Recombinant Myxoma Virus Expressing Walleye Dermal Sarcoma Virus Orfc Is Attenuated in Rabbits

viruses

Article Recombinant Myxoma Virus Expressing Walleye Dermal Sarcoma Virus orfC Is Attenuated in Rabbits

Laura V. Ashton 1, Sandra L. Quackenbush 1, Jake Castle 2, Garin Wilson 1, Jasmine McCoy 1, Mariah Jordan 1 and Amy L. MacNeill 1,* 1 Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA; [email protected] (L.V.A.); [email protected] (S.L.Q.); [email protected] (G.W.); [email protected] (J.M.); [email protected] (M.J.) 2 Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-970-297-5112

Received: 7 April 2020; Accepted: 6 May 2020; Published: 8 May 2020

Abstract: The poxvirus, myxoma virus (MYXV) has shown efficacy as an oncolytic virus (OV) in some cancer models. However, MYXV replication within murine cancer models and spontaneous canine sarcomas is short-lived. In mice, successful treatment of tumors requires frequent injections with MYXV. We hypothesize that treatment of cancer with a recombinant MYXV that promotes apoptosis could improve the efficacy of MYXV. The orfC gene of walleye dermal sarcoma virus (WDSV), which induces apoptosis, was recombined into the MYXV genome (MYXVorfC). A marked increase in apoptosis was observed in cells infected with MYXVorfC. To ensure that expression of WDSV orfC by MYXV does not potentiate the pathogenesis of MYXV, we evaluated the effects of MYXVorfC inoculation in the only known host of MYXV, New Zealand white rabbits. Virus dissemination in rabbit tissues was similar for MYXVorfC and MYXV. Virus titers recovered from tissues were lower in MYXVorfC-infected rabbits as compared to MYXV-infected rabbits. Importantly, rabbits infected with MYXVorfC had a delayed onset of clinical signs and a longer median survival time than rabbits infected with MYXV. This study indicates that MYXVorfC is attenuated and suggests that MYXVorfC will be safe to use as an OV therapy in future studies.

Keywords: poxvirus; myxoma virus; attenuated; rabbits; oncolytic

1. Introduction There is a growing interest in using viruses to eliminate cancers. The first oncolytic virus (OV) approved for use in the United States is marketed for treatment of melanoma in humans [1]. Although there are ongoing clinical trials testing the efficacy of oncolytic virotherapy in many types of human cancers, new OVs that are safer and more globally effective are actively being researched. The poxvirus, MYXV is an excellent candidate oncolytic virotherapeutic because, unlike other OVs, it does not cause disease in humans or other vertebrates, with the exception of rabbits [2–9]. In spite of its species specificity, MYXV productively infects cultured cancer cells from several animal species [10–13]. In culture, data suggest that MYXV can replicate in neoplastic cells which have activated Akt [11,14] and lack appropriate Type I interferon responses to virus infection [12,15]. In rodent cancer models, MYXV treatment has eliminated some glioma xenografts [16] and reduced tumor burden of different allografts [17–20], but to date allografts have not been eliminated by MYXV treatment alone. We hypothesize that a recombinant MYXV with an enhanced oncolytic effect in cell culture will be a more potent anti-cancer therapy. However, before a new MYXV OV is tested in cancer models, its safety profile must be determined in its natural host, rabbits.

Viruses 2020, 12, 517; doi:10.3390/v12050517 www.mdpi.com/journal/viruses Viruses 2020, 12, 517 2 of 16

This study compares the pathogenesis of wild-type MYXV to a recombinant MYXVorfC virus in New Zealand white rabbits. MYXVorfC expresses walleye dermal sarcoma virus (WDSV) OrfC, which encodes a pro-apoptotic protein. In cell culture, the orfC protein disrupts mitochondrial function and cells exhibit characteristics of apoptosis [21]. It is possible that orfC expression contributes to the tumor regression observed during clinical resolution of WDSV infection. It also is likely that the immune response to WDSV in infected ﬁsh plays a critical role in tumor regression [21,22]. Our data indicate that MYXVorfC replicates and enhances apoptosis in permissive cell cultures. In rabbits, clinical signs of virus infection are delayed and median survival time is increased in animals inoculated with MYXVorfC as compared to MYXV. One of six rabbits survived infection with MYXVorfC, while all six rabbits infected with MYXV succumbed to disease. These data indicate MYXVorfC is pro-apoptotic in cell culture and is attenuated in rabbits. These properties support using MYXVorfC in animal models of cancer to determine if MYXVorfC is a more eﬀective OV than wild-type MYXV.

2. Materials and Methods

2.1. Cell Culture Rabbit-kidney epithelial (RK-13, ATCC CCL-37) cells originated in Dr. Richard Moyer’s laboratory. The cancer cells evaluated (Morris soft tissue sarcoma (STS) and King osteosarcoma (OSA)) were isolated from canine cancer patient tumors that were surgically removed at the University of Illinois and Colorado State University Veterinary Teaching Hospitals, respectively. Clients from both institutions gave written permission for cells to be isolated. Cells were validated as canine origin by short tandem repeat analysis [23]. Cell growth media was comprised of Minimal Essential Medium with Earle’s salts and 2 mM L-glutamine supplemented with 2 mM L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin (GE Healthcare, Marlborough, MA, USA); 0.1 mM nonessential amino acids and 1 mM sodium pyruvate (Corning, Corning, NY, USA); and 10% fetal bovine serum (FBS; VWR Life Science Seradigm, Radnor, PA, USA). Cells were maintained in a water-jacketed incubator at 5% CO2 and 37 ◦C.

