Jonatas Abrahao, Universidade Federal de Minas Gerais, Brazil

#### Reviewed by:

Jonas Dutra Albarnaz, University of Cambridge, United Kingdom Rodrigo Araújo Lima Rodrigues, Universidade Federal de Minas Gerais, Brazil

\*Correspondence:

Lidia Szulc-D ˛abrowska<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 29 September 2017 Accepted: 06 December 2017 Published: 19 December 2017

#### Citation:

Szulc-D ˛abrowska L, Struzik J, Cymerys J, Winnicka A, Nowak Z, Toka FN and Gierynska M (2017) ´ The in Vitro Inhibitory Effect of Ectromelia Virus Infection on Innate and Adaptive Immune Properties of GM-CSF-Derived Bone Marrow Cells Is Mouse Strain-Independent. Front. Microbiol. 8:2539. doi: 10.3389/fmicb.2017.02539 Ectromelia virus (ECTV) belongs to the Orthopoxvirus genus of the Poxviridae family and is a natural pathogen of mice. Certain strains of mice are highly susceptible to ECTV infection and develop mousepox, a lethal disease similar to smallpox of humans caused by variola virus. Currently, the mousepox model is one of the available small animal models for investigating pathogenesis of generalized viral infections. Resistance and susceptibility to ECTV infection in mice are controlled by many genetic factors and are associated with multiple mechanisms of immune response, including preferential polarization of T helper (Th) immune response toward Th1 (protective) or Th2 (non-protective) profile. We hypothesized that viral-induced inhibitory effects on immune properties of conventional dendritic cells (cDCs) are more pronounced in ECTVsusceptible than in resistant mouse strains. To this extent, we confronted the cDCs from resistant (C57BL/6) and susceptible (BALB/c) mice with ECTV, regarding their reactivity and potential to drive T cell responses following infection. Our results showed that in vitro infection of granulocyte-macrophage colony-stimulating factor-derived bone marrow cells (GM-BM—comprised of cDCs and macrophages) from C57BL/6 and BALB/c mice similarly down-regulated multiple genes engaged in DC innate and adaptive immune functions, including antigen uptake, processing and presentation, chemokines and cytokines synthesis, and signal transduction. On the contrary, ECTV infection upregulated Il10 in GM-BM derived from both strains of mice. Moreover, ECTV similarly inhibited surface expression of major histocompatibility complex and costimulatory molecules on GM-BM, explaining the inability of the cells to attain full maturation after Toll-like receptor (TLR)4 agonist treatment. Additionally, cells from both strains of mice failed to produce cytokines and chemokines engaged in T cell priming and Th1/Th2 polarization after TLR4 stimulation. These data strongly suggest that in vitro modulation of GM-BM innate and adaptive immune functions by ECTV occurs irrespective of

whether the mouse strain is susceptible or resistant to infection. Moreover, ECTV limits the GM-BM (including cDCs) capacity to stimulate protective Th1 immune response. We cannot exclude that this may be an important factor in the generation of non-protective Th2 immune response in susceptible BALB/c mice in vivo.

Keywords: ectromelia virus, conventional dendritic cells, Th polarization, immunosuppression, viral evasion strategies

#### INTRODUCTION

The poxviruses are large DNA viruses that are undoubtedly masters of immune evasion, and have evolved to modulate and inhibit the host immune and inflammatory responses. The poxvirus genome encodes multiple classes of immunomodulatory proteins that act either intracellularly (virostealth and virotransducers) or extracellularly (viromimetics: virokines and viroceptors) and antagonize or compete with molecules critically involved in the host antiviral response. In fact, due to diversity of these genes, a single immunomodulatory protein that is shared by all poxviruses has not been identified yet. Moreover, a unique repertoire of immunomodulatory proteins encoded by each virus species allows it to successfully evade the immune response and survive in its natural host (Stanford et al., 2007). Highly host-specific survival strategy is employed especially by members of the Orthopoxvirus genus that exhibit a narrow host range and co-evolved with their natural host, e.g., variola virus (VARV, the causative agent of smallpox) in human and ectromelia virus (ECTV, the causative agent of mousepox) in mice. Meantime, other orthopoxviruses, such as vaccinia (VACV), monkeypox (MPXV), and cowpox (CPXV) viruses, which have a broad host range, are able to infect many different mammalian species and may contribute to the unpredictable outcome of infection in a new host species, e.g., MPXV in humans (McCollum and Damon, 2014). Therefore, a better understanding of the immunomodulatory mechanisms used by orthopoxviruses in their natural hosts is especially important for a full knowledge of their immune evasion strategies employed to control the host immune system.

The mousepox model is an excellent small animal model to study pathogenesis of smallpox, a disease that, despite being eradicated from the globe, now represents one of the most dangerous bioterrorism threats to human society. Smallpox is considered by Centers for Disease Control and Prevention (CDC) in Atlanta as a category A bioterrorism agent due to its easy dissemination, transmission from person to person, and high mortality rates (Riedel, 2005). ECTV shares with VARV several common properties, including: narrow host range and co-evolution with the natural host, high infectivity at low dose, and viral transmission and replication. Moreover, both viruses cause severe diseases with similar pathogenesis, aspects of pathology and immune response, and high mortality rates (Stanford et al., 2007). Therefore, mousepox model is extensively used to study basic questions in immune response regulation during generalized viral infections to eventually develop new prophylactic and therapeutic treatments against orthopoxviruses (Parker et al., 2010).

Within inbred strains of mice there is a genetically determined resistance to severe mousepox. C57BL/6 [H-2b ] mice are resistant to the lethal form of disease, whereas BALB/c [H-2d ] mice are fully susceptible to ECTV infection and usually succumb to disease between 7 and 9 days post-footpad infection. Genetic resistance is controlled by at least four autosomal dominant genes called rmp (resistance to mousepox), which are involved in regulation of some aspects of innate immunity (Brownstein and Gras, 1995). Additionally, during the infection resistant and susceptible strains of mice develop different types of the T helper (Th) cytokine immune responses: C57BL/6 mice generate a protective Th1 immune response accompanied by strong cytotoxic T lymphocyte (CTL) activity, whereas BALB/c mice generate a non-protective Th2 immune response, which is associated with a weak/absent CTL activity (Chaudhri et al., 2004).

The central role in driving T cell responses is played by dendritic cells (DCs), the most potent antigen presenting cells (APCs). Depending on lineage, maturation stage, and activation status, DCs release different polarizing signals, the most important of which are cytokines and chemokines that selectively promote the generation of Th1, Th2, Th17, or regulatory T cells (Tregs) (Kaiko et al., 2008). Immature DCs, characterized by low expression of antigen presenting [major histocompatibility complex (MHC) I, MHC II, CD1d] and costimulatory (CD80, CD86, and CD40) molecules induce anergy in antigen-specific naïve T cells or generate Foxp3+ induced Tregs in the presence of transforming growth factor (TGF)-β. Additionally, immature or semi-mature DCs are able to induce regulatory Foxp3− IL-10+ Tr1 (type 1 regulatory T) cells. Th2-polarizing DCs have semi-mature state associated with increased expression of antigen presenting and costimulatory molecules, and inability to secrete polarizing cytokines, such as IL-12p70 (Th1) or IL-6 and IL-23 (Th17). Moreover, IL-10 produced by DCs has been associated with propagation of Th2 immunity, however, this cytokine preferentially blocks DC maturation and induces anergy or Tr1 cells. Fully matured DCs, upon lipopolysaccharide (LPS) or CpG oligonucleotide treatment, produce IL-12p70 and possess strong capacity to polarize T cells toward Th1 profile (Lutz, 2016).

As masters of immune evasion, orthopoxviruses are able to control DC functions important for induction of an antiviral immune response. In general, they inhibit numerous functions of these cells, including antigen uptake and presentation, maturation, pro-inflammatory response, and capacity to activate T cells (Engelmayer et al., 1999; Jenne et al., 2000; Hansen et al., 2011; Szulc-D ˛abrowska et al., 2017). However, ECTV,

unlike VACV and CPXV, can productively infect conventional DCs (cDCs) in vitro (Szulc-D ˛abrowska et al., 2017) and in vivo (Sei et al., 2015), what indicates strong adaptation capacity of ECTV to the natural host immune cells. It has been shown that cDCs derived from resistant C57BL/6 and susceptible BALB/c mouse strains may differentially react during viral (Pejawar et al., 2005) and bacterial (Jiang et al., 2010) infections in vitro. Upon infection, cDCs from C57BL/6 mice underwent higher functional maturation and/or were able to stimulate more potent CD8+ T cell response than cells from BALB/c mice (Pejawar et al., 2005; Jiang et al., 2010).

We therefore confronted the cDCs from resistant (C57BL/6) and susceptible (BALB/c) mice regarding inter-strain differences in reactivity and potential to drive T cell responses following in vitro infection with ECTV. Our results showed that ECTV similarly affects innate and adaptive immune functions of granulocyte-macrophage colony-stimulating factor (GM-CSF) derived bone marrow cells (GM-BM) obtained from both mouse strains. GM-BM infected with ECTV exhibited a profound down-regulation in expression of many genes involved in maturation and activation of DCs, with the exception of Il10, which was up-regulated in a strain independent manner. Moreover, ECTV impaired production of chemokines and cytokines engaged in regulation of Th1 and Th2 immune responses, as well as decreased maturation marker expression on the surface of C57BL/6 and BALB/c GM-BM. Collectively, our data suggest that ECTV-infected GM-BM have strong potential to silence the Th immune response, independently of their genetic background.

#### MATERIALS AND METHODS

#### Animals

Male C57BL/6 (H-2b ) and BALB/c (H-2d ) mice (8–12 weeks old) were purchased from the animal facility at Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw, Poland. After arrival, animals were kept for 7 days for acclimatization in the animal facility at the Faculty of Veterinary Medicine under controlled temperature and humidity with free access to food and water. Experimental procedures on animals were approved by the 3rd Ethical Committee for Animal Experimentation at Warsaw University of Life Sciences—SGGW (permission no. 34/2012) and were performed in accordance with institutional Guidelines for Care and Use of Laboratory Animals.

