Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663 - PubMed (original) (raw)

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663

Yutaka Terada et al. J Virol. 2019.

Abstract

Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed growth kinetics similar to those shown by the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h postinfection. Furthermore, we found that A72 cells derived from canine fibroblasts permitted FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.IMPORTANCE Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is a lethal disease in cats. Tissue culture-adapted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.

Keywords: coronavirus; viral replication.

Copyright © 2019 American Society for Microbiology.

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Figures

FIG 1

FIG 1

Constructing type I FCoV strain C3663 cDNA clones. (A) Schematic diagram illustrating the strategy for constructing infectious cDNA clones bearing the full-length genome of type I FCoV strain C3663. The full-length C3663 sequence was divided into 11 fragments (Fr1 to Fr11), and each fragment was sequentially assembled into the plasmid backbone. The asterisk (*) indicates the site of the genetic marker. (B) Nucleotide sequence of the C3663 genome between nt 9826 and nt 9837. The EcoRI restriction site is underlined. rFCoV was mutated from GAATTC to GAGTTT for disruption of the EcoRI site (ΔEcoRI) to use as a genetic marker. Mutated nucleotides are shown in gray boxes with white letters. (C) Confirmation of the genetic marker in the rC3663 genome by EcoRI treatment. RT-PCR products of parental virus C3663 and rC3663 that amplified a region that included the genetic marker were treated with EcoRI. The treated samples were subjected to electrophoresis to confirm disruption of the EcoRI site in the rC3663 genome. (D) Sequence analysis of C3663 and rC3663 at the genetic marker site. The EcoRI restriction site and ΔEcoRI genetic marker are underlined. (E) Growth kinetics of parental virus C3663 and rC3663 in Fcwf-4 cells. Each virus was inoculated onto Fcwf-4 cells at an MOI of 0.01 and incubated for 24, 48, and 72 h. Viral titers of culture supernatants were measured by plaque assays using Fcwf-4 cells. LOD, limit of detection. The data represent means ± standard deviations (SD) of results from three independent experiments. (F) Northern blot analysis for detecting viral RNA in C3663-infected or rC3663-infected Fcwf-4 cells. Total RNAs from Fcwf-4 cells infected with parental C3663 or rC3663 were extracted and subjected to electrophoresis. The transferred viral RNAs were hybridized with DIG-labeled RNA targeting ORF 7b and 3ʹ-UTR. gRNA, genomic RNA; sgRNA, subgenomic RNA.

FIG 2

FIG 2

Construction and characteristics of the reporter virus carrying the Nluc gene. (A) Nluc gene replacement occurred at ORF 3abc, resulting in pBAC-FCoV-C3663-Nluc. The Nluc gene replaced the sequence from the start codon of ORF 3a to 71 nt upstream of the ORF 3c stop codon to retain the TRS of the E gene. Light gray, gray, and white boxes indicate nonstructural proteins, structural proteins, and accessory proteins, respectively. TRS, transcription regulatory sequence. (B) Luciferase activity of rC3663-Nluc-infected cells. rC3663-Nluc and rC3663 were inoculated onto Fcwf-4 cells at an MOI of 0.01 and incubated for 24, 48, and 72 h. Infected cells were lysed, and luciferase activity was measured. Experiments were carried out in triplicate. (C) Growth kinetics of rC3663-Nluc. rC3663-Nluc and rC3663 were inoculated into Fcwf-4 cells at an MOI of 0.01 and incubated for 24, 48, and 72 h. Viral titers of culture supernatants were measured by plaque assays using Fcwf-4 cells. LOD, limit of detection. (D and E) Evaluation of the inhibitory effects of (D) CsA and (E) lopinavir on rC3663-Nluc. Fcwf-4 cells were inoculated with rC3663-Nluc at an MOI of 0.01. After adsorption, the viruses were removed and replaced by culture medium with or without different concentrations of (D) CsA or (E) lopinavir. After incubation for 24 h, levels of luciferase activities (black circle) or viral RNA (white triangle) were measured. The experiments were carried out in triplicate. (F) Compound screening using rC3663-Nluc and evaluation of the cytotoxicity of protease inhibitors by MTT assays. A total of 68 protease inhibitors were used in this screening. Virus was added at an MOI of 0.01 onto cultured Fcwf-4 cells with a 10 μM concentration of each protease inhibitor or DMSO and further cultured for 24 h. CsA (10 μM) was used as a positive control. Infected cells were lysed, and levels of Nluc activities were measured (bar graphs). For MTT assays, seeded Fcwf-4 cells were cultured with DMEM containing 10% FBS and a 10 μM concentration of each compound for 24 h. Then, cultured cells underwent MTT assays and the absorbance was measured at 570 nm (line graph). The data represent means ± SD of results from three independent experiments.