2.2. Viruses Wild-type MYXV (Lausanne strain) and recombinant MYXV-red (originally designated vMyx-tdTr [24]) was originally acquired from Dr. Grant McFadden. MYXVorfC was constructed by recombining a 2385 base pair (bp) PCR product into wild-type MYXV between the genetic open reading frames M135 and M136 using a pBluescript plasmid vector (Figure1). Plasmid constructs were amplified by transformation into Escherichia coli DH5α chemically competent cells (Rapid5-a, Hardy Diagnostics, Santa Monica, CA, USA). Successful construction was confirmed using restriction enzyme digests and Sanger sequencing. DNA for transfection was prepared using PCR OneTaq Mastermix (New England Biolabs, Ipswitch, MA, USA) containing 0.5 µM of M13 forward primer (50 GTA AAA CGA CGG CCA GT 30), 0.5 µM of M13 reverse primer (50 CAG GAA ACA GCT ATG ACC 30), and 1.0 µg plasmid DNA. Amplification was performed in a thermocycler under the conditions: 94 ◦C for 1 min, followed by 30 cycles of 94 ◦C for 30 sec, 52 ◦C for 1 min, and 68 ◦C for 5 min, and a final 68 ◦C for 5 min step. The PCR product contained DNA sequences of: (1) the 50 fragment of MYXV M135, (2) tandem dimer tomato red (tdTomato) under the transcriptional control of a synthetic early/late poxvirus promoter (vvSynE/L), (3) hemagglutinin (HA)-tagged WDSV orfC under the control of a late poxvirus promoter (p11), and (4) the 30 fragment of MYXV M136. Transfection of the PCR fragment was performed using a modified method described by Rice et al. 2011 [25]. Briefly, RK-13 cells were infected at a multiplicity of infection (moi) of 0.01 with wild-type MYXV, transfected with 0.2 µg of PCR product DNA, and combined with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) to potentiate recombination of the PCR product and viral DNA. Cells were scraped into growth media at 72 h post-inoculation (hpi), centrifuged at 400 g for 15 min, washed in phosphate buffered saline × (PBS), re-suspended in media lacking FBS, frozen and thawed 3 times, and sonicated. Viral lysates Viruses 2020, 12, 517 3 of 16

Viruses 2020, 12, x FOR PEER REVIEW 3 of 17 were serially diluted in media lacking FBS and incubated on RK-13 cells for 30 min. A solid overlay of overlay of 1 part 2× growth media and 1 part 1% agarose was placed on the infected cells. Viral foci 1 part 2 growth media and 1 part 1% agarose was placed on the infected cells. Viral foci that formed that formed× were screened for fluorescent red protein expression using a 560/40 nm bandpass were screened for fluorescent red protein expression using a 560/40 nm bandpass excitation filter and a excitation filter and a Leica DMI4000B inverted microscope. Fluorescent foci were isolated and Leica DMI4000B inverted microscope. Fluorescent foci were isolated and expanded in RK-13 cells. expanded in RK-13 cells. The process of picking foci and growing viruses was repeated 9 times until The process of picking foci and growing viruses was repeated 9 times until only foci that expressed red only foci that expressed red fluorescent protein were observed. Viral purification was confirmed fluorescent protein were observed. Viral purification was confirmed using PCR and next generation using PCR and next generation sequencing. For injection into rabbits, viruses were grown and titered sequencing. For injection into rabbits, viruses were grown and titered in RK-13 cell cultures. Cellular in RK-13 cell cultures. Cellular debris was removed by sucrose pad purification as previously debris was removed by sucrose pad purification as previously described [26]. described [26].

Figure 1. Plasmid design. Diagram (SnapGene, GSL Biotech, San Diego, CA, USA) of the modiﬁed pBluescipt plasmid containing the WDSV orfC gene and the tdTomato reporter gene ﬂanked by MYXV Figure 1. Plasmid design. Diagram (SnapGene, GSL Biotech, San Diego, CA, USA) of the modified M135R and MYXV M136R. pBluescipt plasmid containing the WDSV orfC gene and the tdTomato reporter gene flanked by 2.3. ViralMYXV Growth M135R Curves and MYXV M136R.

2.3. ViralGrowth Growth media Curves was removed from wells of RK-13 cells when they were 80% conﬂuent. Cells were inoculated with MYXV or MYXVorfC in media lacking FBS (moi = 0.1, n = 10 per group). Cells were Growth media was removed from wells of RK-13 cells when they were 80% confluent. Cells incubated with virus for 1 h at 5% CO2 and 37 ◦C. Viral inoculum was removed, cells were rinsed were inoculated with MYXV or MYXVorfC in media lacking FBS (moi = 0.1, n = 10 per group). Cells were incubated with virus for 1 h at 5% CO2 and 37 °C . Viral inoculum was removed, cells were rinsed with PBS, and growth media was added to wells. Cells were scraped into growth media at

Viruses 2020, 12, 517 4 of 16 with PBS, and growth media was added to wells. Cells were scraped into growth media at designated time-points post-inoculation, frozen and thawed 3 times, and sonicated. Plaque assays were then performed to determine the number of infectious virions per mL of media. To perform plaque assays, viral lysates were serially diluted in media lacking FBS and incubated on RK-13 cells for 30 min. A solid overlay of 1 part 2 growth media and 1 part 1% agarose was placed on the infected cells. Viral foci × were counted 4 days later. The log of plaque/focus-forming units (pfu) per mL was calculated and plotted versus time.

2.4. Detection of Exogenous Protein Production by MYXVorfC Fluorescence from tdTomato protein expression by MYXVorfC was detected using a 560/40 nm bandpass excitation filter and a Leica DMI4000B inverted microscope. Production of the HA-tagged OrfC protein was detected using a Western immunoblot. Briefly, RK-13 cells were grown in 35 mm diameter plates to 90% confluency and inoculated with MYXVorfC (moi = 0.5). Infected cells were collected into cell lysis buffer at several time points pi. Total protein concentration was determined with a standard Bradford assay and 20 µg of protein from each cell lysate was separated using SDS-PAGE (10%). The SDS-PAGE-separated proteins were transferred to a nitrocellulose membrane. Membranes were incubated in blocking buffer for 1 h. Membranes were washed then incubated with rat anti-HA IgG (Milipore Sigma, St Louis, MO, USA) diluted 1:1000 in blocking buffer overnight at 4 ◦C. Membranes were washed then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-rat IgG antibody (ImmunoReagents, Raleigh, NC, USA) diluted 1:1000 in blocking buffer. A chemiluminescent Western immunoblot detection kit (GE Healthcare, Marlborough, MA, USA) was used to detect antigen-antibody complexes. These experiments were repeated a minimum of three times.

2.5. Apoptosis in Cell Culture Growth media was removed from wells of RK-13 cells and cancer cells when they were 80% confluent. Cells were inoculated in media lacking FBS without virus (mock), with MYXV, or with MYXVorfC (moi = 1, n 22 per group). Cells were incubated with virus for 1 h at 5% CO and 37 C ≥ 2 ◦ then growth media was added to the wells. A RealTime-Glo Annexin V Apoptosis and Necrosis Assay kit (Promega, Madison, WI, USA) was used per the manufacturer’s instructions to label cells expressing phosphatidylserine. Luminescence and fluorescence (485 nm excitation/525 nm emission) were detected using a BioTek Synergy H1 microplate reader. The percent luminescent signal from cells undergoing apoptosis and the percent fluorescent signal from partially ruptured (necrotic) cells were graphed relative to mock-infected controls. The ratio of percent apoptotic to percent necrotic cells was also calculated and graphed.

