Viruses, Cell Lines and Plasmids
VS-FCV SH/14 strain (GenBank, KT000003.1) was isolated and characterized by our laboratory in Shanghai, China in 2014 [7]. Cat kidney cell line (CRFK cell, ATCC CCL-94, RPMI 1640 medium) was preserved by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The plasmid pKS-EF/HDrz carrying the hEF-1α promoter and the hepatitis D ribozyme core sequence was constructed by our laboratory.
Construction of revers genetics system of VS-FCV
First, total RNA was extracted from cell cultures infected with VS-FCV SH/14 strain using uniQ-10 column total RNA extraction kit (Shanghai Bioengineering Co., LTD., China) according to the instructions. Then, HM87-21KS primer (5'GAGATGCCATGCCGACCCT283') was used as reverse transcription primer, and M-MLV reverse transcription kit (Promega, USA) was used to synthesize the first strand of cDNA. Then, using the cDNA as template, 3 pairs of primers, namely SHINF1/SHINF2, SHINF3/SHINF4 and SHINF5/HM87-21KS (Table 1) were used to amplify the full-length genome of VS-FCV SH/14 strain by Phanta® Max Super-Fidelity DNA polymerase (Nanjing Vazyme Biotechnology Co., LTD., China). The resultant PCR products were clarified, sequenced and designated as SHI, SHII and SHIII, respectively. Meanwhile, pKS-EF/HDrz was used as template, and a pair of primers KS-HM1192 and KS1171 (Table 1) were used to amplify the vector sequence by PCR. The product, designated as KS-HM fragment have length of 5028bp consistent with the expected length. Finally, the purified PCR products (SHI, SHII, SHIII and KS-HM fragment) were joined by ClonExpress® MultiS One Step Cloning Kit (Nanjing Vazyme Biotechnology Co., LTD., China) using DNA seamless linking technique, and the DNA fragments ligand was transformed into E. coli competent cells (DH5α). After amplification, plasmid DNA was extracted. The recombinant plasmid containing the full-length genome of VS-FCV SH/14 strain was obtained and named as pKS-SH14 (Fig.1A). To analyze the effect of the length of poly (A) tail on rescue viral infectivity, primers (HM87-21K) containing different numbers of T bases were used to construct several infectious clones with a poly (A) tail containing 24 A, 27 A, 29 A, 30 A and 38 A respectively. These infectious clones are named as pKS-SH14A24, pKS-SH14A27, pKS-SH14A29, pKS-SH14A30, and pKS-SH14A38, respectively. All the recombinant plasmids were identified by DNA sequencing.
Table 1
Primers used in this study.
Fragments
|
Primers
|
Primer Sequences (5'-3')
|
Product length (bp)
|
SH I
|
SH-INF1
|
TTCCATTTCAGGTGTCGTGAGTAAAAGAAATTTGAGACAATGTCTCAA
|
2499
|
SH-INF2
|
AGCCCAGGCCAAATCAAACACCGAATTAACGG
|
SH II
|
SH-INF3
|
TGTTTGATTTGGCCTGGGCTCTTCGCCGT
|
2884
|
SH-INF4
|
TTTAAGCACGTTAGCGCAGGTTGAGC
|
SH III inf
|
SH-INF5
|
CCTGCGCTAACGTGCTTAAATATTATGATTGGGATCCCC
|
2406
|
SH-INF6
|
TGGAGATGCCATGCCGACCCTGGAGATGCCATGCCGAC
|
|
HM87-21KS
|