FIG 3

FIG 3

Investigation of the infectivity of type I FCoV in canine-derived cell lines. (A and B) Infectivity of rC3663-Nluc in canine-derived cell lines. Fcwf-4 cells (A) and canine-derived A72 as well as MDCK and DH82 cells (B) were subjected to mock inoculation or were inoculated with rC3663-Nluc (Nluc) at an MOI of 0.1 and incubated for 24, 48, and 72 h. After incubation, infected cells were lysed and Nluc activities were measured. The experiments were carried out in triplicate. (C) Cytopathic effects in Fcwf-4 and A72 cells infected with rC3663-Nluc. Fcwf-4 and A72 cells were subjected to mock inoculation or were inoculated with rC3663-Nluc (Nluc) at an MOI of 0.1 and incubated for 24, 48, and 72 h. (D) Real-time RT-PCR for the evaluation of viral RNA replication. Fcwf-4, A72, MDCK, and DH82 cells were inoculated with rC3663 at an MOI of 0.01. Total RNA was extracted from the infected cells, and real-time RT-PCR targeting the 3ʹ-UTR was carried out. (E) Growth kinetics of rC3663 in Fcwf-4, A72, MDCK, and DH82 cells. rC3663 was used to inoculate the cells at an MOI of 0.01, and the cells were incubated for 24, 48, and 72 h. The culture supernatants were collected at each time point, and viral titers were measured by plaque assays using Fcwf-4 cells. LOD, limit of detection. (F) Detection of rC3663 N protein in Fcwf-4 and A72 cells by IFA. rC3663 was used to inoculate Fcwf-4 and A72 cells at an MOI of 0.1. Infected cells were incubated for 48 h. Then, infected cells were fixed with 4% paraformaldehyde. Fixed cells were treated with mouse anti-FCoV N monoclonal antibody (primary antibody) and CF488-conjugated anti-mouse IgG (secondary antibody). (G) Western blot analysis for the detection of rC3663 N protein. Cell lysates of Fcwf-4, A72, MDCK, and DH82 cells infected with rC3663 were subjected to Western blot analysis using anti-FCoV N monoclonal antibody (a-N) and anti-actin antibody (a-actin). “Short” and “Long” indicate short and long exposure times, respectively. The data represent means ± SD of results from three independent experiments.

FIG 4

FIG 4

Investigation of resistance to type I FCoV infection. (A) Strategy for construction of polymerase dead mutant cDNA clones (pBAC-FCoV-C3663-Nluc-PolDead [PolDead]). The asterisk (*) indicates the active site of viral RNA-dependent RNA polymerase (RdRp; nsp12). The diagram represents the nucleotide sequence of the C3663 genome between nt 14592 and nt 14612; mutated nucleotides are shown in gray boxes with white letters. pBAC-FCoV-C3663-Nluc (rC3663-Nluc) or pBAC-FCoV-C3663-Nluc-PolDead (PolDead) was transfected into seeded MDCK cells. Transfected cells were incubated for 24, 48, and 72 h. (B) At each time point, the transfected cells were lysed and levels of Nluc activities measured. As an internal control, a firefly luciferase reporter plasmid (pcDNA3.1-fluc) was cotransfected with BAC plasmids. Nluc activity was normalized to the activity of firefly luciferase. (C) The culture supernatants were collected at each time point, and viral titers were measured by plaque assays using Fcwf-4 cells. LOD, limit of detection. The experiments were carried out in triplicate. (D) Total RNA was extracted from transfected MDCK cells, and the levels of viral RNA were determined by real-time RT-PCR. (E) Cell lysates of transfected MDCK cells were subjected to Western blot analysis using anti-FCoV N monoclonal antibody (a-N) and anti-actin antibody (a-actin). The data represent means ± SD of results from three independent experiments.

FIG 5

FIG 5

Aberrant expression of viral RNA of type I FCoV in A72 cells. (A) Northern blot analysis for the detection of viral RNAs in rC3663-infected, parental C3663-infected, or Yayoi-infected A72 and Fcwf-4 cells. Total RNA was extracted from infected cells and subjected to electrophoresis. Viral RNAs were then hybridized with DIG-labeled RNA targeting ORF 7b and 3ʹ-UTR. gRNA, genomic RNA; sgRNA, subgenomic RNA. (B) Diagram illustrating the DIG-labeled RNA probes used in Northern blot analysis. (C) Northern blot analysis using S, 3abc, M, and N probes for detecting viral RNA in rC3663-infected Fcwf4-cells. The arrow indicates an unknown RNA signal. (D) Northern blot analysis for detecting viral RNA in rC3663-infected Fcwf-4 and A72 cells. First lane, 0.05 μg of total RNA extracted from infected Fcwf-4 cells; second lane, 2 μg of total RNA extracted from infected A72 cells.

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References

    1. Weiss SR, Navas-Martin S. 2005. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev 69:635–664. doi:10.1128/MMBR.69.4.635-664.2005. - DOI - PMC - PubMed
    1. Woo PC, Huang Y, Lau SK, Yuen KY. 2010. Coronavirus genomics and bioinformatics analysis. Viruses 2:1804–1820. doi:10.3390/v2081803. - DOI - PMC - PubMed
    1. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (ed). 2012. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Academic Press, London, United Kingdom.
    1. Biek R, Ruth TK, Murphy KM, Anderson CR Jr, Johnson M, DeSimone R, Gray R, Hornocker MG, Gillin CM, Poss M. 2006. Factors associated with pathogen seroprevalence and infection in Rocky Mountain cougars. J Wildl Dis 42:606–615. doi:10.7589/0090-3558-42.3.606. - DOI - PubMed
    1. Hofmann-Lehmann R, Fehr D, Grob M, Elgizoli M, Packer C, Martenson JS, O’Brien SJ, Lutz H. 1996. Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east Africa. Clin Diagn Lab Immunol 3:554–562. - PMC - PubMed

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