Fig 1.
Phylogenetic analysis of nucleotide sequences of the 80-bp fragment (excluding primer sequences) of 3Dpol gene of a picornavirus identified from rodents in this study.
The strain RASM14A that was successfully isolated in 3T3 cells is in bold. The trees were constructed by neighbor-joining method with bootstrap values calculated from 1000 trees. Only values greater than 700 are indicated. The scale bar indicates the estimated number of substitutions per 50 nucleotides.
Table 1.
Rodents tested for picornaviruses in the present study.
Table 2.
Comparison of amino acid identities between the predicted proteins P1, P2 and P3 of “Rosavirus B” and “Rosavirus C” and those of other representative picornavirus species.
Table 3.
Coding potential and putative proteins of “Rosavirus B” and “Rosavirus C” compared to Rosavirus A.
Fig 2.
Phylogenetic analysis of the amino acid sequences of P1, P2 (excluding 2A) and P3 (excluding 3A) of the novel rodent picornavirus species with genomes completely sequenced.
The strains identified in this study are shown in colors representing different host species of street rodents, Norway rats (strain number RNXXX) (brown), and wild rodents, Coxing's white-bellied rats (NCXXX) (green), roof rats (RRXXX) (blue), chestnut spiny rats (NFXXX) (purple) and Indochinese forest rats (RAXXX) (red) respectively. The strain RASM14A that was successfully isolated in 3T3 cells is shown in bold. The present and other known rodent picornaviruses are indicated by a pictorgram of rodent. The trees were constructed by maximum-likelihood method with bootstrap values calculated from 100 trees and only those >70% are shown. The scale bar indicates the estimated number of substitutions per 2 or 5 amino acids. BPV-1, bat picornavirus 1 (HQ595340); BPV-2, bat picornavirus 2 (HQ595342); BPV-3, bat picornavirus 3 (HQ595344); CanPV-1, canine picornavirus (JN831356); FePV-1, feline picornavirus (JN572117); FMDV-C, foot-and-mouth disease virus—type C (NC_002554); MuKV-1, murine kobuvirus 1 (JF755427).
Fig 3.
Predicted type II-like IRES structures of rosavirus C RRTPC2A.
The stem-loop motifs/domains are labeled sequentially from A to N. Domains E/F and L/M/N were only observed in some strains of Rosavirus B and C. The AUG start codon was in bold and underlined.
Table 4.
Comparison of genomic and protein features of “Rosavirus B” and “Rosavirus C” to those of Rosavirus A and other representative genera in the Picornaviridae family.
Table 5.
Estimation of nonsynonymous and synonymous substitution rates in the genomes of “Rosavirus B” and “Rosavirus C.”
Fig 4.
(A) 3T3 cells infected with rosavirus C RASM14A showing cytopathic effects with rounded and refractive cells rapidly detaching from the monolayer at day 5 after incubation, compared to (B) uninfected cells. (C) Immunofluorescence staining of 3T3 cells infected with rosavirus C RASM14A, compared to (D) uninfected cells using guinea pig antiserum against VP1. (E) Negative contrast electron microscopy of ultracentrifuged deposit of 3T3 cells culture-grown rosavirus C RASM14A, showing non-enveloped picornaviral particles of around 25–30 nm in diameter, bar = 50 nm.
Fig 5.
Western blot analysis for detection of antibodies against purified His6-tagged recombinant VP1 proteins of rosavirus B strain RNCW1002091R from a Norway rat and rosavirus C strains, RASK8F from an Indochinese forest rat and NCGX12IN from a Coxing's white-bellied rat (~40-kDa) in rodent serum samples.
Representatives of results are shown. Lanes: 1, Norway rat serum sample positive for antibody against rosavirus B strain RNCW1002091R VP1 protein; 2, Indochinese forest rat serum sample positive for antibody against rosavirus C strain RASK8F VP1 protein; 3, Coxing's white-bellied rat serum sample positive for antibody against rosavirus C strain NCGX12IN VP1 protein; 4, rosavirus B-VP1 antibody-positive Norway rat serum sample against rosavirus C strain NCGX12IN VP1 protein; 5, rosavirus C-VP1 antibody-positive Coxing's white-bellied rat serum sample against rosavirus B strain RNCW1002091R VP1 protein; 6, positive control (anti-His antibody); 7, negative control.
Fig 6.
Viral loads by qRT-PCR of RT-PCR-positive tissues from sacrificed mice challenged with rosavirus C RASM14A by oral (A) and intracerebral (B) inoculation. Numbers above the bars indicate the number of tested mice with RT-PCR-positive tissues.
Table 6.
RT-PCR and western blot analysis of mice challenged with rosavirus C RASM14A.
Fig 7.
Histological changes and immunohistochemical staining of mice lung and liver tissues on day 3 after oral or intracerebral inoculation of rosavirus C RASM14A with micrographs at magnification 200×.
A-C, Representative images of H&E staining of lung sections of control mouse (A) and infected mice showing alveolar fluid exudation (B) and increased cellularity, interstitial infiltration and alveolar wall thickening (C); D-F, Representative images of H&E staining of liver sections of control mouse (D) and infected mice showing hepatocyte degeneration (E) and lymphocytic/monocytic inflammatory infiltration and giant cell formation (arrow) (F); G-L, Immunohistochemical staining of lung and liver sections using guinea pig anti-serum against rosavirus C VP1 protein antibody; G and J are lung and liver sections from control mice with inoculation of uninfected cell culture medium respectively; H and I, lung sections of infected mice showing bronchiolar and bronchial epithelial cells positively stained with rosavirus C VP1 protein in brown colour (insert, magnification 400×); K and L, liver sections of infected mice showing positive staining of rosavirus C VP1 protein in hepatocytes with brown colour (insert, magnification 400×).