TGGAGATGCCATGCCGACCCTTTTTTTTTTTTTTTTTTTTTTTTTTT
|
RT primer
|
KS-HM
|
KS-HM1192
|
GGGTCGGCATGGCATCTC
|
5028
|
KS1171
|
TCACGACACCTGAAATGGAAGA
|
FCV-VP1
|
FCV-JD1
|
ACTACATTGTTGGTGAGGTGG
|
798
|
FCV-JD2
|
CTTGAAACACAAAGGGTCGAA
|
KSHLC
|
KSH LC1
|
GATGAGGCTGGAAAGATTTTCCA
|
12745
|
KSH LC2
|
CCAATGCATTGGTGGCACTA
|
MCHLC
|
mCherry LC1
|
TAGTGCCACCAATGCATTGGATGGTGAGCAAGGGCGAGG
|
826
|
mCherry LC2
|
AAAATCTTTCCAGCCTCATCTCTAGATCCGGTGGATCCCG
|
MCJD
|
mCherry1
|
ACTTCCTGAATTCGGGACTGT
|
885
|
mCherry2
|
TCGACTGCAGAATTCGAAGCT
|
SHd3UTR
|
SHd3UTR1
|
GGGTCGGCATGGCATCTCCACCT
|
1099
|
SHd3UTR2
|
GACCGAGTACAAGCCCACGGTGC
|
SHd3UTRinf
|
SHd3UTRinf1
|
GCAATTCGGCTATCATATAGGAATTTGTTTAAAATTTGAAAAAAAAAAA
|
1194
|
SHd3UTRinf2
|
ACAAGGAGACGACCTTCCATGACCGAGTACAAGCCCACGG
|
KSHd3UTR
|
KSHd3UTR1
|
ATGGAAGGTCGTCTCCTTGTGA
|
≥11547
|
KSHd3UTR2
|
CTATATGATAGCCGAATTGCATTTAA
|
The part in italics in primer sequence is homologous arm.
Construction of infectious clone carrying mCherry gene
To easily relate the effect of gene deletion or recombination on rescuing viruses, the mCherry gene was inserted into the LC coding region of FCV genome using the following strategy. Firstly, using pKS-SH14 as template, a pair of primers (KSH LC1 and KSH LC2) (Table 1) were used to amplify the KSHLC fragment by PCR,and the expected PCR product length was 12745bp; Using pmCherry-C1 plasmid as template, a pair of primers (mCherry LC1 and mCherry LC2) (Table 1) were used to amplify MCHLC fragment by PCR, and the expected length of PCR product was 826bp. Then, the purified KSHLC and MCHLC fragments were joined by ClonExpress®II One Step Cloning Kit (Nanjing Vazyme Biotechnology Co., LTD., China) using DNA seamless linking technique. After being transformed into E. coli competent cells (DH5α), a new recombinant plasmid named pKS-SH14mCherry was obtained (Fig.1B).
Construction of VS-FCV SH/14 mutant with deletion of 3' UTR
To investigate the relationship between 3' UTR and replication or infectivity of VS-FCV, VS-FCV SH/14 mutant with 3' UTR deletion was constructed. Specific methods are as follows: Firstly, pKS-SH14LC-Mcherry as template, PCR method was adopted to amplification KSHd3UTR fragment with primer KSHd3UTR1 and KSHd3UTR2 (Table 1). Using pKS-SH14 as template, SHd3UTR fragment were amplified by PCR with primer SHd3UTR1 and SHd3UTR2 (Table 1). Then, the fragment SHd3UTR as template, SHd3NCRinf fragment was amplified by PCR with primer SHd3UTRinf1 and SH d3UTRinf2 (Table 1). Since KSHd3UTR and SHd3UTRinf fragments have the same homologous arm, the DNA seamless connection technology can be applied. They were joined by ClonExpress®II One Step Cloning Kit (Nanjing Vazyme Biotechnology Co., LTD., China) by DNA seamless linking technique. After being transformed into E.coli competent cells (DH5α), a new recombinant plasmid named pKS-SH14mCherrydel3UTR with 3' UTR deletion was obtained (Fig.1B).
