First isolation, in-vivo and genomic characterization of zoonotic variegated squirrel Bornavirus 1 (VSBV-1) isolates

Abstract

The variegated squirrel bornavirus 1 (VSBV-1), a member of the family Bornaviridae, was discovered in 2015 in a series of lethal human infections. Screening approaches revealed kept exotic squirrels as the putative source of infection. Infectious virus was successfully isolated by co-cultivation of infected primary squirrel cells with permanent cell lines. For in vivo characterization, neonatal and adult Lewis rats were inoculated either intracranially, intranasally or subcutaneously. After 4.5 months, three out of fifteen neonatal intracranially inoculated rats were VSBV-1 genome positive in the central nervous system without showing clinical signs. Pathohistological examination revealed a nonpurulent encephalitis. While infection of immune incompetent rats (neonatal) using the type species of mammalian bornaviruses, the Borna disease virus 1, proceed to an immune tolerant status, VSBV-1 infection could result in inflammation of neuronal tissue. Sequencing showed minor adaptations within the VSBV-1 genome comparing to the viral genomes from infected squirrels, cell cultures or rat tissues. In conclusion, we were able to generate the first VSBV-1 isolates and provide in vivo animal model data in Lewis rats revealing substantial differences between VSBV-1 and BoDV-1. Furthermore, the presented data are a precondition for insights into the transmission and pathogenesis of this novel zoonotic pathogen.

Genomic and Micro-Evolutionary Features of Mammalian 2 orthobornavirus (Variegated Squirrel Bornavirus 1, VSBV-1)

“…Although the function of these residues remains to be determined, mutations in the G protein and/or L protein (polymerase) have exhibited enhanced neurovirulence in the related Borna Disease virus 1 (BoDV-1) in adult and newborn Lewis rats [20]. In a recent study, following isolation from primary cell cultures and passage in Lewis rats, VSBV-1 was shown to be genetically highly stable [21]. However, within three VSBV-1 isolates examined, at least one AA exchange occurred in the viral glycoprotein.…”

Development of a nonhuman primate model for mammalian bornavirus infection

“…BoDV-1 strain “H24” ( 31 ) and VSBV-1 strain “Theodore” ( 25 ) from infected Lewis rat brains were used for inoculum preparation. Rat brains were homogenized in 2 ml MEM (H) + MEM (E) media and sonicated (Brandson, Sonifier 450, Emerson, St Louis, MO, USA).…”

“…RNA was eluted in 50 µl total volume. Bornaviral RNA was detected by RT–qPCR using a VSBV-1 specific (Squirrel mix 10.1) ( 25 ) or a BoDV-1 specific assay (BoDV-1 mix 1) ( 17 ) and the qScript XLT one-step RT-qPCR ToughMix Kit (Quanta BioSciences, Gaithersburg, MD, USA). In each run, standard dilutions of quantified RNA standards were run in parallel, to calculate copy numbers in the samples.…”

“…All RT-qPCRs were performed on a Rotorgene Q (Rotorgene Q software version 2.3.1; Qiagen). Dideoxy chain-termination sequencing for VSBV-1 and sequence analysis was done as described elsewhere ( 3 , 25 ). For BoDV-1, primers are given in Table S1 (Supplementary Material) .…”

“…BoDV-1 has a long research history and several small animal models have been investigated with the Lewis rat being the best-established model ( 11 , 23 , 24 ). To date, VSBV-1 infection studies have been conducted in Lewis rats ( 25 ), but not in nonhuman primates (NHPs). Several decades ago, two research groups infected NHPs with BoDV-1 by the intracerebral route resulting in severe neurological signs 4 to 8 weeks after infection, but detailed further investigations into viral tissue distribution and route of infection were not performed ( 26 , 27 ).…”