSP3, NSP5) were more distantly related and represented novel genotypes; and that the VP1 gene was distant from all known cognate genes of mammalian and avian rotaviruses (, 4). In the phylogeny estimated from VP7 nucleotide sequences, Bat/KE4852/07 occupied a well-supported (posterior probability 0.94) terminal group with G9 rotavirus strain t203 (AY003871), although the bat strain was connected by a long branch to the terminal node (, panel E). Bayesian analysis of VP7 amino acid sequences yielded the same result.
Figure 3 Phylograms indicating genetic relationships of partial or complete nucleotide sequences of A) viral protein 1 (VP1) partial, B) VP2, C) VP4 partial, D) VP6, and E) VP7 of bat rotavirus strain Bat/KE4852/07 (boldface) from Kenya with representatives of (more ...)
The VP6 phylogenetic estimate grouped Bat/KE4852/07 with the I9 genotype TUCH strain (EF583013) (posterior probability 0.81) and a long terminal branch (, panel D). Phylogenetic analysis of VP2 sequences resulted in well-resolved phylogeny with Bat/KE4852/07 and occupied an intermediate lineage between C1 genotype rotaviruses and a clade containing C2, C3, and C5 viruses (, panel B). Analysis of NSP3 sequences indicated weak support (posterior probability 0.57) for monophyly of the bat rotavirus with T1 and T6 genotype viruses, and Bat/KE4852/07 was separated from these other mammalian viruses by a relatively long terminal branch (, panel B). Bayesian analysis of NSP5 sequences indicated strong support (posterior probability 1.0) for monophyly of the bat rotavirus with mammalian rotaviruses in genogroups H1, H2, H3, and H5. However, intergenogroup relationships within this clade were not resolved (, panel D).
Figure 4 Phylograms indicating genetic relationships of partial or complete nucleotide sequences of A) nonstructural protein 2 (NSP2), B) NSP3, C) NSP4, and D) NSP5 of bat rotavirus strain Bat/KE4852/07 (boldface) from Kenya with representatives of known human (more ...)
Bayesian analysis of partial VP4 sequences yielded a phylogenetic estimate that places the bat rotavirus strain within a clade containing all genotype P viruses (posterior probability 1.0) and occupying a lineage shared with human P strains 6809/ARN and CAU214 (, panel C). The phylogenetic estimate obtained by using NSP4 gene nucleotide data supported the monophyletic origin of Bat/KE4852/07 with human strain I321 (AF165066 genotype E2), bovine strain WC3 (AY050273, genotype E2), and simian strain PTRV (FJ422140, genotype E2) (, panel C).
For partial VP1 sequences, Bayesian analyses of nucleotide and amino acid data did not produce well-resolved phylogenies. However, in each tree the longest terminal branch was between the bat rotavirus and the ancestral node (, panel A). Genetic distance separating Bat/KE4852/07 from the other rotaviruses was longer than the distance between avian rotaviruses (genotypes R4 and R6) and mammalian rotaviruses. For NSP2, the bat rotavirus gene occupied an intermediate position between avian rotaviruses (genotypes N4 and N6) and mammalian rotaviruses (genotypes N1, N2, N3, and N5) and showed maximum posterior probability support (, panel A).
Additional Screening Results
After characterization of Bat/KE4852/07, we processed an additional 39 E. helvum bat fecal swab samples from Kenya and screened them for rotaviruses by using VP6 RT-PCR. Three additional samples (Bat/KE5096/07, Bat/KE5105/07, and Bat/K5175/07) were positive for rotavirus. These 3 samples were obtained in Maseno, Kenya, which is ≈20 km from Vihiga. Given the population dynamics and migratory patterns of this species, bats from both roosts likely interact, at least during certain times of the year. VP6 sequences for Bat/KE5096/07, Bat/KE5105/07, and Bat/K5175/07 samples were 100% identical to VP6 sequence of Bat/KE4852/07. In an attempt to obtain a complete genomic sequence for the bat rotavirus, we will analyze this virus by using sequence-independent deep sequencing.