**Enhanced Cell-Based Detection of Parvovirus B19V Infectious Units According to Cell Cycle Status**

**Céline Ducloux 1,**†**, Bruno You 1,**†**, Amandine Langelé 1,2, Olivier Goupille 2, Emmanuel Payen 2, Stany Chrétien <sup>2</sup> and Zahra Kadri 2,\***


## Academic Editor: Giorgio Gallinella

Received: 27 July 2020; Accepted: 29 September 2020; Published: 18 December 2020

**Abstract:** Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage. Two of the clones tested, B12 and E2, reached sensitivity levels higher than those of UT7/Epo-S1 and CD36<sup>+</sup> erythroid progenitor cells. These findings highlight the importance of cell cycle status for sensitivity to B19V, and we propose a promising new straightforward cell-based method for quantifying B19V infectious units.

**Keywords:** B19 parvovirus; detection; erythroid cells; cell cycle; permissivity

## **1. Introduction**

Human Parvovirus B19 (B19V), a member of the genus *Erythroparvovirus* of the Parvoviridae family, is a widespread virus that is pathogenic to humans [1]. The genome of B19V is a linear 5.6-kb single-stranded DNA, packaged into a 23–28 nm non-enveloped icosahedral capsid [2]. Replication occurs in the nucleus of infected cells, via a double-stranded replicative intermediate and a rolling hairpin mechanism. B19V infection has been associated with a wide spectrum of diseases, ranging from erythema infectiosum during childhood (known as the "fifth disease" and characterized by a common "slapped-cheek" rash) [3,4], to arthropathies [5], severe anemia [6] and systemic manifestations involving the central nervous system, heart and liver, depending on the immune competence of the host [7]. Productive B19V is restricted to human erythroid progenitor cells [8], and its clinical manifestations are linked to the destruction of infected cells [9]. Indeed acute B19V infection can cause pure red-cell aplasia in patients with pre-existing hematologic disorders leading to high levels of erythrocyte turnover (e.g., in sickle cell disease or thalassemia patients) [10–12], and in immunocompromised or transplanted

patients [13]. The virus is transmitted via respiratory secretions and feto-maternal blood transfers. During pregnancy, infection with B19V can cause non-immune fetal hydrops, congenital anemia, myocarditis and terminal heart failure, leading to spontaneous abortion or stillbirth of the fetus [14,15]. The high prevalence of B19V infection in the general population and the large number of blood donations used in the manufacture of plasma-derived factor concentrates favors high levels of contamination. Few reports [16–18] of clinical B19V infection resulting from the transfusion of contaminated blood components or infusions of plasma-derived medicinal products suggests that measures to reduce the transmission risk (e.g., nucleic acid testing (NAT) and/or virus removal/inactivation steps) are effective. This efficacy has led to NAT becoming the gold standard for testing products of biological origin [19]. Indeed, to counteract B19V blood-borne patient's contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing did not correctly reflect B19V infectivity [17,20].

Reducing the risk of B19V infection is mandatory for suppliers of blood-derived products worldwide [21]. The elimination of viruses must be assessed in processes for the production of plasma-derived medical products, but B19V DNA quantification may be inadequate: viral DNA can persist in the serum for months after acute infection, and its levels are therefore not necessarily correlated with infectivity [20,22]. The use of titration-based B19V infectivity assays is therefore essential. Moreover, the last few decades have seen the development of regenerative therapies based on Hematopoietic or Mesenchymal Stem Cells (HSC or MSC) from bone marrow and synovium donors, respectively. According to the guidelines ensuring clinical grade of human stem cells, one of the major safety concerns is detecting latent viruses in cell sources [13,23]. Stem cells seem to act as a latent reservoir for B19 infection [24]. If viral contamination is overlooked at initial screening, then the virus may be amplified during culture before transplantation, through the reactivation of latent B19V [25]. For all these reasons, a practical and sensitive in vitro method for assessing B19V infectivity is required. However, efforts to develop such methods have been hampered by the lack of suitable B19-sensitive cell lines.

B19V displays a marked tropism for erythroid progenitor cells (EPC), but there is still no well-established cell line for B19V infection. The UT7/Epo-S1 cell line [26], an erythropoietin (Epo)-dependent subclone derived from the mega-karyoblastoid cell line UT-7 [27], is the most widely used cell model, because of its high sensitivity to B19V replication and transcription [28]. However, B19V infection is limited to a small number of cells (1%–9%, versus 30%–40%% for primary or immortalized erythroid progenitor cells) [29–31].

Here, we compared the sensitivities of a number of different erythroid cell lines to B19 infection with that of UT7/Epo-S1. We generated stable clones with graduated permissivity to B19V from a single parental cell line. Using the FUCCI (fluorescent ubiquitination cell cycle indicator) system to analyze cell cycle, we demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage, and characterized two clones, B12 and E2, with sensitivity to B19V up to 35 time higher than that of UT7/Epo-S1.

## **2. Materials and Methods**

## *2.1. Cell Lines*

Three distinct UT-7/Epo cell lines were used: (1) UT7/Epo-S1, a clone of UT7/Epo [32], was obtained from Dr Kazuo Sugamura (Tohoku University Graduate School of Medicine, Japan). (2) UT7/Epo-APHP and UT7/Epo-Cl3, a subclone isolated from UT7/Epo-APHP, were a gift from Dr Morinet (APHP: Assistance Publique - Hôpitaux de Paris, Saint Louis Hospital). (3) UT7/Epo-STI cells were derived from UT-7/GM cell line and were maintained at low passage, with stringency for erythroid features [33]. UT7 cell lines were maintained at 37 ◦C, under an atmosphere containing 5% CO2, in alpha minimum essential medium (αMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine (Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 U/mL recombinant human (rh) Erythropoietin

(rh-Epo, Euromedex, RC213-15). Where specified, 0.5 μM JQ1 (Sigma-Aldrich, France) or 2 ng/mL TGF-β (Peprotech, France) was added to the culture medium for two days before B19V infection. TF1 and TF1-ER erythroleukemia cells [34] were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FCS, 2 mM L-glutamine (Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 U/mL rh-Epo or 25 ng/mL human granulocyte macrophage colony-stimulating factor (GM-CSF, Peprotech). KU812Ep6 cells [35], a gift from Dr Sakai (Health Science Research Resources Bank, Tokyo, Japan), were maintained in RPMI-1640, 2 U/mL rh-Epo, 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin and Insulin Transferrin Selenium-X supplement (ITS-X, Gibco), at 37 ◦C, 5% CO2. Human embryonic kidney (HEK) 293T and NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin.

## *2.2. CD36*<sup>+</sup> *Erythroid Progenitor Cell (EPC) Line Generation*

Umbilical cord blood (CB) units from normal full-term deliveries were obtained, with the informed consent of the mothers, from the Obstetrics Unit of Saint Louis Hospital, Paris, and collected in placental blood collection bags (Maco Pharma, Tourcoing, France). Blood mononuclear cells were purified by Ficoll density gradient separation (Leucosep, Greiner Bio-one) and Hanks medium (Thermo-Fisher). Low-density cells were recovered and enriched for CD34<sup>+</sup> cells by automated cell sorting (CD34 isolation kit and autoMACS System, Miltenyi Biotec). CD34<sup>+</sup> cells were cultured in serum-free expansion medium: Iscove's Modified Dulbecco's Medium (IMDM), 15% BIT 9500 (BSA-Insulin-Transferrin, Stem Cell Technologies), 60 ng/mL rh-Stem Cell Factor (SCF), 10 ng/mL rh- interleukin-3 (IL-3), 10 ng/mL rh-IL-6, 2 U/mL rh-Epo, 100 U/mL penicillin and 100 μg/mL streptomycin. After seven days of culture, CD36<sup>+</sup> cells were isolated with biotin-coupled anti-CD36 antibody and anti-biotin microbeads on an autoMACS System. CD36<sup>+</sup> EPCs were obtained by lentivirus-mediated transduction with the *hTERT* and *E6*/*E7* genes from human papillomavirus type 16, as previously described [36], and were grown in expansion medium to generate a continuous CD36<sup>+</sup> EPC line.

## *2.3. B19 Virus Stock and Cell Inoculation*

Plasma samples containing high titers of infectious B19V from asymptomatic donors at blood donation were provided by the Etablissement Français du Sang (EFS). Determination of anti-B19V IgG and IgM in plasma samples was performed using respectively LIAISON® Biotrin Parvovirus B19 IgG (cat. N◦ 317000) and IgM (cat. N◦ 317010) (Diasorin S.A., Antony, France). According to manufacturer's instructions, samples were analyzed by Chemiluminescence ImmunoAssay (CLIA) and obtained results were compared to provided negative and positive controls. Plasma samples were determined to be qualitatively negative for both B19V IgG and IgM, with a viral titer of 1011 B19V DNA genome equivalent (ge)/mL. The infection assay was performed using a protocol similar to that previously described [37]. Briefly, cells were maintained in exponential growth condition by dilution to 0.3 <sup>×</sup> 106 cells/mL the day before infection. On the day of infection, cells were washed and diluted in FCS-free medium without Epo, at a density of 10<sup>7</sup> cells/mL. B19V inoculation was carried out in a 96-well plate, with 10 μL of cell suspension (105 cells) and 50 μL of a 100-fold dilution of B19V plasma (109 ge/mL), corresponding to a mean of 500 ge/cell. The cells were then incubated at 4 ◦C for 2 h, and then at 37 ◦C, for 1 h, under an atmosphere containing 5% CO2. We added 140 μL of complete medium and maintained the cells in culture until 72 h. Where specified, we added chloroquine (CQ) to the complete medium, at a final concentration of 25 μM. Cell viability was assessed by Trypan blue exclusion test (0.4% in PBS, Thermo Fisher Scientific), by counting blue and total cells under a microscope, with a hemocytometer. After correction for the dilution factor, viability was calculated as follows: percent of viable cells = (1 − (number of blue cells/number of total cells)) × 100. At 24, 48 or 72 h post infection (hpi), cells were centrifuged (8 min at 300× *g*), supernatants were discarded and cell pellets were frozen at −80 ◦C until analysis.

## *2.4. Fucci2a Lentivirus Production and Cell Transduction*

The Fucci2a DNA sequence [38] (RDB13080, RIKEN BioSource Center) was synthetized into the LTGCPU7 lentiviral vector backbone [39] without the puromycin resistance-gene cassette, and under the control of the EF1α promoter and enhancer (GenScript). Lentiviral particles were produced by the transient transfection of HEK293T cells with the five-plasmid packaging system, by PEIpro (Polyplus transfection), as previously described [40]. These particles were then concentrated by ultracentrifugation. Infectious titers were determined in NIH-3T3 cells. We transduced 0.5 <sup>×</sup> 106 UT7/Epo-STI cells with FUCCI particles at a mean of infection of 10 in 200 μL of complete medium, and the cells were kept at 37 ◦C for 4 h. Cells were subsequently diluted at 0.1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL. On days 6 and 9 post-transduction, cells were analyzed by cytometry for the expression of FUCCI proteins.

#### *2.5. UT7*/*Epo-FUCCI Clones Generation*

UT7/Epo-FUCCI refers further to a UT7/Epo-STI pool expressing FUCCI. UT7/Epo-FUCCI clones were isolated in a U-bottom 96-well plate, by limiting dilution, with one seeded cell per well in 100 μL of complete medium. Cells were visualized by microscopy, and wells containing more than one cell or non-fluorescent cells were excluded. Clones were then separately expanded with an assigned name corresponding to their location on the plate. After expansion, each clone was considered further as a new cell line. A cell bank of 156 isolated clones was constituted (stored at 80 ◦C in 90% FCS, 10% DiMethyl SulfOxide (DMSO)). To reach exponential growth, cells were diluted at 0.3 <sup>×</sup> 10<sup>6</sup> cells/mL. The day after dilution, isolated clones were subjected to FUCCI expression profiling. The stability of the cell cycle profiles of the isolated clones was controlled both sequentially, for at least five independent cultures, and for 10 passages of the same culture.

