<i>IFNAR2</i>-dependent gene expression profile induced by IFN-α in <i>Pteropus alecto</i> bat cells and impact of <i>IFNAR2</i> knockout on virus infection

<div><p>Bats are important reservoirs of many viruses, which are capable of infecting the host without inducing obvious clinical diseases. Interferon and the downstream interferon regulated genes (IRGs) are known to act as the first line of defense against viral infections. Little is known about the transcriptional profile of genes being induced by interferon in bats and their role in controlling virus infection. In this study, we constructed <i>IFNAR2</i> knockout bat cell lines using CRISPR technology and further characterized gene expression profiles induced by the most abundant IFN-α (IFN-α3). Firstly, we demonstrated that the CRISPR/Cas9 system is <a href="http://dict.cn/practicable" target="_blank">applicable</a> for bat cells as this represents the first CRIPSR knockout cell line for bats. Our results showed the pleiotropic effect of IFN-α3 on the bat kidney cell line, PaKiT03. As expected, we confirmed that <i>IFNAR2</i> is indispensable for IFN-a signaling pathway and plays an important role in antiviral immunity. Unexpectedly, we also identified novel <i>IFNAR2</i>-dependent IRGs which are enriched in pathways related to cancer. To our knowledge, this seems to be bat-specific as no such observation has been reported for other mammalian species. This study expands our knowledge about bat immunology and the cell line established can provide a powerful tool for future study into virus-bat interaction and cancer biology.</p></div

Verification of <i>IFNAR2</i> KO in two cell lines obtained from two independent gRNAs.

<p>(A) Sanger sequencing was performed to validate the location and nature of the deletion events. Left: The location of gRNA and PAM motif are given in blue and red, respectively. The deletion regions are highlighted in gray. Right: Chromatogram of DNA sequence spanning the deletion region. Quantitative RT-PCR (B) and western blot (C) analyses were performed to confirm the functional phenotype of the clones.</p

Effect of <i>IFNAR2</i> KO on H1N1 infection in bat cells.

<p>Cells were treated with IFN-α3 for 2 hrs before infected with H1N1 at MOI of 0.1. Cells and Culture supernatants were harvested at 48 hrs post infection. Gene expression was determined by measuring mRNA level using qPCR (A) and the data were normalized against the expression level of the housekeeping gene SNRPD3. Virus titers were determined by plaque assay in BHK cells (B). Error bars indicate standard deviations from three independent experiments.</p

The top-ranked diseases enriched by IPA analysis for the unknown IRGs.

<p>The top-ranked diseases enriched by IPA analysis for the unknown IRGs.</p

Number of DEGs induced by IFN-α3 in wild-type PaKiT03 and <i>IFNAR2</i> knockout cell lines.

<p>Number of DEGs induced by IFN-α3 in wild-type PaKiT03 and <i>IFNAR2</i> knockout cell lines.</p

Canonical pathways analysis for the up-regulated total IRGs (A) and the up-regulated unknown IRGs (B) using IPA software.

<p>Statistical significance is represented by -log (P-Value), and values exceeding 1.30 (indicated by the dotted lines) are considered significant (P<0.05). The ratio represents the percentages of genes enriched to the total number of genes in each category.</p

Additional file 1: Figure S1. of Detection of alpha- and betacoronaviruses in rodents from Yunnan, China

Geographical map of Jianchuan country and the sampling areas. Figure S2. Alignment and predicted domains and cleavage sites of AcCoV-JC34 spike protein. NTD, N-terminal domain; RBD, receptor-binding domain; HR, heptad repeat; TM, transmembrane anchor. The signal peptide corresponds to residues 1 to 19. The cleavage sites were indicated by arrows. (PDF 223 kb

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus - Fig 8

<p><b>Analysis of receptor usage by immunofluorescence assay (A) and real-time PCR (B).</b> Virus infectivity of Rs4874, WIV1-Rs4231S and WIV1-Rs7327S was determined in HeLa cells with and without the expression of human ACE2. ACE2 expression was detected with goat anti-human ACE2 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI (49,6-diamidino-2-phenylindole).The columns (from left to right) show staining of nuclei (blue), ACE2 expression (green), virus replication (red) and the merged triple-stained images, respectively.</p

Functional characterization of diverse ORF8 and ORF8a proteins of bat SARSr-CoVs.

<p>(A) The ORF8 proteins of SARS-CoV and bat SARSr-CoVs induces the ATF6-dependent transcriptional activity. HeLa cells were transiently transfected with the pcAGGS expression plasmids of the ORF8 of SARS-CoV GZ02, bat SARSr-CoV Rf1, WIV1 and Rf4092 and the reporter plasmid 5×ATF6-GL3 for 40h. Control cells were co-transfected with the reporter plasmid and the empty pCAGGS vector for 24h, and treated with or without TM (2μg/ml) for an additional 16h. The cell lysates were harvested for dual luciferase assay and data are shown as the average values from triplicate wells. (B) The ORF8a proteins of SARS-CoV and bat SARSr-CoV triggered apoptosis. 293T cells were transfected with the expression plasmids of the ORF8a of SARS-CoV Tor2 and bat SARSr-CoV Rs4084 and a pcAGGS vector control for 24h. Apoptosis was analyzed by flow cytometry after annexin V staining and the percentage of apoptotic cells were calculated. Data are shown as the average values from triplicate cells. Error bars indicate SDs. * <i>P</i><0.05.</p

Similarity plot based on the full-length genome sequence of civet SARS CoV SZ3.

<p>Full-length genome sequences of all SARSr-CoV detected in bats from the cave investigated in this study were used as reference sequences. The analysis was performed with the Kimura model, a window size of 1500 base pairs and a step size of 150 base pairs.</p