2.6. Rabbits All animal procedures were approved by the Institutional Animal Care and Use Committee at Colorado State University (protocol #17-7708A). Twelve 8-week-old, female, New Zealand white rabbits were purchased from Western Oregon Rabbit Company (Philomath, OR, USA) and were acclimated for 5 days. Prior to starting the study, all rabbits were deemed healthy by physical examination including determination of heart rate and respiratory rate; auscultation of the heart and lungs; palpation of the abdomen and lymph nodes; measurement of weight and rectal temperature; evaluation of the injection site, haired skin, and mucous membranes for evidence of lesion formation; and assessment of food and water intake, grooming behavior, and mentation. An area of fur on the lateral aspect of the upper right thigh was shaved and disinfected with an accelerated hydrogen peroxide solution. Rabbits were inoculated with virus via an intradermal injection of 50 pfu of sucrose pad puriﬁed wild-type MYXV (n = 6) or MYXVorfC (n = 6) in 100 µL PBS. The area was cleaned with an accelerated hydrogen peroxide solution after the virus is administered. Rabbits were given physical examinations daily. The lesion length, width, and height at the site of inoculation were measured daily. Lesion volume was Viruses 2020, 12, 517 5 of 16 calculated as volume = length width height. The clinical scoring system outlined in Rice et al., JVI × × 2014 was used [27]. Animals were euthanized as soon as signs of respiratory distress were observed; if rectal temperature was less than 36 ◦C; if the rabbit appeared mentally dull with poor response to noise or light; if the rabbit stopped grooming, eating, or drinking; or at Day 21 post-inoculation.

2.7. Sample Collection Prior to euthanasia, rabbits were anesthetized with a freshly-mixed combination of 50 mg/kg ketamine and 10 mg/kg xylazine by intramuscular injection into the proximal and lateral aspect of the left thigh. Fully anesthetized rabbits (unresponsive to noxious stimulation, such as pinching the toes with forceps) were administered an overdose of intracardiac pentobarbital (120 mg/kg) to ensure they were deceased. After euthanasia, a necropsy was performed to collect skin lesions and all internal organs. Two sections of each tissue were collected; one was ﬂash frozen in liquid nitrogen for isolation of virus and detection of viral DNA and one was preserved in 10% buﬀered formalin for histopathology.

2.8. Detection of Virus in Tissues Flash frozen tissue sections were weighed and homogenized. Samples were serially diluted in media without FBS, sonicated, and inoculated onto RK-13 cells for 30 min. A solid overlay of 1 part 2 × growth media and 1 part 1% agarose was placed on the infected cells. Viral foci were counted 4–6 days later. Pfu per mg of tissue calculated. Additionally, DNA was isolated from sample homogenates using a tissue DNA isolation kit (DNeasy Blood and Tissue Kit, QIAGEN, Hilden, Germany). Next, 5 µL of DNA was used in PCR reactions with two primer sets. A set of primers that detect a multigenic region of the MYXV genome that includes M032R, M033R, M034L (DNA polymerase), and M035R was used to detect MYXV in tissues (Forward 50CAC CCT CTT TAG TAA AGT ATA CAC C 30, Reverse 50GAA ATG TTG TCG GAC GGG 30). An 800 bp product is detected for both MYXV and MYXVorfC with these primers. A second set of primers was also used (Forward 50 ACA TAC GAC ATC GGA CAG CA 30, Reverse 50CGT CGA TCG CTG TGT AAG AA 30) that covered the region of the gene insert (M135-M136); a 320 bp product is expected for wild-type MYXV and a 1547 bp product is expected for MYXVorfC. Ampliﬁcation was performed in a thermocycler under the conditions: 94 ◦C for 1 min, followed by 30 cycles of 94 ◦C for 30 sec, 55 ◦C for 1 min, 72 ◦C for 2 min and a ﬁnal 72 ◦C for 10 min elongation step.

2.9. Histopathology Formalin-fixed tissue was paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histologic evaluation. The extent of inflammation, necrosis, and edema observed in skin lesions histologically was graded as previously described [20]. Similarly, additional sections were used to detect apoptosis using a TUNEL (TdT-mediated dUTP Nick End Labeling) assay (Click-iT TUNEL, Life Technologies, Carlsbad, CA, USA) and the extent of apoptosis was graded.

2.10. Statistical Analysis Median survival time was calculated using Kaplan–Meier survival curves. The apoptosis to necrosis ratio in RK-13 cells; clinical scores, respiratory rate, heart rate, body temperature, and lesion volume in rabbits; virus titers in tissues; and the extent of inﬂammation, necrosis, edema, and apoptosis in histologic sections were compared using t-tests. All data were analyzed using GraphPad Prism version 8.1.0 software (GraphPad, San Diego, CA, USA).

3. Results

3.1. Validation of MYXVorfC Construction A diagram of the PCR product introduced between genes M135 and M136 to create the recombinant MYXVorfC virus is shown in Figure2A. Restriction enzyme digestions of MYXVorfC indicated the Viruses 2020, 12, 517 6 of 16 Viruses 2020, 12, x FOR PEER REVIEW 6 of 17 unexpectedpredicted DNA genetic fragment changes lengths. were caused MYXVorfC during sequence recombination analysis of indicated the PCR thatfragment no unexpected into wild- genetictype MYXVchanges (Figure were caused2B). during recombination of the PCR fragment into wild-type MYXV (Figure2B).

Figure 2. Design andand sequencesequence analysis analysis of of MYXVorfC. MYXVorfC. (A )(A Diagram) Diagram (SnapGene, (SnapGene GSL, GSL Biotech, Biotech, San Diego,San Diego,CA, USA) CA, of USA the) PCR of the product PCR that product was transfected that was transfected into MYXV-infected into MYXV RK-13-infected cells. RK (B-)13 Alignment cells. (B) of Alignmentthe sequence of mapsthe sequence from 130,000–135,000 maps from 130,000 bp in–135,000 puriﬁed bp recombinant in purified MYXVorfCrecombinant DNA MYXVorfC as compared DNA to asMYXV compared (AF170726.2) to MYXV using (AF170726.2 next generation) using sequencing.next generation The numbersequencing. of times The eachnumber nucleotide of times was each read nucleotideby the sequencer was read is represented by the sequencer in gray. (Cis) rTheepresented expected in PCR gray. insert (C) was The successfully expected PCR recombined insert was into successfullyMYXV to create recombined MYXVorfC. into Wild-type MYXV to MYXV create sequence MYXVorfC. was Wild not detected-type MYXV in the sequence puriﬁed MYXVorfC was not Virusesdetectedvirus 2020, sample. 12 ,in x FORthe purified PEER REVIEW MYXVorfC virus sample. 7 of 17

