Construction of eGFP-Tagged Senecavirus A for Facilitating Virus Neutralization Test and Antiviral Assay

Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus Senecavirus in the family Picornaviridae. The SVA CH-LX-01-2016 was isolated from Guangdong Province of China in 2016. In this study, a recombinant SVA CH-LX-01-2016 was constructed using reverse genetics, and proven to be able to express efficiently an enhanced green fluorescent protein (eGFP) in vitro. This eGFP-tagged recombinant SVA (rSVA-eGFP) exhibited a high capacity for viral replication. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay. The rSVA-eGFP-based VNT was used to detect eight porcine serum samples, out of which four were determined to be neutralization titer-positive. Subsequently, two antiviral drugs, ribavirin and apigenin, were assayed for evaluating both effects against the rSVA-eGFP in vitro. The result showed that only the ribavirin exhibited an anti-SVA activity.


Introduction
Senecavirus A (SVA), previously known as Seneca Valley virus, is an emerging pathogen in China [1,2]. It is a non-enveloped, positive-sense and single-stranded RNA virus, and classified into the genus Senecavirus in the family Picornaviridae. The SVA was incidentally isolated from the PER. C6 (transformed fetal retinoblast) cell line in 2002, and had evidently been introduced into the cell culture through the use of contaminated fetal bovine serum or porcine trypsin [3]. SVA infection is characterized by porcine vesicular lesions on coronary bands, snout and oral cavities, clinically indistinguishable from those caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus and vesicular stomatitis virus [4,5].
Although the SVA has been silently circulating in pig herds of the USA since 1988, SVA-caused vesicular disease was not identified in pigs until 2007 in Canada [6]. The second case of SVA infection was identified in the USA in 2010 [7]. At the end of 2014 and the beginning of 2015, increasing outbreaks in weaned and adult pigs were reported in different geographical regions of Brazil [3,8]. Afterwards, SVA emergences were reported in more countries, including China [2], Thailand [9], Vietnam [10] and Colombia [11]. Since its emergence in China in 2015, SVA has affected several provinces, such as Guangdong [12], Fujian [13], Hubei [14] and Heilongjiang [15]. SVA has only one serotype and is a unique species in the genus Senecavirus [16]. SVAs are icosahedral virions without envelopes. The diameter of the virus particles is approximately 27 nm. The SVA prototype, SVV-001 strain (Genbank access No.: NC_011349), has an approximately 7300-nt-long genome that contains a 5 untranslated region (UTR) of 666 nt, followed by a single long open reading frame (ORF) consisting of 6543 nt, encoding a 2181 aa polyprotein. This polyprotein can potentially be cleaved into 12 polypeptides in the standard picornavirus "L-4-3-4 (L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D)" layout. The L region encodes the leader protein that can be cleaved from the nascent polyprotein precursor. The P1 region codes for four structural proteins (VP4, VP2, VP3, and VP1): the P1 polypeptide is cleaved by the 3C protease to form VP0, VP3 and VP1, and subsequently a maturation cleavage of VP0 occurs to form the VP2 and the internally located VP4. The P2 and P3 regions encode three (2A, 2B and 2C) and four (3A, 3B, 3C and 3D) nonstructural proteins, respectively. A 3 UTR of 71 nt is followed by a poly(A) tail of unknown length [17].
Reverse genetics as a platform can be used for genetic manipulation of viral cDNA to construct desired virus phenotype. Different SVA strains have been used to develop reverse genetics systems for studying viral pathogenesis [18], oncolytic activity [19] and recombination mechanism [20]. The SVA CH-LX-01-2016 was an isolate, emerging in Guangdong Province of China in 2016 [21]. Its reverse genetics system has been constructed previously at our laboratory, and proven to be efficient for rescuing recombinant SVAs to express foreign proteins. In this study, we constructed a recombinant SVA (rSVA) that could express an enhanced green fluorescent protein (eGFP) in cells. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay.