## *2.6. Flow Cytometry Analysis of Cell Cycle Status*

FUCCI-2a stable transduction in UT7-FUCCI cells allows the expression of two fluorescent proteins (m-Venus and m-Cherry) fused with specific cell cycle proteins (respectively Geminin and Cdt1). Fusion proteins are continuously expressed, but as Cdt-1 and Geminin are ubiquitinated and degraded by the proteasome at specific cell cycle stages, resting fluorescence reflects cell cycle status: during G1 phase, the geminin-mVenus fusion protein is degraded, while the Cdt1–mCherry fusion is expressed resulting in red-fluorescence. During G1/S transition, both proteins are present in the cells and the cell nuclei appear yellow as the green and red fluorescence overlay. In the S, G2, and M phases, the Cdt1–m-Cherry fusion is degraded, leaving only the geminin–m-Venus fusion and resulting in a green-fluorescence signal. This dynamic color change from red to yellow to green serves as an indicator of the progression through cell cycle and division. Fucci2a bicistronic expression was monitored with an LSRFortessa cytometer (BD Biosciences, Le Pont de Claix, France). Fluorescent fusion proteins were detected with the 488 nm blue laser and a 530/30 nm bandpass filter (B530/30) for mVenus-hGeminin, and the 590 nm yellow laser and a 610/20 nm bandpass filter (Y610/20) for mCherry-hCdt1. For alternative monitoring of the cell cycle according to DNA content, cells were stained with the permeable DNA dye Hoechst 3342 (10 μg/mL) for 1 h at 37 ◦C, and immediately analyzed for DNA content with the 355 nm violet laser and a 450/40 nm bandpass filter (V450/40). FACSDiVa and FlowJo X software (BD Biosciences, Le Pont de Claix, France) were used to operate the instrument and for data analysis, respectively.

## *2.7. RNA Extraction and Duplex RT-qPCR*

Total RNA was extracted from cell pellets with the RNeasy 96 QIAcubeHT kit and a QIAcubeHT machine, according to the manufacturer's instructions. The extraction step included DNase treatment for 15 min, to decrease the risk of genomic DNA amplification during PCR. Real-time reverse transcription-quantitative PCR (RT-qPCR) was performed with the *Taq*man Fast Virus one-step PCR kit (Applied Biosystems). B19 VP2 transcripts were amplified with the sense primer B19-21 5- -TGGCAGACCAGTTTCGTGAA-3- (nts 2342-2361), the antisense primer B19-22 5- -CCGGCAAACTTCCTTGAAAA-3- (nts 3247-3266) and the probe B19-V23 5- -VIC-CAGCTGC CCCTGTGGCCC-3- (nts 3228-3245). For control and normalization with respect to the number of cells, we used a duplex strategy. A target sequence of the spliced beta actin transcript was selected and amplified with the sense primer actin-S 5- -GGCACCCAGCACAATGAAG-3- , the antisense primer actin-AS 5- GCCGATCCACACGGAGTACT-3 and the probe actin-FAM 5- -FAM-TCAAG ATCATTGCTCCTCCTGAGCGC-3- . Reactions were performed on 5 μL of extracted RNA with the Quant Studio 3 PCR system. The reaction began with reverse transcription at 48 ◦C for 15 min, followed by inactivation of the reverse transcriptase and activation of the polymerase by heating at 95 ◦C for 10 min, followed by 40 cycles of 15 s at 95 ◦C and 30 s at 60 ◦C. The PCR program was optimized for amplification of the VP2 spliced transcripts rather than the VP2 genomic sequence (Figure 1A).

**Figure 1.** Comparison of the B19V sensitivity and permissiveness of hematopoietic cell lines. (**A**) B19V transcription profile (adapted from Ganaie et al., J Virol. 2018 [7]). The major transcription unit of the B19V duplex genome (GenBank accession no. AY386330) is shown to scale at the top, with the P6 promoter, 2 splice donors (D1, D2) and 4 acceptors (A1 to A4) sites. In blue, mRNA encoding the VP2 viral proteins, with nucleotides (nts). At the bottom, the primers and probe used for the RT-PCR amplification of VP2. (**B**) Bone marrow-derived primary Erythroid Progenitor Cells (CD36<sup>+</sup> EPCs), human leukemic cell lines (TF1, TF1-ER, UT7/Epo-APHP, UT7/Epo-STI) and isolated clones (KU812Ep6, UT7/Epo-cl3 and UT7/Epo-S1) were seeded in triplicate and inoculated with or without B19V in culture medium supplemented with Epo (2 U/mL)(/Epo), or granulocyte macrophage colony-stimulating factor (GM-CSF) (25 ng/mL)(/GM) for TF1 and TF1-ER. When specified, cells were cultivated with (+) or without (-) Chloroquine (CQ, 25 μM). No CQ treatment was applied to CD36+EPC. 72 h post-infection, cells were pelleted and lysed. RNA was extracted and analyzed by RT-qPCR for VP2 to quantify B19 viral genome expression, and for β-actin for cell number normalization. For each cell line, the results without B19V correspond to the negative control. Relative B19V threshold cycle (Ct) values were normalized relative to b-actin Ct and expressed according to the 2−ΔΔCt method with normalization against mean VP2 expression for UT7/Epo-S1 cells without CQ (*n* = 6). Results are presented as means + SEM of 3 independent experiments. \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS = No Significance. ND = Not detected.

## *2.8. Statistical Analysis*

Values are expressed as mean ± SEM. Data were determined to be normally distributed as the max and the min values in each data set were <3.sd from the mean. Data were analyzed using Prism 6.0 (GraphPad Software) by one-tailed Student's t-test or one-way ANOVA with Bonferroni post-test (α < 0.05). Parameters measured over multiple time points were analyzed with two-way ANOVA with Bonferroni post-test and time was within subject factor. The significance level displayed on figures are as follows: \* *p* <0.05, \*\* *p* <0.01, \*\*\* *p* <0.001 and "NS" means no significance. Samples and experiment sizes were determined empirically to achieve sufficient statistical power.

### **3. Results**

To assess and compare the degree of permissivity to B19V, hematopoietic cell lines were infected with B19V and maintained for 72 h. Where specified, chloroquine (CQ) was added to boost virus entry and prevent the degradation of incoming viruses through a blockade of lysosome transfer [41]. Active transcription of the B19V genome in host cells was evaluated by RT-qPCR for the *VP2* capsid gene (Figure 1A), with normalization to beta-actin gene expression. As a reference to calculate relative B19 mRNA expression, the value for UT7/Epo-S1 without chloroquine was set to 1 (Figure 1B). As previously reported, UT7/Epo-S1 and KU812Ep6 cells were less permissive to B19V than CD36<sup>+</sup> EPCs. Chloroquine treatment markedly enhanced UT7/Epo-S1 sensitivity (five-fold), but without reaching the level obtained for CD36<sup>+</sup> EPCs. VP2 expression was undetectable in both the parental TF1 erythroleukemia cell line and a TF1-ER cell line expressing a full Epo-receptor, under the control of GM-CSF or Epo, with or without chloroquine treatment. Among all UT7/Epo cell lines tested, UT7/Epo-STI was the UT7/Epo cell line tested with the highest sensitivity to B19V, with B19 mRNA levels 11.8 ± 0.2 times higher than those in UT7/EpoS1. Sensitivity was enhanced by chloroquine treatment and reaches an equivalent level compared to CD36<sup>+</sup> EPCs (UT7/Epo-STI <sup>+</sup> CQ: 25.8 <sup>±</sup> 4.9 vs. CD36<sup>+</sup> EPCs 21.49 <sup>±</sup> 2.7).

This increase in sensitivity was not due to resistance to B19V-induced cytotoxicity (Figure 2A). The expression kinetics of UT7/Epo-STI B19V were similar to those for CD36<sup>+</sup> EPC, with a maximum reached at 72 h post-infection for both cell lines (Figure 2B).

**Figure 2.** Comparison of B19V sensitivity of hematopoietic cell lines. (**A**) Cell viability was assessed 72 h post-infection. The results shown are the means + SD of three independent experiments. (**B**) UT7/Epo-STI cells and CD36<sup>+</sup> EPCs were cultured in triplicate, with or without B19V, for 72 h. At 24, 48 and 72 h post-inoculation, cells were collected by centrifugation. RNA was extracted from the cell pellet and VP2 mRNA levels were analyzed to quantify B19 viral DNA expression, and β-actin mRNA levels were analyzed for cell number normalization. For each cell line, results without B19V correspond to the negative control. Relative B19V threshold cycle (Ct) values were normalized relatively to the β-actin (log B19V/actin). The results shown are the means + SEM of three independent experiments. \*\*\* *p* <0.001; NS = No Significance.

As sensitivity to B19V is directly linked to maturation stage, we therefore subjected UT7/Epo-STI cells to the chemical (JQ1) or hormonal (TGF-β) induction of erythroid differentiation two days before B19V infection. Both treatments decreased B19V infection by a factor of about 10, to levels similar to those obtained for UT7/Epo-S1 (Figure 3).

**Figure 3.** B19V-sensitivity of UT7/Epo-STI cells is linked to maturation stage. UT7/Epo-STI cells were cultured for 48 h before inoculation with B19V, without (-) or with JQ1 (0.5 μM) or TGF-β (2 ng/mL). 72 h post-inoculation, relative levels of B19V VP-2 mRNA were evaluated with UT7/Epo-S1 cells as the reference.

We chose to select clones according to cell cycle status, to increase sensitivity to B19V. UT7/Epo-STI cells were transduced with FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) lentiviral particles to generate the UT7/Epo-FUCCI cell line (Figure 4A). The FUCCI cell cycle sensor allows cell cycle analysis of living cells. The UT7/Epo-FUCCI cell line presents three different color profiles, from green, corresponding to the S, G2 and M phases, to red, consequent to G1 phase, with a green plus red (yellow) overlay indicating the G1-to-S transition. We checked that these dynamic color changes correctly represented progression through the cell cycle and division, by staining the DNA content of UT7/Epo-FUCCI cells with Hoechst stain (Figure 4B). An overlay of the DNA staining and FUCCI profiles resulted in a perfect match between the cell cycle status assigned by the FUCCI technique and that assigned on the basis of DNA content: G1 (red) FUCCI cells were detected at a DNA content of 2N, whereas cells at S-G2-M (green) had DNA content peaks of 2N to 4N, consistent with the expected replication of the DNA replication before mitosis. The G1/S transition phase (yellow) population was located at the 2N peak, with a slight shift from G1 cells. Overall, these results confirm that FUCCI is an appropriate cell cycle indicator for UT7/Epo cells.

**Figure 4.** Generation of a UT7/Epo-STI cell line with stable expression of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI). (**A**) Experimental design for the generation of the UT7/Epo-FUCCI cell line. Bottom: Two-color cell cycle mapping with the FUCCI2a Cell Cycle Sensor and right, flow cytometry analysis of exponentially growing UT7/Epo-STI and UT7/Epo-FUCCI cells. The profile shown corresponds to one representative experiment. (**B**) DNA content and FUCCI profiles for the same sample. Exponentially growing UT7/Epo-FUCCI cells were stained with Hoechst 33342. DNA content (Hoechst on the *x*-axis; cell count on the *y*-axis) and FUCCI proteins (m-Venus on the *x*-axis; m-Cherry on the *y*-axis) were concomitantly evaluated by flow cytometry. Bottom: Overlay of gated cell cycle populations, as determined by FUCCI analysis with DNA content profile.

We then generated different UT7/Epo-FUCCI clones, each obtained by limiting dilution and culture from a single fluorescent cell. Unlike the UT7/Epo-FUCCI pool, these clones were generated from single cells and 100% of the cells were therefore transduced: the colorless cells of the FUCCI profile correspond to the early G1 (eG1) phase and were included in the G1 phase for the purposes of this analysis. We isolated 156 independent clones and expanded each as new sub-lines. FUCCI-negative clones, accounting for one third of the cells isolated, were excluded. We studied the cell cycle status of FUCCI-positive clones. We defined three types of cell cycle profile in a total of 97 clones: (1) 54 clones presented a cell cycle with more than 60% of the cells in G1 phase (55.7% of clones); (2) 29 clones presented a balanced distribution of cells between the G1 and S/G2/M phases (29.9% of clones); (3) 14 clones had a high percentage of cells in the S/G2/M phases (14.4% of clones). With the aim of analyzing these three types of cell cycle profile, we selected 11 isolated clones in regard to the diversity of their cell cycle patterns at exponential growth (Figure S1).