#### Virus

The Moscow strain of ECTV (ATCC, VR-1374) was obtained from American Type Culture Collection (Manassas, VA, United States). The virus was propagated on African green monkey kidney (Vero) cells (ATCC, CCL-81) maintained in DMEM high glucose (HyClone, Logan, UT, United States) supplemented with 5% fetal bovine serum (FBS; HyClone) and 1% antibiotic–antimycotic solution (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B; Sigma–Aldrich, St. Louis, MO, United States). The virus stock was purified by sucrose cushion centrifugation. Briefly, virus suspension was layered onto 36% sucrose in 1 mM Tris pH 9.0 and centrifuged at 30,000 × g for 60 min at 4◦C. After purification, virus stock infectivity was determined by plaque formation assay (PFU/ml) on Vero cell monolayer. Inactivation of the virus was performed by a 30 min exposure to UV radiation (120 W, 320 nm) at a working distance of 12 cm from the UV lamp. The absence of plaque formation in the Vero cell monolayer indicated complete inactivation of the virus.

#### Generation and Enrichment of GM-BM

The protocol used to generate GM-BM was similar to that used by Lutz et al. (1999). Bone marrow was flushed with cold RPMI-1640 medium from femurs and tibias of mice and erythrocytes were removed using ammonium chloride buffer. Cells were then washed and plated at 1 × 106 /well in a six-well plate in RPMI-1640 medium containing 10% heat-inactivated FBS, 1% antibiotic solution (100 U/ml penicillin and 100 mg/ml streptomycin; Sigma–Aldrich), 50 µM 2-mercaptoethanol (Sigma–Aldrich), and 20 ng/ml recombinant mouse (rm) GM-CSF (R&D Systems, Minneapolis, MN, United States). Fresh medium with 20 ng/ml rmGM-CSF was added at day 3 of culture and then was partially replaced every 2 days. On day 8 post-culture, cDCs were enriched using MACS CD11c+ labeled magnetic beads (Miltenyi Biotec, Auburn, CA, United States). After MACS separation GM-BM cultures were evaluated for surface expression of cellular markers using the following monoclonal antibodies (mAbs): anti-mouse CD11c-BV421 (N418, Armenian Hamster IgG2; BioLegend, San Diego, CA, United States), anti-CD11b-BV605 (M1/70, rat IgG2b), anti-I-A/I-E-BV711 (M5/114.15.2, rat IgG2b; both from BD Biosciences, San Jose, CA, United States) and anti-CD205-PE-Cy7 (NLDC-145, rat IgG2a; BioLegend) (**Supplementary Figure S1**).

#### GM-BM Infection and Treatment

MACS-separated CD11c+ cells (purity ≥95%) were infected with live ECTV at multiplicity of infection (MOI) of 1. In some experiments, cells were exposed to UV-inactivated ECTV (uvi-ECTV) at MOI of 1 (before inactivation). As a control, non-infected cells were cultured in parallel in complete RPMI-1640 medium. Additionally, non-, ECTV-, and/or uvi-ECTV-exposed cells were left untreated or were treated for 24 h with 1 µg/ml LPS (Escherichia coli 0111:B4; Sigma–Aldrich), which is a Toll-like receptor (TLR)4 agonist used as a positive control for fully matured cells.

#### Plaque Assay

Vero cells cultured on 24-well plates were treated with 10-fold serial dilutions of ECTV stocks obtained from C57BL/6 and BALB/c GM-BM at 4, 12, and 24 hpi. After 5 days, plaques were counted under Olympus IX71 inverted microscope. After counting, Vero cell monolayers were stained with 0.3% crystal violet and air dried.

#### Measurement of Apoptosis

The apoptotic rate of GM-BM was measured using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences),

according to the manufacturer's protocol. Briefly, cells were washed twice with cold phosphate-buffered saline (Sigma–Aldrich) and resuspended in 1× binding buffer at a concentration of 1 × 106 cells/ml. Then, 100 µl of the solution (containing 1 × 105 cells) was stained with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) and incubated for 15 min at room temperature in the dark. Finally, cells were resuspended in 400 µl of 1× binding buffer and analyzed immediately by flow cytometry. Viable cells (no measurable apoptosis) are Annexin V-FITC and PI negative, early apoptotic cells (membrane integrity is present) are Annexin V-FITC positive and PI negative, whereas late apoptotic cells (end stage apoptosis and death) are Annexin V-FITC and PI positive.

#### Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The expression of selected genes involved in innate and adaptive immune functions of GM-BM was evaluated using real-time reverse transcription polymerase chain reaction (RT-PCR), as previously described (Dolega et al., 2017; Szulc-D ˛abrowska et al., 2017) with minor modifications. Briefly, RNA was extracted from 1 × 106 mock- or ECTV-infected GM-BM, untreated or treated with LPS for 24 h using Qiagen RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, United States), as recommended by the manufacturer. Additionally, on-column DNase I digestion of genomic DNA was performed using RNase-Free DNase Set (Qiagen). The RNA concentration and purity was assessed using the Take-3 system on Epoch BioTek spectrophotometer and analyzed in Gen5 software (BioTek Instruments, Inc., Winooski, VT, United States). Total RNA (1 µg) was applied for cDNA synthesis using the RT2 First Strand Kit (Qiagen) according to the manufacturer's protocol. Before RT, genomic DNA was additionally removed by incubation in GE2 buffer for 5 min at 42◦C. For analysis of selected genes involved in regulation of innate and adaptive immune properties of GM-BM the Mouse Dendritic and Antigen Presenting RT2 Profiler PCR Array (Qiagen) was used according to the recommendation of the manufacturer. Briefly, 550 ng cDNA was mixed with RT2 SYBR Green Mastermix (Qiagen) and aliquoted into the 96-well RT2 Profiler PCR array plate, containing lyophilized RT2 qPCR primers for a set of 84 related genes, five housekeeping genes (Actb, B2m, Gapdh, Gusb, and Hsp90ab1), three Reverse Transcription Controls (RTC), three Positive PCR Controls (PPC), and one Mouse Genomic DNA Contamination (MGDC) control (**Supplementary Table S1**). Amplification was performed in ABI 7500 thermocycler (Life Technologies, Carlsbad, CA, United States) at 95◦C for 10 min, 40 cycles of 95◦C for 15 s and 60◦C for 1 min. Fluorescence data were collected each cycle after the 1 min step at 60◦C. Amplification data were acquired through SDS Software (Applied Biosystems).

#### Data Quality Control, Normalization, and Analysis

Obtained data fulfilled the criteria of PCR array reproducibility, RT efficiency, and genomic DNA contamination. If the average PPC Ct was 19 ± 3 and no two arrays had an average PPC Ct > 2 away from one another, then the samples passed the criteria for the PCR array reproducibility. If 1Ct (AVG RTC − AVG PPC) was ≤5, then the samples passed the criterion for RT efficiency. If Ct (MGDC) was ≥35, then the samples passed the criterion for genomic DNA contamination.

Three independent biological experiments were performed for each experimental group. The normalization was performed using the most stable genes/gene in the PCR array data set, identified by software at the Qiagen Data Analysis Center. The Ct values for these genes were geometrically averaged and used for the calculation of 11Ct values. The data are presented as fold change (2−11Ct) which is the normalized gene expression (2−1Ct) in the test sample divided by the normalized gene expression (2−1Ct) in the control sample. Fold regulation represents fold change results in a biologically meaningful way. Fold change values greater than one indicate a positive- or an up-regulation, and the fold regulation is equal to the fold change. Fold change values less than one indicate a negative or down-regulation, and the fold regulation is the negative inverse of the fold-change. The P-values were calculated based on a Student's t-test of the replicate 2−1Ct values for each gene in the control group and treatment groups. Significance was assessed at ∗P ≤ 0.05 and ∗∗P ≤ 0.01.

#### Immunophenotyping

Multi-color immunophenotyping was performed as previously described (Szulc-D ˛abrowska et al., 2017). Cell surface maturation markers were stained with the following mAbs used in appropriate combinations: anti-H-2D[b]-PE (KH95, mouse IgG2b), anti-H-2D[d]-PE (34-2-12, mouse IgG2a), anti-I-A/I-E-BV711, anti-CD40-APC (3/23, rat IgG2a), anti-CD80-APC (16-10A1, Armenian hamster IgG2), anti-CD83-PE (Michel 19, rat IgG1) (all from BD Biosciences), and anti-CD86-PerCP-Cy5.5 (GL-1, rat IgG2a; eBioscience). Chemokine receptors were stained with anti-CD191(CCR1)-APC (643854, Rat IgG2b; R&D Systems), anti-CD195(CCR5)-PE (C34-3448, rat IgG2c; BD Biosciences), and anti-CD197(CCR7)-PerCP/Cy.5.5 (4B12, rat IgG2a; BioLegend) mAbs. Additionally, GM-BM were labeled for CD11c marker using anti-CD11c-BV421 mAbs. Appropriate isotype controls (purchased from BD Biosciences) were used to determine the percentages of cells expressing the respective markers. Moreover, to obtain proper gating strategies the Fluorescence Minus One samples were included as negative controls.

#### Intracellular Staining

Non-, uvi-ECTV-, or ECTV-infected GM-BM were left unstimulated or were stimulated with LPS for 24 h in a 24-well plate at a density of 5 × 105 cells/well. Brefeldin A (6 µg/ml; BD Biosciences) was added for the last 5 h of culture. Cells were then collected and stained with anti-CD11c-BV421. Intracellular staining of cytokines was performed using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. The following mAbs were used for cytokine detection: anti-tumor necrosis factor (TNF)-FITC (MP6-XT22, rat IgG1), anti-IL-12(p40/p70)-APC (C15.6, rat IgG1; both from BD Biosciences) and anti-CCL3 [macrophage inflammatory protein 1 alpha (MIP-1α)]-PE (DNT3CC; rat IgG2a; eBioscience, San Diego, CA, United States). In some experiments, cells were stained intracellularly for the presence of ECTV antigens using a Cytofix/Cytoperm kit and rabbit polyclonal Abs anti-ECTV-FITC, obtained as previously described (Szulc-Dabrowska et al., 2016). The staining procedure also included appropriate isotype controls obtained from BD Biosciences and eBioscience.

#### Flow Cytometry Analysis

fmicb-08-02539 December 17, 2017 Time: 11:57 # 5

The population of live cells was gated based on size and granularity according to forward (FSC-A) and side (SSC-A) scatter profile. GM-BM enriched in cDC population were gated as FSChigh and CD11c+. Twenty thousand gated cell events were acquired from each specimen and analyzed on a BD FACSCanto II and BD LSRFortessa flow cytometers (Becton Dickinson). Data were analyzed with FACSDiva 7.0 software (Becton Dickinson).