Construction of Plasmid
The rSVA cDNA clone (see Subheading 2.1) was modified to construct another one, rSVA-eGFP cDNA clone ( Figure 1A), containing an eGFP ORF (Genbank access No.: KY295913) but without a stop codon at its 3 end, which was fused with a Thosea asigna virus 2A (T2A) sequence (Genbank access No.: JN717245). The eGFP-T2A fusion sequence was inserted between the 2A and the 2B of rSVA cDNA clone using an In-Fusion ® Cloning kit (Takara, Dalian, China). The rSVA-eGFP cDNA sequence was flanked by the T7 promoter and the Not I recognition sequences at its 5 and 3 ends, respectively. In order to enhance the transcription efficiency of T7 promoter, three guanine (G) residues were added to its 3 end [23].

Rescue of rSVA-eGFP
The rSVA-eGFP cDNA clone was linearized by digestion with Not I restriction endonuclease, followed by agarose gel electrophoresis for extraction of the linearized plasmid from gel by a gel extraction kit (TIANGEN, Beijing, China). The purified cDNA was used for transfection of BSR cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) according to the manufacturer's instruction. Briefly, BSR cells were seeded into a 12-well plate 1 d before transfection. Twenty-four hours later, a cell monolayer at 70-90% confluency was transfected with the linearized cDNA clone (1.5 µg/well). Transfected cells were cultured at 37 • C with 5% CO 2 in DMEM supplemented with 10% FBS, and observed by a fluorescence microscope at 24, 48 and 72 h post transfection (hpt). The BSR cell culture was harvested at 72 hpt, and subjected to one freeze-and-thaw cycle to collect supernatant for five serial blind passages in BSR cells.  Figure 1. Construction of rSVA-eGFP cDNA clone for rescuing eGFP-expressing recombinant SVA. Schematic representation of rSVA-eGFP cDNA clone (A). The eGFP-T2A fusion sequence is inserted between the 2A and 2B genes in the backbone of rSVA cDNA clone, flanked by the T7 promoter and the Not I recognition sequences at its 5 and 3 ends, respectively. Two pairs of primers, F1/R1 and F2/R2 (black arrow-marked), are designed for RT-PCR analyses on rSVA-eGFP genome and on foreign sequence, respectively. Schematic representation of two self-cleavage sites (red arrow-marked) in the rSVA-eGFP polyprotein (B). Agarose gel electrophoresis of Not I-linearized rSVA-eGFP cDNA clone (C). The rSVA-eGFP cDNA clone-transfected BSR cell monolayers at 24, 48 and 72 hpt (D). BSR cell monolayers infected with the rescued rSVA-eGFP at P1, P2, P3 and P4 at 24 hpi (E). BF: bright field (the same as following figures).

RT-PCR Analysis
The culture supernatant of rSVA-eGFP at passage 5 (P5) was harvested for extracting viral RNA by a Viral RNA/DNA Extraction Kit (Takara, Dalian, China). The extracted RNA was used as template for RT-PCR analysis using a High Fidelity One Step RT-PCR Kit (Takara, Dalian, China). The forward/reverse primers, F1/R1 (Table 1 and Figure 1A), were designed for amplifying a 693-bp fragment. The RT-PCR reaction underwent 45 • C for 10 min, 94 • C for 2 min and then 30 cycles at 98 • C (10 s), 55 • C (15 s) and 68 • C (10 s). The extracted RNA was simultaneously subjected to PCR analysis as a control using the F1/R1 primer pair. The PCR reaction contained 2 × PrimeSTAR Max Premix (Takara, Dalian, China) and underwent 30 cycles at 98 • C (10 s), 55 • C (5 s) and 72 • C (10 s). RT-PCR and PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing for analyzing the RT-PCR product.