We evaluated sensitivity to B19V of exponentially growing selected clones as previously described (Figure 5A). Permissivity ranged from 1-fold to 35-fold relative to UT7/Epo-S1. Three populations were assigned: group I, with a sensitivity close to that of UT7/Epo-S1 (six clones); group II, gathering clones with UT7/Epo-FUCCI-like permissivity (three clones); group III, containing clones B12 and E2, displaying remarkable sensitivity to B19V infection. Interestingly, classification based on B19V sensitivity seemed to group together clones with similar cell cycle patterns (Figure 5B). The cell cycle profiles of group I clones displayed a predominance of the G1 phase. Group II clones displayed a balance between the G1 and S/G2/M phases, as observed for the original UT7/Epo-FUCCI pool. Finally, the S/G2/M cell population predominantly represents the group III profile, with 82% and 75.8% for B12 and E2, respectively.

**Figure 5.** Improvement of B19V sensitivity and permissiveness according to cell cycle status. (**A**) UT7/Epo-S1 cells (S1), UT7/Epo-STI cells expressing the FUCCI system (FUCCI) and 11 UT7/FUCCI-derived isolated clones were inoculated with B19V. Relative levels of B19V mRNA were determined 72h post-infection, with UT7/Epo-S1 as the reference, and cell lines were classified on the basis of B19V sensitivity as group I for S1-equivalent clones, group II for FUCCI-equivalent clones, and group III for highly permissive clones. The results shown are the means + SD of 3 independent experiments for groups I and II, and *n* = 9 for group III clones. (**B**) The cell cycle status of exponentially growing FUCCI cell lines and isolated clones was assessed by flow cytometry. The results shown are the means + SEM of three independent measurements.

We evaluated the correlation between cell cycle stage and B19V sensitivity, by analyzing the correlation of the coefficient of determination (R2) obtained for eG1, G1, G1/S and S/G2/M with B19V mRNA levels (Figure S2 and Figure 6). For G1 cell cycle parameter, R<sup>2</sup> was low, with values of 0.3743 for early G1 (eG1) and 0.5148 for G1.

**Figure 6.** Comparison of relative levels of B19 mRNA (as in Figure 5A) and cell cycle status (as in Figure 5B). Each dot corresponds to the mean result for a single cell line or clone (*n* = 3), classified to groups I (white marks -), II (orange marks •) and III (red marks •). Logarithmic regression analysis and R2 values are presented.

The highest value was obtained for the S/G2/M phase of the cell cycle, with R<sup>2</sup> = 0.8642, demonstrating an excellent agreement between the percentage of cells in S/G2/M stage and B19V sensitivity (Figure 6).

Results obtained show that E2 and B12 are the most permissive clones. In order to assess the stability of their cell cycle profile, E2 and B12 were submitted to serial culture passage and cultivated for up to 80 days. Cell cycle profiles were analyzed by flow cytometry according to FUCCI expression (Figure 7 and Figure S3).

**Figure 7.** S/G2/M cell percentage of E2 and B12 clones during serial culture passage. UT7/Epo-FUCCI clone E2 and B12 were maintained in culture during 80 days after thawing with up to 25 culture passages. At indicated time points, cell cycle was evaluated by flow cytometry according to specific FUCCI protein expression. The graph represents the evolution of the percentage of cells in S/G2/M cell cycle status during cell culture, as calculated in Fig S3A (E2) and B (B12) Results shown are the means of three independent measurements.

E2 and B12 clone showed a decrease of percentage of S/G2/M cells from, respectively, 75.8% and 82 % at day 7, to 60.4 and 53.4% at day 80, suggesting a higher stability of E2 cell line. B19V permissivity stability was also analyzed throughout serial culture passage and compared to UT7/Epo-S1 reference (Figure 8).

**Figure 8.** B19V permissivity throughout serial culture passage—UT7/Epo-S1 cell line (**A**) E2 (**B**) and B12 (**C**) clones were maintained in culture during 67 days after thawing with up to 21 culture passages. At indicated time points, B19V permissivity was evaluated by measuring levels of VP2 mRNA and normalized with β-actin mRNA by the 2−ΔCt method. The results shown are the means + SD of 3 independent measurements.

While UT7/Epo-S1 and B12 clone showed a decrease of sensitivity to B19V, E2 clones' permissivity seems to be stable at high culture passage.

Overall, our results identify two highly permissive UT7 clones, B12 and E2, and show that the S/G2/M phase is essential for B19V sensitivity.

### **4. Discussion**

Most of the currently available approaches focus on the detection of B19V DNA, but there is a need for a suitable in vitro method for the direct quantification of virion infectivity, for use in assessing neutralizing antibodies, to evaluate viral inactivation assays or in antiviral research field. However, efforts to develop such methods have been hampered by the lack of suitable B19-sensitive cell lines in vitro. We describe here a new cell model with high sensitivity to B19V infection. As expected, hematopoietic cell lines of different origins were heterogeneous but, surprisingly, our results also demonstrate considerable variability among cell lines derived from the same patient, all named UT-7/Epo. This variability of B19V sensitivity may depend on erythroid stage, B19V entry receptor expression and/or the activation of specific signaling pathways [7]. Our findings highlight the need for tracking criteria to ensure the stability of the cell line used. As we show here that B19V sensitivity is linked to S/G2/M cell cycle status, we propose the use of cell cycle status to define the optimal cells for selection and as a keeper of clone stability. This study proposes an improved cellular model for the detection of B19V infectious units, with a sensitivity up to 35 times higher than previously achieved.

B19V has an extremely strong tropism for human erythroid progenitor cells. Since the discovery that B19V inhibits erythroid colony formation in bone marrow cultures by inducing the premature apoptosis of erythroid progenitor cells [42], numerous approaches and studies attempt to find a method of virus culture in vitro. Primary [43,44] or immortalized [36] CD36<sup>+</sup> erythroid progenitor cells (EPC) derived from hematopoietic stem cells were the most permissive cell models for B19V infection. CD36<sup>+</sup> EPCs reflect the natural etiologic B19V cell host, but the main problem with the use of this model is the difficulty in obtaining a continuously homogeneous cell line, with respect to differentiation stage, proliferation rate and metabolic activity. Moreover, the reagents and cytokines required for cell culture (SCF, Il-3, Il-6, Epo) preclude the use of CD36<sup>+</sup> EPCs for routine B19V cell-based detection methods. To counteract this lack of suitability, cancer cell lines constitute a sound, practical, cost-effective alternative model, overcoming these difficulties. In recent years, many cancer cell lines

have been tested, but only a few erythroid leukemic (KU812) [35] or mega-karyoblastoid cell lines (UT-7) [26] with erythroid characteristics support B19V replication. In our study, we chose also to investigate TF-1 permissivity. The TF-1 cell line is derived from the bone marrow aspirate of an erythroleukemic patient [45]. These cells display marked erythroid morphological and cytochemical features common to CD36<sup>+</sup> EPCs, and the constitutive expression of globin genes highlights the commitment of the cells to the erythroid lineage [46]. Surprisingly, Gallinella et al. showed that TF-1 cells allow only B19V entry, with impaired viral genome replication and transcription, as shown by the presence of single-stranded DNA, and the absence of double-stranded DNA and RNA in B19V-infected TF-1 cells [47]. As previously described, no B19V RNA was detectable in the TF-1 cell line. The cellular factors involved in the transcriptional activation of the B19V promoter contribute to the restriction of permissiveness. Two factors, erythropoietin (Epo) and STAT-5, are key factors involved in B19V replication and transcription [48]. TF-1 cells express a truncated and mutated form of the Epo receptor [49], leading to impaired STAT-5 activation [34]. In the TF-1-ER cell line, stable ectopic expression of a full-length Epo receptor restores Epo-induced proliferation and STAT-5 activation. Here, despite Epo receptor signaling and STAT-5 activation, we found no evidence of B19V transcription, reflecting the involvement of unknown processes in the molecular mechanisms controlling B19V permissivity.

The first cell line reported to be permissive for B19 infection was an Epo-dependent subclone of UT-7, a mega-karyoblastoid cell line [32]. In 2006, Wong et al. published a comparative study of B19V sensitivity and permissivity in various cell lines [22]. They obtained evidence for the B19V infection of UT7/Epo and KU812Ep6 cells, although the percentage of B19V-positive cells was low (<1% immunofluorescent B19V<sup>+</sup> cells). UT7/Epo-S1, a subclone of UT7/Epo obtained by limiting dilution and screening for B19V susceptibility [26], had the highest sensitivity, with approximately 15% of the cells staining positive for B19V [29]. Permissivity is restricted to a subset of cells, but the degree of viral DNA replication in these cells is similar to that in EPCs [50,51]. Since its characterization, the UT7/Epo-S1 cell line has been widely used to investigate the molecular mechanisms of B19V infection and to develop antiviral strategies against B19 [28]. We used UT7/Epo-S1 as a reference and compared the sensitivity of UT7/Epo cells from different laboratories. B19V permissivity seemed to be similar in the various UT7/Epo cells, but UT7/Epo-STI cells displayed levels of B19V gene expression almost 10 times higher than those in UT7/Epo-S1 cells. UT7/Epo-STI cells have been cultured with great care to ensure the preservation of their erythroid features, and they undergo erythroid differentiation following treatment with JQ1, a Bet-domain protein inhibitor [33] or TGF-β1 [52]. However attempts to characterize cell lines have been hampered by the heterogeneity of continually evolving multiple sub-clonal leukemic populations, as revealed at the cytogenetic level by the unstable karyotype documented at various time points for UT-7: at the admission of the patient to hospital (44 chromosomes, XY), at the first cell line characterization (92 ± 6 chromosomes, XXYY) [27], in subsequent publication (82 ± 4 chromosomes, XXYY [53] and in our own cell line in 2017 (72 ± 13 chr., XXYY; unpublished data). This karyotype heterogeneity highlights the presence of heterogeneous subclones within cell lines and might account for the variation of B19V sensitivity among UT-7 cell lines and clones.

The cell cycle is known to be crucial for erythroid differentiation, ensuring precise coordination of the critical differentiation process by Epo and erythroid-specific transcription factors [54,55]. We decided to select clones on the basis of cell cycle status. The FUCCI system represents a convenient approach to track cell cycle as its readability allows analysis of living cells at a single cell level [56]. By using clones with different cell cycle status, we demonstrated a strong correlation between S/G2/M cell cycle status and permissivity. A total RNA analysis, with a comparison of transcriptomes from highly permissive (group III) and less sensitive (group I & II) clones would shed more light on the molecular mechanisms involved in B19V infection. Along with karyotype, study of permissive versus restrictive clones could lead to the discovery of key molecular factors for B19V permissivity.

B19V has been shown to induce cell cycle arrest at G2 phase [26,57], but the importance of cell cycle status for B19V entry has not been investigated. A complex combination of multiple factors, including differentiation stage, specific cell cycle status, surface receptor and co-receptor, signaling pathways and transcription factors, may account for the difficulty of identifying the best cellular model for completion of the B19 viral cycle. We describe here two clones, E2 and B12, with a permissivity for B19 up to 35 times higher than that of the previously described references. By comparison with their less sensitive counterparts (groups I & II), these new highly permissive cell models (group III) constitute a potential advance towards understanding the crucial molecular determinants of B19V infectivity.

In addition to the use of E2 and B12 clones to investigate the molecular mechanisms of B19 infection, cell-based methods can be used for the detection/quantification of B19 infectious units, at low levels (<10<sup>4</sup> DNA geq), in human fluids and tissues. There is a need for a practical in vitro method for the direct quantification of virion infectivity, as applied to the screening and/or assessment of neutralizing antibodies, antiviral drugs, and viral inactivation assays.

In the context of plasma-derived medicinal products, due to the lack of a suitable in vitro culture assay for B19, animal parvoviruses are currently used as a model for B19V, to assess B19 viral reduction during manufacturing processes. However, it remains unclear whether these models accurately reflect the behavior of B19V. Yunoki et al. reported an unexpected greater resistance of B19V than of canine parvoviruses (CPVs) to heating [58]. Mani et al. pointed up a discrepancy between animal and human parvovirus in a comparative study, describing the remarkable stability of the B19 viral genome in its encapsidated state [59]. Animal model parvoviruses display a certain resistance to heat inactivation [60,61] and pH stability [62], but comparative studies have indicated that they may behave differently from human B19 [2]. As E2 and B12 were the most sensitive cells in our study, with a permissivity up to 35 times higher than that of previously established references, they could allow the use of human parvovirus for the testing of viral inactivation processes, and the results of these tests would reflect the behavior of the native human virus.

