Active Case Finding of Current Bornavirus Infections in Human Encephalitis Cases of Unknown Etiology, Germany, 2018-2020

Abstract

Human bornavirus encephalitis is a severe and often fatal infection caused by variegated squirrel bornavirus 1 (VSBV-1) and Borna disease virus 1 (BoDV-1). We conducted a prospective study of bornavirus etiology of encephalitis cases in Germany during 2018-2020 by using a serologic testing scheme applied along proposed graded case definitions for VSBV-1, BoDV-1, and unspecified bornavirus encephalitis. Of 103 encephalitis cases of unknown etiology, 4 bornavirus infections were detected serologically. One chronic case was caused by VSBV-1 after occupational-related contact of a person with exotic squirrels, and 3 acute cases were caused by BoDV-1 in virus-endemic areas. All 4 case-patients died. Bornavirus etiology could be confirmed by molecular methods. Serologic testing for these cases was virus specific, discriminatory, and a practical diagnostic option for living patients if no brain tissue samples are available. This testing should be guided by clinical and epidemiologic suspicions, such as residence in virus-endemic areas and animal exposure.

Keywords: BoDV-1; Germany; VSBV-1; active case finding; bornavirus; bornavirus disease virus 1; case definition; epidemiology; etiology; human encephalitis; infections; meningitis/encephalitis; serologic analysis; variegated squirrel bornavirus 1; viruses; zoonoses.

Figure 1

Serologic testing scheme for human bornavirus encephalitis, Germany, 2018–2020. Scheme was based on serologic screening and confirmatory assays and in conjunction with a case definition for variegated squirrel bornavirus 1 (VSBV-1) and Borna disease virus 1 (BoDV-1) encephalitis (Table 1) was diagnosed. Screening of serum samples and cerebrospinal fluid for bornavirus-reactive IgG was conducted by using an indirect immunofluorescence antibody test. A persistently BoDV-1–infected cell line was used with uninfected cells of the same cell line as controls (Vero cells or Crandell-Rees feline kidney cells). For confirmation of a positive IFAT screening result, a line blot with recombinant VSBV-1 and BoDV-1 phosphoprotein proteins was used in our study, but alternative assays, such as WB or ELISA with recombinant antigen(s) or antigen(s) derived from infected cells, might also be appropriate after sufficient validation. Adequate control serum samples from confirmed human VSBV-1 and BoDV-1 encephalitis cases and a pooled serum of 20 healthy blood donors were used for the IFAT and the line blot. IFAT, indirect immunofluorescence antibody test; IB, immunoblot; WB, Western blotting.

Figure 2

Germany showing locations of residences of human case-patients who had encephalitis and other conditions and were tested for bornavirus etiology, 2018–2020. Among 103 encephalitis cases with unknown etiology, 4 bornavirus cases were found: 1 chronic VSBV-1 infection in northern Germany (case 1) and 3 BoDV-1 infections in southern Germany (cases 2, 3, and 4). Encephalitis cases without a bornavirus etiology are indicated as green circles. Among 121 cases without a clinical history of encephalitis but for whom a bornavirus serologic analysis was requested, no bornavirus infections were detected (blue circles). Purple indicates regions known to be endemic for BoDV-1. BoDV-1, Borna disease virus 1; VSBV-1, variegated squirrel bornavirus 1.

Figure 3

Reactivity of samples from BoDV-1- and VSBV-1-infected patients with homologous and heterologous bornavirus P antigens, Germany. Bornavirus indirect immunofluorescence antibody test–positive serum and cerebrospinal fluid samples were tested by using the Euroimmun (https://www.euroimmun.com) line blot with BoDV-1 P and VSBV-1 P antigens (BoDV-1: 52 samples from 14 patients; VSBV-1: 3 samples from 2 patients). Samples originated from patients with laboratory-confirmed BoDV-1 or VSBV-1 infection (,–; this study). Results are indicated as arbitrary units. Dotted line indicates cutoff value of 16 as defined by the manufacturer. BoDV-1, Borna disease virus 1; P, phosphoprotein; VSBV-1, variegated squirrel bornavirus 1.

Figure 4

Phylogenetic analysis of BoDV-1 nucleotide sequences from virus-endemic areas, Germany. Shown are partial bornavirus sequences (nucleoprotein gene to phosphoprotein gene, 1,824 nt, representing genome positions 54–1877 of BoDV-1 reference genome U04608), including BoDV-1 sequences from animals and humans in virus-endemic regions in Germany, Austria, Switzerland, and Liechtenstein. BoDV-2 was used as an outgroup. Analysis was performed by using the neighbor-joining algorithm and the Jukes–Cantor distance model in Geneious Prime (https://www.geneious.com) and the tree was rooted for the VSBV-1 clade. Human sequences are indicated in black. Sequences of cases 1–4 included in this study are indicated in bold. Values at branches represent support in 1,000 bootstrap replicates. Only bootstrap values >70 at major branches are shown. Cluster designations, host, and geographic origin are indicated according to previously published studies (,,,,–23). Colors and numbers at right indicate clusters. Scale bar indicates nucleotide substitutions per site. AUT, Austria: UA, Upper Austria; ST, Styria. GER, Germany: BB, Brandenburg; BW, Baden-Wuerttemberg; BY, Bavaria; HE, Hesse; NI, Lower Saxony; RP, Rhineland-Palatinate; SN, Saxony; ST Saxony-Anhalt. LIE, Liechtenstein. SUI, Switzerland: GR, Grisons; SG, St. Gall.