Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene

Journal Pre-proof Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene Jie Tao, Benqiang Li, YingShi, Jinghua Chen, Guoqiang Zhu, Xiaohui Shen, Huili Liu

PII:

S0166-0934(19)30496-3

DOI:

https://doi.org/10.1016/j.jviromet.2020.113842

Reference:

VIRMET 113842

To appear in:

Journal of Virological Methods

Received Date:

11 November 2019

Revised Date:

26 February 2020

Accepted Date:

29 February 2020

Please cite this article as: Tao J, Li B, YingShi, Chen J, Zhu G, Shen X, Liu H, Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene, Journal of Virological Methods (2020), doi: https://doi.org/10.1016/j.jviromet.2020.113842

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene

Jie Tao1,2,4, Benqiang Li1,2,4, YingShi1,2,4,Jinghua Chen1,2,4, Guoqiang Zhu3, Xiaohui Shen1,2,4, Huili

of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences,

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1Institute

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Liu1,2,4*

2Shanghai

Key Laboratory of Agricultural Genetic Breeding, Shanghai 201106, China

of Veterinary Medicine, and Jiangsu Co-innovation Center for Prevention and Control of

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3College

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Shanghai, China

Engineering Research Center of Pig Breeding, Shanghai 201302, China

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4Shanghai

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Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China

* Correspondence: Huili Liu

BeiDi Rd 2901, Institute of Animal Science & Veterinary Medicine, Shanghai Academy of

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Agricultural Sciences, Shanghai, China E-mail: [email protected] Tel: 86 021 62202473 Fax: 86 021 62207858

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Running Title

Highlights

An exogenous PEDV S antigen inserted between the seventh and eighth amino acids of the capsid gene in the

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BVDV genome could be expressed successfully.

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Abstract

Infectious bovine viral diarrhea virus (BVDV) cDNA clones have been used for the expression of classical swine

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fever virus (CSFV) genes for immune prevention and control. However, can it be used for the expression of an allogenetic fragment? To answer this question, a BVDV chimeric virus expressing the spike (S) antigen fragment of

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porcine epidemic diarrhea virus (PEDV) was constructed. Antigen S 499-602 was inserted into pig-derived BVDV-2 infectious cDNA clone pASH28 in tandem by overlapping PCR, located between the seventh and eighth amino acids at the N-terminus of the capsid (C) protein of BVDV. Indirect immunofluorescence assay confirmed that the chimeric virus vASH-dS312 containing double S499-602 sequences was successfully assembled, which could react with the monoclonal antibody (MAb) against BVDV E2 and PEDV S proteins. Further western blot analysis confirmed that the exogenous

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S499-602 double protein could be stably expressed. Next, the chimeric virus vASH-dS312 was administered to BALB/C mice either orally or by intramuscular injection. The immunized mice were healthy and showed no signs of toxicity. IgG against BVDV and PEDV antibodies could be detected in the mice administered vASH-dS312 by intramuscular injection, which had neutralization activity against BVDV and PEDV. Thus, this study reported a new insertion site in the BVDV infectious cDNA clone that could successfully express an allogenetic antigen.

Keywords: BVDV; pig; PEDV; chimeric virus; vaccine carrier; immunogenicity

Running Title

Because of efficient humoral and cellular immunity, various DNA viruses, such as poxvirus (Sánchez-Sampedro et al., 2015; Torres-Domínguez et al., 2019), adenovirus (Humphreys et al., 2018; Morris et al., 2016), herpes virus (Agelidis et al., 2019; Johnston et al., 2016), and vesicular stomatitis virus (Ruedas et al., 2017; Stein et al., 2019), have been used as vectors in vaccine research. Advancement of technology in vaccinology and immunology promoted the development of live attenuated RNA virus vaccines. Many RNA viruses have the potential to accommodate foreign genes as ideal vaccination vector candidates. Furthermore, the insertion and expression of foreign gene- based reverse genetic

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strategies will enhance and extend the function of these RNA virus vaccines in vaccinology and immunology. Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus in the Flaviviridae family with certain

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serological cross reactions (Saegerman et al., 2018). BVDV has a wide host spectrum, including bovine, pig, sheep, goat, and wild animals. Recently, BVDV infection in swine has often occurred due to contaminated fetal serum or cell-prepared

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vaccines (Gatto et al., 2018). Pigs infected with BVDV often present atypical clinical symptoms of growth retardation, diarrhea, and reproductive disturbance (Tao et al., 2013; Mósena et al., 2020). Notably, it can induce immunosuppression

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and persistent infection, thereby causing considerable economic loss (Deng et al., 2015). Reports have confirmed that BVDV can also be used as a recombinant virus vector to deliver exogenous genes and provide specific immune

