Characterization of Novel Rhabdoviruses in Chinese Bats

Abstract

Bats, the second largest order of mammals worldwide, harbor specific characteristics such as sustaining flight, a special immune system, unique habits, and ecological niches. In addition, they are the natural reservoirs of a variety of emerging or re-emerging zoonotic pathogens. Rhabdoviridae is one of the most diverse families of RNA viruses, which consists of 20 ecologically diverse genera, infecting plants, mammals, birds, reptiles, and fish. To date, three bat-related genera are described, named Lyssavirus, Vesiculovirus, and Ledantevirus. However, the prevalence and the distribution of these bat-related rhabdoviruses remain largely unknown, especially in China. To fill this gap, we performed a large molecular retrospective study based on the real-time reverse transcription polymerase chain reaction (RT-qPCR) detection of lyssavirus in bat samples (1044 brain and 3532 saliva samples, from 63 different bat species) originating from 21 provinces of China during 2006-2018. None of them were positive for lyssavirus, but six bat brains (0.6%) of Rhinolophus bat species, originating from Hubei and Hainan provinces, were positive for vesiculoviruses or ledanteviruses. Based on complete genomes, these viruses were phylogenetically classified into three putative new species, tentatively named Yinshui bat virus (YSBV), Taiyi bat virus (TYBV), and Qiongzhong bat virus (QZBV). These results indicate the novel rhabdoviruses circulated in different Chinese bat populations.

Keywords: China; RT-qPCR; bat; brain; complete genome; ledantevirus; lyssavirus; phylogeny; rhabdovirus; vesiculovirus.

Figure 1

Repartition of the bat samples analyzed in this study and collected in 21 provinces of China (in grey and back) during the period 2006–2018, with the total number of samples indicated per province. The two provinces in black (Hainan and Hubei) indicate the presence of positive samples. Magnifications of these provinces are presented, with the position and the name of the positive sample caves or sites indicated with black dots, as well as the different rhabdovirus identified (TYBV: Taiyi bat virus, YSBV: Yinshui bat virus, and QZBV: Qiongzhong bat virus).

Figure 2

Schematic genomic organization of the six Chinese bat rhabdoviruses. The grey arrows represent the five canonical open reading frames (ORFs) (N, P, M, G, and L), and the black arrows indicate the position of the putative additional ORFs. The nucleotide and amino acid lengths of each ORF are indicated.

Figure 3

Phylogenetic classification of the sic Chinese bat rhabdoviruses. A maximum likelihood phylogenetic tree was done using MEGA7.0 on the full amino-acid sequence of the L protein including 155 previously reported rhabdoviruses from GenBank, using the LG+G+I+F model and with 1000 bootstrap replicates. Bat-related rhabdoviruses are indicated by a dedicated symbol. Unclassified rhabdoviruses are indicated by asterisks. The Chinese bat rhabdoviruses described in this study are indicated in grey. All bootstrap proportion values (BSP) > 80% are specified. Scale bar indicates nucleotide substitutions per site.

Figure 4

Phylogenetic classification of the six Chinese bat rhabdoviruses. A maximum likelihood phylogenetic tree was done with PhyML3.0 on the nucleotide complete genome sequences, including representative members of the Vesiculovirus genus (a) and of the Ledantevirus genus (b), using the GTR+G+I model with a bootstrap of 1000 replicates. The main animal reservoirs for each virus are indicated by specific cartoons and the Chinese bat rhabdoviruses described in this study are indicated in grey. All bootstrap proportion values (BSP) > 80% are specified. Scale bar indicates nucleotide substitutions per site.

Figure 5

Identification of the nucleotide and amino acid mutations between the four isolates (1017, D170001, D170022, and D170190) of putative YSBV species. The nucleotide mutations are indicated in bold and the amino acid mutations in grey.