#### Enzyme-Linked Immunosorbent Assay

CD11c+-enriched GM-BM were plated into a 24-well plate at a density of 2 × 104 cells/well. After treatment with medium (mock), uvi-ECTV, or live ECTV, cells were incubated with or without LPS for 4, 8, 12, 18, and 24 h. In some experiments, cells were additionally stimulated with 3 mM ATP for 6 h to induce IL-1β and IL-18 release (Mehta et al., 2000). The concentration of cytokines and chemokines in culture supernatants was quantified using available commercial sandwich enzyme-linked immunosorbent assay (ELISA), according to the instructions given by the manufacturers. TNF, IL-6, IL-10, IL-12p40, IL-12p70, and CCL2/monocyte chemoattractant protein 1 (MCP-1) were detected using BD OptEIA ELISA sets (BD Biosciences). CCL3/MIP-1α and CCL5/RANTES (regulated upon activation normal T cell expressed and secreted) were quantified using Quantikine ELISA Kits (R&D Systems). IL-18 and IL-15R/IL-15 complex were assessed using Platinum ELISA and ELISA Ready-SET-Go!, respectively (eBioscience). The absorbance was read at 450 nm using Epoch Microplate Spectrophotometer. Each plate contained its own standard curve for determination of cytokine/chemokine levels in the supernatants.

#### Nitric Oxide detection

Nitric oxide (NO) formation can be investigated by measuring nitrite (NO2 −), which is one of two primary, stable, and non-volatile breakdown products of NO. The amount of nitrite (µM/ml) in culture supernatants was assessed using the Griess Reagent System (Promega, Madison, WI, United States), according to the manufacturers' protocol. Briefly, an equal volume of supernatant was mixed with the Sulfanilamide Solution and incubated for 10 min at room temperature in the dark. Then, N-1-naphthylethylenediamine dihydrochloride solution was added and incubated for additional 10 min. The absorbance was measured at 520 nm using Epoch Microplate Spectrophotometer. A standard curve generated with sodium nitrite was used to calculate the nitrite concentration in the supernatants.

#### Statistical Analysis

Data are presented as mean ± standard deviation (SD) from at least three independent biological replicates. Normal distribution of variables was assessed using the Shapiro–Wilk W-test. If the data were normally distributed and the variances were homogeneous, then two-independent (unpaired) Student's t-test was applied for group comparison. Some data were analyzed using two-dependent (paired) Student's t-test. If the data were non-normally distributed, then the Wilcoxon signed-rank or Mann–Whitney U-tests were applied for comparison of paired and unpaired values, respectively (STATISTICA 6.0 software, StatSoft Inc., Tulsa, OK, United States). Statistical significance was assessed at ∗P ≤ 0.05 and ∗∗P ≤ 0.01.

#### RESULTS

#### The Kinetics of ECTV Replication Cycle Are Similar in GM-BM Derived from C57BL/6 and BALB/c Mice

Our previous study showed that ECTV is able to productively infect murine GM-BM, including cDCs, with subsequent release of progeny virions (Szulc-D ˛abrowska et al., 2017). In the present study, we compared the kinetics of ECTV replication cycle in GM-BM derived from resistant C57BL/6 and susceptible BALB/c mice. Cells were infected with ECTV at MOI = 1 and after 4, 12, and 24 hpi the percentage of ECTV+ cells was determined by intracellular staining and flow cytometry analysis (**Figure 1A**). The mean percentage of ECTV+ cells during the first 12 h of virus replication was comparable between both strains of mice (**Figure 1B**). At 24 hpi, the percentage of ECTV+ cells was slightly higher in C57BL/6 comparing to BALB/c mice (75 vs. 63%, respectively). LPS treatment of ECTV-infected GM-BM derived from both mouse strains significantly (P ≤ 0.05) increased the percentage of ECTV+ cells during the entire virus replication cycle. It suggests that TLR4-agonist stimulation positively regulates the tempo of virus reproduction in GM-BM infected at MOI of 1 (Szulc-D ˛abrowska et al., 2017). Additionally, to confirm that ECTV infection of GM-BM is productive, we performed a plaque assay to determine quantify infectious viral particles (**Figure 1C**). At 4 hpi, the number of infectious virions was 4.2 × 103 and 5 × 103 PFU/ml in C57BL/6 and BALB/c GM-BM cultures, respectively. At 12 and 24 hpi, the virus titers in both cultures increased around 100- and 500-fold, respectively, and reached 5 × 105 and 7 × 105 PFU/ml at 12 hpi, and 3.2 × 106 and 2.6 × 106 PFU/ml at 24 hpi in C57BL/6 and BALB/c GM-BM cultures, respectively (**Figure 1C**). LPS treatment slightly increased the number of infectious virus particles, especially at 24 hpi and is agreement with flow cytometry results.

Our previous study also showed that 10-day culture of GM-BM and later infected with ECTV at MOI = 5 exhibited a high percentage (more than 30%) of early and late apoptotic cells during later stages of infection (Szulc-D ˛abrowska et al., 2017). To minimize the apoptotic rate in GM-BM cultures, in the present study we used 8-day culture of GM-BM and later infected with

ECTV at MOI = 1. Under such conditions, we observed only a small induction of apoptosis in GM-BM at 24 hpi in the absence of LPS (**Figure 1D**). Meanwhile, ECTV infection of GM-BM in the presence of LPS slightly (but not significantly) increased the percentage of apoptotic cells compared with LPS-treated mockor uvi-ECTV-infected cells (**Figure 1D**). It is likely that LPS prevents the apoptotic effect induced by the virus infection, since LPS has been shown to inhibit caspase 3-dependent apoptosis in immune cells (Russe et al., 2014).

#### Generalized Down-Regulation of Genes Involved in Innate and Adaptive Immune Functions Is Comparable in GM-BM-Derived from C57BL/6 and BALB/c Mice

Before assessment of ECTV impact on gene expression profile, we first compared the gene expression between uninfected GM-BM derived from C57BL/6 and BALB/c mice to determine interstrain differences in genes involved in innate and adaptive immune functions (**Figure 2**). These genes, encoding proteins that regulate biological properties of DCs, were divided into seven categories: (1) antigen uptake, (2) antigen presentation, (3) chemotaxis, (4) chemokines and cytokines, (5) cytokine receptors, (6) other cell surface receptors, and (7) signal transduction.

Before calculating 2−11Ct values, the data were normalized to the geometric mean of the four most stable housekeeping genes: ActB, Gapdh, Gusb, and Hsp90ab1. The scatter plots of the mRNA expression profiles of untreated and LPS-treated C57BL/6 vs. BALB/c GM-BM are presented in **Figures 2A,D**, respectively. The scatter plots compare the normalized expression of every gene on the array between two groups by plotting them against one another to quickly visualize large gene expression changes. As shown in **Figure 2A**, C57BL/6 compared to BALB/c GM-BM exhibited up-regulation of 25 genes, including Ccl5, Ccl8, Ccl17, Cxcl10, Cd40, Fas, Fcer2a, Fcgr1, Il-12b, Irf7, Thbs1, and Tlr9, and down-regulation of four genes, Cd1d2, Cd2, Fcgrt, and Icam2. LPS treatment of C57BL/6 and BALB/c GM-BM for 24 h resulted in up-regulation of 42 and 41 genes, and down-regulation of 13 and 15 genes, respectively (**Figures 2B,C**). Up-regulated genes were those known to influence maturation of DCs and included genes for cell surface receptors (e.g., Cd1d1, Cd1d2, Cd40, Cd80, Cd86, H2-DMa, Tlr1, Tlr2), chemokines and cytokines (e.g., Ifn, Il10, Il12a, Il12b, Tnf, Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl8, Ccl12, Ccl17, Ccl17, Cxcl1, Cxcl2, Cxcl10) and signal transduction (e.g., Irf7). Cd28, Cd36, Cd209a, and Clec4b2 were the commonly down-regulated genes after LPS exposure of GM-BM from both mouse strains. Interestingly, C57BL/6 GM-BM treated with LPS for 24 h showed up- and down-regulation of 31 and 5 genes, respectively, compared to BALB/c GM-BM treated with LPS (**Figure 2D**). Among up-regulated genes there were those primarily engaged in the antigen uptake (Tap2) and presentation (Cd4, Cd40, Cd80, Cd86, H2-DMa, Thbs1) and chemokine and cytokine production (Ccl5, Ccl7, Ccl8, Ccl12, Cxcl2, Cxcl10, Cxcl12). Moreover, TLR9 mRNA was significantly (P ≤ 0.01) up-regulated in C57BL/6 compared with BALB/c GM-BM after LPS-treatment. Collectively, it may suggest that C57BL/6 GM-BM are at a higher maturation state than BALB/c GM-BM after TLR4 agonist stimulation.

The gene expression comparison between uninfected and infected cells untreated or treated with LPS was made after data normalization to Cdkn1a, due to its uniform expression across treatment groups in the experiments. Differentially expressed genes are presented by volcano plots, which summarize fold-change in gene expression and statistical significance in four different experimental groups (**Figure 3**). ECTV infection of GM-BM derived from C57BL/6 and BALB/c mice resulted in a global down-regulation of 75 and 74 genes, respectively, among which 52 and 50 were significantly (P ≤ 0.05) repressed (**Figures 3A,B**). Profound inhibition in gene expression was observed in all gene categories in cells from both mouse strains. Only Il10 gene was significantly (P ≤ 0.05) up-regulated in infected cells. Moreover, ECTV infection inhibited LPS-induced up-regulation of mRNA transcripts for many genes involved in activation and maturation of GM-BM. LPS-treated ECTV-infected cells derived from C57BL/6 and BALB/c mice displayed down-regulation of 65 and 63 genes, respectively, compared to LPS-treated uninfected cells (**Figures 3C,D**). Among these genes 45 and 48, respectively, were significantly (P ≤ 0.05) down-regulated. Taken together, our data clearly demonstrate that ECTV induces global gene repression in GM-BM of both strains and this repression is maintained during TLR4 agonist treatment.

