Novel B19-Like Parvovirus in the Brain of a Harbor Seal

<div><p>Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (<i>Phoca vitulina</i>) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family <i>Anelloviridae</i> were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus <i>Erythrovirus</i>, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and <i>in vitro</i> replication. Using <i>in situ</i> hybridization, we showed for the first time that a parvovirus of the genus <i>Erythrovirus</i> was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus <i>Erythrovirus</i> can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection.</p> </div

Detection of Seal parvovirus DNA by ISH.

<p>Pictures of H&E stained tissues of seal 12-410 of the spleen (A), the parenchyma of the cerebrum with a blood vessel (B), the Virchow-Robin space of the cerebellum (C) and the parenchyma of the cerebellum adjacent to the meninges (D). Red dots that indicate the presence of Seal parvovirus DNA were detected in the red pulp of the spleen (E, I), blood vessels in the brain parenchyma of the cerebrum (F, J), the Virchow-Robin space (G, K) and the brain parenchyma adjacent to the meninges of the cerebellum (H, L). A, B, C, D, E, F, G, K: original magnification 200x. I, J, K, L: original magnification 1000x.</p

Replication of Seal parvovirus <i>in</i><i>vitro</i>.

<p>Bone marrow cells of seal were incubated with (open symbols) or without (closed symbols) canine erythropoietin and inoculated with either PBS (diamond) or 2µl (circles) or 20µl (squares) of brain tissue homogenate from the parvovirus positive seal. At various time points after inoculations, samples were collected for counting of live cells by FACSanalysis (A) or real time-PCR using primers and probe specific for the Seal parvovirus (B). The detection limit of the assay was indicated with ND (not detectable).</p

Genome organization and phylogenetic analysis of Seal anelloviruses 2 and 3.

<p>A, B Genome organization of the two novel anelloviruses, Seal anellovirus 2 (A) and Seal anellovirus 3 (B). The location of the three main ORFs was indicated (black line). C. Phylogenetic neighbor-joining tree with <i>p</i>-distance and 1,000 bootstrap replicates of the deduced amino acid sequences of the ORF1 genes of representative viruses of the family <i>Anelloviridae</i>. Genbank accession numbers: HsTTMDV1 (Homo sapiens torque teno midi virus 1): NC_009225, HsTTMDV2 (Homo sapiens torque teno midi virus 2): NC_014093, HsTTMV1 (Homo sapiens torque teno mini virus 1): NC_014097, HsTTMV2 (Homo sapiens torque teno mini virus 2): NC_014086, PtTTV14 (Pan troglodytes torque teno virus 14): NC_014077, HsTTV1 (Homo sapiens torque teno virus 1): NC_007013, HSTTV10 (Homo sapiens torque teno virus 10): GU797360, CfTTV10 (Canis familiaris torque teno virus 10): NC_014071, TbTTV14 (Tupaia belangeri chinensis torque teno virus 14): AB057358, SsTTV1 (Sus sucrofa torque teno virus 1): AY823990, SeAV2 (Seal anellovirus 2): KF373760 , SeAV3 (Seal anellovirus 3): KF373758, SeAv TFFN (Seal anellovirus TFFN/USA/2006): NC_015212, ZcAV (Zalophus californianus torque tenovirus): FJ459582, FcTTV4 (Felis catus torque teno virus 4): AB076003, PRA4 (Felis catus anellovirus PRA 4): EF538878, PRA1 (Felis catus anellovirus PRA1): EF538877.</p

Genome organization and phylogenetic analysis of Seal parvovirus.

<div><p>A. Genome organization of Seal parvovirus. Indicated were the location of the major ORFs (grey) and the location of the start- and stopcodons on the nucleotide level counted from the 5’ end of the partial seal parvovirus genome. B. Phylogenetic neighbor-joining tree with <i>p</i>-distance and 1,000 bootstrap replicates of the deduced amino acid sequences of the VP2 genes of various viruses of the subfamily <i>Parvovirinae</i>. Genbank accession numbers: Canine parvovirus 2a: JQ996152, Porcine parvovirus Tai’an: FJ853421, Mouse parvovirus 2: NC_008186, Fox parvovirus: KC692368, AMD (Aleutian Mink disease) parvovirus: GU183264, Gray fox amdovirus: JN202450, Bufavirus-2 BF 39: JX027297, Human parvovirus 4: AY622943, Swine parvovirus H-1: AB076669, Bovine parvovirus 3: AF406967.</p> <p>Seal parvovirus: KF373759, Chipmunk parvovirus: GQ200736, Pig tailed macaque parvovirus: AF221123, Rhesus macaque parvovirus: AF221122, Simian parvovirus: U26342, Human parvovirus B19: NC_000883, Muscovy duck parvovirus: NC_006147, Adeno-associated virus-2: NC_001401, Porcine bocavirus 5: JN831651, Canine minute virus SH1: FJ899734, Human bocavirus 3: HM132056.</p></div

Evaluation of the presence of lesions by MRI and histology.

<p>A.Transverse T2-weighted MR image of the brain of seal 12-410. No gross abnormalities were detected in the brain parenchyma (the subdural hypointense areas are compatible with air due to post-mortem preparation). B, C, D. Perivascular cuffing with inflammatory cells distending the Virchow-Robin space in the brain parenchyma (B, C) and in the meninges (D). The infiltrates consisted mainly of mononuclear cells. E. Infiltrates of neutrophils and mononuclear cells in the liver parenchyma. F. Megakaryocytes and rubricytes in the spleen associated with extramedullary hematopoiesis. H&E stained slides, original magnification B, C, D: 200x; E, F: 400x.</p