Characterization of a novel G3P[3] rotavirus isolated from a lesser horseshoe bat: a distant relative of feline/canine rotaviruses

Abstract

Bats are considered important animal reservoirs for many viruses pathogenic to humans. An approach based on viral metagenomics was used to study gut specimens from 78 insectivorous bats in Yunnan Province, China. Seventy-four reads were found to be related to group A rotavirus (RVA). Further reverse transcription-PCR screening and viral isolation on cell cultures confirmed the presence of a novel RVA strain, named RVA/Bat-tc/MSLH14/2012/G3P[3], in 1 (6%) of 16 lesser horseshoe bats. Full genomic sequencing analyses showed that MSLH14 possessed the genotype constellation G3-P[3]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is akin to human and animal rotaviruses believed to be of feline/canine origin. Phylogenetic analysis indicated that VP7 was most closely related to bovine RVA strains from India, whereas VP4 was most closely related to an unusual human RVA strain, CMH222, with animal characteristics isolated in Thailand. The remaining gene segments were only distantly related to a range of animal RVA strains, most of which are believed to be related to feline/canine RVAs. Experimental infection showed that bat RVA strain MSLH14 was highly pathogenic to suckling mice, causing 100% mortality when they were inoculated orally with a titer as low as 5 × 10² 50% tissue culture infective doses. As this virus is not closely related to any known RVA strain, it is tempting to speculate that it is a true bat RVA strain rather than a virus transmitted between species. However, further screening of bat populations, preferably juvenile animals, will be crucial in determining whether or not this virus is widely distributed in the bat population.

Fig 1

(A) Bayesian analysis of 818-bp segments of VP7 from G3 RVA strains suggests that MSLH14 (filled triangle) diverged in 1982 from an ancestor most closely related to bovine RVA strains in India. (B) Phylogenetic analysis of a 2,254-bp VP4 segment of MSLH14 (filled triangle) and other representative RVA strains suggests that MSLH14 is most closely related to CMH222.

Fig 2

Phylogenetic analyses of VP1 (A), VP2 (B), VP3 (C), and VP6 (D) of RVA/Bat-tc/MSLH14/2012/G3P[3] (filled triangles) and other representative RVA strains. Sequences from fruit bat strain RVA/Bat-wt/KEN/KE4852/07/2007/G25P[6] are indicated by open triangles. Only the partial VP1 sequence of strain KE4852 (open triangles) was determined, thereby preventing the classification of this gene segment (32).

Fig 3

Phylogenetic analyses of NSP1 (A), NSP2 (B), NSP3 (C), NSP4 (D), and NSP5 (E) of MSLH14 (filled triangles) and other representative RVA strains. Fruit bat RVA strain KE4852 is indicated by open triangles.

Fig 4

Cells inoculated with RVA-positive samples. (A) Mock-infected Marc145 cells show a normal appearance, and no bright fluorescence was noted by IFA. (B) CPE generated by RVA/Bat-tc/MSLH14/2012/G3P[3] in Marc145 cells, showing many detached infected cells connected by cytoplasmic filaments and colored by bright fluorescence in the cytoplasm. (C) Mock-infected BFK cells. (D) CPE generated by MSLH14 in BFK cells.

Fig 5

PAGE analysis (A) and morphological observation (B) of strain RVA/Bat-tc/MSLH14/2012/G3P[3]. (A) Typical 4-2-3-2 gene segregation pattern of RVA strains. From left to right, the bonds are VP1, VP2, VP3, VP4, NSP1, VP6, NSP3, VP7, NSP2, NSP4, and NSP5. (B) Many particles lacking VP4 spikes are visible, along with some DLPs lacking VP4 and VP7 and SLPs or cores.

Fig 6

Weight changes and survival curves of mice after oral administration of RVA/Bat-tc/MSLH14/2012/G3P[3]. (A) Group Y inoculated with 5 × 10⁵ TCID₅₀ of virus. (B) Group D inoculated with 5 × 10² TCID₅₀ of virus. (C) Mock-infected group inoculated with MEM.