#### Expression of Genes Involved in Antigen Uptake and Presentation

In the antigen uptake category, Cd44, Icam1, Icam2, and Rac1 were mostly significantly (P ≤ 0.05) down-regulated in untreated or LPS-treated ECTV-infected C57BL/6 and BALB/c GM-BM in comparison with their uninfected counterparts (**Figure 4A**). The mean expression level of these genes was more than 10-fold repressed. The level of Rac1 repression was significantly (P ≤ 0.05) higher in infected C57BL/6 than BALB/c cells. Meanwhile, Cdc42 was not significantly up-regulated in LPS-treated ECTV-infected GM-BM from C57BL/6 mice compared to LPS-treated uninfected cells (**Figure 4A**).

Of the 16 genes analyzed in the antigen presentation category, ECTV infection resulted in significantly (P ≤ 0.05) down-regulation of 11 mRNA transcripts in C57BL/6 and BALB/c GM-BM (**Figure 4B**). B2m, Cd1d1, Cd74, Cd80, Cd86, Fcgrt, H2-DMa, Tapbp, and Thbs1 were significantly (P ≤ 0.05) repressed in cells from both mouse strains, whereas Cd28, Cd40Ig, and Cd1d2, Cd40 were significantly down-regulated in C57BL/6 and BALB/c GM-BM, respectively. The level of Cd74 repression was significantly (P ≤ 0.05) higher in infected BALB/c than C57BL/6 cells. LPS treatment of mock-infected C57BL/6 and BALB/c GM-BM resulted in up-regulation of the following genes: Cd1d1, Cd1d2, Cd209a, Cd40, Cd80, Cd86, Tapbp, and Thbs1. Meanwhile, treatment of ECTV-infected cells with LPS did not up-regulate genes, as observed in mock-infected LPS-treated GM-BM from the two mouse strains. After stimulation with LPS, infected C57BL/6 and BALB/c GM-BM still exhibited a profound down-regulation of 10 following genes: B2m, Cd1d1,

Cd1d2, CD40, Cd74, Cd86, Fcgrt, H2-DMa, Tapbp, and Thbs1. In all cases the most repressed genes were Cd1d1 and Thbs1, and their mean expression levels were ≥300-fold decreased. The repression level of mRNA transcript for CD40 was significantly (P ≤ 0.05) higher in LPS-stimulated ECTV-infected C57BL/6 than BALB/c cells. The mRNA expression of CD80 was not significantly (P > 0.05) down-regulated (**Figure 4B**). Only Cd4 was differentially regulated in LPS-treated ECTV-infected GM-BM from the two strains of mice. Cd4 was significantly (P = 0.009) up-regulated in BALB/c cells, whereas in C57BL/6 cells its expression remained unchanged.

#### Expression of Genes Involved in DC Chemotaxis

The four important genes in this group were significantly (P ≤ 0.05) down-regulated in ECTV-infected GM-BM from both mouse strains (**Figure 4C**). These genes were Ccl5, Ccr1, Ccr2, and Ccr5. Ccr1 was significantly (P ≤ 0.05) more repressed in infected C57BL/6 than BALB/c cells. The same set of genes were also down-regulated in LPS-treated ECTV-infected GM-BM compared to LPS-treated uninfected cells. However, in cells from C57BL/6 mice, the repression of Ccr2 and Ccr5 was not statistically significant (P > 0.05). Although the down-regulation of Cxcr4 in GM-BM from both mouse strains was not significant, statistical analysis revealed that this gene was considerably repressed in C57BL/6 mice.

### Expression of Genes Involved in Chemokine and Cytokine Production

The next category of analyzed genes concerned those engaged in the synthesis of chemokines (**Figure 5A**) and cytokines (**Figure 5B**). Among the 15 chemokine genes assayed, seven

genes in both C57BL/6 and BALB/c GM-BM were significantly (P ≤ 0.05) down-regulated (**Figure 5A**) following ECTV infection. Ccl4, Ccl5, Ccl17, and Ccl20 were repressed in cells from both mouse strains, whereas Ccl2, Ccl3, Ccl8 and Ccl12, Cxcl2, Cxcl12 were decreased in C57BL/6 and BALB/c GM-BM, respectively (**Figure 5A**). LPS treatment of mock-infected cells up-regulated 12 genes (Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl8, Ccl12, Ccl17, Ccl20, Cxcl1, Cxcl2, and Cxcl10) in GM-BM from both mouse strains (**Figures 2C,D**). Meanwhile, LPS-treated ECTV-infected cells showed significant (P ≤ 0.05) down-regulation of seven mRNA transcripts compared to LPS-treated uninfected cells (**Figure 5A**). GM-BM from both mouse strains exhibited repression of Ccl2, Ccl3, Ccl5, and Ccl8, whereas Ccl12, Cxcl1, Cxcl10 and Ccl4, Ccl17, Cxcl2 were significantly (P ≤ 0.05) decreased in cells from C57BL/6 and BALB/c mice, respectively. ECTV-infected C57BL/6 GM-BM displayed significant (P ≤ 0.05) down-regulation of Ccl2, Cxcl12 and Ccl2, Ccl3, Ccl8 than BALB/c cells, cultured without or with LPS, respectively.

Among the 13 genes analyzed in the cytokine group, Ifng, Mif, Tgfb1, and Tnf were significantly (P ≤ 0.05) repressed in GM-BM of both mouse strains (**Figure 5B**). The level of Mif expression was significantly (P ≤ 0.05) decreased in C57BL/6 cells. Il12a, Il16, and Flt3l were also down-regulated, however, significant (P ≤ 0.05) repression of these genes was observed only in C57BL/6 cells. On the contrary, the expression of Il12b was significantly (P ≤ 0.01) decreased in BALB/c GM-BM. Interestingly, ECTV infection significantly (P ≤ 0.05) up-regulated the expression of Il10 in cells from both mouse strains (**Figure 5B**).

Uninfected GM-BM from C57BL/6 and BALB/c mice displayed up-regulation of Ifng, Il6, Il10, Il12a, Il12b, and Tnf after LPS treatment for 24 h (**Figures 2C,D**). Meantime, LPS-stimulated ECTV-infected cells exhibited significant (P ≤ 0.05) repression of Il6, Il10, Il12a, Mif, and Tgfb1, compared to uninfected cells incubated with TLR4 agonist (**Figure 5B**). Moreover, LPS-treated ECTV-infected GM-BM from C57BL/6 and BALB/c mice showed down-regulation of Il16 and Flt3l or

Ifng and Tnf, respectively. Csf2 was significantly up-regulated only in BALB/c GM-BM after ECTV infection and LPS treatment.

#### Expression of Genes for Cytokine and Other Cell Surface Receptors

In the cytokine receptors category, Ccr1, Ccr2, Ccr3, Ccr5, Csf1r, and Lyn were significantly (P ≤ 0.05) down-regulated in untreated or LPS-treated ECTV-infected GM-BM from both mouse strains, compared to their uninfected counterparts (**Figure 6A**). Ccr1, Cxcr4, and Lyn were significantly (P ≤ 0.05) more repressed in C57BL/6 than BALB/c GM-BM. Moreover, Ccr9 and Flt3 were significantly decreased only in infected BALB/c cells.

The majority of other analyzed genes in the cell surface receptors category were down-regulated in ECTV-infected GM-BM from both mouse strains (**Figure 6B**). Cd36, Fcgr1, Lrp1, Tlr1, and Tlr2 were significantly (P ≤ 0.05) repressed in cells from both mouse strains and such inhibitory effect was independent of LPS stimulation. Cd2, Tlr7, and TLr9 were also down-regulated in both strains, however, after LPS stimulation Tlr7 and TLr9 were significantly (P ≤ 0.05) repressed only in C57BL/6 GM-BM. On the contrary, Fcer2a was significantly (P ≤ 0.05) up-regulated in ECTV-infected C57BL/6 and BALB/c GM-BM treated with LPS (**Figure 6B**). LPS stimulation of mock-infected cells resulted in up-regulation of Cd40, Fcgr1, Tlr1, and Tlr2 (**Figures 2C,D**).

#### Expression of Genes Involved in Signal Transduction

The last analyzed group concerned 12 genes involved in signal transduction (**Figure 6C**). The following 8 genes were mostly significantly (P > 0.05) down-regulated in ECTV-infected GM-BM derived from both strains of mice and independent of LPS treatment: Fas, Irf7, Itgam, Itgb2, Nfkb1, Ptprc, Rela, and Stat3. Moreover, Cebpa and Relb were significantly (P > 0.05) down-regulated in GM-BM from both and only BALB/c mouse,

respectively. Cebpa, Irf7 and Stat3 were significantly (P > 0.05) more repressed in infected C57BL/6 than BALB/c GM-BM (**Figure 6C**).

#### Mouse Strain-Independent Effect of ECTV-Infection on Cytokine and NO Production by GM-BM

cDCs play a key role in driving T cell responses by eliciting polarizing signals, the most important of which are cytokines that selectively promote the generation of Th1 or Th2 cells. We next confronted the GM-BM obtained from resistant C57BL/6 and susceptible BALB/c mice regarding their Th-polarizing cytokine profile under ECTV infection in vitro. The analyzed cytokines and chemokines are engaged in T cell activation and involved in regulation of Th1 or Th2 polarization during the adaptive immune responses.

TNF-α contributes to DC activation and maturation and is required for subsequent induction of optimal T cell responses (Zhang et al., 2003). Uninfected GM-BM from C57BL/6 and BALB/c mice produced TNF-α at similar low levels, however, after LPS stimulation, BALB/c cells secreted higher amounts of TNF-α than C57BL/6 cells (**Figure 7A**). During LPS treatment, the concentration of TNF-α in culture

supernatants from C57BL/6 and BALB/c GM-BM started to decrease at 24 h post-stimulation and was two- and fourfold lower, respectively, than at 4 h post-stimulation. Meantime, ECTV infection of GM-BM from both strains of mice profoundly inhibited TNF-α secretion, even after LPS treatment.

IL-6 produced by DCs in response to TLR recognition of microbial products has been demonstrated to regulate T cell activation by overcoming the suppressive effect of Tregs (Pasare and Medzhitov, 2003). Similar to TNF-α, mock- or uvi-ECTV-infected GM-BM from both mouse strains secreted comparable low levels of IL-6. After LPS stimulation, GM-BM of both strains of mice produced high amounts of IL-6 and from 8 h post-stimulation, DCs from BALB/c mice produced higher levels of IL-6 than those from C57BL/6 mice (**Figure 7A**). ECTV-infected GM-BM untreated or treated with LPS were able to secrete IL-6, however, the amounts were significantly (P > 0.01) lower compared to those secreted by mock- or uvi-ECTV-infected LPS-untreated or -treated GM-BM.