Growth curve kinetics of wild-type MYXV and MYXVorfC in RK-13 cells were statistically indistinguishable (Figure 3), indicating that insertion of the PCR product did not negatively affect MYXVorfC replication in permissive RK-13 cells. Next, protein expression of orfC and tdTomato by MYXVorfC was assessed. Red fluorescence was detected in RK-13 cell cultures inoculated with MYXVorfC (Figure 4). The number of cells expressing tdTomato was similar when MYXV-red and MYXVorfC infections were compared (Figure 4A). However, subtle morphologic differences were noted. Some MYXVorfC-infected cells contained large cytoplasmic vacuoles, which were not observed in MYXV-red-infected cells (Figure 4B). OrfC protein expression was detected by 12 hpi using Western blot analysis (Figure 5). Together, these data indicate that tdTomato and orfC were successfully inserted into wild-type MYXV to create a recombinant MYXVorfC that expresses detectable amounts of tdTomato and orfC proteins.

Figure 3. Virus growth curves. Data points represent the average of ten replicates. No significant Figure 3. Virus growth curves. Data points represent the average of ten replicates. No significant differences were observed in the growth of wild-type MYXV, MYXV-red, or MYXVorfC (moi = 0.1, error differences were observed in the growth of wild-type MYXV, MYXV-red, or MYXVorfC (moi = 0.1, bars = SEM). error bars = SEM). Growth curve kinetics of wild-type MYXV and MYXVorfC in RK-13 cells were statistically indistinguishable (Figure3), indicating that insertion of the PCR product did not negatively a ffect MYXVorfC replication in permissive RK-13 cells. Next, protein expression of orfC and tdTomato by MYXVorfC was assessed. Red fluorescence was detected in RK-13 cell cultures inoculated with MYXVorfC (Figure4). The number of cells expressing tdTomato was similar when MYXV-red and

Figure 4. Expression of tdTomato by recombinant MYXV (moi = multiplicity of infection). (A) Representative images of infected RK-13 cells 24 hpi (100× magnification). No significant differences in the number of infected cells were observed. (B) Morphologic differences in viral plaques grown on RK-13 cells (moi = 0.01, 48 hpi). Some MYXVorfC-infected cells contained large cytoplasmic vacuoles (arrows) which were not detected in cells infected with MYXV-red (800× magnification). Data were repeatable (n > 3).

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MYXVorfC infections were compared (Figure4A). However, subtle morphologic di ﬀerences were noted. Some MYXVorfC-infected cells contained large cytoplasmic vacuoles, which were not observed in MYXV-red-infected cells (Figure4B). OrfC protein expression was detected by 12 hpi using Western blotFigure analysis 3. Virus (Figure growth5). Together,curves. Data these points data represent indicate the that average tdTomato of ten andreplicates. orfC wereNo significant successfully inserteddifferenc intoes wild-type were observed MYXV in tothe create growth a recombinantof wild-type MYXV, MYXVorfC MYXV that-red, expresses or MYXVorfC detectable (moi = amounts0.1, of tdTomatoerror bars and= SEM). orfC proteins.

FigureFigure 4. ExpressionoftdTomatobyExpression of tdTomato recombinant by recombinant MYXV(moi MYXV=multiplicityofinfection). (moi = multiplicity of (A infection).)Representative (A) Representativeimages of infected images RK-13 of infected cells 24 RK hpi-13 (100 cellsmagnification). 24 hpi (100× magnification). No significant No diff significanterences in differences the number × inof the infected number cells of infected were observed. cells were (B observed.) Morphologic (B) Morphologic differences indifferences viral plaques in viral grown plaques on RK-13 grown cells on RK(moi-13= cells0.01 (moi, 48 hpi).= 0.01, Some 48 hpi). MYXVorfC-infected Some MYXVorfC- cellsinfected contained cells contained large cytoplasmic large cytoplasmic vacuoles vacuoles (arrows) Viruses(arrows)which 2020, were1 2whi, x FOR notch were PEER detected not REVIEW detected in cells infected in cells withinfected MYXV-red with MYXV (800 -redmagnification). (800× magnification). Data were Data repeatable were 8 of 17 × repeatable(n > 3). (n > 3).

Figure 5. Figure 5. Western immunoblot. Hemagglutinin Hemagglutinin-tagged-tagged orfC orfC was was detected detected in inRK RK-13-13 cell cell lysates lysates by by12 12 hpi (moi = 0.5). Data were repeatable (n = 3). hpi (moi = 0.5). Data were repeatable (n = 3).

3.2. Apoptosis Is Induced By MYXVorfC The purpose of using MYXV to express the orfC protein was to enhance apoptosis in infected cancer cells. Apoptosis was greatly increased in RK-13 cells infected with MYXVorfC as compared to MYXV-red infected or mock-infected cells (Figure 6).

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Figure 6. Percent (A) apoptosis and (B) necrosis relative to mock-infected controls in RK-13 cells and Figure 6. Percent (A) apoptosis and (B) necrosis relative to mock-infected controls in RK-13 cells and two cancer cells (Morris STS and King OSA). (C) The ratio of percent apoptosis and percent necrosis two cancer cells (Morris STS and King OSA). (C) The ratio of percent apoptosis and percent necrosis is also shown. Apoptotic and necrotic cells were signiﬁcantly increased after MYXVorfC infection is also shown. Apoptotic and necrotic cells were significantly increased after MYXVorfC infection as as compared to mock-controls or MYXV-red infection (n 22, moi = 1, 24 hpi, error bars = SEM, compared to mock-controls or MYXV-red infection (n ≥ 22, moi≥ = 1, 24 hpi, error bars = SEM, p-values p-values 0.0016) ≤ 0.0016).≤

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3.3.3.3.3.3. MYXVorfC MYXVorfC MYXVorfC Is Is IsAttenulated Attenulated Attenulated in in inRabbits Rabbits Rabbits AsAsAs expected, expected, expected, all all all New NewNew Zealand ZealandZealand white whitewhite rabbits rabbits infected infected with with with 50 50 50pfu pfu pfu wild wild wild-type-type-type MYXV MYXV MYXV needed needed needed to tobe to be euthanizedbeeuthanized euthanized due due to due torespiratory respiratory to respiratory distress. distress. distress. One One of of Onesix six rabbits ofrabbits six infected rabbits infected with infected with 50 50 pfu with pfu of ofMYXVorfC 50 MYXVorfC pfu of MYXVorfC survived. survived. Thesurvived.The median median The survival survival median time time survival of ofrabbits rabbits time inoculated of inoculated rabbits inoculatedwith with MYXVorfC MYXVorfC with MYXVorfC was was significantly significantly was signiﬁcantly greater greater than than greater that that ofthan ofrabbits rabbits that inoculated of inoculated rabbits inoculatedwith with MYXV MYXV with (Figure (Figure MYXV 7). 7). The (Figure The median median7). Thesurvival survival median times times survival for for MYXV MYXV times- and- for and MYXVorfC MYXV- MYXVorfC and- - infectedMYXVorfC-infectedinfected rabbits rabbits were were rabbits9 and9 and 12 were 12 Days Days 9 andpi pi(dpi)12 (dpi) Days, respectively., respectively. pi (dpi), respectively.