Indirect Immunofluorescence Assay (IFA) and Ultrathin Section
BSR cells were infected (MOI = 1) with the rSVA-eGFP for 12 h, and then fixed in 4% paraformaldehyde at room temperature for 30 min. After fixation, cells were washed four times with PBS, and then permeated with 0.4% Triton X-100 at room temperature for 30 min. After permeation, cells were washed three times with PBS and blocked in blocking solution (Takara, Dalian, China) at 37 • C for 1 h. Subsequently, cells were incubated with the anti-VP1 monoclonal antibody (MAb, 1: 400 in blocking solution) at 37 • C for 2 h. After incubation with the primary antibody, cells were washed three times with PBS and incubated with the Alexa Fluor ® 555 conjugate (Thermo Fisher, Waltham, MA, USA) (1: 250 in blocking solution) at 37 • C for 1 h. Cells were washed three times with PBS, coated with 90% glycerin, and visualized under the fluorescence microscope.
BSR cells were infected (MOI = 1) with the rSVA-eGFP for 12 h, and then collected to prepare ultrathin section for transmission electron microscope (TEM) observation at the Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Table 1. Two pairs of primers for RT-PCR analyses on enhanced green fluorescent protein-tagged recombinant SVA (rSVA-eGFP).

Primers
Sequences ( BSR cells were seeded into ten 35-mm dishes (7 × 10 5 cells/dish) for culture at 37 • C for 2 h, and then all cell monolayers were inoculated (MOI = 1) with the rSVA-eGFP for further incubation at 37 • C. The dishes were randomly removed from the incubator per hour for observation of green fluorescence until all dishes were removed.

Plaque Assay
Serial 10-fold dilutions were made by mixing 120 µL of rSVA-eGFP with 1080 µL of DMEM. One mL of each dilution was seeded to individual wells of a 6-well plate containing confluent BSR cells. The plate was incubated at 37 • C for 1.5 h before the layer of 1% low melting point agarose (in DMEM with 10% FBS) was added. After 3 d incubation at 37 • C, fluorescent cytopathic effects (CPE) on cell monolayers were observed by the fluorescence microscope. The cell monolayers were fixed in 4% paraformaldehyde and then stained with 0.2% crystal violet. Plaque morphology was observed after washing the plate with tap water, and compared with that of the rSVA.

Growth Curve
Growth kinetics of the P5 rSVA-eGFP were compared with that of the P5 rSVA in vitro. Briefly, BSR cells were plated into four 35-mm dishes (2 × 10 6 cells/dish) for incubation at 37 • C for 2 h. The rSVA-eGFP was inoculated into all dishes (MOI = 0.001) for incubation at 37 • C for 2 h, and then supernatants were replaced with DMEM for further incubation at 37 • C. At 0, 24, 48, 72 h post infection (hpi), any of dishes was randomly removed from the incubator and subjected to one freeze-and-thaw cycle to collect supernatant for viral titration by 50% tissue culture infective dose (TCID 50 ) assay. The viral titer was calculated by the Spearman-Kärber equation [24].

