Specimen collection and treatment
The lung tissue sample was obtained from one diseased piglet with clinical symptoms of severe respiratory disease in Henan province, China. The tissue sample was chopped, homogenized with Dulbecco’s modified Eagle’s high glucose medium (DMEM), and subjected to freezing-thawing cycles. After centrifugation, the supernatants were obtained for pathogens detection and viral isolation.
Swine pathogens detection
The viral genome was extracted from the supernatants using a commercial viral DNA/RNA extraction kit (TaKaRa,Dalian, China). Viral cDNA was generated using the revert aid first stranded cDNA synthesis kit (Thermo Fisher scientific, USA). The presences of potential pathogens leading to severe respiratory disease in piglets including PCV2, PCV3, Pseudorabies virus, Classical swine fever virus, and swine influenza virus, etc. were detected by real-time PCR assays using commercial kits (Wuhan Keqian Biology Co., Ltd, Wuhan, China), respectively.
Viral isolation and identification by indirect immunofluorescence assay
PRRSV isolation on marc-145 cells was performed according to previous studies [11–12]. Briefly, the supernatants of lung tissue were passed through 0.22 µm filters (millipore, Billerica, MA, USA). Subsequently, monolayers of marc-145 cells were incubated with the supernatants for 2 h, which were cultured with cell maintenance media (DMEM contained with 2% fatal bovine serum (Gibco, USA), 100 ug/mL streptomycin, and 100 IU/mL penicillin) in a 37℃ incubator containing 5% CO2. The cells and supernatants were harvested until the obvious cytopathic effects (CPEs) were monitored, and subjected to three freezing-thawing cycles. Finally, the supernatants were obtained and the infectious virus was purified via plaque purification assay on Marc-145 cells [11].
The infectious viruses (designed as HeN-ZZ) were further identified via indirect immunofluorescence assay (IFA). Briefly, monolayers of marc-145 cells were cultured in 6-well plates and infected with the purified viruses. After 24 hours post infection, cells were fixed with the 4% paraformaladehyde for 15 min, and then permeabilized with 0.1% TritonX-100 for 10 min. Subsequently, the cells were incubated with 3% BSA for 1 h, and then incubated with the primary PRRSV N antibody (1:2000) overnight at 4℃. The cells were washed with PBS for five times and incubated with the goat anti-rabbit IgG secondary antibody (1:3000) for 1 h in a dark box. Finally, the cells were washed with PBS for five times and observed using the fluorescence microscope.
Full-length genome sequencing, and biological characteristics analysis
The viral RNA genome of PRRSV HeN-ZZ strain were obtained using the TRIzol Reagent according to the manufacturer’s instructions. Subsequently, the RNA genome was sent to Shanghai Tanpu Biotechnology Co., Ltd for next-generation sequencing on the Illumina iseq 100 platform (2×150 bp). Finally, the complete PRRSV genome sequences including the 5’ and 3’ ends was assembled according to the PRRSV reference strains.
Twenty full-length PRRSV genome sequences with different lineages were download from the NCBI database (Supplementary Table 1). the nucleic acid sequences of HeN-ZZ (GenBank accession number OR250810) and the reference PRRSV strains were aligned for comparison via DNAStar software. Phylogenetic tree was generated on the basis of the complete genome sequences of HeN-ZZ and the reference strains to explore the evolutionary position of HeN-ZZ strain, using the neighbor-joining method (Bootstrap = 1000) in MEGA 7.0 software. Moreover, the potential recombinant events occurred in the genome of HeN-ZZ strain were analyzed using the SimPlot version 3.5.1 recombinant analysis software.
MTT assay
The cytotoxicity of ASP (sigma, USA) to marc-145 cells was analyzed via MTT assay. Briefly, nearly 60% confluence of marc-145 cells in 96-well plate were exposed to different concentrations of ASP or DMEM for 48 h. 10 µL of MTT solution was added into each well and incubated for 4 h. Subsequently, the supernatants were removed, the cells were incubated with DMSO (150 µL) for 10 min. Finally, the relative cell activity of marc-145 cells in each well was measured via determining the absorbance value (OD490 nm) using a spectrophotometer according to a previous study [15].
RT-qPCR
Total RNA was extracted from the cells using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Subsequently, equal amounts of RNA (nearly 1 µg) were reverse transcripts into cDNA using a commercial kit (Thermo Fisher scientific, USA). Real-time PCR(qPCR) assay was performed to determine the relative mRNA levels of PRRSV ORF7, IL-6, IL-8, TNF-α, and IL-1α genes using the GAPDH gene as a endogenous control, the primer information was shown in Supplementary Table 2.
TCID50 assay
Nearly 60% confluence of marc-145 cells seeded in 96-well plates were infected with the serial 10-fold dilations of the virus in six replicates. After five days incubation, viral titer were calculated via observing the cytopathological changes, and determined based on the Reed-Muench method, which was shown as the 50% tissue culture infectious dose (TCID50)/mL.
The effects of ASP on PRRSV infection in vitro
Nearly 70% confluence of marc-145 cell in 6-well plate were pre-treated with different concentrations of ASP or DMEM for 2 h, and then infected with HeN-ZZ strain (MOI = 0.1). After incubation for 2 h, the cells were treated with the corresponding concentrations of ASP before removing the supernatants. At the 24, 48, and 72 hours post infection (hpi), the supernatants were collected for determining the viral titers, and the cells were obtained to analyze the relative mRNA levels of PRRSV ORF7 gene via RT-qPCR.
The model of antiviral action assays
Marc-145 cells were seeded in 6-well plates and divided into four groups to investigate the antiviral steps of ASP against PRRSV infection.
Inactivation assay
106 TCID50 PRRSV were incubated with equal volume of ASP (20 µg/mL) at 37 ℃ for 2 h. Mar-145 cells were infected with the mixture at 37 ℃ for 2 h for virus entry, subsequently, the supernatants were replaced with the cell maintenance media. At 48 hpi, the cells were obtained to analyze the relative mRNA levels of PRRSV ORF7 gene via RT-qPCR.
Attachment assay
Marc-145 cells were pre-treated with DMEM or ASP (20 µg/mL) at 4 ℃ for 1 h, and incubated with 106 TCID50 PRRSV at 4 ℃ for 2 h for viral attachment. The supernatant was removed, and the maintenance media was added into the plate. At 48 hpi, the cells were subjected to analyze the relative viral mRNA level via RT-qPCR.
Entry assay
Marc-145 cells were incubated with 106 TCID50 PRRSV at 4 ℃ for 2 h for viral attachment. After removing the supernatants, cells were incubated with DMEM with or without ASP (20 µg/mL) at 37 ℃ for 2 h to permit virus entry. Then, the cells were washed with PBS for three times, and incubated with the maintenance media for 48 h. Finally, total RNA was extracted from the cells, and sent to analyze viral mRNA level via RT-qPCR.
Replication assay
Marc-145 cells were infected with 106 TCID50 PRRSV at 37 ℃ for 2 h to allow virus entry. Subsequently, the cells were washed with PBS, and incubated with the maintenance media with or without ASP (20 µg/mL) at 37 ℃ for 48 h. The viral mRNA level of cells was analyzed via RT-qPCR.
Statistical analysis
The GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA) was used for graph creation and analyzing the statistical significance of data between different groups with the t-test approach. P-value༜0.05 was considered statistically significant.