Figure 7. The median survival time of rabbits infected with 50 pfu MYXV was significantly shorter Figure 7. The median survival time of rabbits infected with 50 pfu MYXV was significantly shorter thanFigure that 7. of The rabbits median infected survival with time 50 pfu of MYXVorfCrabbits infected ((n = 6with per 50 group, pfu MYXVp-value was= 0.014). significantly shorter thanthan that that of ofrabbits rabbits infected infected with with 50 50pfu pfu MYXVorfC MYXVorfC ((n (( =n 6 = per 6 per group, group, p- valuep-value = 0.014). = 0.014). Development of clinical signs of disease (including respiratory distress and dissemination of virus to formDevelopmentDevelopment edematous, of ulcerativeofclinical clinical signs skinsigns of lesions) ofdisease disease was (including (including slightly delayedrespiratory respiratory in rabbits distress distress infected and and dissemination withdissemination MYXVorfC of of virus(Figurevirus to 8 to form). form Slightly, edematous, edematous, but significantly, ulcerative ulcerative lower skin skin lesions) clinical lesions) wasscores was slightly wereslightly observed delayed delayed in in MYXVorfC ra bbits rabbits infected infected rabbits with withon MYXVorfCDaysMYXVorfC 5, 6, and(Figure (Figure 8 after 8). 8). Slightly, virus Slightly, injection but but significantly, significantly, (Figure9A). lower These lower clinical diclinicalfferences scores scores were were were attributed observed observed in to inMYXVorfC the MYXVorfC delay in rabbitsdevelopmentrabbits on on Days Days of 5, secondary 5,6, 6,and and 8 after8 lesionsafter virus virus on injection mucusinjection ( membranesFigure (Figure 9A). 9A). These and These ear differences differences pinnae in were rabbitswere attributed attributed infected to withtothe the delay in development of secondary lesions on mucus membranes and ear pinnae in rabbits infected MYXVorfCdelay in development (Figure9B) and of detectionsecondary of lesions fever (body on mucus temperature membranes> 40 ◦andC, Figure ear pinnae 10A). in Small rabbits diff erencesinfected withinwith mean MYXVorfC MYXVorfC respiratory (Figure (Figure rate 9B) (Figure 9B) and and 10detection B)detection and heartof offever fever rate (body (Figure (body temperature temperature10C) were > found 40> 40 °C on°C, Figure, certain Figure 10A). dpi,10A). butSmall Small no differencesdefinitivedifferences trendsin inmean mean were respiratory respiratory seen over rate time. rate (Figure No(Figure significant 10B 10B) and) and di heartff hearterences rate rate in(Figure ( lesionFigure 10C) volume 10C) were were were found found calculated, on on certain certain but dpi,thedpi, averagebut but no no definitive volume definitive of trends the trends primary were were seen lesion seen over ofover rabbits time. time. No infected No significant significant with MYXVorfC differences differences decreased in inlesion lesion involume animalsvolume were thatwere calculated,survivedcalculated, beyond but but the Daythe average average 12 pi (Figure volume volume 10 ofD). of the These the primary primary data indicate lesion lesion that of of rabbits MYXVorfC rabbits infected infected is less with pathogenic with MYXVorfC MYXVorfC than decreasedwild-typedecreased MYXV.in in animals animals that that survived survived beyond beyond Day Day 12 12 pi pi (Figure (Figure 10D) 10D). These. These data data indicate indicate that that MYXVorfCMYXVorfC is isless less pathogenic pathogenic than than wild wild-type-type MYXV. MYXV.

FigureFigureFigure 8. 8. 8.RepresentativeRepresentative Representative images images images of of ofgross gross gross skin skin skin lesions lesions lesions 9 9 days9 days days after after after intradermal intradermal intradermal inoculation inoculation inoculation of of of50 50 50pfu pfu pfu MYXVMYXVMYXV or or orMYXVorfC MYXVorfC MYXVorfC on on on the the the right right right thigh. thigh thigh.

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Figure 9. Differences in disease progression in rabbits after intradermal inoculation of 50 pfu MYXV or MYXVorfC (n = 6 per group). (A) Average daily clinical score (error bars = SEM, *p-values < 0.035). Figure 9. Diﬀerences in disease progression in rabbits after intradermal inoculation of 50 pfu MYXV or Figure(B) Day 9. Differencesthat development in disease of primaryprogression and insecondary rabbits after skin intradermal lesions was inoculation first observed of 50 on pfu individual MYXV MYXVorfC (n = 6 per group). (A) Average daily clinical score (error bars = SEM, * p-value < 0.035). orrabbits. MYXVorfC (n = 6 per group). (A) Average daily clinical score (error bars = SEM, *p-values < 0.035). (B) Day that development of primary and secondary skin lesions was ﬁrst observed on individual rabbits. (B) Day that development of primary and secondary skin lesions was first observed on individual rabbits.