VNT of Porcine Sera
Eight serum samples, collected from SVA-infection-like pigs, were kindly provided by Dr. Bo Ni, China Animal Health and Epidemiology Center, and by Dr. Guimei Li, Qingdao Agricultural University. A standard VNT was carried out in two 96-well plates, as described in literature [25] with a minor modification. The experiment was independently repeated three times. Briefly, the serum was diluted 1:5 after heat-inactivation at 56 • C for 30 min, and then was two-fold serially diluted (1:10 to 1:1280) in DMEM. One hundred µL of rSVA-eGFP (10 3 TCID 50 / mL ) was mixed with 100 µL of a given dilution of serum in wells of a 96-well plate, eight replicate wells per dilution. The last three columns were 8-well positive control (positive serum mixed with 100 TCID 50 rSVA-eGFP), negative control (negative serum mixed with 100 TCID 50 rSVA-eGFP) and mock (virus-and serum-free DMEM), respectively. Besides these three controls, a series of 8-well rSVA-eGFP controls without serum were arranged in the first four columns of another 96-well plate as follows: 100 TCID 50 /well, 10 TCID 50 /well, 1 TCID 50 /well and 0.1 TCID 50 /well.
All 96-well plates for VNTs of eight sera were incubated at 37 • C for 1.5 h. Cell suspension was added to each well (3 × 10 4 cells/well), and then all plates were incubated at 37 • C for 48 h for the serum titration. If the rSVA-eGFP was correctly diluted in the rSVA-eGFP control wells, green fluorescence would be emitted from all wells with 100 and 10 TCID 50 /well dilutions, from approximately half the wells with 1 TCID 50 /well dilution, but not from the wells with 0.1 TCID 50 /well dilution. The neutralization titer was the dilution of serum that neutralized rSVA-eGFP in half the wells of a given column. A neutralization titer of ≥ 20 would be regarded as neutralization titer-positive. The concentration of ribavirin that reduced the viability of BSR cells to 50% of the control was estimated as the 50% cytotoxic concentration (CC 50 ). The MTT assay was used to determine BSR cell viability. Briefly, BSR cells were seeded in a 96-well plate at a density of 2 × 10 4 cells/well, and incubated at 37 • C for 3 h. Supernatants were replaced with DMEM containing different concentrations of ribavirin (0, 8, 16, 32, 64, 128, 256, 512, 1024 and 2048 µM) (Solarbio, Beijing, China), five replicate wells per dilution. The ribavirin-treated cells were incubated at 37 • C for 24 h. Subsequently, MTT (Sangon, Shanghai, China) solution was added to each well in a final concentration of 0.5 mg/mL, and then the 96-well plate was incubated at 37 • C. After 4 h incubation, supernatants were replaced with dimethyl sulfoxide (DMSO, 100 µL/well) to dissolve violet formazan crystals. The plate was read using a microplate reader at 570 nm wavelength. The CC 50 value for ribavirin was calculated by nonlinear regression fitting using the GraphPad Prism software (Version 7.0).
Based on the result of cytotoxicity assay, the ribavirin was tested for its antiviral activities at different concentrations. Briefly, BSR cells were seeded in a 24-well plate at a density of 10 6 cells/well and incubated at 37 • C for 3 h. Subsequently, the cells were inoculated with rSVA-eGFP (MOI = 0.1) and incubated at 37 • C for 1.5 h. Supernatants were replaced with DMEM containing different concentrations of ribavirin (0, 4, 8, 16, 32 and 64 µM). After incubation at 37 • C for 24 h, ribavirin-treated cells were observed by the fluorescence microscope. The 24-well plate was subjected to two freeze-and-thaw cycles to collect supernatants for TCID 50 assays of rSVA-eGFP. Antiviral activity of ribavirin was expressed as the 50% effective concentration (EC 50 ), which was calculated by nonlinear regression fitting using the GraphPad Prism software to determine the drug concentration required to achieve 50% of viral titer reduction [26].

Statistics
Experiments were performed three times with reproducible results. The GraphPad Prism software was used for statistical analysis by two-tailed Student s t-test. The data shown are the means ± standard deviations (SD) of three independent experiments. Statistical significance: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Construction and Characterization of rSVA-eGFP cDNA Clone
The backbone of rSVA-eGFP cDNA clone was the rSVA (CH-LX-01-2016) cDNA clone. As shown in Figure 1A, the eGFP-T2A fusion sequence was inserted between the 2A and 2B sequences of the rSVA cDNA clone by the In-Fusion ® recombineering technology. To facilitate linearization of the recombinant plasmid, the Not I recognition sequence was added to the 3 end of rSVA-eGFP cDNA clone. During translation of the rSVA-eGFP polyprotein, the ribosome initially skipped at the "TNPG↓P" motif of SVA 2A protein, and then did once again at another "TNPG↓P" motif in the T2A protein. Two consecutive events of ribosomal skipping led to releasing an eGFP, flanked by one extra N-terminal proline and a C-terminal T2A cleavage product ( Figure 1B).