Next, we analyzed cytokines and chemokines engaged in Th1 polarization, such as: IL-12p40, IL-12p70, IL-15/IL-15Rα, IL-18, CCL3/MIP-1α, and CCL5/RANTES (**Figures 7A,B**). C57BL/6 GM-BM produced larger amounts of IL-12p40 and IL-12p70 than BALB/c GM-BM, especially after LPS treatment

(**Figure 7A**). ECTV-infection significantly (P > 0.01) decreased the production of both cytokines by LPS-untreated or -treated GM-BM of both mouse strains. Meantime, CCL3 was secreted at higher levels by uninfected BALB/c than C57BL/6 cells, especially in response to the ligand for TLR4 (**Figure 7A**). At 12 hpi with ECTV, GM-BM from both mouse strains produced less CCL3 than mock- or uvi-ECTV infected cells, whereas at 24 hpi those cells showed significantly (P > 0.01) increased level of CCL3 compared to mock- or uvi-ECTV-treated cells. However, GM-BM infected with live ECTV exhibited the suppression of TLR4 agonist-induced secretion of CCL3. CCL5 production was higher by C57BL/6 GM-BM, however, LPS stimulation induced robust secretion of comparable large amounts of CCL5 by cells from both mouse strains (**Figure 7A**). The secretion of CCL5 was >2 fold reduced in ECTV-infected C57BL/6 and BALB/c GM-BM, either in the absence or presence of LPS.

IL-15/IL-15Rα complex was produced at similar levels by C57BL/6 and BALB/c GM-BM, even after LPS-stimulation for 24 h (**Figure 7B**). ECTV infection significantly (P > 0.01) reduced the level of this complex in GM-BM from both mouse strains at 24 hpi. IL-18 was also secreted at comparable amounts by uninfected cells from both strains of mice, however, after stimulation with LPS for 24 h and ATP for additional 6 h, BALB/c GM-BM produced fourfold higher levels of IL-18 than C57BL/6 GM-BM (**Figure 7B**). ECTV-infection significantly (P > 0.01) inhibited TLR4 agonist + ATP-induced IL-18 production.

Because NO selectively enhances Th1 cell proliferation (Niedbala et al., 1999), we additionally checked the influence of ECTV infection on NO production by GM-BM from C57BL/6 and BALB/c mice (**Figure 7B**). Uninfected GM-BM from both mouse strains produced low levels of NO. After LPS treatment for 24 h, cells produced elevated levels of NO. In addition, after TLR4 agonist stimulation C57BL/6 GM-BM produced larger amounts of NO than BALB/c GM-BM. However, ECTV-infected cells from both mouse strains exhibited a significant (P > 0.01) decrease in NO production in response to LPS stimulation.

Additionally to Th1-polarizing cytokines, we evaluated the effect of ECTV infection on GM-BM capacity to produce cytokines that positively regulate Th2 polarization: IL-10, CCL2/MCP-1 and IL-1β, which has also Th1-polarizing properties (**Figures 7A,B**). IL-10 was undetectable in mock-, uvi-ECTV-, or ECTV-infected cultures of GM-BM from both mouse strains, indicating that ECTV is not able to stimulate IL-10 secretion by these cells (**Figure 7A**). After LPS treatment, uninfected BALB/c GM-BM produced twofold higher amounts of IL-10 than C57BL/6 cells. ECTV infection significantly (P > 0.01) decreased the production of IL-10 by LPS-treated GM-BM from both mouse strains. CCL2 was secreted at similar levels by C57BL/6 and BALB/c GM-BM, however, after LPS treatment cells from C57BL/6 mice produced slightly more CCL2 compared to BALB/c GM-BM (**Figure 7A**). ECTV infection significantly (P > 0.01) decreased the level of CCL2 secreted by GM-BM from both strains of mouse in response

to LPS. Meanwhile, BALB/c GM-BM produced more IL-1β compared to C57BL/6 cells, especially after LPS treatment (**Figure 7B**). Further stimulation of cells with ATP additionally increased the level of secreted IL-1β. Similar to other cytokines, ECTV infection significantly (P > 0.01) reduced the level of IL-1β in supernatants of GM-BM from both mouse strains cultured under different conditions.

Intracellular staining of selected cytokines revealed that ECTV-infection also reduced the percentage of cells producing TNF-α and IL-12p40/p70 (**Figure 7C**). After LPS treatment the percentage of GM-BM from both mouse strains producing TNF-α, IL-12p40/p70, and CCL3 was significantly (P > 0.05) decreased. Uninfected GM-BM from C57BL/6 and BALB/c mice produced more TNF-α and IL-12p40/p70 or CCL3, respectively, in response to LPS treatment. Collectively, our data indicate that C57BL/6 and BALB/c GM-BM are able to produce higher amounts of Th1- and Th2-polarizing cytokines, respectively, especially in response to TLR4 agonist stimulation. ECTV infection causes a profound inhibition of the production of both Th1 and Th2-polarizing cytokines by GM-BM in a strain-independent manner, indicating that there is no differences in the reactivity of cDCs from resistant C57BL/6 and susceptible BALB/c mice to the virus.

#### The in Vitro Effect of ECTV-Infection on the Expression of MHC and Costimulatory Molecules on GM-BM Is Mouse Strain-Independent

We compared the influence of ECTV infection on the expression of MHC class I and II molecules, as well as costimulatory molecules, including CD40, CD80, and CD86, and CD83, on GM-BM generated from bone marrow progenitor cells of different mouse strains (**Figure 8**). GM-BM derived from C57BL/6 mice expressed lower levels of H-2Db molecules than BALB/c cells H-2Dd molecules. However, C57BL/6 cells expressed higher level of I-A/I-E, CD40 and CD80 molecules

ECTV infection and/or LPS treatment on maturation marker expression in GM-BM from C57BL/6 and BALB/c mice at 24 hpi. Below each histogram set is a table with mean values (±SD) for a given marker from at least three independent experiments. The expression is presented as mean fluorescent intensity (MFI) or the percentage of positive cells for a given marker (paired Student's t-test; ∗P < 0.05, ∗∗P < 0.01).

than those from BALB/c mice. Moreover, a higher percentage of GM-BM expressing CD83 and CD86 molecules was found in GM-BM derived from C57BL/6 than BALB/c mice (**Figure 8**). This suggests that GM-BM derived from C57BL/6 mice mature more efficiently than those from BALB/c mice. LPS treatment increased the expression of all tested molecules on GM-BM from both mouse strains in a maturation-dependent manner (**Figure 8**).

ECTV infection modulated the expression of all tested maturation molecules on GM-BM from both mouse strains. Infected cells from C57BL/6 and BALB/c mice showed significantly (P > 0.01) reduced mean fluorescent intensity

(MFI) of H-2Db and H-2Dd molecules, respectively, even after LPS treatment (**Figure 8**). The level of MFI for H-2D molecules was significantly more decreased in infected C57BL/6 compared to infected BALB/c cells, untreated (sevenfold vs. twofold; P = 0.0015) or treated (15-fold vs. 5-fold; P = 0.0024) with LPS. Meanwhile, in ECTV-infected cells we observed a significant (P ≤ 0.05) increase in the percentage of I-A/I-E-positive cells, however, after LPS stimulation the percentage of such cells was reduced compared to mock-infected LPS-treated cells. Additionally, ECTV infection significantly (P ≤ 0.05) reduced the expression of CD40 and CD80 costimulatory molecules on GM-BM from both mouse strains, even in the presence of LPS. Moreover, upon infection the percentage of CD83+ cells significantly (P ≤ 0.05) decreased in C57BL/6 and BALB/c GM-BM cultures. On the contrary, the percentage of CD86+ cells was significantly (P ≤ 0.05) increased in ECTV-exposed compared to mock-exposed cells from both mouse strains and in BALB/c cells treated with ECTV + LPS compared to cells treated only with LPS (**Figure 8**).

#### Chemokine Receptor Expression in Down-Regulated in ECTV-Infected GM-BM from C57BL/6 and BALB/c GM-BM

Our last question concerned the effect of ECTV infection on the expression of cell surface chemokine receptors that are differentially regulated upon maturation. CCR1 and CCR5 are reported to be reduced, and CCR7 is reported to be upregulated on the surface of mature DCs (Le Nouën et al., 2011). Our results show that GM-BM from BALB/c mice expressed higher levels of chemokine receptors characteristic for immature DCs, compared to C57BL/6 cells (**Figure 9**). BALB/c cultures contained a higher percentage of CCR1+ cells and expression of CCR5 on their surface was increased. On the contrary, C57BL/6 cultures had a higher percentage of CCR7+ GM-BM. After infection with ECTV, cells from both mouse strains exhibited reduced percentage of CCR1+ and CCR7+ cells and decreased expression of CCR5, compared to mock-infected cells. After LPS treatment, the expression of chemokine receptors changed in a maturation dependent manner, i.e., the percentage of CCR1+ and CCR7+ cells decreased and increased, respectively, and MFI for CCR5 was low. Meanwhile, stimulation of ECTV-infected GM-BM with LPS resulted in the reduction of the percentage of CCR7+ cells and MFI for CCR5 expression, compared to LPS-treated cells. Taken together, our results indicate that ECTV impairs expression of chemokine receptors on GM-BM and, therefore, it is not excluded that their potential to respond to chemokines regulating their migration is limited.