Given the severity of B19V infection in immunocompromised patients, the development of antiviral strategies and drugs directed against B19V is of the highest relevance [28]. Depending on the immune state of the infected patient, acute infections can be clinically severe, and an impaired immune response can lead to persistent infections. The administration of high-dose intravenous immunoglobulins (IVIG) is currently considered the only available option for neutralizing the infectious virus [23,63]. In addition to the use of IVIG, the discovery of antiviral drugs with significant activity against B19 would offer important opportunities in the treatment and management of severe clinical manifestations. Two factors have critically limited the search for compounds to date. Firstly, the lack of a standardized and sensitive in vitro cell culture model has hampered advances in this field. Due to its usefulness, practicability and sensitivity, our cell model could replace the use of CD36<sup>+</sup> EPCs and UT7/Epo-S1 cells in the discovery and evaluation of antiviral candidate compounds. Secondly, antiviral research requires native B19 infectious particles. However, B19V particles from viremic patients limit the feasibility of high-throughput screening against the available chemical libraries. No appropriate system for cell culture and in vitro virus production are available to date. UT-7 cells have been reported to produce infectious viral particles in vitro [43,64], but only a few UT-7 cells are infected and virions are produced in small numbers [65–67]. The strategies used here for the selection of clones permissive for B19V could also be used to select highly productive clones. Our most permissive clones, E2 and B12, could be challenged and assessed for in vitro viral production.

## **5. Conclusions**

Altogether, we propose here an improved cell model with a high degree of permissivity to B19V, allowing the sensitive detection of infectious particles of B19. This finding opens up challenging new perspectives for basic research on the B19V life cycle. It may also offer opportunities for improving key steps in a number of critical applied approaches, including the sensitive evaluation of B19V virions in manufactured blood-derived products, and new strategies for B19V production in vitro.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/12/12/1467/s1, Figure S1: Cell cycle profile of the exponentially growing UT7/Epo-FUCCI cell line and clones, Figure S2: Comparison of relative levels of B19 mRNA and percentage of cells in respective cell cycle status. Figure S3: Cell cycle stability of E2 and B12 clones during serial culture passage.

**Author Contributions:** Conceptualization, C.D., B.Y. and Z.K.; Methodology, C.D., A.L. and Z.K.; Validation, C.D., B.Y. and Z.K.; Formal Analysis, Z.K.; Investigation, C.D., A.L. and Z.K..; Resources, C.D., A.L., B.Y., E.P. and Z.K.; Data Curation, C.D., A.L. and Z.K.; Writing—Original Draft Preparation, Z.K.; Writing—Review & Editing, C.D., B.Y., A.L., O.G., E.P., S.C. and Z.K.; Visualization, C.D. and Z.K.; Supervision, B.Y., S.C. and Z.K.; Project Administration, Z.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. This work was supported by the *Commissariat à l'Energie Atomique et aux Energies Alternatives* (CEA).

**Acknowledgments:** The authors acknowledge the RIKEN BioResource Center (RIKEN BRC) for providing the pCAG-Fucci2a vector (provided by Richard Mort) and the National Heart, Lung and Blood Institute (NHLBI) for UT7/Epo-S1 cells (provided by Kazuo Sugamura and Susan Wong). We thank Morinet and Capron for expertise of B19 inoculation; Anne-Sophie Gallouët and Mario Gomez-Pacheco (Infectious Disease Models and Innovative Therapies-IDMIT-Fontenay-aux-Roses, France) for their help and support for cytometry studies, Corinne Bergeron (LFB) for developing the RNA extraction and qPCR protocol; Valérie Barlet and Isabelle Dupont (EFS Grand Est, Metz-Tessy site) for providing the B19-positive plasma sample.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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## *Review* **AMDV Vaccine: Challenges and Perspectives**

**Nathan M. Markarian <sup>1</sup> and Levon Abrahamyan 2,\***


**Abstract:** Aleutian mink disease virus (AMDV) is known to cause the most significant disease in the mink industry. It is globally widespread and manifested as a deadly plasmacytosis and hyperglobulinemia. So far, measures to control the viral spread have been limited to manual serological testing for AMDV-positive mink. Further, due to the persistent nature of this virus, attempts to eradicate Aleutian disease (AD) have largely failed. Therefore, effective strategies to control the viral spread are of crucial importance for wildlife protection. One potentially key tool in the fight against this disease is by the immunization of mink against AMDV. Throughout many years, several researchers have tried to develop AMDV vaccines and demonstrated varying degrees of protection in mink by those vaccines. Despite these attempts, there are currently no vaccines available against AMDV, allowing the continuation of the spread of Aleutian disease. Herein, we summarize previous AMDV immunization attempts in mink as well as other preventative measures with the purpose to shed light on future studies designing such a potentially crucial preventative tool against Aleutian disease.

**Keywords:** AMDV; Aleutian disease; mink parvovirus; Aleutian mink disease virus; vaccine

## **1. Introduction**

In the mid-1900s, some American mink farmers reported cases of a novel disease causing an enlargement of kidneys, the spleen and lymph nodes in the blue–gray variety of mink known as the Aleutian mink [1]. With time, the number of cases of this disease, called Aleutian disease (AD), rapidly increased throughout many ranches and the disease was eventually discovered to be due to a virus, as opposed to the initial false presumption of being a genetic disorder [2–5]. Today, AD is the most significant disease in the worldwide mink industry since it is responsible for causing infertility and the loss of animals, leading to low fur quality and, ultimately, significant financial losses for farmers [6]. The causative agent of this deadly and widespread disease is the Aleutian mink disease virus (AMDV), which is categorized as an *Amdoparvovirus* genus, one of the six genera of the *Parvovirinae* subfamily. This subfamily is part of the *Parvoviridae* family that belongs to the *Picovirales* order [7].

AMDV, being a parvovirus, is a single-stranded DNA virus possessing a small genome of around 4.8 kb in size with two large open reading frames (ORFs), among which are the left ORF at nucleotide positions 116–1975, the right ORF at nucleotide position 2241–4346, three smaller central ORFs and palindromic structures at both the 3 and 5 termini [3,8,9]. The left ORF encodes for three non-structural proteins (NS1, NS2 and NS3) that play a role in regulating gene expression and viral replication, whereas the right ORF codes for two viral capsid proteins (VP1 and VP2) serving as the key proteins for viral tropism and pathogenesis [10]. As for the palindromic structures at both 3 and 5 termini, these can fold into hairpin telomeres essential in the replication process [11]. Structurally, AMDV

**Citation:** Markarian, N.M.; Abrahamyan, L. AMDV Vaccine: Challenges and Perspectives. *Viruses* **2021**, *13*, 1833. https://doi.org/ 10.3390/v13091833

Academic Editor: Giorgio Gallinella

Received: 2 August 2021 Accepted: 9 September 2021 Published: 14 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

virions contain a predominantly negative strand DNA genome, measuring around 20 to 25 nm in diameter, are spherical, non-enveloped and are composed of 60 self-assembling proteins, including both VP1 and VP2 at a 1:9 ratio [11–13].

As for cell tropism, AMDV infects alveolar type II epithelial cells in mink kits and in macrophages for persisting infections, as well as B and T lymphocytes [14–16]. To enter macrophages and alveolar type II epithelial cells, AMDV has been reported to utilise antiviral antibodies using an Fc-receptor-mediated mechanism, which is known as an antibody-dependent enhancement of infection (ADE) [17,18]. In the case of other parvoviruses, many receptors are used to infect the target cells such as the transferrin receptor, bound by the canine and feline parvoviruses (CPV and FPV) and the erythrocyte P antigen for human parvovirus B19, whereas sialic acid is the main receptor for AMDV, minute virus of mice (MVM) and porcine parvovirus (PPV), respectively [19–22]. Upon receptor binding, parvoviruses are uptaken by clathrin-mediated endocytosis and slowly trafficked toward the cell nucleus, escaping the endosomal compartments with the help of the crucial phospholipase A2 (PLA2) motifs present on the VP1 capsid protein [23,24]. The PLA2s are enzymes that produce lysophospholipids and fatty acids from phospholipid substrates. Amdoparvoviruses, such as AMDV, on the other hand, are the only viruses in the *Parvovirinae* subfamily that do not contain this particular motif in the VP1 protein, and so, the endolysosomal trafficking remains to be clarified in future studies [24–26].

Upon the delivery of the single-stranded DNA genome of parvoviruses to the nucleus via the nuclear pore complex, the host cell DNA polymerase attaches itself to the 3 terminal hairpin telomere of the genome to convert it into a double-stranded DNA [27]. During the S-phase of the host cellular cycle, from the P3 promoter, a precursor mRNA (premRNA) is produced which is followed by polyadenylation and splicing to generate six distinct mRNAs named R1, R1 , R2, R2 , Rx and Rx . From these, R1 and R1 mRNAs encode for the NS1 protein; R2, which is the most abundant mRNA produced, encodes for the viral capsid proteins VP1, VP2 and the NSP2. The R2 mRNA encodes for NS2, while the NS3 protein, whose function is not yet clear, is encoded from the Rx and Rx mRNAs [28–31]. In a permissive infection, the NS1 protein is cleaved by caspase proteins at two particular sites being 224INTD↓S228 and 282DQTD↓S286, which facilitates the localization of the full-length NS1 protein to the nucleus to initiate viral replication. This localization has been proposed to be possible by the oligomerization of cleaved NS1 protein with full-length NS1 [30,32,33]. In some cases, AMDV infection can be persistent where this caspase-mediated cleavage event of NS1 does not take place, resulting in a lower amount of viral DNA replicated and a limited amplification of mature virions [34,35]. NS1, with its helicase, DNA binding, ATPase and endonuclease domains, binds covalently to specific ACCA motifs at 5 end of one of the strands of the double-stranded DNA genome and then performs a single-strand nick. This enables a process known as rolling hairpin replication (RHR) in which the terminal hairpin is synthesized and rearranged many times allowing the replication fork to be reversed and to generate many copies of the single-stranded DNA genome [36,37]. The latter is then bound by the VP1 structural protein when it enters the nucleus and, together with VP2, AMDV virions are then assembled to, subsequently, exit out of the nuclear pore complex and from the cell [20]. In a permissive infection, where the NS1 protein was cleaved prior to replication, the mature virions exit the cell by inducing cell lysis [38]. A proposed AMDV viral cycle is summarized in Figure 1.

At both the nucleotide and amino acid sequence levels, AMDV has an unusually high genetic variability where several strains have been identified each having a varying degree of severity of the disease [39,40]. In the case of the Utah 1, Ontario, Danish -K and United strains, these are known to be highly pathogenic, whereas SL3 and Pullman strains are moderately virulent and AMDV-G strain is non-pathogenic [41–43]. In newborn farmed mink (*Mustela vison*), Aleutian disease manifests as a fatal acute interstitial pneumonia with a mortality rate higher than 90% for mink infected with highly virulent strains and ranging from 30% to 50% in low-virulence strains (in infected newborn mink). The most serious form of the disease, however, is persistent and occurs in adults, where it is mediated by the immune system, causing plasma cell proliferation (plasmacytosis), an abnormal amount of gamma globulins present in the blood (hypergammaglobulinemia), the formation of infectious immune complexes and inflammation of kidneys (glomerulonephritis) [3,44–46]. AMDV is transmitted horizontally by air and contact between farms and also vertically from mother to offspring [46–48]. It has been shown that AMDV can also infect different species such as ferrets, otters, polecats, foxes, racoons, skunks, dogs, cats, and mice, but the disease has only been reported to be manifested in mink. Since this virus can infect other animals, the latter can be regarded as possible reservoirs facilitating AMDV spread [49–53].