Rescue and Passaging of rSVA-eGFP
The Not I-linearized rSVA-eGFP cDNA clone was subjected to electrophoresis ( Figure 1C) and purification for transfection of BSR cells. At 24 hpt, a small number of cells had begun to emit green fluorescence, and the proportion of fluorescence-emitting cells was growing over time ( Figure 1D). The cDNA-transfected cells were harvested at 72 hpt, and subjected to one freeze-and-thaw cycle to collect supernatant for serial blind passages, during which the eGFP was always expressed in BSR cells ( Figure 1E). Meanwhile, virus-infected cell monolayers showed typical CPEs, characterized by rounding and detachment of cells from flask ( Figure 1E, BF).

RT-PCR
Total RNA was extracted from the P5 rSVA-eGFP at 24 hpi ( Figure 2A) and analyzed by RT-PCR to confirm its identity. An expected band of 693-bp amplicon was observed only on the RT-PCR lane by agarose gel electrophoresis ( Figure 2B, Lane RT-PCR). The PCR control ( Figure 2B, Lane PCR) indicated no potential cDNA contamination affecting the RT-PCR analysis. The Sanger sequencing showed that the P 5-based RT-PCR product was identical to the 693-bp sequence.

IFA
To assess expression of the SVA VP1, the IFA was performed using the VP1 MAb and the Alexa Fluor ® 555 conjugate. Bright red fluorescence was visible on the virus-infected cell monolayer but not on the mock cells. Serial treatments (4% paraformaldehyde, 0.4% Triton X-100 and so on) for cells by the IFA did not significantly affect eGFP activity in cells, and most cell contours with red and green fluorescence could be merged together ( Figure 2C).

Ultrathin Section
The TEM observation of ultrathin section revealed a typical morphological criterion of picornaviruses [27][28][29] in an rSVA-eGFP-infected cell. Virus particles, unsure whether or not fully matured, appeared as round-, oval-or hexagon-shaped opaque bodies, closely packed together and surrounded by a small mass of light-colored material in a cellular vesicle ( Figure 2D). These opaque bodies inside the cell showed an average diameter of 20 nm, less than that (27 nm) of purified SVA virions by CsCl-gradient centrifugation [17].

Timeline of Fluorescence Appearance
The rSVA-eGFP was inoculated into BSR cells (MOI = 1) to observe fluorescence appearance from 0 to 10 hpi. The result showed that virus-infected cells began to emit green fluorescence at 6 hpi. Fluorescence became increasingly bright from 6 hpi to 9 hpi, and was relatively stable after 9 hpi ( Figure 2E).

Plaque Assay
After 3 d incubation at 37 • C, a typical CPE with fluorescence was apparent on the cell monolayer ( Figure 2F). Plaque morphology of the rSVA-eGFP was further determined by plaque assay. A single plaque was visible on the cell monolayer infected with the rSVA-eGFP at the highest dilution, morphologically similar to those induced by its parental virus (rSVA), but slightly blurred and not easily distinguished from a blue background ( Figure 2G).

Growth Curve
At the indicated time points, virus-infected cell cultures were frozen and thawed once to collect supernatant for titration in BSR cells. The growth curve of the P5 rSVA-eGFP was compared with that of the P5 rSVA ( Figure 2H). Both viruses, albeit identified as identical initial titers at 0 hpi, showed a slight difference in growth kinetics during 72 h. Their final titers were similar to each other at 72 hpi.