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protection (Arenhart et al., 2016). Reimann et al.(2004) replaced the E2 gene of the CP7 strain of BVDV with the E2 gene of the Alfort/187 strain of CSFV, and the resultant chimeric virus could protect pigs from fatal CSFV infection

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(Reimann et al., 2004), which indicates that BVDV can be feasibly used as a vaccine platform. In addition, our previous study confirmed that swine infected with BVDV-2 strain SH-28 revealed no clinical symptoms or detoxification, which demonstrated that it might be used as a carrier of exogenous genes. Viral diarrhea is presently known to have an immense influence on the pig industry, and porcine epidemic diarrhea virus (PEDV) is one of the main pathogens causing porcine diarrhea (Sung et al., 2019). In recent years, the prevalence of

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PEDV has increased, and existing vaccines do not efficiently protect the immune system (Chen et al., 2019). The S protein of PEDV is a peplomer glycoprotein that is located on the surface of viral particles, and it can mediate the production of neutralizing antibodies, which implies that it is a target for vaccine research. Nevertheless, the S protein is a highly glycosylated protein, and in vitro expression of recombinant S protein usually results in the loss of immunogenicity (Sun et al., 2016). Therefore, in this study, we aimed to evaluate the efficiency of the infectious cDNA clone of pig-derived BVDV-2 (GenBank No. HQ258810.1) as a vector to express the PEDV S antigen.

Running Title First, using the PEDV strain (JS-2/2015, GenBank No. KX534206) as a research target, the fragment S499-602 was amplified using overlapping PCR and then inserted into the infectious cDNA clone pASH28 plasmid in tandem between the seventh and eighth amino acids at the N-terminus of the nucleocapsid (C) protein (Fig. 1). First, SphI-F and C-N7-SR1 primers were used to amplify the C-N7-S-1 fragment, and primers C-N7-S-F4/SphI-R were used to amplify C-N7-S-4 using pASH28 as the template. Second, C-N7-S-2 and C-N7-S-3 were amplified from the PEDV cDNA using C-N7-SF2/C-N7-S-R2 and C-N7-S-F3/C-N7-S-R3 primers, respectively. Then, C-N7-S was amplified using the C-N7-S-1, CN7-S-2, C-N7-S-3 and C-N7-S-4 fragments as templates with SphI-F/SphI-R primers. Finally, pASH28 was digested

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with SphI and homologously recombined with the C-N7-S fragment using the In-Fusion®HD Cloning Kit (Takara, Japan). After enzyme digestion identification and sequencing, a final recombinant infectious cDNA clone pASH-dS312 harboring

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double PEDV S499-602 fragments at the N-terminus of the C gene was assembled. The recombinant infectious cDNA clone pASH-dS312 was extracted using the QIAGEN Plasmid Midi Kit. After linearizationby SbfI, the recombinant infectious

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cDNA plasmids were gel purified followed by in vitro transcription using Ambion T mMESSAGE mMachine Kits (Thermo Fisher Scientific, Waltham, MA, USA). MDBK cells in 6-well plates were grown to 80% confluence and

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transfected with 2 μg of in vitro transcripts and 2μLof DMRIE-C Transfection Reagent (Invitrogen Corporation) according to the manufacturer’s instructions. After culturing for 8 h at 37°C in 5% CO2, transfected cells were maintained

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in DMEM containing 2% FBS. The cells were harvested at 84 h post infection for continuous passage culture. The resultant chimeric virus was referred to as vASH-dS312. The supernatant of the rescued virus (3rd passage) was collected, followed by RT-PCR detection using SphI-F/SphI-R primers. A 1889 bp fragment was amplified (Fig. 2A) and

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sequenced, which confirmed that the exogenous PEDV S antigen was inserted at the correct site with no amino acid mutation. Furthermore, an indirect immunofluorescence assay was carried out. MDBK cells were cultured in 24-well plates and infected with 0.1 MOI vASH-dS312 or SH-28. At 48 h post infection, the cellular supernatants were discarded, and the cells were gently washed thrice with PBS containing 0.05% Tween-20 (PBST). Thereafter, they were fixed with

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cold methanol for 20 min at −20 °C and washed as aforementioned, blocked with 10% bovine serum albumin (BSA), followed by incubation for 1 h at 37 °C with monoclonal antibodies (MAbs) against BVDV and PEDV. After washing thrice with PBST, the cells were incubated with FITC-conjugated goat anti-mouse IgG antibody. Eventually, they were examined under a fluorescence microscope (Zeiss, Germany). The results confirmed that specific green fluorescence against BVDV E2 and PEDV S MAbs could be detected in the cytoplasm of the cells inoculated with vASH-dS312 (Fig. 2B). Next, the cells infected with 0.05 MOI vASH-dS312 or SH-28 were collected at different times postinoculation, and