### DISCUSSION

The inter-strain differences in the reactivity of DCs to the antigen exposure/infection contributes to various types of adaptive immune responses and may partially determine the outcome of a disease (Liu et al., 2000, 2002). Therefore, in the

FIGURE 9 | ECTV infection similarly affects the expression of chemokine receptors on GM-BM from C57BL/6 and BALB/c mice. Representative flow cytometry histograms of chemokine receptor expression on GM-BM following the infection with ECTV and/or treatment with LPS at 24 hpi. Below each histogram set is a table with mean values (±SD) for a given marker from at least three independent experiments. The expression is presented as mean fluorescent intensity (MFI) or the percentage of positive cells for a given marker. Statistical comparisons were between mock- and ECTV-exposed DCs and between LPS- and ECTV + LPS-exposed DCs (paired Student's t-test; ∗P < 0.05, ∗∗P < 0.01).

present study, we investigated the differences in the response of C57BL/6 and BALB/c GM-BM to ECTV infection by analysis of their maturation degree, chemokine receptor expression and production of different types of cytokines/chemokines engaged in T cell activation, polarization, and function. In addition, we analyzed the expression of key genes involved in maturation and activation of DCs. We found that in vitro ECTV modulation of GM-BM innate and adaptive immune properties occurred independently of the mouse strain susceptibility to infection. In cells from both strains of mice, ECTV infection contributed to the profound suppression of polarizing signals to prime Th1 immune response. Furthermore, contrary to expectations, a more pronounced viral inhibitory effect was observed in resistant C57BL/6 GM-BM, despite their higher potential to stimulate Th1 response under physiological conditions. These results indicate strong adaptation capacity of ECTV to the natural host DCs and its direct inhibitory effect on DC properties is irrespective of whether they are derived from resistant or susceptible mouse strain.

Several in vivo studies have reported that Th1 and Th2 immune responses prevail in C57BL/6 and BALB/c mice, respectively, under physiological conditions (Trunova et al., 2011), stress (Palumbo et al., 2010), or infection (Belkaid et al., 2002; Sacks and Noben-Trauth, 2002; Chaudhri et al., 2004; Watanabe et al., 2004). The immune response of different types of cells from these mouse strains, including DCs and macrophages, may influence the development of Th1 and Th2 adaptive immunity (Liu et al., 2002; Watanabe et al., 2004). It has been shown that DCs from C57BL/6 mice produce higher levels of IL-12 and IL-15 than DCs from BALB/c mice during early stages of Listeria monocytogenes infections (Liu et al., 2000). Moreover, DCs isolated from spleens of naïve C57BL/6 mice preferentially expressed a higher level of TLR9 mRNA, whereas those from BALB/c mice expressed a higher level of mRNAs for TLR2, -4, -5, and -6. In response to microbial ligands for TLR2 (lipoprotein), TLR4 (LPS), TLR2/6 (zymosan), and TLR9 (CpG), DCs from C57BL/6 and BALB/c mice produced larger amounts of IL-12p40 and CCL2, respectively. Additionally, DCs from C57BL/6 mice exhibited a more mature phenotype than BALB/c DCs due to higher expression of maturation markers, such as CD40 and CD86, and signal transducer and activator of transcription 4 (Stat4)—a key intermediate in IL-12 signaling pathway and Th1 cell development (Liu et al., 2002).

Our in vitro studies revealed that GM-BM derived from C57BL/6 mice displayed higher expression of Ccl5, Cd40, Il-12b, Irf7, Thbs1, and Tlr9 than BALB/c cells. Moreover, upon LPS stimulation, Cd80, Cd86, Ccl5, and Tlr9 were more up-regulated in C57BL/6 than BALB/c GM-BM. Additionally, C57BL/6 cells produced higher levels of IL-12p40 and IL-12p70, whereas BALB/c cells secreted more TNF-α, IL-6, and IL-10, especially after LPS treatment. Higher expression of maturation markers, such as MHC II, CD40, and CD80, and large percentage of cells expressing CD83, CD86, and CCR7 was observed in C57BL/6 cultures. Overall, our data indicate that GM-BM from resistant C57BL/6 mice are more matured than cells from susceptible BALB/c cells and possess a higher potential to stimulate Th1 response. Inter-strain differences in maturation state were also observed between GM-BM from BALB/c, C57BL/6, and atopic prone NC/Nga mice (Koike et al., 2008). The latter had greater expression of MHC class II, CD80, CD86, and CD11c compared to cells from BALB/c and C57BL/6 mice, as well as preferentially stimulated Th2 cytokine immune response. Therefore, it is suggested that the genetic background may enhance the differentiation and function of DCs and may be partially related to the development or aggravation of allergic/atopic diseases (Koike et al., 2008). On the contrary, resting BMDCs generated with GM-CSF and IL-4 from bone marrow progenitor cells of C57BL/6 and BALB/c mouse strains displayed no differences in the expression of CD40, CD80, CD86, MHC II, and TLR2 (Jiang et al., 2010).

It has been proposed that differences in innate and adaptive immune functions of DCs underline additional mechanisms responsible for resistance or susceptibility of C57BL/6 or BALB/c mice to L. monocytogenes (Liu et al., 2000, 2002). Moreover, DC capacity to differentiate naive T cells into functional Th1 or Th2 effector cells during Leishmania major infection is cell-intrinsic and Th2 polarization is not restricted to H2-d mice, since DCs from BALB/c and B10.D2 DCs (both H2d) exhibited differences in maturation and ability to induce Th differentiation (Filippi et al., 2003). In our in vitro study, the influence of ECTV infection on C57BL/6 and BALB/c GM-BM functions was comparable. Cells from both strains of mice exhibited a profound immunosuppression due to the productive virus replication (Szulc-D ˛abrowska et al., 2017). Interestingly, GM-BM from resistant C57BL/6 mice had a higher percentage of ECTV+ cells compared to BALB/c cells at 24 hpi, suggesting that the viral spread is more efficient in these cells at later stages of infection. Moreover, upon ECTV infection 11 genes (Rac1, Ccl2, Cxcl12, Mif, Ccr1, Cxcr4, Lyn, Fcgr1, Cebpa, Irf7, and Stat3) were significantly more repressed in C57BL/6 cells compared to BALB/c cells, whereas the latter only had two genes (Ccl3 and Cd74) more down-regulated compared to C57BL/6 cells. Additionally, infected GM-BM from C57BL/6 mice displayed a more expressive reduction in MHC I expression than BALB/c GM-BM. It is not excluded that slightly increased virus inhibitory effect on C57BL/6 GM-BM function is associated with a higher rate of ECTV dissemination within these cells. The ability of ECTV to replicate productively in GM-BM is a manifestation of its high adaptation capacity to the natural host immune cells, since other orthopoxviruses, such as CPXV and VACV abortively infect human DCs (Engelmayer et al., 1999; Jenne et al., 2000; Hansen et al., 2011).

Our studies revealed that ECTV infection in GM-BM derived from C57BL/6 and BALB/c mice down-regulated many genes involved in antigen uptake and processing, chemokine and cytokine synthesis, receptor expression, and signal transduction. The inhibitory effect on gene expression was also observed after TLR4 agonist treatment. In general, the analyzed genes were regulated in the same way in cells from both strains of mice and we did not observe any strain-specific response in GM-BM upon in vitro ECTV-infection. On the contrary, peritoneal macrophages from C57BL/6 and BALB/c mice infected with ECTV exhibited differential regulation of 14 innate antiviral genes, which were up-regulated in C57BL/6 and down-regulated

in BALB/c cells, suggesting that these variations in gene expression may partially contribute to resistance or susceptibility to severe mousepox (Dolega et al., 2017). Meantime, several interstrain variations in GM-BM functionality have been observed during paramyxovirus simian virus 5 (SV5) infection (Pejawar et al., 2005). Firstly, GM-BM from C57BL/6 mice were much more permissive to SV5 infection than cells derived from BALB/c mice. Secondly, despite the production of a similar panel of cytokines, cells differed in the maturation state: C57BL/6 cells up-regulated the expression of CD40, CD80, and CD86, whereas BALB/c cells displayed increase only in CD40 and CD86 expression. Thirdly, SV5-matured C57BL/6 GM-BM were more potent to activate naïve CD8+ T cells than SV5-matured BALB/c cells (Pejawar et al., 2005). Similar inter-strain differences were observed in BMDCs during infection with Chlamydia muridarum (Jiang et al., 2010). After in vitro infection, BMDCs from C57BL/6 mice underwent higher functional maturation than cells from BALB/c mice. This was reflected by higher expression of MHC class II and costimulatory molecules (CD40, CD80, and CD86) and greater production of IL-12. On the contrary, BALB/c BMDCs secreted more IL-23, IL-6, IL-10, and TNF-α than C57BL/6 cells (Jiang et al., 2010). Overall, the data described above demonstrate that genetically defined differences in functionality between DCs from C57BL/6 and BALB/c mice may be phenotypically expressed during exposure to the microbial agent/infection, however, this reactivity also may depend on the nature of the agent and the host–agent immunobiology.

ECTV infection of GM-BM derived from C57BL/6 and BALB/c mice led to a profound repression of a set of chemokine and cytokine genes involved in Th1 [Ccl3, Ccl4, Ccl5 (Lebre et al., 2005), Cxcl2 (Seow et al., 2008), Cxcl10 (Lebre et al., 2005), Ifng, Il12a, Il12b, and Il16 (Lynch et al., 2003)] and Th2 [Ccl2 (Liu et al., 2002), Ccl8 (Lech and Anders, 2013), Ccl11 (Dixon et al., 2006), Ccl17 (Belperio et al., 2004), Cxcl12 (Piao et al., 2012), Mif (Das et al., 2011), and Tgfb1 (Maeda and Shiraishi, 1996)] immune response regulation. But, ECTV infection up-regulated mRNA transcript for IL-10 in GM-BM of both strains of mice. Despite the increase in gene expression, the level of IL-10 remained undetectable in the culture supernatants during 24 h incubation. We cannot exclude that GM-BM were cultured at too low cell density to detect IL-10 in our experimental model. On the other hand, it is possible that ECTV does not stimulate IL-10 production by GM-BM, similar to CPXV, which has been shown not to induce the secretion of IL-10 by different types of human DCs: monocyte-derived DCs, myeloid DCs, and plasmacytoid DCs (Hansen et al., 2011).

IL-10, first described as a Th2-polarizing cytokine, is an anti-inflammatory cytokine that suppresses pro-inflammatory response leading to increased pathogen dissemination and/or reduced pathology. IL-10 secreted by DCs may act in an autocrine manner to inhibit production of chemokines (such as CCL2, CCL5, CCL12, CXCL8, and CXCL10) and pro-inflammatory cytokines (such as IL-1α/β, IL-6, IL-12, IL-18, and TNF-α). Moreover, IL-10 may directly inhibit proliferation and production of IL-2, IFN-γ, IL-4, IL-5, and TNF-α by CD4+ T cells, thus it regulates both Th1 and Th2 immune responses (Couper et al., 2008). Additionally, IL-10 induces long-lasting T cell anergy and promotes the differentiation of naïve T cells into Tr1 cells in humans and mice (Gregori et al., 2010). The importance of cellular IL-10 in immune system regulation is supported by the fact that several viruses, including seven members of the Poxviridae family, encode orthologs of cellular IL-10, called viral IL-10s (vIL-10s), which have been acquired by viruses from their host during evolution (Ouyang et al., 2014).