eGFP Stability during Passaging
Fluorescence intensity inside virus-infected cells was gradually weakening after the tenth rSVA-eGFP passage. Especially in the P16-infected cell monolayer, there were only sporadic cells that could emit green fluorescence ( Figure 3A, P16). The F1/R1-based RT-PCR showed that although the level of eGFP expression was gradually weakening after the tenth passage, the 693-bp-specific product was successfully amplified from all viruses (P6, P8, P10, P12, P14 and P16) ( Figure 3B). However, the other RT-PCR analysis using the F2/R2 primer pair revealed that the brightness of 1004-bp-specific band ( Figure 3C, yellow arrowhead-marked) was gradually decreasing from the P6 to P10, and finally unobservable from the P12 to P16, whereas another band ( Figure 3C, red arrowhead-marked) instead gradually became bright from the P12 to P16.
All six products of the F2/R2-based RT-PCR were subjected to Sanger sequencing, indicating that a 597-bp sequence ( Figure 3D, black letters) was deleted from the eGFP ORF from P12 to P16. Consequently, the eGFP ORF only retained a 120-bp fragment ( Figure 3D, red letters), out of which nine nucleotides derived from the eGFP 5 end and the others derived from its 3 end. The P16 sequencing chromatogram was shown in Figure 3E, in which the black arrow illustrated a possible site, where the 597-bp sequence was deleted.

rSVA-eGFP-Based VNT
The sera were collected from rSVA-immunized mice for VNT. The result showed that the formalin-inactivated rSVA elicited a high-titer neutralizing antibody response in most mice. Neutralization titers ranged from 320 to 1280, and all pre-immunization sera were negative. Sera with neutralization titer greater than 640 could be used as positive controls for developing the rSVA-eGFP-based VNT in 96-well plates. Figure 4A was a schematic representation of 96-well plate format designed for performing rSVA-eGFP-based VNT. Eight serum samples were collected from pigs with suspected SVA infection, and then analyzed by the developed VNT. The result showed that four samples (S1, S2, S3 and S4) were positive for rSVA-eGFP neutralization, and the others (S5, S6, S7 and S8) had no neutralizing activity ( Figure 4B).

Ribavirin
The effect of ribavirin on BSR cell viability was determined by the MTT assay ( Figure 5B). The CC 50 value was 58.2 µM for the ribavirin (Figure 5C), calculated by nonlinear regression fitting using the GraphPad Prism software. Based on the CC 50 value, the ribavirin was tested for its antiviral activities at different concentrations. Ribavirin-treated cell monolayers were observed at 24 hpi in a randomly selected field-of-view of fluorescent microscope. As the ribavirin concentration increased, there was a gradual decrease in the proportion of green cells ( Figure 5A). All ribavirin-treated cell cultures were titrated by TCID 50 assay at 24 hpi. Like observation under the fluorescence microscope ( Figure 5A), both the absolute titer ( Figure 5D,E) and the relative titer ( Figure 5F,G) gradually decreased with the increase in ribavirin concentration. The antiviral effect of ribavirin was quantitatively measured by determination of the EC 50 value, estimated at 3.45 µM ( Figure 5G). Effectiveness of an antiviral drug was generally described in terms of the selectivity index (SI, CC 50 /EC 50 ), which was 16.87 in this study.

Apigenin
Apigenin was dissolved in DMSO for preparing its stock solution. Cytotoxicities of apigenin ( Figure 6B) and DMSO ( Figure 6D) were separately determined in BSR cells by the MTT assay. The CC 50 values for apigenin and for DMSO were 80.02 µM ( Figure 6C) and 0.10% ( Figure 6E), respectively, calculated by nonlinear regression fitting after the MTT assay. The apigenin was tested for its antiviral activities at different concentrations, and DMSO was used as controls at corresponding concentrations to those of apigenin. Apigenin-treated cell monolayers were observed at 24 hpi in a randomly selected field-of-view of fluorescent microscope.
However, unlike the ribavirin, the apigenin did not show gradual decreases in the proportion of green cells with serial increases in drug concentration ( Figure 6A). Interestingly, the mean size of fluorescence-emitting cells was larger in the 118.4 µM apigenin group than those in the other groups. In comparison with other DMSO controls, the group of 0.128% DMSO revealed a notable reduction in the proportion of green cells ( Figure 6A). All apigenin-and DMSO-treated cell cultures were titrated by TCID 50 assay at 24 hpi, showing that, instead of inhibiting rSVA-eGFP proliferation, all apigenin-treated groups exhibited high viral titers, compared with their DMSO controls ( Figure 6F,G). Although the titer decreased dramatically in the 118.4 µM apigenin group, this concentration (118.4 µM) was higher than the CC 50 value (80.02 µM) of apigenin.

