Genetic diversity within human erythroviruses: identification of three genotypes

Abstract

B19 virus is a human virus belonging to the genus Erythrovirus: The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus Erythrovirus, and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3.

FIG.1.

Phylogenetic relationships among erythroviruses. (A) Phylogenetic relationships based on full-length genome sequences (4,339 bp). Phylogenetic analysis was performed by using the neighbor-joining algorithm based on the Kimura two-parameter distance estimation method (similar results were obtained by using the maximum-parsimony method). Significant bootstrap values (in percentages) are indicated for 10³ neighbor-joining/10³ maximum-parsimony replicates. In the left panel simian parvovirus sequences (rhesus macaque parvovirus [RMPV], pig-tailed macaque parvovirus [RTMPV], and long-tailed macaque parvovirus [LTMPV]) are included and the chipmunk parvovirus (ChPV) sequence is used as an outlier. The right panel shows the nucleotide distances between human erythroviruses only. (B) Phylogenetic relationships among human erythroviruses based on nucleotide (DNA) and amino acid (AA) distances for the NS1, VP1, and VP2 genes. Distances between sequences were analyzed by using the neighbor-joining algorithm in the Phylip package (based on the Kimura 2 parameter distance estimation method for nucleotides and Dayhoff PAM matrix for amino acids). The phylogenetic trees have topologies similar to those observed with full-length sequences.

FIG. 2.

Sequence alignment in the p6 promoter region (nucleotides 102 to 324 of the Pvbaua sequence). Major potential binding sites for transcription factors are boxed as described by Gareus et al. (16). Compared to the B19 virus p6 sequence, the D91.1, V9, and Lali sequences contain numerous substitutions, and deletions are observed in the fourth GC box located just upstream of the TATA box.

FIG. 3.

Phylogenetic relationships among B19 virus and V9-related viruses based on the NS1-VP1u sequences (994 bp). Phylogenetic analysis was performed by using the neighbor-joining algorithm based on the Kimura 2 parameter distance estimation method. Significant bootstrap values (in percentages) are indicated for 10³ neighbor-joining/10³ maximum-parsimony replicates. The human erythrovirus sequences are distributed into three distinct clusters corresponding to genotype 1 (prototype strain Pvbaua), genotype 2 (prototype strain Lali), and genotype 3 (prototype strain V9). For clarity, the names of the B19 virus sequences included in genotype 1 (Pvbaua, Pvb19nsvp, Pvb19×528, Pvb19×560, Pvb19×599, Pvbpro, HV, R43, R227, R308, CDC105, and CDC156) are omitted from the figure.

FIG. 4.

Amino acid alignment of the VP1u neutralizing epitope (aa 46 to 93 of the Pvbaua sequence). Numbers indicate genotypes as defined in Fig. 3. Compared to genotype 1 viruses (B19 virus strains), an N68S substitution is shared by genotype 2 (prototype strain Lali) and genotype 3 (prototype strain V9) viruses, and an additional N72D substitution is shared by genotype 2 viruses (arrowheads).