It has been shown that CPXV induces in vitro secretion of IL-10 by BMDCs and RAW 264.7 macrophages at 24 hpi. Moreover, CPXV is able to induce higher IL-10 production in vivo than VACV (Spesock et al., 2011). Experiments with IL-10-deficient mice have indicated that after intranasal CPXV infection these mice exhibited similar weight loss and viral burdens as wild-type mice. However, IL-10-deficient mice were more susceptible to CPXV reinfection, because increased viral loads were observed in their lungs, what corresponded with lower antibody and CD8+ T cell responses compared to wild-type mice. The role of IL-10 during CPXV infection is probably beneficial for the virus, and IL-10 suppresses immunopathology in the lungs because IL-10-deficient mice after re-challenge with CPXV displayed greater bronchopneumonia than wild-type mice (Spesock et al., 2011). Meanwhile, using recombinant VACV expressing mouse IL-10 (mIL-10) it has been shown that in immunocompetent mice mIL-10 expressed from the VACV genome affected natural killer (NK) and virus-specific CTL activity, whereas in severe combined immunodeficient (SCID) mice VACV-mIL-10 infection resulted in increased NK cell activity and higher degree of virus clearance compared to infection with control VACV (Kurilla et al., 1993). In agreement with these studies, van Den Broek et al. (2000) have demonstrated that IL-10-deficient mice showed dramatically reduced viral titer in ovaries after intraperitoneal infection, suggesting a rapid viral clearance. Together, these data indicate that IL-10 is a potent cytokine in suppressing the immune response against VACV and may be a dominant factor for susceptibility to acute VACV infection (van Den Broek et al., 2000). On the hand, more recently it has been found, using multiphoton intravital microscopy imaging, that IL-10 produced locally in the skin of VACV-infected mice contributed to viral clearance, probably by shaping the innate immune response within the inflamed tissue and/or by reducing virus-induced inflammation (Cush et al., 2016). The up-regulation of IL-10 mRNA level observed in ECTV-infected GM-BM may therefore suggest that these cells possess a stronger ability to damp of the immune response and/or cross-regulate Th1 and Th2 immune responses. It is highly possible that IL-10 may be an important factor in the generation of non-protective Th2 immune response in susceptible BALB/c mice in vivo.

Our studies revealed that ECTV infection impairs inflammatory response and maturation of GM-BM in a strain-independent manner. At 24 hpi GM-BM from C57BL/6 and BALB/c mice displayed an altered production of several cytokines and chemokines with the exception of CCL3, which was produced at a higher concentration. Interestingly, mRNA transcript for CCL3 was down-regulated at this time point in ECTV-infected cells. It is known that mRNA expression does not always correlate with protein levels in mammalian

cells (Vogel and Marcotte, 2012). Moreover, ECTV infection of GM-BM from both mouse strains down-regulated expression of MHC class I, and CD40 and CD80 co-stimulatory molecules, but increased the percentage of MHC II+ and CD86+ cells. Possibly, bystander non-infected GM-BM underwent partial maturation at the same time being the main source of CCL3. It has been demonstrated that highly attenuated modified vaccinia virus Ankara (MVA) induces phenotypic and functional maturation of bystander DCs resulting in production of a large array of cytokines and chemokines involved in T cell activation and recruitment, and regulation of inflammatory response, including CCL3 (Pascutti et al., 2011). However, ECTV encodes vCCI decoy receptor, EVM1, which is an abundantly secreted glycoprotein during infection and can bind the CC chemokines to form highly stable complexes with CCL3 and CCL5 (Arnold and Fremont, 2006). Our results also showed that the inhibition of inflammatory response and maturation of C57BL/6 and BALB/c GM-BM caused by ECTV was most pronounced after LPS treatment. This observation is in agreement with previous in vitro studies showing that other orthopoxviruses, such as VACV and CPXV severely affected maturation and activation of cDCs (Engelmayer et al., 1999; Jenne et al., 2000; Hansen et al., 2011).

As a master of immune inhibitory strategies, ECTV also altered the expression of chemokine receptors, such as CCR1, CCR5, and CCR7 on the C57BL/6 and BALB/c GM-BM surface, what may lead to their impaired migration (Jang et al., 2006). Humrich et al. (2007) have demonstrated that VACV infection targets chemokine-induced migration of DCs at multiple functional levels. VACV-infected mature DCs showed inability to migrate toward the lymphoid chemokines CCL19 and CXCL12 without apparent alterations in expression of surface chemokine receptors CXCR4 and CCR7. In fact, in VACV-infected immature or uninfected bystander DCs there is decreased or increased, respectively, expression of the inflammatory chemokine receptors CCR1 and CXCR1, which abrogates or intensifies their migration toward CCL3 and CCL5. Moreover, VACV-infected and uninfected bystander DCs are not able to up-regulate CCR7 expression after LPS treatment suggesting their disability to undergo chemokine receptor switch (Humrich et al., 2007). Additionally, poxviruses encode membrane cytokine and chemokine receptors to evade host immune responses (Felix and Savvides, 2017).

Taken together, our results indicate that in vitro ECTV infection of GM-BM, including cDCs, leads to their functional impairment independently of the genetic background of mice from which they were generated. ECTV-employed host-specific

#### REFERENCES

strategies to evade host antiviral immune response allow the virus to control GM-BM independently of the host resistance or susceptibility to severe mousepox. Moreover, our study confirms that ECTV is a master of immune inhibitory strategies showing a wide array of mechanisms for disrupting the innate and acquired immune functions of GM-BM. Better understanding of the virus interactions with cDCs, the most potent APCs, may help to elucidate additional mechanisms responsible for resistance or susceptibility to mousepox and that can lead to rational design means and ways of containing virus infection.

#### AUTHOR CONTRIBUTIONS

LS-D conceived and designed the study. LS-D, JC, ZN, and FT conducted real-time PCR experiments. LS-D, AW, and MG performed flow cytometry analysis. LS-D and JS performed ELISA. LS-D, JS, FT, and MG analyzed and interpreted the data. LS-D prepared figures and wrote the draft of the manuscript. All authors reviewed and approved the manuscript.

#### ACKNOWLEDGMENTS

This work was supported by grant No. UMO-2012/05/D/NZ6/02916 to LS-D from the National Science Center in Poland. Results were partially presented at the 17th International Congress of Immunology—ICI 2016, 21–26 August 2016, Melbourne, VIC, Australia, in which LS-D's participation was supported by KNOW (Leading National Research Centre) Scientific Consortium "Healthy Animal – Safe Food," decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2017.02539/full#supplementary-material

FIGURE S1 | Characteristics of C57BL/6 and BALB/c GM-BM before and after MACS separation of CD11c+ cells. (A) Representative dot plot demonstrating gating strategy of isotype controls and CD11c and I-A/I-E staining. (B) Representative histograms demonstrating MFI of CD11c, CD11b, and CD205 expression on C57BL/6 and BALB/c GM-BM before and after MACS separation.

TABLE S1 | Gene list description of Mouse Dendritic and Antigen Presenting Cell RT2 Profiler PCR Array (Qiagen).

cell-mediated immunity determine genetic resistance to mousepox. Proc. Natl. Acad. Sci. U.S.A. 101, 9057–9062. doi: 10.1073/pnas.0402949101

human metapneumovirus. PLOS Pathog. 7:e1002105. doi: 10.1371/journal.ppat. 1002105

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer RALR and handling Editor declared their shared affiliation.

Copyright © 2017 Szulc-D ˛abrowska, Struzik, Cymerys, Winnicka, Nowak, Toka and Gierynska. This is an open-access article distributed under the terms of the Creative ´ Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Serological Evidence of Orthopoxvirus Circulation Among Equids, Southeast Brazil

Iara A. Borges1 \*, Mary G. Reynolds2 , Andrea M. McCollum2 , Poliana O. Figueiredo1 , Lara L. D. Ambrosio1 , Flavia N. Vieira1 , Galileu B. Costa1 , Ana C. D. Matos1 , Valeria M. de Andrade Almeida1 , Paulo C. P. Ferreira1 , Zélia I. P. Lobato1 , Jenner K. P. dos Reis1 , Erna G. Kroon1 and Giliane S. Trindade1

1 Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Centers for Disease Control and Prevention (CDC), Atlanta, GA, United States

#### Edited by:

Bernard La Scola, Aix-Marseille Université, France

Reviewed by: Filippo Turrini, Vita-Salute San Raffaele University, Italy Subir Sarker, La Trobe University, Australia

> \*Correspondence: Iara A. Borges<EMAIL_ADDRESS>
> > Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 12 December 2017 Accepted: 21 February 2018 Published: 08 March 2018

#### Citation:

Borges IA, Reynolds MG, McCollum AM, Figueiredo PO, Ambrosio LLD, Vieira FN, Costa GB, Matos ACD, de Andrade Almeida VM, Ferreira PCP, Lobato ZIP, dos Reis JKP, Kroon EG and Trindade GS (2018) Serological Evidence of Orthopoxvirus Circulation Among Equids, Southeast Brazil. Front. Microbiol. 9:402. doi: 10.3389/fmicb.2018.00402 Since 1999 Vaccinia virus (VACV) outbreaks involving bovines and humans have been reported in Brazil; this zoonosis is known as Bovine Vaccinia (BV) and is mainly an occupational disease of milkers. It was only in 2008 (and then again in 2011 and 2014) however, that VACV was found causing natural infections in Brazilian equids. These reports involved only equids, no infected humans or bovines were identified, and the sources of infections remain unknown up to date. The peculiarities of Equine Vaccinia outbreaks (e.g., absence of human infection), the frequently shared environments, and fomites by equids and bovines in Brazilian farms and the remaining gaps in BV epidemiology incited a question over OPV serological status of equids in Brazil. For this report, sera from 621 equids - representing different species, ages, sexes and locations of origin within Minas Gerais State, southeast Brazil – were examined for the presence of anti-Orthopoxvirus (OPV) antibodies. Only 74 of these were sampled during an Equine Vaccinia outbreak, meaning some of these specific animals presented typical lesions of OPV infections. The majority of sera, however, were sampled from animals without typical signs of OPV infection and during the absence of reported Bovine or Equine Vaccinia outbreaks. Results suggest the circulation of VACV among equids of southeast Brazil even prior to the time of the first VACV outbreak in 2008. There is a correlation of OPVs outbreaks among bovines and equids although many gaps remain to our understanding of its nature. The data obtained may even be carefully associated to recent discussion over OPVs history. Moreover, data is available to improve the knowledge and instigate new researches regarding OPVs circulation in Brazil and worldwide.