Discussion
SVA infection was an emerging disease that recently affected numerous pig farms in different provinces of China. The SVA CH-LX-01-2016 was an isolate that had emerged in Guangdong Province of China in 2016 [21]. Its reverse genetics system has been constructed previously at our laboratory, and proven to be efficient for rescuing recombinant SVAs to express foreign proteins, such as luciferase. In this study, we modified the cDNA clone of SVA CH-LX-01-2016 to rescue successfully the eGFP-expressing recombinant virus for facilitating VNT and antiviral assay.
Inserting a foreign gene into the SVA cDNA clone is the first step to rescue a recombinant virus. In this study, the eGFP-T2A fusion sequence was inserted between SVA 2A and 2B genes to construct the rSVA-eGFP cDNA clone ( Figure 1A). The T2A peptide can mediate "cleavage" of a polyprotein during its translation in eukaryotic cells [30], accordingly releasing an eGFP-T2A fusion protein that should not obviously affect functions of other SVA proteins. Generally, it was not difficult to construct a recombinant cDNA clone, whereas we found that both the rSVA-eGFP and the rSVA cDNA clones were often unstable in E.coli JM109 for an unknown reason. Therefore, recombinant cDNA clones should be extracted immediately from E.coli once they have been identified by PCR analysis.
In vitro transcription of RNA genome is a common way to rescue recombinant picornaviruses [31][32][33][34][35]. The BSR cell line is permissive to SVA infection, and can provide the T7 RNA polymerase to initiate RNA transcription. Therefore, in this study, BSR cells were directly transfected with the Not I-linearized rSVA-eGFP cDNA clone, instead of with in vitro transcription products. The primary transcription product was flanked by "GGG" at its 5 end and possible Not I-digested residues at its 3 end, both of which did not affect rescue of the rSVA-eGFP, and with replication of the genome RNA in cells, both of which would probably be selectively "ignored" to generate correct 5' and 3' termini. When recovered from the cDNA clone, the P0 virus showed a weak adaptability in BSR cells. This cell-adapted ability was gradually improved with serial passaging in vitro. Green fluorescence was always visible during passaging (P0 to P5), and meanwhile the rSVA-eGFP gradually improved itself in growth performance to evolve a high-fitness phenotype in cells.
The identity of rSVA-eGFP was analyzed by RT-PCR, IFA and ultrathin section at the P5. Although the green fluorescence always appeared on cell monolayers during passaging, the RT-PCR analysis was still necessary for detecting recovery of recombinant virus. Meanwhile, PCR as a control was indispensable for eliminating a possible interference of residual cDNA clone. The morphological analysis of SVA was based on the ultrathin section, showing a number of opaque body-like virions, probably mixed with procapsids [36], closely packed together inside a cellular vesicle. Such an intracellular viral morphology was different from that of virions purified from culture supernatant.
Expression of eGFP by the rSVA-eGFP was initially observable at 6 hpi, and was relatively stable after 9 hpi, while the typical CPE would usually appear after 12 hpi. In order to observe complete cell morphology, IFA should be performed during 10 to 12 hpi. In this study, the P5 rSVA-eGFP was chosen for comparing its growth curve with that of its parental virus (P5 rSVA). Based on a quasispecies theory of RNA viruses [37], the same number of viral passages is a prerequisite for comparing their growth kinetics. The eGFP-T2A sequence is a nonstructural gene during rSVA-eGFP replication, and therefore its insertion theoretically affects viral growth kinetics to some extent. Actually, both viruses showed only a slight difference in their growth curves ( Figure 2H).