Rescue of progeny virus in CRFK cells by directly transfecting infectious clone
When CRFK reached a confluency of approximately 70%, the cells were transfected with 2~5 μg recombinant plasmid using Lipofectamine 2000 (Invitrogen). After 4 h, cells were replenished with fresh growth medium. The cells were then placed in an incubator containing 5% CO2 and continued to be cultured at 37 ℃ and observed.
Identification and characterization of rescued progeny virus by RT-PCR
The total RNA of the rescue recombinant viruses was extracted and purified using a RNeasy extraction kit (Qiagen). The first strand of cDNA was then synthesized using FCV-JD2 (Table 1) as a reverse transcription primer. Then primers FCV-JD1 and FCV-JD2 were used to amplify the partial sequence of VS-FCV VP1 by PCR. To identify the rescue rVS-FCV SH/14mCherry, primer mCherry1 and mCherry2 (Table 1) were used to amplify the partial mCherry gene from the cells infected with rescue virus by RT-PCR. Finally, the PCR products were analyzed and sequenced on 1.5% agarose gel.
Immunofluorescence Assay (IFA)
Cells pre-infected with recombinant virus were fixed in 3.7% paraformaldehyde in PBS (pH 7.5) at room temperature for 30 min and were subsequently permeabilized by incubation in methanol at -20 °C for 30 min. The fixed cells were blocked with 5% (w/v) nonfat milk in PBST buffer for 3 h at 4 °C and were then stained with a primary mAb specific of VS-FCV VP1 (1:500 dilution) for 2 h at 37 °C. After washed for three times for 10 min each, the cells were incubated with a secondary antibody against IgG conjugated to fluorescein isothiocyanate (FITC) (No. F0382, Sigma-Aldrich) in PBST buffer for 1 h at room temperature. Finally, after washed for three times for 10 min each, the samples were observed under a fluorescence microscope equipped with a video documentation system (ZEISS, Germany).
Electron microscopy detection of rescue viruses.
CRFK cell cultures were collected after infection with rVS-FCV SH/14 and rVS-FCV SH/14mCherry for 8 hours. Then the viruses were purified by differential centrifugation, resuspension and precipitation with PBS, conventional negative staining was performed, and the virions were observed under JEM-1200EX electron microscope and photographed.
Plaque assay
According to the method described in the references [20], plaque assay was carried out to determine the morphological biology of the parental and recombinant viruses. Firstly, a total of 1× 105 CRFK cells were seeded in 6-well plates for 12 h until about 90% confluent growth. Secondly, the medium was removed and washed three times with PBS. The virus suspension was inoculated onto the CRFK cells and incubated at 37 °C for 1 h. Thirdly, the cells were overlayed with 2 mL of 0.9% methylcellulose (Sigma M0512) in RPMI-1640 containing 2% FBS and 100 U/mL streptomycin/penicillin, and then incubated at 37°C for 2~3 days. Fourth, the methylcellulose was removed and the cells were fixed with 4% paraformaldehyde overnight at room temperature and stained with 0.2% Crystal Violet dissolved in 10% of ethanol and 90% of PBS. Finally, the crystal violet was washed off with water and dried naturally. The formation of the plaque was observed and photographed.
One-step growth curve
To compare the difference in replication capacity between the rescue virus and the parental virus, and to analyze the effect of 3' UTR deletion on VS-FCV replication, we measured the one-step growth curves of different viruses. In short, CRFK cells (1 × 106) were infected with VS-FCV SH/14, rVS-FCV SH/14 and rVS-FCV SH/14mCherry at a multiplicity of infection (MOI) of 5 TCID50 units. Cells were incubated with viruses at 37 °C for 1 h, then washed three times with 2 mL PBS to remove unbound viral particles. Extra 2 mL of fresh RPMI-1640 medium containing 2% FBS were added to each well. Supernatants were collected at 1h, 2h, 4h, 6h, 8h, 10h, 12h and 14h post infection (p.i.). The virus titers were titrated by TCID50 assay in triplicates and the one-step growth curves were formulated using GraphPad Prism 7.00 software.