Figure 10. Average daily measurements during physical examination of rabbits infected with 50 pfu of MYXVFigure or 10. MYXVorfC Average (ndaily= 6 permeasurements group). Small, during but statisticallyphysical examination significant of di rabbitsfferences infected were observed with 50 pfu on a fewof MYXV days when or MYXVorfC average ( A(n) = body 6 per temperature, group). Small, (B but) respiratory statistically rate, significant and (C) heartdifferences rate were were compared. observed No significant differences were observed when (D) average daily lesion volumes were calculated (error Figureon a few10. Average days when daily average measurements (A) bod yduring temperature, physical (Bexamination) respiratory of rate,rabbits and infected (C) heart with rate 50 pfu were bars = SEM, * p-values < 0.046). ofcompared. MYXV or MYXVorfCNo significant (n = 6differences per group). were Small, observed but statistically when (D significant) average differencesdaily lesion were volumes observed were calculated (error bars = SEM, *p-values < 0.046). 3.4. MYXVorfCon a few days Tissue when Tropism average Is Not (A) Altered body temperature, (B) respiratory rate, and (C) heart rate were compared. No significant differences were observed when (D) average daily lesion volumes were 3.4.Dissemination calculatedMYXVorfC (error Tissue of bars wild-type Tropism = SEM, *Isp MYXV -Notvalues Altered and< 0.046). MYXVorfC during infection of rabbits appeared similar. Viral DNADissemination was detected of inwild skin,-type lungs, MYXV heart, and spleen, MYXVorfC liver, andduring kidney infection in all MYXV-infectedof rabbits appeared rabbits similar. and most3.4.Viral MYXVorfC MYXVorfC-infected DNA was Tissue detected Tropism rabbits in skin, Is Not (Table lungs, Altered1 ).heart, Viral spleen, titers (indicatingliver, and kidney the presence in all MYXV of infectious-infected virions) rabbits were significantly lower in the primary lesions, lungs, and spleen of the MYXVorfC-infected rabbits Dissemination of wild-type MYXV and MYXVorfC during infection of rabbits appeared similar. when compared to MYXV-infected rabbits (Figure 11). Viral DNA could be detected in tissues with Viral DNA was detected in skin, lungs, heart, spleen, liver, and kidney in all MYXV-infected rabbits

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Viruses 2020, 12, x FOR PEER REVIEW 12 of 17 titers as low as 2.5 pfu/mg. All tissues with titers > 35 pfu/mg were positive for viral DNA by PCR usingand both most primer MYXVorfC sets. -infected rabbits (Table 1). Viral titers (indicating the presence of infectious virions) were significantly lower in the primary lesions, lungs, and spleen of the MYXVorfC-infected Tablerabbits 1. whenDetection compared of MYXV to MYXV or MYXVorfC-infected rabbits by PCR (Figure in rabbit 11). tissues Viral collectedDNA could at thebe detected time of euthanasia in tissues fromwith titers rabbits as inoculated low as 2.5 withpfu/mg. 50 pfu All MYXV tissues or with MYXVorfC titers > 35 (n =pfu/mg6 per group).were positive for viral DNA by PCR using both primer sets. Viral DNA Targets of Primers Table 1. Detection of MYXV or MYXVorfC by PCR in rabbit tissues collected at the time of euthanasia Virus Tissue Multigene M135-M136 from rabbits inoculated with 50 pfu MYXV or MYXVorfC (n = 6 per group). MYXV Primary lesion 6/6 (100%) 6/6 (100%) Viral DNA Targets of Primers Virus TissueSecondary lesionMultigene 6/6 (100%) 6/6 (100%)M135-M136 MYXV Primary lesionHeart 6/6 6/6 (100%) (100%) 6/6 (100%)6/6 (100%) Secondary lesionKidney 6/6 6/6 (100%) (100%) 6/6 (100%)6/6 (100%) Heart 6/6 (100%) 6/6 (100%) Liver 6/6 (100%) 6/6 (100%) Kidney 6/6 (100%) 6/6 (100%) Liver Lung6/6 6/6 (100%) (100%) 6/6 (100%)6/6 (100%) Lung Spleen6/6 6/6 (100%) (100%) 6/6 (100%)6/6 (100%) MYXVorfCSpleen Primary lesion6/6 6/6 (100%) (100%) 6/6 (100%)6/6 (100%) MYXVorfC Primary lesion 6/6 (100%) 6/6 (100%) Secondary lesion 6/6 (100%) 6/6 (100%) Secondary lesion 6/6 (100%) 6/6 (100%) Heart Heart3/6 3/6 (50%) (50%) 3/6 (50%)3/6 (50%) KidneyKidney 5/6 5/6 (83%) (83%) 5/6 (83%)5/6 (83%) Liver 6/6 (100%) 5/6 (83%) Liver 6/6 (100%) 5/6 (83%) Lung 6/6 (100%) 5/6 (83%) Spleen Lung 64/6/6 (100%)(67%) 5/6 (83%)3/6 (50%) Spleen 4/6 (67%) 3/6 (50%)

Figure 11. Average virus titer in rabbit tissues. Tissue homogenates from MYXV- and MYXVorfC-infectedFigure 11. Average rabbits virus titer at the in timerabbit of tissues. euthanasia Tissue containedhomogenates replicating from MYXV virus- and (n MYXVorfC= 6 per group).- Signiﬁcantlyinfected rabbits lower at titers the time of MYXVorfC of euthanasia were contained extracted replicating from primary virus (n lesions,= 6 per group). lung,and Significantly spleen (error bars = SEM, * p-values < 0.05) Viruses 2020, 12, x FOR PEER REVIEW 13 of 17

lower titers of MYXVorfC were extracted from primary lesions, lung, and spleen (error bars = SEM, Viruses 2020, 12, 517 12 of 16 *p-values < 0.05).

3.5. MYXV and MYXVorfC Induce Similar Histologic Lesions No overt histologic abnormalities were appreciated in heart or kidney sections. Viral pneumonia was observed in lung tissue from all rabbitsrabbits with mild to moderate edema and didiffuseffuse suppurative inflammation.inflammation. Sloughing Sloughing of of bronchial bronchial epithelial epithelial cells into the bronchial bronchial lumen lumen was was noted. noted. Mild hyperplasia of perivascular lymlymphoidphoid tissue of the lungs was observed in both MYXVMYXV-- and MYXVorfC-infectedMYXVorfC-infected rabbits. rabbits. Evidence Evidence of of disease disease in the liver liver was was more more variable. variable. Mild didiffuseffuse lymphocytic inflammation inflammation was was observed observed in in the the livers livers of of 67% (4/6) (4/6) MYXV MYXV-infected-infected rabbits and 33% (2(2/6)/6) MYXVorfC-infected MYXVorfC-infected rabbits. rabbits. Mild Mild diffuse diffuse mixed inflammation mixed inflammation (including (including neutrophils, neutrophils, histiocytes, lymphocytes,histiocytes, lymphocytes, and plasma and cells) plasma was cells) observed was observed in the liver in ofthe 50% liver (3 of/6) 50% MYXVorfC-infected (3/6) MYXVorfC-infected rabbits. Norabbits. inflammation No inflammation was observed was observed in the liver in the of the liver remaining of the remaining 33% (2/6) 33% MYXV-infected (2/6) MYXV and-infected 17% (1and/6) MYXVorfC-infected17% (1/6) MYXVorfC rabbits.-infected Tissue rabbits. sections Tissue fromsections primary from primary and secondary and secondary skin lesions skin oflesions all rabbits of all hadrabbits the had classic the classic appearance appearance of myxomatosis of myxomatos ini Europeans in European rabbits. rabbits. A A large large focus focus of of full-thicknessfull-thickness dermal necrosis was associated with several large reactive fibroblasts fibroblasts and myxedema ( (FigureFigure 12).12). Fibroblasts and epithelial cells sometimes contained a lar largege basophilic cytoplasmic viral inclusion. Marked suppurative inflammationinflammation was present within the necrotic lesion and moderate mononucle mononuclearar inflammationinflammation surroundedsurrounded the the damaged damaged tissue. tissue. The The average average number number of TUNELof TUNEL positive positive cells cells in ten in 40ten × objective40× objective microscopic microscopic fields fields in sections in sections of primary of primary skin lesions skin lesions was not was significantly not significantly different different in MYXV- in andMYXV MYXVorfC-infected- and MYXVorfC rabbits-infected (Figure rabbits 13 ). (Figure The secondary 13). The lesion secondary from one lesion MYXVorfC-infected from one MYXVorfC rabbit- wasinfected beginning rabbit towas heal beginning with extension to heal of with the epidermisextension underof the aepidermis layer of fibrin under and a cellularlayer of debris.fibrin and cellular debris.