The eGFP-expressing picornaviruses are generally unstable and prone to losing their foreign sequences by a recombination mechanism [20,38]. In this study, the eGFP could be stably expressed in BSR cells up to approximately eight virus passages. Likewise, our previous research showed that a luciferase-expressing SVA could maintain luciferase expression only for nine passages. Owing to the unstable characteristics of eGFP during serial passaging, the rSVA-eGFP was generally restricted to three to five passages for VNT and antiviral assay.
Conventional VNT, albeit time-consuming, is sensitive and specific, thus serving as a prescribed test for international trade of some animal diseases. As a diagnosis of SVA infection, both specificity and sensitivity of the conventional VNT were slightly higher than those of the competitive ELISA [39]. In this study, the rSVA-eGFP-based VNT shortened the incubation period of 96-well plate to 48 h, much less than that of the conventional VNT. Moreover, this novel method was more sensitive and specific than the conventional one using wild-type SVA, because a final VNT result of readout would depend on green fluorescence, instead of CPE. According to our previous study, wild-type SVA-caused CPE was generally too unapparent in some wells to determine a correct result via the conventional VNT. More importantly, since there was only one serotype of SVA worldwide, the novel VNT could be broadly used for clinical diagnosis.
A crucial aspect of the VNT is that the rSVA-eGFP inoculum should be maintained at approximately 100 TCID 50 /well. If significantly greater or less than 100 TCID 50 /well, the final readout for neutralization titer would be correspondingly less or greater than an actual value. Therefore, serial inoculum controls (100, 10, 1 and 0.1 TCID 50 /well) must be arranged in a 96-well plate ( Figure 4A, column 13 to 16). The developed VNT was used for detecting eight serum samples, out of which four sera were diagnosed neutralization titer-positive, implying SVA endemic in a given area of China.
A variety of eGFP-tagged viruses have been widely used for antiviral screening [26,[40][41][42][43][44]. In this study, the rSVA-eGFP was also proven to be a fast and efficient tool for antiviral assay. The effect of antiviral drug could be visually displayed by variation in eGFP intensities, and then would be quantified by the TCID 50 assay, by which the decrease in virus titer could be determined 48 h later. Such a time-saving TCID 50 assay has a great potential in high-throughout screening of anti-SVA drug candidates.
Ribavirin is a synthetic nucleoside analogue that structurally resembles guanosine [45], and can interfere with the synthesis of viral mRNA. It has been demonstrated that ribavirin is a common anti-picornavirus drug with low cytotoxicity [46][47][48][49][50]. Its antiviral effect was generally described in terms of the SI value. A high SI value indicates that a given drug preserves cell viability by reducing the level of pathogenic viruses, while low cytotoxicity is observed [51]. A SI value ≥ 4 is generally considered suitable for an antiviral drug [52]. The SI value was 16.87 for ribavirin in this study, suggesting that it was an effective anti-SVA drug with a relatively safe profile.
The other drug analyzed in this study was apigenin, a plant-derived flavonoid, which was practically insoluble in water, but soluble in DMSO. The apigenin has been demonstrated to have the ability to inhibit replication of some picornaviruses, such as FMDV [53] and enterovirus-71 [54,55]. However, we did not find its anti-SVA effect by the rSVA-eGFP-based antiviral assay. Interestingly, in comparison with their corresponding DMSO controls, the apigenin-treated groups exhibited high virus titers instead ( Figure 6F,G).
In conclusion, the rSVA-eGFP was constructed here using the developed platform of reverse genetics. The eGFP could be efficiently expressed in BSR cells but was relatively unstable during serial passaging. Its green fluorescence-tracked characteristics made the rSVA-eGFP-based TCID 50 assay faster and more accurate than the conventional method did so, thus having a great potential in VNT and antiviral assay. Using the rSVA-eGFP, a total of four serum samples were diagnosed neutralization titer-positive, and the ribavirin was demonstrated to possess anti-SVA activity.