**Figure 1.** Proposed viral life cycle of Aleutian mink disease virus (AMDV). (1) The virus attaches and binds to the sialic acid receptor on host cells, triggering uptake by clathrin-mediated endocytosis. (2) Endolysosomal trafficking toward the nucleus. (3) Viral single-stranded DNA (ssDNA) genome is delivered into the nucleus. (4) Conversion of ssDNA into dsDNA by host cell DNA polymerase (DNA pol). (5) Transcription of double-stranded DNA (dsDNA) produces precursor mRNA (pre-mRNA) in the nucleus. (6) Splicing and polyadenylation of pre-mRNA yields six mature mRNAs, which exit the nucleus for (7) translation of viral proteins (only NS1, VP1 and VP2 shown). (8) Specifically, for AMDV: cleavage of NS1 protein by caspase proteins to facilitate localization of full-length NS1 to nucleus during permissive replication. (9) Full-length/cleaved NS1 oligomer enters the nucleus to covalently bind to the 5 end of dsDNA, and (10) performs a nick. (11) Rolling hairpin replication (RHR) generates many copies of ssDNA genome. (12) Together, the VP1 and VP2 proteins enter the nucleus and (13) bind the ssDNA genome to form a mature AMDV virion. (14) Exit of AMDV virion from nucleus. (15) Release of AMDV virions through cell lysis. Created with BioRender.com (accessed on 15 August 2021).

Unfortunately, there are not any efficient methods to control the spread of this disease as AMDV is resistant to physical and chemical treatments, which is why farmers manually identify infected mink and kill those screened positive [18]. The diagnosis of AD mink takes place by looking for clinical signs and the detection of AMDV antibodies, which can be performed using non-specific methods such as iodine serum plate agglutination, where the excess of the gamma globulins levels in the serum can be visualized since certain gamma globulins can form complexes with iodine [54]. However, currently, more specific approaches are available such as counterimmunoelectrophoresis (CIEP), which involves using an electric current to assess binding resulting from the migration of unbound

AMDV antigens towards the specific AMDV antibodies [55,56]. In recent times, this approach has been shown to be successful in reducing the prevalence of infected mink in Nova Scotia Canada, but has failed in eradicating AMDV from most of the infected farms [57]. Despite its advantages, CIEP is not a very sensitive diagnostic technique, can yield false negative results and is very time consuming [58,59]. In contrast, the enzymelinked immunosorbent assay (ELISA) is another approach that is frequently used, which is based on detecting anti-AMDV antibodies in blood with the advantage of being automated and more sensitive [60]. Both CIEP and ELISA are very comparable and in good agreement, according to a study by Dam-Tuxen et al., where both techniques were evaluated in Danish mink [61]. Although being a more sensitive approach, ELISA has been shown to be a less accurate technique than CIEP, as reported by Farid and Rupasinghe (2016) [62]. Alternatively, the polymerase chain reaction (PCR) is another specific method that is used as a supplementary test, and this allows the detection of viral DNA by amplifying AMDV gene fragments [63]. A drawback of this method is that it does not detect all virus strains since AMDV has a great genetic diversity and is constantly mutating [64,65]. Another strategy to eliminate the harmful effects of AMDV is by selecting mink that are tolerant to this virus and this can be performed by identifying mink with a low antibody titer, which is performed in Europe and North America [58,66–69]. The selection for tolerance can also be performed by characterizing genomic regions of AD-tolerant mink [70]. Despite the different measures aimed to control the spread of AMDV and its harmful effects on mink, the virus is, unfortunately, still persistent on farms, which increases the demand of more effective AD preventative measures, as well as effective therapies.

Throughout the years, vaccines have been proven to be ingenious tools to be used against highly infectious pathogens and, thus, have been successful in eradicating diseases such as rinderpest in cows [71]. Consequently, one can assume that a plausible approach to combat against the persistent and highly consequential AMDV is to immunize mink against this virus. In fact, there were many attempts of researchers trying to develop an AMDV-vaccine using different approaches with a wide range of results obtained. For this reason, in this article, we review the several attempts of AMDV-vaccine design and the challenges faced in the process to provide a thorough understanding for future studies in the goal of eradicating this deadly disease.

## **2. AMDV Preventative Measures**

Aleutian disease is one of the most threatening diseases in modern day mink farming. Further, domesticated mink escaping farms either by accident or with the help of animal activists pose a threat to wild populations of mink which could act as reservoirs of AMDV [72]. For this reason, the development of a novel AMDV vaccine is of crucial importance. In the meantime, it is of great interest to use alternative strategies to limit the viral spread and avoid huge financial losses to farmers and protect feral mink populations from potential spillovers of the virus. One strategy that is used for temporarily controlling the pathogen in farms is by the mandatory testing of mink to have an early detection and surveillance of AMDV. Common approaches of AMDV diagnosis consist in using serological testing techniques such as ELISA and counterimmunoelectrophoresis (CIEP) or PCR [73,74]. PCR is not as widely used as a routine serological testing method for herd screening due to a short-lived viral replication and viremia which causes negative PCR, while AMDV is sequestered in organs, making viral detection more challenging [44,58,75–77]. An additional reason for this is the cost of an essential DNA extraction step, which requires the clearance of residual inhibitors that would lead to the loss of sample DNA if not removed [78,79]. Additionally, in a recent study, it was shown that CIEP turned out to be more accurate than conventional PCR testing in black American mink infected with AMDV [75]. When taken together, early diagnostic approaches of AMDV-positive mink could provide reinforcing tools to help limit viral spread. Indeed, these have been somewhat effective in several countries, including Spain, where farms with AD have been reduced from 100% in 1980 to around 25% in 2019 [68].

Despite this encouraging statistic, however, the eradication of the disease remains challenging and has failed in many countries, including Canada, Denmark, and others [57,63,68,80]. This can be explained by the reinfection of farms due to viral persistence and by contaminated human individuals visiting farms, leading to a failure of eradication systems [81]. For this reason, some researchers explored the idea of immunomodulation in AMDV-infected mink. In fact, this approach has been investigated in a variety of animals, where the host immune system is modulated to be enhanced against infectious diseases. This can be achieved with numerous substances such as cytokines, microbial products, traditional medicinal plants, nutraceuticals and pharmaceuticals [82]. An example of immunomodulators are β-glucans, which are readily used in sheep and swine production systems [83]. For Aleutian mink disease, this strategy has also been explored by some researchers. This is the case of a study by Kowalczyk et al. [84] where female brown AMDV-positive mink were given methisoprinol (isoprinosine; inosiplex; inosine pranobex), a drug with immunopotentiating properties for which antiviral and immunostimulatory effects on Aujeszky's and Newcastle disease have been reported by various groups [84]. Interestingly, the methisoprinol treatment of mink displayed a lower number of the AMDV DNA copies in the spleen and lymph node, as well as a higher fecundity compared to control mink [85]. More recently, Farid and Smith investigated the effect of kelp meal (*Ascophylum nodosum*) in mink infected with AMDV. From this study, it was shown that the supplementation of mink with kelp meal for 451 days did not improve the mink immune response to infection or virus replication. Interestingly, lower levels of blood, urea, nitrogen (BUN) and creatine were noticed, which suggested an improved kidney function [86].

Alternatively, another initiative for reducing the effects of AD on chronically infected farms is by passive immunity, which can provide immediate, but short-lived, protection to humans and animals [87]. In the context of Aleutian disease, the effect of the passive transfer of anti-ADV gamma globulins to newborn mink infected with a highly virulent strain of AMDV has already been investigated by Alexandersen et al. [88]. From their research, an acute AMDV infection was prevented by the passive antibodies, but mink still manifested a chronic infection [88]. The mechanism of the modulation from acute to chronic AMDV was then assessed in vitro, where infected alveolar type II cells treated with antibodies showed lower levels of AMDV replication and transcription intermediates. This was also noted in antibody-treated kits in vivo, which allowed the authors to suggest a role of antiviral antibodies in developing persistent infection in mink [88]. Furthermore, antiviral drugs are regarded as an important strategy to control the spread. Indeed, this strategy is very common and used for many infectious agents in humans and animals, such as human immunodeficiency virus (HIV) and Feline Herpes Virus (FHV) [89]. For AMDV, unfortunately, there is no approved antiviral treatments available for use; however, recently, Lu et al. [90] attempted a novel antiviral treatment using the "magnetic beads-based systemic evolution of ligands by exponential enrichment" (SELEX) strategy to generate aptamers for the AMDV VP2 protein. From their results, it was shown that, in vitro, the micromolar concentrations of the aptamers for AMDV VP2 protein were able to specifically inhibit by half the AMDV production by infected cells [90]. More investigation is, therefore, required in future studies in designing an adequate antiviral for AMDV.

Another strategy to prevent the harmful effects of AMDV is to select mink having low titers of anti-AMDV-protein antibodies in their blood, which is a common practice in many countries [58,66–69]. Besides this classical selection method, an alternative approach has been recently studied by Karimi et al. which is based on characterizing host-genomic patterns of AD-tolerant mink with the intention to select those that could be tolerant or resistant to AMDV. More specifically, the authors performed a sequencing approach to focus on genomic regions related to host specific immune responses. Using this strategy, two regions of the mink genome were shown to be strongly selected and these contained important genes involved in the immune response, viral–host interaction, reproduction and liver regeneration [70]. Additionally, since AMDV is a rapidly evolving pathogen, it is of great importance to be informed about current and past circulating strains to be

able to develop epidemiological models, predictions and forecasting of the strains that may cause serious outbreaks. This knowledge would help us in devising an efficient and practical control strategy against this virus in the future, by setting up a database similar to the global initiative on sharing all influenza data (GISAID), which has been especially useful in the ongoing coronavirus disease-19 (COVID-19) pandemic in tracking novel variants [91]. Some studies have in fact performed phylogenetic analyses of the AMDV genomes deciphered by genetic sequencing. This was performed in order to develop new tools for outbreak investigation, the determination of virulence markers and development of more sensitive diagnostic tests in the future [92–99]. As an example of such work, Canuti et al. [93] studied the evolutionary dynamics of AMDV from 2004 to 2014 using sequences obtained from many regions of the world, including North America and Europe. From these, a very high viral genetic diversity as well as high rates of co-infection were noted in Newfoundland, Canada. Interestingly, a low level of diversifying selection was shown on structural proteins, which was explained to be due to the specific cell-entry mechanism of AMDV, which uses antibodies to enter cells. Finally, it was concluded that a great amount of circulating viruses on farms has the capacity to recombine and increase viral diversity by co-infections [93]. Therefore, summarizing, these methods could be of great use to control the spread of AMDV, while actively working on a development of effective vaccine candidates. A summary of these preventative measures is shown in Table 1.


**Table 1.** Some of the AMDV Preventative Measures Proposed: Diagnostic and Reduction Methods.

Diagnostic methods are used for detection, surveillance, for early detection and to control the spread. Immunomodulator molecules can be used to enhance the host immune system against infectious diseases. a—counterimmunoelectrophoresis. b—enzyme-linked immunosorbent assay. c—polymerase chain reaction.

#### **3. AMDV Vaccine Attempts**

## *3.1. Inactivated Vaccine*

The use of inactivated and live attenuated virus-based vaccines dates back to more than a century. The principle of an inactivated viral vaccine consists of exposing a virus to chemical or physical agents, which disable its infectivity while allowing it to stimulate an immune response [100]. An example of this is by using formaldehyde, a cross-linking agent of amino acids, or, more modern approaches such as ascorbic acid or hydrogen peroxide, have been shown in recent years [101]. Currently, there are many inactivated

viral vaccines available and approved for usage against viruses, including Hepatitis A, Japanese encephalitis virus, poliovirus, rabies and SARS-CoV-2 [102–105]. A study by Karstad et al., in 1963 [106], was one of the first attempts to develop an AMDV vaccine using this approach. These Canadian researchers used the inoculations of formalin-treated suspensions of tissues of AMDV-infected mink as a candidate vaccine [106]. It was shown that upon inoculating mink with this vaccine candidate, no plasmacytosis was noted in contrast to those inoculated with untreated diseased tissue. However, formalin-treated tissue failed to protect inoculated mink from the challenge with a virulent inoculum. Further, it was also noted that mink receiving three doses of the formalin-treated tissue suspension subcutaneously were not able to stimulate the immune system, i.e., the mink experienced plasmacytosis upon challenge [106]. Less than 10 years later, Porter et al. [107] tried a similar approach, where formaldehyde was used to inactivate AMDV-infected spleen and liver tissue homogenates. The inactivated homogenate was then administered to pastel mink and performed in parallel with the administration of a control group. From this, it was shown that there was no change in gamma globulin levels due to AMDV or the control group 33 days after inoculation. However, upon the challenge with a 102 and 10<sup>5</sup> tissue culture infective dose (TCID50), the gamma globulin levels in AMDV-vaccinated mink were slightly greater than the control group. Surprisingly, it was also reported that mink vaccinated against AMDV displayed more tissue lesions and enhanced plasmacytosis than the mink receiving the control vaccine, where both groups were challenged with the same dose of the virus. The authors then evaluated the effect of antibodies on this AD enhancement by giving 250 mg or 1 g of normal mink IgG antibodies to infected mink. A significantly enhanced disease was observed with acute necrotizing lesions. In contrast, this was not observed in infected mink receiving a lower dose of normal mink IgG, and in uninfected mink receiving both anti-AMDV IgG and normal mink IgG. Thus, it was suggested that antibody levels in the AMDV-vaccinated mink were not necessarily higher than in the control, since it was not noted as a significant number of lesions in the former compared to the latter. Moreover, based on immunofluorescence data, the authors suggested that observed enhanced pathology was due to antibody-complementinduced cytolysis followed by the complement-mediated attraction of polymorphonuclear leukocytes [107].

