Characterization of a novel species of adenovirus from Japanese microbat and role of CXADR as its entry factor

Abstract

Recently, bat adenoviruses (BtAdVs) of genus Mastadenovirus have been isolated from various bat species, some of them displaying a wide host range in cell culture. In this study, we isolated two BtAdVs from Japanese wild microbats. While one isolate was classified as Bat mastadenovirus A, the other was phylogenetically independent of other BtAdVs. It was rather related to, but serologically different from, canine adenoviruses. We propose that the latter, isolated from Asian parti-colored bat, should be assigned to a novel species of Bat mastadenovirus. Both isolates replicated in various mammalian cell lines, implying their wide cell tropism. To gain insight into cell tropism of these BtAdVs, we investigated the coxsackievirus and adenovirus receptor (CXADR) for virus entry to the cells. We prepared CXADR-knockout canine kidney cells and found that replication of BtAdVs was significantly hampered in these cells. For confirmation, their replication in canine CXADR-addback cells was rescued to the levels with the original cells. We also found that viral replication was corrected in human or bat CXADR-transduced cells to similar levels as in canine CXADR-addback cells. These results suggest that BtAdVs were able to use several mammalian-derived CXADRs as entry factors.

Figure 1

Isolation of BtAdVs from Japanese microbats. We collected 164 fecal samples from ten bat species captured in six prefectures in Japan. Bat common names and the numbers of samples are described for each prefecture. BtAdV-Mm32 and -Vs9 were isolated from the bats shown underlined, respectively (A). Both isolates were negatively-stained when observed by transmission electron microscope (B).

Figure 2

Phylogenetic classification of BtAdVs. A neighbor-joining tree was generated based on full-length genomic sequences (A) or DNA polymerase amino acid sequences (B). Bootstrap values are shown at the major nodes. Scale bar indicates the number of substitutions per site. All BtAdVs are underlined and novel isolates in this study are shown in bold.

Figure 3

Pathogenicity of BtAdVs in mice. BALB/c mice (4-week old, n = 3) were intranasally inoculated with 10⁵ PFU of BtAdV-Mm32, -Vs9, or PBS (mock). Then, their body weights were measured daily for 21 days. Asterisks (*) reveal significant differences compared to mock-infected mice (p < 0.05 by Dunnett’s test).

Figure 4

Antigenic cross-reactivity between BtAdVs and CAdVs. Viral neutralization titers were determined in homologous or heterologous combinations using BtAdV-Mm32, -Vs9, CAdV1, or CAdV2 and their respective antisera by plaque reduction assays in MDCK cells. An 80% reduction value in plaque numbers was used as neutralization titer (PRNT₈₀). The data are reported as the mean titers with standard deviations for three independent experiments (A). BtAdV-Mm32, -Vs9, CAdV1, or CAdV2 were inoculated into cells at an MOI of 1. At 24 hpi, the cells were fixed and permeabilized. After blocking, each antiserum was used as the primary antibody in homologous or heterologous combinations. We used a secondary antibody conjugated with a fluorophore (Alexa 488) and observed the cells by fluorescent microscopy (B).

Figure 5

Viral growth kinetics in various mammalian cell lines. BtAdV-Mm32, -Vs9, CAdV1, or CAdV2 were inoculated into MDCK, Vero, A549, CRFK, MDBK, PK-15, FBKT, and DemKT1 cells at an MOI of 0.01. Following this, the supernatants were collected daily for 6 dpi. Viral titers were determined by plaque assay in MDCK cells. The data are reported as the mean titers with standard deviations for three independent experiments.

Figure 6

Viral growth in canine (c) CXADR-KO cells. cCXADR was not detected in three lines of CXADR-KO cells, unlike wild type (WT) cells, by a western blot analysis using a mouse anti-CXADR monoclonal antibody (CXADR E1). Plasmid-expressed cCXADR was used as a positive control (PC). ACTB was used as a loading control in each lane with the same amount of sample (A). WT or cCXADR-KO cells were inoculated with BtAdV-Mm32, -Vs9, CAdV1, or CAdV2 at an MOI of 1. At 24 hpi, virus antigens were detected by IFA using each antiserum under a fluorescence microscope (B). Numbers of IFA positive cells in panel B were quantified and reported as the mean values with standard deviations for three independent experiments. Asterisks (*) reveal significant differences compared to WT cells (p < 0.001 by Dunnett’s test) (C). WT or cCXADR-KO cells were inoculated with each virus at an MOI of 1 and the supernatants were collected and titrated daily for 6 days post-infection. The data are reported as the mean values with standard deviations for three independent experiments. Asterisks (*) reveal significant differences compared to WT cells (*p < 0.05; **p < 0.01 by Student’s t test) (D).

Figure 7

Viral growth in CXADR-addback or -transduced cells. Expressions of canine (c), human (h), or bat (b) CXADRs were rescued in these addback or transduced cells as revealed by western blot analysis using a mouse anti-CXADR monoclonal antibody (CXADR E1). Plasmid-expressed cCXADR was used as a positive control (PC). ACTB was used as a loading control in each lane with the same amount of sample (A). WT, cCXADR-KO, -addback, hCXADR or bCXADR-transduced cells were inoculated with each virus at an MOI of 1. At 24 hpi, viral antigens were detected by IFA using each antiserum under a fluorescence microscope (B). Numbers of IFA positive cells in panel B were quantified and reported as the mean values with standard deviations for three independent experiments. Asterisks (*) reveal significant differences compared to cCXADR-KO cells (p < 0.001 by Dunnett’s test) (C). WT, cCXADR-KO, -addback, hCXADR or bCXADR-transduced cells were inoculated with each virus at an MOI of 1 and the supernatants were collected and titrated daily for 6 days post-infection. The data are reported as the mean values with standard deviations for three independent experiments. Asterisks (*) reveal significant differences compared to cCXADR-KO cells (*p < 0.05; **p < 0.01 by Student’s t test) (D).

Figure 8

Viral attachment to WT, cCXADR-KO, -addback, hCXADR or bCXADR-transduced cells. Each of the viruses was inoculated to these cells followed by incubation for 1 h at 4 °C. Then, the cells were lysed and the DNA was extracted. The relative viral loads were measured by real-time PCR (n = 3). Asterisks (*) reveal significant differences compared to cCXADR-KO cells (*p < 0.05; **p < 0.01 by Dunnett’s test).