Figure 12. Photomicrographs of skin from the lateral thigh of rabbits injected with ( A,A,D) saline [28] [28],, ((BB,,E)) 5050 pfupfu MYXV, oror ((CC,,FF)) 5050 pfupfu MYXVorfC.MYXVorfC. SamplesSamples were collected at the time of euthanasia, processed, andand stainedstained withwith H&EH&E (A,(A, B,B, andand CC magniﬁcationmagnification 1010× objective; D, E,E, and F magniﬁcationmagnification × 4040× objective). ×

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Figure 13. TUNEL staining in primary skin lesions. (A) Positive (brown nuclei) and (B) negative Figure 13. TUNEL staining in primary skin lesions. (A) Positive (brown nuclei) and (B) negative controlFigure samples13. TUNEL were staining adequate. in primary Positive skin TUNEL lesions. staining (A) Positivewas observed (brown in nuclei)both (C ) and MYXV-infected (B) negative control samples were adequate. Positive TUNEL staining was observed in both (C) MYXV-infected andcontrol (D) samples MYXVorfC-infected were adequate. rabbits Positive (magnification TUNEL staining 20 objective; was observedn = 6 per in both group). (C) (MYXVE) The- averageinfected and (D) MYXVorfC-infected rabbits (magnification 20×× objective; n = 6 per group). (E) The average numberand (D) ofMYXVorfC TUNEL positive-infected cells rabbi in tents (magnification 40 objective high-power20× objective fields; n = (hpf) 6 per was group). calculated. (E) The The average mean number of TUNEL positive cells in ten 40×× objective high-power fields (hpf) was calculated. The mean dinumberfference of between TUNEL TUNELpositive stainingcells in ten in primary40× objective lesions high of- MYXV-power fields and MYXVorfC-infected (hpf) was calculated. rabbits The mean was difference between TUNEL staining in primary lesions of MYXV- and MYXVorfC-infected rabbits notdifference statistically between significant TUNEL (error staining bars =inSEM) primary lesions of MYXV- and MYXVorfC-infected rabbits was not statistically significant (error bars = SEM). was not statistically significant (error bars = SEM). 3.6. MYXVorfC Stimulated Follicular Hyperplasia in Lymphoid Tissues 3.6. MYXVorfC Stimulated Follicular Hyperplasia in Lymphoid Tissues 3.6. MYXVorfCSpleen and Stimulated lymph nodes Follicular from uninfectedHyperplasia rabbitsin Lymphoid and rabbitsTissuesinfected with MYXV had minimal Spleen and lymph nodes from uninfected rabbits and rabbits infected with MYXV had minimal lymphoidSpleen reactivity and lymph (Figure nodes 14 fromA,B). uninfected In contrast, rabbits MYXVorfC-infected and rabbits infected rabbits with developed MYXV had lymphoid minimal lymphoid reactivity (Figure 14A,B). In contrast, MYXVorfC-infected rabbits developed lymphoid hyperplasialymphoid reactivity (Figure 14 (FigureC) suggestive 14A,B). In of contrast, a more activated MYXVorfC adaptive-infected systemic rabbits immune developed response lymphoid to hyperplasia (Figure 14C) suggestive of a more activated adaptive systemic immune response to the thehyperplasia virus. (Figure 14C) suggestive of a more activated adaptive systemic immune response to the virus. virus.

FigureFigure 14.14. RepresentativeRepresentative photomicrographs of of splenic splenic lymphoid lymphoid follicles follicles from from rabbits rabbits injected injected with with (FigureA(A)) saline, saline, 14. (RepresentativeB ()B 50) 50 pfu pfu MYXV, MYXV, photomicrographs or ( C or) 50 (C pfu) 50 MYXVorfC. pfu of MYXVorfC. splenic Samples lymphoid Samples were follicles collected were from collected at the rabbits time at of theinjected euthanasia. time with of Marked(euthanasia.A) saline, follicular (BMarked) 50 hyperplasia pfu follicular MYXV, washyperplasia or ( notedC) 50 in pfu was rabbits MYXVorfC. noted infected in rabbits Samples with infected MYXVorfC were with collected (H&E, MYXVorfC at magnification the (H&E, time of 20euthanasia.magnificationobjective). Marked 20× objective). follicular hyperplasia was noted in rabbits infected with MYXVorfC (H&E, magnification× 20× objective). 4.4. DiscussionDiscussion 4. Discussion DevelopmentDevelopment of of safe, safe, eeffffective OVs could revolutionize cancer cancer treatment, treatment, particularly particularly for for neoplasmsneoplasmsDevelopment thatthat areare refractoryrefractory of safe, eff totoective currently OVs available could revolutionizetherapies. MYXV MYXV cancer is is nonpathogenic nonpathogenic treatment, particularly in in humans humans [6] for[6 ] andneoplasmsand showsshows thatpotentialpotential are refractory as a safe to OV currently [2 [299,30],30].. However, However,available therapies.the the effectiveness effectiveness MYXV of is of MYXVnonpathogenic MYXV is is somewhat somewhat in humans limited. limited. [6] WeandWe predict showspredict thatpotential that OVs OVs that asthat a inducesafe induce OV apoptosis apoptosis[29,30]. However, of of tumor tumor cellsthe cells effectiveness quickly quickly can canexpose ofexpose MYXV the the is immune immunesomewhat response response limited. to tumorWeto tumorpredict antigens antigens that more OVs more rapidlythat induce rapidly and negate apoptosis and negate limitations of tumor limitations of rapidcells ofquickly removal rapid can of removal MYXVexpose of (which the MYXV immune we (which hypothesize response we istohypot due tumorhesize to the antigens anti-viral is due more responseto the rapidly anti induced - andviral negate by response healthy limitations induced cells within of by rapid the healthy tumor removal cells microenvironment). of within MYXV the (which tumor With we thishypotmicroenvironment). inhesize mind, we is designed due With to thisthe a pro-apoptotic, in anti mind,-viral we responsedesigned recombinant a induced pro MYXV,-apoptotic, by MYXVorfC. healthy recombinant cells MYXV, within MYXVorfC. the tumor microenvironment).MYXVorfCMYXVorfC containscontains With ananthis HA-taggedHA in mind,-tagged we orfC designed gene isolated isolateda pro-apoptotic, from from WDSV. WDSV. recombinant WDSV WDSV orfC orfCMYXV, may may MYXVorfC. pro promotemote seasonalseasonalMYXVorfC tumor tumor regressioncontains regression an in HA fishfish-tagged infected orfC with gene WDSV WDSV isolated by by from inducing inducing WDSV. cytochrome cytochrome WDSV orfC C C releasemay release pro from frommote mitochondriaseasonalmitochondria tumor [[21]21 regression].. Expression in of fish orfC infected by by MYXVorfC MYXVorfC with WDSV was was placed placed by inducing under under the the cytochrome control control of of a C alate late release poxvirus poxvirus from promotermitochondriapromoter elementelement [21]. (vaccinia(vacciniaExpression p11) of orfC with bythe MYXVorfC expectation was that that placed complete complete under replication replication the control of of ofMYXVorfC MYXVorfC a late poxvirus and and promoterproduction element of infectious (vaccinia virions p11) would with the occur expectation before significant that complete apoptosis replication was indu of cedMYXVorfC in infected and production of infectious virions would occur before significant apoptosis was induced in infected