## *3.2. DNA Vaccine*

Before AMDV was known to be the causative agent of Aleutian Disease, one study by Basrur and Karstad [108] investigated the effects of infection with DNA, extracted from the spleen tissue of mink with Aleutian disease. An in vitro inoculation of mink testis cells with viral DNA demonstrated a change in cellular morphology and abortive growth, which were absent in controls. This finding was also evaluated in vivo, on nine-month-old standard dark male mink, where plasmacytosis was observed in most of animals inoculated with the viral DNA. In contrast, the control group did not demonstrate such an effect, leading the authors to believe that the disease was of viral origin [108]. In more recent times, the use of DNA derived from viruses or other pathogens has been of great interest in the development of vaccines. This immunization method is referred to as DNA vaccination that consists of administering foreign viral protein-coding plasmid DNA into animals of interest [109,110]. Using the host cellular translation machinery, antigen proteins are then generated in situ followed by MHC-I and MHC-II pathways and the induction of CD8+ and CD4+ T cells, leading to an antigen-specific immunity [109–111]. DNA vaccines have been used in human clinical trials against infectious agents, for cancer immune therapies and for asthma and allergies as well as gene therapies for chronic diseases [112–114]. For animals, there are several DNA vaccines created against a variety of viruses. Some DNA vaccines have already been approved to be used, such as the first commercial DNA vaccine against H5N1 for chickens and the West Nile Virus vaccine for horses [115–117].

In the case of AMDV, there have been several attempts to develop a vaccine, but to date, none of them have been successful. One of those attempts was conducted by the

group of Castelruiz et al. (2005), which designed a vaccine candidate based on an AMDV NS1 protein-coding plasmid (pNS1). This approach was first tested in vitro, where a high expression of the NS1 protein was revealed after transfecting Crandell feline kidney cells (CRFK) with the NS1 expressing plasmid. After confirming the NS1 protein expression in cell culture, the vaccine candidate was evaluated in vivo using three different groups of non-Aleutian female mink. In the first group, the animals were immunized with pNS1, followed by a booster inoculum containing recombinant NS1 protein (named "DNA + protein" group). The second group was identical to the first, with the exception of omitting the NS1-protein inoculation step (named "DNA only"). Finally, the third group received an empty plasmid, which did not contain the NS1 gene (named "control" group). After each of these groups were challenged, the "DNA + protein" vaccine cohort was shown to be the only group to exhibit antibodies against the NS1 protein at the time of challenge. With time, this was also observed in the other cohorts and, more specifically, at 12 weeks post infection, the "DNA only" group displayed a significantly higher anti-NS1 titer than the "control" group. A similar result was reported for all groups, when assessing anti-VP1/2 antibodies, with the only difference of not observing any VP1/2 antibodies on the day of challenge with AMDV. Interestingly, after 1 month of challenging mink with the virus, the "DNA+ protein" group exhibited higher levels of CD8+ T lymphocytes than the two other groups. This effect was suggested to be due to a memory response after an increase in interferon γ-producing lymphocytes for the same group was observed between 4 and 8 weeks post-challenge. A mild vaccine effect was suggested when noting that the "control" group displayed 10% higher gamma globulin levels than the other groups 8 weeks post-challenge. In terms of post-challenge deaths, most of the "control" group succumbed, where two of the latter were not likely to be AD related. In contrast, in the "DNA only" and "DNA + protein" groups, most of the animals survived. From the results of both AMDV NS1 DNA vaccinated groups, it was finally concluded that a mild vaccine effect was induced, but it was only partially protective, since there were still mink that died during the experiment [118]. Later, another attempt was undertaken by Liu et al. [119] to achieve the AMDV DNA-based vaccine (2018). For their vaccine candidate, the AMDV-DL125 strain was selected as a template since it displayed the highest TCID50 from virulent strains assessed in CRFK cells. From this strain, the whole genome was used to generate plasmid-vectored vaccines, by truncating regions of VP2 and NS1 genes using an overlap extension PCR. Similar to the study of Castelruiz et al., these plasmids with truncated regions were then tested in vitro on CRFK cells, where the successful infection of the cells with vaccine plasmids was confirmed by immunofluorescence, in contrast to controls, where no immunofluorescence signal was observed. Next, groups of six-month-old female mink were used to conduct an in vivo study. Most of these groups were administered with distinct DNA vaccine candidates, whereas the rest served as control groups, receiving phosphate-buffered saline (PBS), empty vector and AMDV-inactivated virus, respectively. Similar to Castelruiz et al., the anti-AMDV antibody levels in each group were shown to increase with time after challenge with AMDV. Interestingly, at the 24-week timeframe post-challenge, all groups demonstrated similar antibody levels in the serum, except for the inactivated AMDV-administered control group. The latter also displayed rapidly increasing levels of circulating immune complexes (CIC), suggesting that the inactivated AMDV vaccine could help accelerate the development of AD. For other groups, the ones inoculated with VP2 gene carrying plasmid with deletions at nucleotides coding for residues 428–446 and 487–501 (pcDNA3.1-ADV-428–487) had the lowest serum gamma globulin and CIC levels. This was suggested to be the most protective cohort; nonetheless, dead mink in each vaccinated and control group were observed with the earliest death occurring in the AMDV-inactivated group, followed by the others. By the end of the experiment (36 weeks post-challenge), the pcDNA3.1-ADV-428–487 group had the lowest mortality, and this allowed Liu et al. to suggest that this vaccine could be potentially used in mink populations in the future [119].

## *3.3. Subunit Based Vaccine*

In 1981, the Food and Drug Administration (FDA) approved, for the first time, the plasma-derived hepatitis B virus (HBV) Heptavax-B vaccine (Merck) [120]. This was based on a heat-inactivated HBV surface envelope protein (HBsAg) isolated from the blood of asymptomatic HBV-infected patients and was shown to provide good protection in immunized individuals [121]. However, with the imminent HIV/AIDS global epidemic, concerns of infecting vaccinated individuals by blood with other pathogens limited this initiative, leading it to be discontinued in 1990 [122,123]. This encouraged the development of recombinant DNA technology, where the HBsAgs were produced in a yeast culture and this approach was used for the first time by the RECOMBIVAX vaccine (Merck Sharp and Dohme) approved in 1986 [123,124]. Protein subunit-based vaccines such as these primarily induce humoral immunity and exclude pathogens from their production, which reduces the risk of an incomplete activation, pre-existing immunity or causing disease [125,126]. Despite these advantages, the subunit vaccination, on its own, stimulates weaker immunological responses, which is why additional components known as adjuvants are added in vaccine doses [127,128]. Adjuvants, such as aluminium salts, can enhance cell-entry and preserve the structural integrity of the antigen, stimulate macrophages that promote helper T-cell responses, induce CD8+ cytotoxic T-lymphocytes as well as help in slowly releasing the antigens to prolong the exposure time to the immune system [129,130]. Currently, alongside HBV vaccination, the subunit-based approach is actively being tested for use in a variety of human pathogenic viruses such as Ebola and HIV [131–133].

In the case of Aleutian mink disease virus, this approach was investigated by Aasted et al. in 1998, where recombinant VP1/2 and NS1 proteins of AMDV-G were used to inoculate nine-month-old black female mink. Mink vaccinated with VP1/2 were challenged with AMDV which resulted in a higher death rate, more extreme hypergammaglobulinemia, higher NS1 and VP1/2 titers and higher counts of CD8-positive lymphocytes in lower peripheral blood compared to unvaccinated mink. The authors suggested that the observed outcome was caused by the antibody-dependent enhancement (ADE) of infection, a key mechanism in the pathophysiological changes of the AD, arguing that AMDV replicating cells are likely to be Fc-receptor positive. In the case of the group of mink vaccinated with a 10-fold higher dose of NS1, a more positive immunization effect was noticed. Throughout 11 months after challenge, lower levels of serum gamma globulin and CD8 positive lymphocytes were noted in addition to the lower death rates compared to a group of the non-vaccinated mink. The researchers did not detect vaccine-induced antibodies for VP1/2 and NS1 using ELISA assays. It was suggested that it could have been due to the induction of antibodies with a low affinity to native AMDV proteins since this higher affinity could have been developed to denatured the antigen protein vaccine present in antigen preparations prior to inoculation [134].

Considering the different vaccination approaches designed throughout the years against AMDV, the most successful attempt can be concluded to be the DNA vaccines, more particularly, those engineered by the group of Liu et al. [119]. Given the limited number of approaches for a successful vaccine design, more testing and the use of recent technological advancements in vaccinology can be employed to reinforce the fight against AMDV. Such methods are further elaborated in the Discussion section. The different vaccination attempts covered in this section against AMDV are summarized in Table 2.


### **Table 2.** AMDV Vaccination Attempts.

## **4. Discussion**

Throughout the years, many animal viruses have emerged and have caused serious financial and public health repercussions in many regions worldwide. A prime example of these is the Aleutian mink disease virus (AMDV), which poses a serious threat to mink since it is the most significant disease in their farming around the world [135]. Therefore, it is of great importance to produce effective treatments and vaccines to prevent significant losses caused by AMDV. As discussed in this review, there have been many attempts of producing vaccines against AMDV using different strategies. From these, three main techniques were used, including inactivated, subunit protein and DNA-based vaccines. In the case of inactivated vaccines, Karstad et al. showed an enhanced disease upon challenging mink that had been inoculated with formalin-treated AMDV-infected tissues, which was also shown by Porter et al. [106,107]. The latter additionally reported higher gamma globulin levels as well as more lesions in challenged vaccinated mink [107]. As for the design of subunit vaccines, Aasted et al. also demonstrated an enhanced disease after challenging VP1/2-vaccinated mink with higher death rates and extreme hyperglobulinemia [134]. In contrast, this effect was not noted in mink when a recombinant NS1 protein was used at a dose of 10-fold greater than that used for the VP1/2 inoculum. Instead, lower levels of gamma globulins and CD8 positive lymphocytes were present in the serum and less deaths were shown [134]. For DNA vaccine candidates, Castelruiz et al. obtained better

results by using an AMDV NS1 coding plasmid either given alone or in combination with recombinant NS1 protein [118]. In fact, for both strategies, a mild vaccine effect as well as partial protection were found [118]. Similarly, a much promising result was reported by Liu et al., who designed seven plasmid-vectored vaccines, which were based on the genome of the infectious AMDV-DL125 strain [119]. From those, the best candidate showed partial protection and a higher efficacy based on lower levels of circulating immune complexes (CIC) gamma globulins in the serum [119]. This was based on the VP2 gene, which had deletions at nucleotides coding for residues 428–446 and 487–501 [119]. From all AMDV vaccination attempts, the most promising ones have been shown to be the DNA-based vaccines. Interestingly, for the other strategies, some authors have previously suggested an antibody-dependent enhancement of disease (ADE), which was used to explain the enhanced disease upon challenge of post-vaccinated mink [106,107,134]. Indeed, AMDV has already been reported to use the Fc-receptor-mediated mechanism to enter cells [17,18]. With this in hand, Bloom et al. investigated the molecular mechanism of ADE in the context of AMDV in vitro [18]. Namely, researchers evaluated effects of mono- and poly-clonal antibodies against short peptides designed from the immunoreactive VP2: 429–524 linear epitope using CRFK and K562\_cells [18]. From their results, the VP2: 428–446 peptideinduced Fc-mediated ADE, the neutralization of AMDV and participated in an immune complex formation. A limited ADE was also noted in the case of the VP2: 487–501 peptide. It was finally concluded that this may be the mechanism by which capsid-based vaccines have the ability of inducing both neutralization and ADE, which agrees with the disease enhancement (vaccine-enhanced disease) shown in the inactivated and subunit protein approaches [18]. Further, this is also consistent with the study by Liu et al., where the exclusion of these sites led to a more efficient vaccine candidate [119]. Therefore, it might be suggested that in future vaccination attempts, sites from the immunoreactive VP2 segment should be excluded for a better response.

