Isolation of a Novel Bat Rhabdovirus with Evidence of Human Exposure in China

Abstract

Bats are well-recognized reservoirs of zoonotic viruses. Several spillover events from bats to humans have been reported, causing severe epidemic or endemic diseases including severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome-CoV (MERS-CoV), henipaviruses, and filoviruses. In this study, a novel rhabdovirus species, provisionally named Rhinolophus rhabdovirus DPuer (DPRV), was identified from the horseshoe bat (Rhinolophus affinis) in Yunnan province, China, using next-generation sequencing. DPRV shedding in the spleen, liver, lung, and intestinal contents of wild bats with high viral loads was detected by real-time quantitative PCR, indicating that DPRV has tropism for multiple host tissues. Furthermore, DPRV can replicate in vitro in multiple mammalian cell lines, including BHK-21, A549, and MA104 cells, with the highest efficiency in hamster kidney cell line BHK-21, suggesting infectivity of DPRV in these cell line-derived hosts. Ultrastructure analysis revealed a characteristic bullet-shaped morphology and tightly clustered distribution of DPRV particles in the intracellular space. DPRV replicated efficiently in suckling mouse brains and caused death of suckling mice; death rates increased with passaging of DPRV in suckling mice. Moreover, 421 serum samples were collected from individuals who lived near the bat collection site and had fever symptoms within 1 year. DPRV-specific antibodies were detected in 20 (4.75%) human serum samples by indirect immunofluorescence assay. Furthermore, 10 (2.38%) serum samples were DPRV positive according to plaque reduction neutralization assay, which revealed potential transmission of DPRV from bats to humans and highlighted the potential public health risk. Potential vector association with DPRV was not found with negative viral RNA in bloodsucking arthropods. IMPORTANCE We identified a novel rhabdovirus from the horseshoe bat (Rhinolophus thomasi) in China with probable infectivity in humans. DPRV was isolated in vitro from several mammalian cell lines, indicating wide host tropism, excluding bats, of DPRV. DPRV replicated in the brains of suckling mice, and the death rate of suckling mice increased with passaging of DPRV in vivo. Serological tests indicated the possible infectivity of DPRV in humans and the potential transmission to humans. The present findings provide preliminary evidence for the potential risk of DPRV to public health. Additional studies with active surveillance are needed to address interspecies transmission and determine the pathogenicity of DPRV in humans.

Keywords: Ledantevirus; bats; cell culture; complete genome; pathogenicity; phylogeny; rhabdovirus.

FIG 1

Genome characterization of DPRV. (A) Genome organization and protein annotation of DPRV. The transcription initiation (TI) and transcription termination (TTI) motifs are boxed in red; TI is shown in red font and TTI in blue font. (B) Amino acid sequence divergence scan of the L gene within subgroup C of ledanteviruses, including the four most related known ledanteviruses (accession number of the reference sequence is shown in Fig. 2).

FIG 2

Maximum likelihood phylogenetic tree based on the nucleotide sequences of nearly full-length (A) and amino acid of L gene (B), N gene (C), and M gene (D) of DPRV, using the complete deletion option and G+I rate and patterns option and a WAG amino acid evolutionary model. Bootstrap support values (>70%) are shown for key nodes. Evolutionary analyses were conducted using the MEGA (version 5.1) program.

FIG 3

Amino acid identities of the L, M, and N genes among members of the Ledantevirus genus. The reference sequences were downloaded from GenBank and used in the phylogenetic tree. Sequence alignment was performed using MEGA5.0, and amino acid identities were calculated using the Sequence Demarcation tool (v1.2).

FIG 4

Quantification of wild DPRV-positive bats, brain tissues of suckling mice, and cell-inoculated DPRV as well as the death rate of suckling mice over time. (A) Tissue quantification of original DPRV-positive bats. (B) Viral loads in brain tissue of suckling mice. (C) The x axis death rate of suckling mice over time across three passages.

FIG 5

CPEs and growth kinetics of DPRV. (A) Negative control of BHK-21 cells. (B) DPRV-induced CPEs in BHK-21 cells. (C) Negative control of A549 cells. (D) DPRV-induced CPEs in A549 cells. (E) Growth kinetics of DPRV in mammalian cell lines. BHK-21, MA104, and A549 cells were infected by DPRV with the equivalent of 1 × 10⁶ viral genomes for 3 to 4 days.

FIG 6

Electron micrograph of a DPRV-infected BHK-21 cell.

FIG 7

IFA and PRNT serological analyses of DPRV antibodies in human serum. (A) Representative IFA images of serum samples positive for DPRV. (B) Representative images of the PRNT assay results. DPRV suspensions were diluted 10⁻⁴ and mixed with serum samples. The positive control was also inoculated with a 10⁻⁴ suspension of DPRV.

FIG 8

Sampling sites in China. Sampling locations of arthropods are colored as purple; Yunnan province where bats, human sera, and arthropods were sampled is colored as green. The maps are based on maps issued by the Chinese Ministry of Civil Affairs and drawn using R.