Viruses 2020, 12, 517 14 of 16 production of infectious virions would occur before significant apoptosis was induced in infected cells. Additionally we engineered MYXVorfC to encode the reporter element tdTomato under the control of an early/late poxvirus promoter (vvSynE/L) to enable us to screen for and plaque purify recombinant virus. Our sequencing data indicated that the HA-tagged orfC gene and the reporter element tdTomato were successfully recombined into wild-type MYXV. The virus growth curve in RK-13 cells was not altered by recombination of tdTomato or HA-orfC into MYXV. Protein expression of tdTomato was observed as early as 8 hpi and HA-tagged orfC was detected by 12 hpi. Importantly, MYXVorfC significantly increased apoptosis in cell culture as compared to wild-type MYXV. These data indicate that MYXVorfC is able to replicate in cells, express exogenous proteins, induce apoptosis, spread to neighboring cells, and continue to replicate until all cells have been infected. Apoptosis did not reduce the virus growth rate because virus particles that are formed during infection remain infectious when the cells loose viability. Next, we infected New Zealand white rabbits with MYXVorfC to ensure that the pathogenicity of the recombinant virus was not more severe than wild-type MYXV. During wild-type MYXV infection, virus replicates to high titers at the primary site of virus inoculation. Within 2 to 4 dpi, viremia occurs allowing for systemic dissemination of infectious viral particles. MYXV localizes to the skin and mucous membranes by 5 dpi, replicates to high titers, and causes secondary myxomatous lesions, fever, and viral pneumonia. This leads to severe obstruction of the respiratory tract, respiratory arrest, and death between 8 and 12 dpi [28]. In comparison, rabbits infected with MYXVorfC had a significantly longer median survival time. Five animals required euthanasia between 11 and 16 dpi, but one animal recovered from MYXVorfC infection. Differences in overall clinical scores were minimal and mostly due to a short delay in the development of secondary lesions and fever in rabbits infected with MYXVorfC. The clinical similarities in infected rabbits are not unexpected, as no MYXV genes were disrupted by insertion of orfC into the virus. Therefore, any attenuation observed was due to expression of orfC by MYXVorfC. Although MYXVorfC infection was attenuated, the dissemination of MYXVorfC was similar to MYXV. Virus DNA and infectious virions were detected for both viruses in all organs tested. However, significantly lower MYXVorfC titers were recovered from the primary skin lesion at the site of inoculation, the lung tissue, and the spleen. This could reflect a mild reduction of MYXVorfC replication in rabbit tissues or increased clearance of the virus by the immune response. MYXVorfC rabbits had clear evidence of lymphoid hyperplasia, which was lacking in MYXV-infected rabbits. This suggests that rabbits mount a more effective immune response against MYXVorfC. Lymphocytic inflammation is also observed during regression of tumors caused by WDSV [22]. If lymphocytic inflammation is induced by orfC expression during oncolytic virotherapy, it could greatly improve outcomes for cancer patients being treated with MYXVorfC. MYXVorfC was engineered to increase apoptosis during infection. This was confirmed using RK-13 cells in tissue culture. In rabbits, the average number of TUNEL positive cells in MYXV-infected primary lesions (26.7 11.7 /hpf) was lower than that of MYXVorfC-infected lesions (37.2 7.1 /hpf). ± ± Although this difference was not statistically significant, a slight increase in TUNEL positive cells was consistently observed in MYXVorfC-infected tissue. It is possible that MYXV-induced apoptosis will be more enhanced in cancerous cells. In conclusion, MYXVorfC is pro-apoptotic in cell culture and attenuated in rabbits. The marked lymphoid hyperplasia observed in rabbits infected with MYXVorfC suggests that expression of orfC allows rabbits to mount a more effective immune response against MYXVorfC than wild-type MYXV. A very mild increase in apoptosis at the site of MYXVorfC inoculation was also observed. It is possible that the combination of increased immunogenicity and apoptosis contribute to the decreased pathogenicity of the virus. Future studies will test MYXVorfC treatment in animal cancer models to determine if MYXVorfC is more effective than MYXV at eliminating cancer.

Author Contributions: Conceptualization, S.L.Q. and A.L.M.; data curation, L.V.A. and A.L.M.; formal analysis, L.V.A. and A.L.M.; funding acquisition, A.L.M.; investigation, L.V.A., J.C., G.W., J.M., M.J., and A.L.M.; Viruses 2020, 12, 517 15 of 16 methodology, A.L.M.; project administration, L.V.A. and A.L.M.; recourses, A.L.M.; supervision, L.V.A. and A.L.M.; validation, L.V.A. and A.L.M.; visualization, L.V.A. and A.L.M.; writing—original draft, A.L.M.; writing—review and editing, L.V.A. and S.L.Q. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Colorado State University College of Veterinary Medicine and Biomedical Sciences and The Animal Health Innovation Fund. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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