Running Title their TCID50 titers were tested by IFA as previously described by drawing the growth curves. vASH-dS312 had a replication efficiency similar to that of SH-28 (Fig. 2C) (Tao et al., 2018). Its virus titer gradually increased as the passages increased (Table 1). To assay the expression of exogenous S polypeptide, the cells were lysed at 48 h postinfection, and western blot analysis revealed that the E2 protein and the exogenous S polypeptide could be detected in the cells infected withvASH-dS312 (Fig. 2D). Then, the replication efficiency of the 3 rd, 5th, 10th, and 20th passaged vASH-dS312 cells was assayed by detection TCID50 and demonstrated that it was improved and stabilized until the 10th generation (Table 1). Furthermore, their complete genomes were sequenced, which confirmed that no amino acid

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mutation was observed within the 20 passages. Finally, vASH-dS312 was administered to the mice via oral or intramuscular injection to evaluate its

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immunogenicity. Twenty 5- to 6-week-old BALB/c mice were randomly divided into five groups (5 mice per group) (Table 2). Mice in group 1 (G1) were used as a negative control. Mice in group 2 (G2) were orally inoculated with 106.0

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TCID50/animal of vASH-dS312. Mice in groups 3 (G3), 4 (G4) and 5 (G5) were intramuscularly inoculated with 106.0 TCID50/animal of vASH-dS312, TGEV-PEDV-PoRV (G5) live vaccine (Harbin Weike Biotechnology Co. Ltd, China) or

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SH-28, respectively. All mice were immunized again two weeks later. Clinical signs of the infected animals were monitored, and fecal swabs were collected daily. Serum samples were taken at 0, 14, 21, and 35 days post-immunization.

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The fecal swabs were detected by RT-PCR using primers against BVDV (FBVDVI-II:5’-CATGCCCATAGTAGGAC3’;RBVDVI-II: 5’-CCATGTGCCATGTACAG-3’). As expected, no virus was shed into the environment, and the immunized mice had no clinical symptoms.

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To further analyze the humoral response, the levels of BVDV-specific and PEDV-specific IgGs were evaluated as follows: the purified SH-28 (or JS-2/2015) antigen in 100 μl of carbonate buffer (30 mM Na2CO3, 70 mM NaHCO3, pH 9.6) was used to coat wells of ELISA plates (Corning) and incubated overnight at 4 °C. The plates were washed three times with 100 μl of washing buffer (PBS, 0.05% [wt/vol] Tween 20) and blocked with 100 μL of 8% BSA for 2 h at

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room temperature. Diluted mouse sera were used to assay100 μl/well. Plates were incubated at 37 °C for 1 h and washed againand then incubated with 100 μl/well of HRP-conjugated goat anti-mouse IgG (Invitrogen) at room temperature for 1h. Subsequently, the plates were washed, and 50 μl/well of TMB substrate (Tiangen) was added. The reaction was stopped by the addition of 50 μl/well H2SO4 (2 M), and the absorbance at 450 nm was measured. Vaccination elicited BVDV-specific antibodies to a higher level in G2 than in G2 since 14 dpi, while both of them were lower than those in G5 (p < 0.05) (Fig. 3A). However, PEDV-specific antibodies were elicited only in G3 and G4 at 35 dpi (p < 0.05) (Fig. 3A).

Running Title Furthermore, the neutralization capability of the antibodies elicited was evaluated. Mouse serum at 35 dpi was inactivated at 56 °C for 30 min and serially diluted in 2-fold dilutions in DMEM. A total of 200 TCID 50 of BVDV (or PEDV) was mixed with serum dilutions and incubated at 37℃ for 1 h. Virus suspensions were added to 1x104 MDBK (or Vero) cells seeded 4 h prior to assay in 96-well plates and incubated for 4 days at 37°C. VNTs were calculated as the reciprocal of the highest dilution that abolished infection. All the mice in the G2, G3 and G5 groups indeed developed BVDV VNTs, while only the mice in the G3 and G4 groups induced PEDV VNTs (Fig. 3B). No VNTs against BVDV or PEDV were detected in the control group. In summary, vASH-dS312 specifically induced antibodies in intramuscular

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immunized mice capable of neutralizing BVDV as well as PEDV. To better investigate cellular and humoral immunity in mice, the total RNA of the peripheral blood at 35 dpi was

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extracted, and the RNA quantity was measured using a UV spectrophotometer. Total RNA was used to generate cDNA by reverse transcription-PCR using the PrimeScript○ R RT Reagent Kit with gDNA Eraser (Takara Bio, Shiga, Japan). The

CT values were normalized using the 2

−△△CT

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transcriptional levels of IFN-γ and IL-4 were detected by real-time PCR using SYBR○ R Premix Ex Taq II (Takara Bio). method. The transcriptional levels of IFN-γ and IL-4 were upregulated in

G2 induced the highest IL-4 mRNA (Fig. 3C).