Another approach to this could be to analyze the full VP2 sequence to find epitopes that can further mediate ADE. One way to achieve this is by generating infectious clones of the virus and identifying capsid residues that are necessary for infecting cells in vitro. In fact, Xi et al. previously used this method to show that VP2 residues 92 and 94 are critical for AMDV replication in vitro [136].

Since there have been few vaccine technologies used to generate AMDV vaccine candidates, other vaccine development strategies or their combinations can be further used such as viral-vectored, virus-like particles and nanoparticle-based vaccines [137]. For example, for some diseases, viral vectors have been successfully used in combination with other vaccine approaches in a strategy called a heterologous prime-boost [138]. Lately, in the context of the current coronavirus-19 pandemic (COVID-19), one particular approach, based on the mRNA technologies, has been used in two licensed vaccines, which has helped significantly reduce infection numbers worldwide [139,140]. Both mRNA-1273 (Moderna) and BNT162b2 (Pfizer—BioNTech) rely on lipid-nanoparticle (LNP)-encapsulated mRNA expressing the pre-fusion stabilized spike glycoprotein, which is translated upon entering the cells [141,142]. With the great efficiency and success brought by these vaccines, a plausible approach could be to design a VP1/2 or an NS1-based mRNA vaccine for AMDV.

As the search for an efficient AMDV vaccine continues, novel methods are currently being explored to enhance vaccine (immunogen) delivery. This is the case of nanoparticle technology, which has been explored in the context of many viruses [143]. In fact, nanovaccines (vaccines, where nanoparticles or nanomaterials are used as carriers) are created by displaying relevant antigenic sites on nanomaterials either by physical entrapment or by covalent binding [144]. Such vaccines have a similar size to the infectious pathogens, which makes it easier to enter the target cells, where they will be degraded and release the antigen over time [145]. In terms of their numerous advantages, these can induce immune responses, could be given intramuscularly or via mucosal sites, and they are stable at room temperature [144,146,147]. One interesting biodegradable nanovaccine approach that has been approved by the FDA is by using liposome-derived nanovesicles as carriers, since

these can enhance the delivery of antigens to cells [148,149]. The use of such liposomal delivery systems has been previously reported to enhance immune responses against many viruses, including Newcastle disease virus [150,151]. Using this approach has also been shown to stimulate both Th1 and Th2 responses in response to an H3N2 influenza subunit vaccine candidate [152]. Another example of nano-vaccines is virus-like particles (VLPs), which mimic native viruses with the exception of not having the capacity to infect and replicate [153]. These are composed of self-assembling viral antigen proteins which can induce a stronger humoral immune response compared to single soluble antigens [154]. In recent years, there have been several approved vaccines using this technology, including Gardasil® and Gardasil9®, against human papilloma virus (HPV), and many others [155].

Alternatively, another new vaccination method that is actively being explored involves the use of cell-derived bi-layered extracellular vesicles (EVs) such as exosomes, microvesicles and apoptotic bodies [156,157]. Indeed, these have great advantages in terms of delivery and can increase overall immunogenicity since they have immunostimulatory molecules on their surfaces such as MHC class I or II molecules [156,158]. Further, exosomes have the capacity to carry pathogen antigens, as was shown by Montaner-Tarbes et al. in a study analyzing exosomes from pigs infected with porcine reproductive and respiratory virus (PRRSV) [159]. From this, a specific reaction was demonstrated in a PRRSV RNA-negative and seropositive pig upon testing the exosome-derived viral proteins [159]. The microvesicle approach has also been tested by many researchers, including Rappazzo et al., where mice were vaccinated with microvesicles expressing ClyA surface protein fused with influenza matrix protein 2 [157,160]. Upon challenge with a virulent mouse-adapted H1N1 influenza strain, vaccinated mice demonstrated a full protection, and the passive transfer of their antibodies to non-vaccinated mice protected the latter when challenged [160]. Microvesical vaccines, such as liposomal nano-vaccine delivery methods, can also be given orally or nasally [161]. With the aforementioned advantages, these novel approaches for designing effective vaccines could be used to vaccinate mink against AMDV. This could be performed by incorporating key AMDV antigens in aerosolbased nanoparticles or EVs, which would help vaccinate a large number of mink rapidly from their cages by simply releasing these vaccines in the air. However, another important challenge faced, when designing AMDV vaccines, is that the virus has an unusually high genetic variability in both NS1 and VP2 proteins [39,40].

To enhance the design of future AMDV vaccines, one strategy can be the constant tracking of current and past circulating strains by phylogenetic analyses, which can help determine key virulence markers [92–97]. This is how Canuti et al. revealed that structural proteins were not under diversifying pressure in sequences analysed in Newfoundland, probably due to the conserved cell-entry mechanism of AMDV [93]. Moreover, in the wait of effective vaccines, there are many methods used and still being developed to control AMDV spread. A commonly used approach is detecting infected mink earlier on by serological testing techniques and by, subsequently, eliminating them [73,74]. Other techniques that have been explored or that are in development include the usage of passive anti-AMDV antibodies, antivirals, immunomodulators and the selection of mink with AMDV-tolerant traits [70,85,86,88,90]. Unfortunately, despite some positive results, these are not enough to eradicate this deadly disease, which is why the design of an effective vaccine is of primordial importance.

To conclude, AMDV poses a great threat to a huge number of mink either in farms or in in the wild and represents a potential risk to other species. In this review, the attempts to design a much-needed vaccine against this virus were covered, as well as the challenges that researchers are faced in designing a novel vaccine. For future studies, novel vaccine technologies should be aimed at, as well as trying to design better preventive measures to control viral spread.

**Author Contributions:** Conceptualization, L.A.; methodology, L.A.; validation, L.A.; data curation, N.M.M. and L.A.; writing—original draft preparation, N.M.M. and L.A.; writing—review and editing, N.M.M. and L.A.; supervision, L.A.; project administration, L.A.; funding acquisition, L.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partially supported by the Mink Research Partnership and Canada Mink Breeders Association grant (RY000238) and by the NSERC Discovery grant RGPIN/04897-2017 to Levon Abrahamyan (Université de Montréal). N. Markarian was a recipient of the NSERC Undergraduate Student Research Award, and he realized an internship under L. Abrahamyan's supervision.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Hossain Farid (Dalhousie University) and John Easley (Mink Research Partnership) for their valuable comments that greatly improved the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Communication* **Construction and Immunogenicity of Virus-Like Particles of Feline Parvovirus from the Tiger**

**Cuicui Jiao 1,2,**†**, Hongliang Zhang 1,**†**, Wei Liu 2,**†**, Hongli Jin 3,4, Di Liu 2, Jian Zhao 2, Na Feng 4,\*, Chuanmei Zhang 1,\* and Jing Shi 1,2,\***


Received: 9 February 2020; Accepted: 13 March 2020; Published: 16 March 2020

**Abstract:** Feline panleukopenia, caused by feline parvovirus (FPV), is a highly infectious disease characterized by leucopenia and hemorrhagic gastroenteritis that severely affects the health of large wild Felidae. In this study, tiger FPV virus-like particles (VLPs) were developed using the baculovirus expression system. The VP2 gene from an infected Siberian tiger (*Panthera tigris altaica*) was used as the target gene. The key amino acids of this gene were the same as those of FPV, whereas the 101st amino acid was the same as that of canine parvovirus. Indirect immunofluorescence assay (IFA) results demonstrated that the VP2 protein was successfully expressed. SDS-PAGE and Western blotting (WB) results showed that the target protein band was present at approximately 65 kDa. Electron micrograph analyses indicated that the tiger FPV VLPs were successfully assembled and were morphologically similar to natural parvovirus particles. The hemagglutination (HA) titer of the tiger FPV VLPs was as high as 1:218. The necropsy and tissue sections at the cat injection site suggested that the tiger FPV VLPs vaccine was safe. Antibody production was induced in cats after subcutaneous immunization, with a >1:2<sup>10</sup> hemagglutination inhibition (HI) titer that persisted for at least 12 months. These results demonstrate that tiger FPV VLPs might provide a vaccine to prevent FPV-associated disease in the tiger.

**Keywords:** feline parvovirus; virus-like particles; VP2 protein; antibodies

Feline panleukopenia, caused by feline parvovirus (FPV), is an acute and highly contagious disease [1]. It clinically manifests as vomiting, high fever, leukopenia and enteritis in infected animals [2]. FPV has been known to induce disease in cats since the 1920s [3]. Since its discovery, FPV has been isolated from cats, raccoons, monkeys, and many different wild and captive carnivores [4,5]. FPV is very stable in the environment, with infectivity persisting for up to one year within contaminated material [6]. Consequently, FPV has a high risk of transmission, posing a great threat to rare wild animals. In addition, FPV is prevalent in the Chinese Siberian tiger (*Panthera tigris altaica*) population [7,8]. On June 6th 2016, an outbreak of fatal FPV infection among captive Siberian tigers in Zhengzhou Zoo in central China was reported [7]. An FPV strain was isolated and identified from a captive Siberian tiger in a wildlife park in the Jilin Province, China [8]. Wild Siberian tigers from the Russian Far East

had a 68% FPV antibody-positivity rate [9]. Vaccination is an effective means to prevent FPV infection. Currently, attenuated vaccines and inactivated vaccines are approved for use against FPV in cats. Attenuated vaccines have been recommended for wildlife use in North American countries, Europe and Asia [10]. A live-attenuated vaccine (Virbac S.A., Carros 06511, France), which combines feline calicivirus (FCV), feline herpesvirus (FHV) and FPV strains, is used to prevent disease in tigers [11]. However, it has been reported that attenuated FPV vaccines with a risk of virulence reversion can cause cerebellar dysplasia in cat fetuses [12]. The antibody level produced by inactivated vaccines is low, the antibody duration is short, and there is a risk of incomplete inactivation. Furthermore, the safety of using inactivated vaccines in tigers remains unknown. Therefore, the development of a novel vaccine for feline parvovirus disease in tigers will provide material reserves and new products for immune protection against important diseases in tigers.

FPV, a single-stranded DNA virus, belongs to the family *Parvoviridae*, subfamily *Parvovirinae*, genus *Protoparvovirus* [13]. The virions have a symmetrical icosahedral structure [14]. The FPV genome contains two open reading frames encoding nonstructural proteins (NS1 and NS2) and structural proteins (VP1 and VP2). The VP2 protein contains the major epitopes that stimulate the production of neutralizing antibodies [15], and mutations in key amino acid sites of the VP2 protein can affect its antigenic characteristics and host range [16,17].

Virus-like particles (VLPs) are composed of multiple copies of one or more recombinant viral structural proteins and are spontaneously assembled into particles without the viral genome [18]. Due to being replication-incompetent and producing nonpathogenic effects, VLPs have been widely used in studies of human and veterinary candidate vaccines [19]. In this study, the parvovirus VP2 gene was identified from a dead Siberian tiger in a wildlife park in the Jilin Province, China [8]. The tiger had symptoms such as diarrhea and vomiting before it died. The VP2 sequence was analyzed and kindly provided by Dr. Wang [8]. We used the baculovirus expression system [20] to produce the VP2 protein of tiger FPV. The VP2 protein can be assembled into VLPs which resemble the natural virus in size and shape. Meanwhile, tigers and cats both belong to the family Felidae. Due to the rarity and importance of the tiger, cats were selected to conduct immunization experiments of the tiger FPV VLPs vaccine. Herein, the tiger FPV VLPs vaccine stimulated cats to produce hemagglutination inhibition (HI) antibody, providing a preliminary basis and technical support for the preparation of new vaccines against tiger (*Panthera tigris altaica*) feline parvovirus disease.