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the immunized groups. Mice in the G3 group induced higher IFN-γ levels than mice in the G2 group; however, mice in

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In this study, we demonstrated the capacity of recombinant BVDV encoding PEDV S glycoprotein at the N-terminus of the C gene. To date, two different strategies to develop recombinant BVDVs have been proposed. First, the

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recombinant BVDVs expressed a foreign gene by replacing the same-sized corresponding gene of BVDV (Hofmann et al., 2003; Reimann et al., 2004; Van Gennip et al., 2000). Second, the foreign genes inserted into the Npro gene of BVDV contain only a small fraction of the Npro gene or miss the whole Npro gene (Fan et al., 2008). This is the first time that a foreign antigen has been inserted into the BVDV genome without deletion of any sequence. However, the expression level of PEDV S antigen in the chimeric virus vASH-dS312 is notvery high, resulting in the weak immunity of vASH-

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dS312, including the low IgG antibody against PEDV and weak neutralization activity. This may be related to the exogenous antigen length and the insertion site in the BVDV genome. Next, we will verify the maximum length of the insertable fragment, and the insertion sites still need to be explored.

Funding

Running Title This work was supported by the National Key Research and Development Program of China (Grant No. 2017YFD0501102) and the Shanghai Agriculture Applied Technology Development Program, China (Grant No.T20170111).

Author Statement

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Dear Respected Editor(s),

Thank you very much for providing the reviewer nice comments and suggestions concerning our manuscript. We have

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revised it. Each of the coauthors has seen and agrees with each of the changes made to this manuscript in the revision and

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to the way his or her name is listed.

Conflict of Interest

Ethical approval

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could be construed as a potential conflict of interest.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Furthermore, this article does not contain any experiments with human subjects or animals performed by any of the authors.

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Running Title Figure legends Fig.1 Schematic depiction of the chimeric virus vASH-dS312. The first large box is the BVDV viral gene, and insertion of the exogenous gene was performed between the two sphI restriction enzymes. The exogenous gene S499-602 was inserted between the seventh and eighth amino acids at the N-terminus of the C gene in tandem, which overlapped with fragments A and B by overlapping PCR (the second box). Then, it was inserted into the BVDV genome by homologous

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recombination with pASH28 digested by SphI (the third box).

Running Title Fig.2 Characterization of the chimeric virus vASH-dS312. (A) The recombined virus vASH-dS312 (3rd passage) and parent virus SH-28 were detected by RT-PCR using SphI-F/SphI-R primers. (B) Indirect immunofluorescence assay of MDBK cells infected at an MOI of 0.1 with vASH-dS312 or SH-28 using the MAb against BVDV E2 protein. Uninfected cells served as mock controls. (C) Growth kinetics of recombinant BVDV on MDBK cells infected at an MOI of 0.05 with vASH-dS312. Titers of samples prepared at the indicated time points postinfection were tested on MDBK cells. (D) Exogenous antigen expression of vASH-dS312 was determined by western blot. Blots were probed using the MAb reactive against BVDV E2

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or PEDV S.

Running Title Fig.3.Immune evaluation of the chimeric virus vASH-dS312 in mice. (A) Induction of antibodies that specifically bind BVDV or PEDV antigens. Sera of mice vaccinated on days 0 and 14 with the indicated viruses were sampled on days 14, 21, and 35 and analyzed for antibodies that bound BVDV or PEDV antigens by ELISA. Antibodies were detected at an optical density of 450 nm. (B) Analysis of neutralizing antibodies. VNTs of the sera sampled on day 35 were tested for complete neutralization of 200 TCID50 of BVDV or PEDV. PBS-inoculated mice served as mock controls. VNTs were calculated as reciprocals of the highest dilution abolishing infectivity. (C) Detection of IFN-γ and IL-4 contents in whole blood sampled on day 35. Total RNA was extracted from the blood, and the expression levels of IFN-γ and IL-4 cytokines

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were evaluated by relative quantitative real-time RT-PCR. The results are expressed as increases in mRNAlevels relative to those in mock cells and were normalized to the expression level of the GAPDH housekeeping gene. *, p<0.05; **,

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<0.01.

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Running Title

Table 1. TCID50 detection of the different passaged recombinant viruses The different passaged chimeric viruses (TCID50/mL) 5th

10th

20th

104.3

105.2

105.6

105.6

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vASH-dS312

3rd

Table 2. Animal groups in the immune test

Volume

Inoculated pathway

DMEM

200 µL

Injection

2

vASH-dS312

106.0TCID50/mL

200 µL

Oral

3

vASH-dS312

106.0TCID50/mL

200 µL

Injection

4

TGEV-PEDV-PoRV live vaccine

/

200 µL

Injection

5

SH-28

106.0TCID50/mL

200 µL

Injection

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1

Viral content

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Vaccination1

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Group