First, the amino acid sites of the parvovirus VP2 gene from a tiger were analyzed. The results showed that most of the amino acid sites were consistent with those of the FPV reference sequence, whereas the 101st amino acid was consistent with that of the canine parvovirus (CPV) reference sequence (Table 1). Therefore, the parvovirus VP2 gene from the tiger was named tiger FPV VP2.


**Table 1.** Amino acid variations in the VP2 protein.

The box shows the difference between Tiger FPV and FPV at 101st amino acid.

Next, we optimized the codons of tiger FPV VP2 for *Spodoptera frugiperda* 9 (Sf9) cells according to codon preference (shown in Supplementary Materials, Sequence 1). Sequence optimization and synthesis were completed by Shanghai Biological Engineering Co., Ltd. The VP2 gene was amplified using the primers shown in Table 2 and cloned into the pFastBac Dual vector (Invitrogen, Carlsbad, CA, USA). The pFBD-dVP2 vector, a donor plasmid containing two copies of the VP2 gene, was successfully constructed. The pFBD-dVP2 donor plasmid was transformed into *E. coli* DH10Bac competent cells (Invitrogen, Carlsbad, CA, USA) to obtain the rBacmid-dVP2 recombinant bacmid.



The bold characters are the sequences of restriction enzyme sites.

The rBacmid-dVP2 was transfected into Sf9 cells and cultured in six-well plates at approximately 80% confluence with Cellfectin II Reagent Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and the cells were then cultured in sf-900 II SFM medium (Gibco, Grand island, NY, USA) at 27 ◦C. It took approximately 96 h to reach an 80% cytopathogenic effect (CPE). The supernatant was collected to obtain recombinant baculovirus, called rpFBD-dVP2 which was passage 1 (P1). The genome was extracted and determined to be correct by PCR. The baculovirus stock titer was measured using a Baculovirus Rapid Titer Kit (TaKaRa, Tokyo, Japan) according to the manufacturer's instructions. RpFBD-dVP2 was continuously passaged to P4 at a multiplicity of infection (MOI) of 0.5, and obvious CPE was observed at 48 h, with large, rounded and shed cells (Figure 1A).

The expression of VP2 protein was verified by an indirect immunofluorescence assay (IFA). Sf9 cells were infected with rpFBD-dVP2 at a multiplicity of infection (MOI) of 0.5 and fixed after two days. A monoclonal antibody against CPV (clone 8H7, HyTest Ltd., Turku, Finland) was used as a primary antibody (1:200), and fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse IgG (Solarbio, Beijing, China) was used as the secondary antibody (1:200). The IFA results showed obvious green fluorescence (Figure 1Ba), and no specific fluorescence was observed in normal Sf9 cells (Figure 1Bb). This result indicated that VP2 protein was expressed in Sf9 cells and could be specifically recognized by the VP2 monoclonal antibody.

Sf9 suspension cells were cultured in triangular flasks with sf-900 II SFM (Gibco, Grand island, NY, USA) at a speed of 120 rpm. To assess the expression of VP2 protein, Sf9 suspension cells were harvested 4 days after infection. The mixture was separated into cells and supernatant at 1751× *g* for 30 min. The cells were lysed with filtered 25 mM NaHCO3, after which the supernatant was harvested by centrifugation. The samples were characterized by SDS-PAGE, and a clear band was observed at 65 kDa (Figure 1C). The results showed that VP2 protein was successfully expressed in Sf9 cells and released into the supernatant after simple cell lysis. A Western blotting (WB) assay was carried out to verify whether the 65 kDa band was the target protein. Samples from SDS-PAGE were transferred to a nitrocellulose (NC) membrane (GE Healthcare, Dassel, Germany). In the WB assay, as the primary antibody, the monoclonal antibody against CPV (clone 8H7, Hytest, Turku, Finland) at a 1:500 dilution was incubated at room temperature (RT) for 1.5 h. Then, the horseradish peroxidase (HRP)-labeled sheep anti-mouse IgG at a 1:5000 dilution was incubated at RT for 1 h. As shown in Figure 1D, the WB analysis results showed that the target protein specifically bound the monoclonal antibody against parvovirus; a specific band was seen at the molecular weight of approximately 65 kDa in lane 2, whereas no specific band was seen in the control sample. These results indicated that the expressed VP2 protein had the capacity to elicit a specific antibody response.

**Figure 1.** Recombinant baculovirus acquisition and the identification of VP2 protein expressed in Sf9 cells. (**A**) Cytopathogenic effect (CPE) of the recombinant rpFBD-dVP2 baculovirus (magnification, 200×). (Aa) Sf9 cells infected with rpFBD-dVP2 displayed typical CPE 48 h after infection, with large, rounded and shed cells, and (Ab) untreated Sf9 cells remained normal. (**B**) Indirect immune fluorescence antibody staining of VP2 protein in Sf9 cells infected with rpFBD-dVP2 (magnification, 200×). (Ba) Obvious green fluorescence was observed in Sf9 cells infected with rpFBD-dVP2, and (Bb) untreated Sf9 cells showed no fluorescence. (**C**) SDS-PAGE analysis of VP2 protein expression. Lane 1, untreated mixture; lane 2, supernatant after centrifugation; lane 3, supernatant after sodium bicarbonate treatment; lane 4, prestained protein markers; lane 5, cells infected with control baculovirus. (**D**) WB analysis of VP2 protein expression. Lane 1, cells infected with control baculovirus; lane 2, cells infected with recombinant baculovirus; lane 3, prestained protein markers.

HA tests were carried out as previously described to detect VP2 expression levels [22]. The HA titer was determined by the highest dilution of tiger FPV VLPs that caused pig erythrocytes agglutination. The results in Figure 2A showed that the HA titer of tiger FPV VLPs reached 1:218. High yields of tiger FPV VLPs could be produced due to the high HA titer [23]. To inspect their morphology, the tiger FPV VLPs were observed by electron microscopy. The results showed that the tiger FPV VLPs were successfully assembled with a diameter of ~22 nm; their morphology was similar to that of natural parvovirus particles (Figure 2B).

**Figure 2.** Determination of HA titer and observation of tiger FPV VLPs morphology. (**A**) HA activity of tiger FPV VLPs. (**B**) Electron micrograph analysis of tiger FPV VLPs. The arrow indicates VLPs, and the bar represents 100 nm.

The supernatant containing VLPs after cell lysis was used to prepare the vaccine. Tiger FPV VLPs were emulsified with Gel 02 adjuvant (Seppic, Paris, France) at a volume ratio of 7:1. To determine whether the vaccine was safe for cats, an overdose vaccination test was carried out on cats. Two 10-week old cats were subcutaneously immunized in the neck with 2 mL of tiger FPV VLPs vaccine and given an immunization boost with the same dose four weeks later. The feces production, behavior, and food and water consumption were observed continuously for 14 days after the last immunization. Fourteen days later, the immunized cats were euthanized. The injection site was dissected to observe whether there were side effects, such as nodules, and the tissue was sectioned to detect pathological changes. No anomalies in the daily observation parameters were observed in cats immunized with tiger FPV VLPs vaccine. There were no sarcomas in the injection site after necropsy (Figure 3Aa), which indicated that the absorption of the vaccine was good and that the tissue sections at the injection site were not abnormal (Figure 3Ab). These results demonstrated that the tiger FPV VLPs vaccine was safe for cats.

**Figure 3.** Safety and immunogenicity assay of tiger FPV VLPs. (**A**) Injection site necropsy and tissue section results. (Aa) There were no sarcomas in the injection site after necropsy. (Ab) The tissue sections were normal. (**B**) The immunogenicity of tiger FPV VLPs in cats was evaluated by HI assay after immunization. Cats were immunized twice via subcutaneous injection at four-week intervals. The immunization group was immunized with tiger FPV VLPs mixed with Gel 02 adjuvant. The control group was immunized with PBS.

To detect the immune effects, two 10-week old cats were subcutaneously immunized with 1 mL of tiger FPV VLPs vaccine, and one cat was given PBS as a control. Identical vaccinations were then repeated at four weeks after the primary immunization. Blood samples were collected at 2, 4, 5, 6, and 7 weeks and at 2, 3, 4, 5, 6, 7, 8, 9, 10, and 12 months after the primary immunization. Antibody levels of tiger FPV in the sera were measured by HI assay using pig erythrocytes, and the assay was performed as described previously [24]. The results, shown in Figure 3B, indicated that the anti-FPV HI titer was detected in the immunized cats two weeks after primary immunization, with a titer of approximately 1:210. After boost immunization, the HI titer was essentially stable at 1:210. The anti-FPV HI titer was still detected 12 months after the primary immunization, and the antibody titer was not less than 1:2<sup>10</sup> (Figure 3B). The second batch of immunization was conducted in other independent tests, which are still under observation. The tendency of HI titer after immunization was the same as above, with

HI titers of 1:29–1:210 six months after immunization (Figure S1). These results showed that in cats, tiger FPV VLPs effectively stimulated the immune response and induced the production of anti-FPV antibodies, which had a good immune effect.

VP2 is the main component of the capsid protein of FPV and determines its antigenicity [25]. The amino acid sites of the VP2 protein from tiger FPV were analyzed in this article. The 101st amino acid was consistent with that of CPV, and the rest of the amino acid variations in the VP2 protein [21] were consistent with those of FPV. It has been speculated that the strain might be the product of a recombination event or mutation in response to selection pressure. Therefore, the parvovirus VP2 gene from a tiger was selected to generate a vaccine for tiger parvovirus, which is more representative than other strains and provides a method to prevent and treat tiger parvovirus disease.

In this study, tiger FPV VLPs were developed using the baculovirus expression system, and the expressed protein was identified, confirming that VP2 protein was expressed in vitro and could self-assemble into VLPs. The recombinant baculovirus infected Sf9 cells, cultured in suspension, could express the VP2 protein at high yields and with a high HA titer, indicating their suitability for production on a large scale. VLPs are generally safer than inactivated and attenuated vaccines due to their lack of viral nucleic acids and infectivity. In addition, Gel 02 adjuvant, which is a water-soluble adjuvant, was used in this study. There were no sarcomas and site effects in cats immunized with the tiger FPV VLPs vaccine emulsified with Gel 02 adjuvant. Historically, HI is considered the gold standard for FPV antibody detection [26]. Generally, a HI titer of ≥1:40 is considered protective against FPV [27]. The highest HI titer in the sera of the cats in this experiment reached 1:213, and the HI titer remained stable at 1:2<sup>10</sup> after 12 months. The successful preparation of tiger FPV VLPs provides a new approach for the prevention and control of tiger parvovirus disease and technical support for the prevention of rare wild animal viral diseases. Due to the rarity and importance of tigers, relevant institutions are particularly cautious about the inoculation of tigers. Therefore, cats of the same family as tigers were selected for the evaluation of the vaccine immunity effect in this study, with the aim of developing a new type of safe and effective vaccine against feline parvovirus disease in tigers and laying a foundation for the prevention, control and treatment of feline parvovirus disease. In the subsequent application, the tiger FPV VLPs vaccine can be used as a single vaccine or a multiple vaccine combined with other vaccines for routine immunizations of tigers in zoos.

In this study, animal experimentation was handled in compliance with the guidelines and protocols of the Welfare and Ethics of Laboratory Animals of China. The animal studies were approved by the Animal Welfare and Ethics Committee of Changchun Sino Biotechnology Co., Ltd.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/12/3/315/s1, Figure S1: The immunogenicity of tiger FPV VLPs in cats was evaluated by HI assay, Sequence S1: The codon-optimized tiger parvovirus VP2 sequences.

**Author Contributions:** N.F., C.Z. and J.S. designed the experiments. C.J. and H.Z. performed the experiments. H.J., D.L. and J.Z. analyzed the data. C.J. and W.L. wrote the article. C.J., H.J. and J.S. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key Research and Development Program of China—Research and Development of Technology for Prevention and Control of Important Diseases and Domestication and Breeding of Rare and Endangered Wild Animals under Grant No. 2017YFD0501704.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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