Keywords: Vaccinia virus, horsepox, bovine vaccinia, Equine Vaccinia, Horse diseases

## INTRODUCTION

After 1980, following cessation of mass smallpox immunization, Vaccinia virus (VACV) – the Orthopoxvirus (OPV) used during the successful World Health Organization Smallpox Eradication Campaign (Fenner and Henderson, 1988) – emerged as a zoonosis in India, Pakistan and Brazil (Essbauer et al., 2010) VACV outbreaks involving dairy cattle and humans were first described in

Brazil in 1999 (Damaso et al., 2000). The number of Bovine Vaccinia (BV) reports continues to increase. BV commonly affects milking cows and cattle workers of small properties. Exanthemas are often located at the udder and teats of cows. All stages of the lesions – macule, papule, vesicle, pustule, ulcer, and scab – are highly contagious and direct or indirect contact with an abrasion or bare skin is enough to cause VACV infection in humans. Humans are often infected during manual milking; exanthemas are usually found on their hands and forearms (Damaso et al., 2000; Trindade et al., 2003).

Vaccinia virus is currently the only known OPV circulating in Brazil and has recently been detected in other South American countries such as Uruguay, Argentina and Colombia. However, other OPVs are known to occur in the Americas (Emerson et al., 2009; Gallardo-Romero et al., 2012). Recently, various species of rodents have been investigated as possible reservoirs for VACV and the virus has been detected in sylvatic and synantropic species (Abrahão et al., 2009, 2010a).

A VACV outbreak in horses occurred at a breeding center at south Brazil in 2008 and constitutes the first national report of VACV infected horses (Brum et al., 2010; Campos et al., 2011). Surprisingly, no relationship or contact to infected bovines was identified and the source of infection is not known up to date. A second and similar outbreak occurred in southeast Brazil during 2011 (Matos et al., 2013). Horses from different properties developed exanthemas on their muzzles and no infected bovine, human or other animal were determined as the possible source of infection, nor were any non-equine species observed to be infected as a result of the outbreak (Matos et al., 2013). The third outbreak reported the occurrence of oral lesions in donkeys and mules from the northeastern region of the country. Molecular findings indicated a VACV from Group 1 as the etiological agent and again no other species but Equus sp. have been connected to this outbreak (Abrahão et al., 2017).

Brazil has the largest herd of horses in Latin America and the third in the world. Together with the mules and donkeys are 8 million head, moving R\$ 7.3 billion, only with the production of horses. Brazil is the eighth largest exporter of equine meat but when it comes to the export of live horses, the numbers are significant and expanded by 524% between 1997 and 2009. The largest Brazilian population of horses is in the Southeast, followed by the Northeast, Midwest, South and North, with one of its main functions being the daily work in agricultural activities, where about five million animals are primarily used for the management of cattle (MAPA).

To better comprehend the relationship between VACV and equids in Brazil, serum from more than six hundred equids from all mesoregions of Minas Gerais (MG) state, southeast Brazil, were analyzed for evidence of OPV exposure. Previous exposure of equids to OPV was evaluated by two different assays: the plaque reduction neutralization test (PRNT) to identify neutralizing antibodies (gold standard), and an enzyme-linked immunosorbent assay (ELISA). This latter method allows for assessment of non- neutralizing anti-OPV IgG antibodies.

This study provides an evaluation of serological evidence of VACV exposure in equids, expanding the epidemiological hypothesis of VACV circulation in Brazil, and potentially in other countries where VACV has been reported but its natural cycle is not well understood. The introduction of VACV into the Americas and its circulation among European horses prior to the 19th century have gained greater attention recently and highlighted the importance of horses in OPVs history (Damaso, 2017; Esparza et al., 2017; Schrick et al., 2017). Insights of the possible correlation of OPVs outbreaks among cows and equids have been evaluated and more data is currently available to improve the knowledge and also the development of control and prevention methods of OPVs circulation worldwide, specially VACV-like viruses.

### MATERIALS AND METHODS

#### Samples

A total of 621 sera were examined from seven mesoregions of MG State. The mesoregions are defined by the animal defense bureau of the state of MG. **Figure 1** describes the abbreviations for regions that are used throughout this report. Four hundred seventy-eight sera from equids (Equus caballus, E. asinus and hybrids) were collected from numerous locations around MG state during the second half of 2003 and first half of 2004 (**Figure 1**). An additional 74 sera of E. caballus from RIV (**Figure 1**) were collected in July 2011, from the second known episode of Equid Vaccinia (EV) in Brazil. The serum specimens were collected approximately 1 month after the onset of symptoms, and clinical diagnoses were confirmed using molecular assays (Matos et al., 2013). The last 69 sera were randomly collected in RII during the second half of 2012 (**Figure 1**). All specimens were donated by the "Departamento de Medicina Veterinaria Preventiva, Escola de Veterinaria, Universidade Federal de Minas Gerais". All clinical specimens were derived from domestic equids on private properties and were collected by a veterinarian according to standard sanitary protocols in agreement to the requirements of national and local livestock agencies, "Ministerio da Agricultura, Pecuaria e Abastecimento" and "Instituto Mineiro de Agropecuaria", respectively. The sampling procedure was submitted and approved by the "Comite de Etica em Experimenação Animal (CETEA)" in accordance to the requirements of animal research of Universidade Federal de Minas Gerais (UFMG), MG state, Brazil (protocol number 131/2010, approval date 8/2010 and expiration date 8/2015).

Sterile vacuum blood collection tubes and needles were used to collect approximately 5 mL of blood from each animal while the animal was restrained with a bridle. The area of the jugular vein was cleaned with cotton soaked in 70% alcohol, and samples were collected from the jugular vein.

The name of the responsible employee and/or owner and the addresses of all properties were collected. Permission for sampling the horses was verbally granted. There was a verbal agreement to maintain confidentiality of the names and specific geographic location of each property. Coordinates from the regions further mentioned in this study are as follows (Datum: WGS84): RI or TRIANGULO

MINEIRO AND ALTO PARANAIBA 19◦ 160 18.4500South and 48◦ 200 59.9500West; RII or CENTRAL MINEIRA AND CENTRO-OESTE DE MINAS 18◦ 510 15.5200South and 44◦ 290 22.8800West; RIII or SUL DE MINAS 21◦ 190 18.5700South and 45◦ 480 09.6700West; RIV or CAMPO DAS VERTENTES AND ZONA DA MATA 21◦ 300 01.9100South AND 43◦ 270 57.2400West; RV or VALE DO RIO DOCE 19◦ 280 22.4400South AND 41◦ 460 46.6200 West; RVI or VALE DO JEQUITINHONHA AND MUCURI 16◦ 580 31.86"South and 41◦ 150 41.4300West; RVII or NORTE E NOROESTE DE MINAS 16◦ 170 40.8700South and 44◦ 410 03.2100West.

### Epidemiologic and Demographic Variables

Four hundred seventy-eight sera were collected for multiple research purposes and there is no record of clinical signs suggestive of OPV acute infection among their database, as well as for the 69 sera randomly collected in RII. All these 547 sera have been submitted to PCR to OPV vgf gene according to Abrahão et al. (2010b) to detect a possible silent DNAmia; none trialed positive (data not shown). The geographic location, species, age, and sex of each equid sampled are also recorded.

The 74 sera (from the second EV outbreak notified in Brazil) were collected from equids with and without OPV-like exanthemas as described by Matos et al. (2013). According to them, viruses were isolated from sampled lesions and DNAmia was detected among several horses approximately 1 month after the first infected equid was noticed. Authors demonstrated through molecular methods a VACV sample to be the etiologic agent of this outbreak. The geographic location, species and sex of each equid sampled are recorded.

The location data for equids was aggregated by state subdivision. Subdivisions (mesoregions) are shown in **Figure 1** along with the absolute number and corresponding percentage of equids evaluated from each area. Since BV has traditionally been associated with dairy production in Brazil, it is helpful to consider dairy production activities in MG in relation to equid sampling locations. Milk producing properties are found throughout MG but several distinctions are found between the different mesoregions. Small dairy properties are concentrated at RIV and RII, where only one municipality had properties with areas larger than or equal to 69,106 Km2 . RIV is the area where the second known case of Brazilian EV occurred (Matos et al., 2013). Although traditionally known for its pure breed beef cattle herds, RI produced between 9 and 100 million of liters of milk during the year of 2006, the highest values found in MG State during the last census. RI properties also occupy the largest areas related to all other mesoregions as nearly 50% of its municipalities have properties with areas1 over 69,106 Km2 .

## Serological Assays

#### Plaque Reduction Neutralization Test

The PRNT was performed according to (Newman et al., 2003). Briefly, six well plates with BSC-40 cell monolayers (ATCC R CRL-2761) were inoculated with a 2.5% serum solution plus

1http://www.ibge.gov.br/

Borges et al. Orthopoxvirus Circulation in Equids, Brazil

150 PFU of VACV Western Reserve (WR) per well. Before infection, sera/WR solutions were incubated overnight at 37oC. To maintain the viability of the virus control, fetal bovine serum (FBS) was added to this solution at the same concentration (2.5%). The cell control contained 2.5% FBS media only. After infection, 1 h of adsorption was followed by the addition of 2 ml of 1% FBS media per well and incubation of all monolayers at 37oC and 5% CO2 for approximately 48 h. After typical VACV-WR cytopathic effects were clearly observed, all monolayers were fixed with 3.7% formaldehyde and stained with 1% crystal violet.

All samples were tested in triplicate and the number of PFU in each well was enumerated. Positive sera (positive for neutralizing antibodies) were defined as those samples that had PFU below the 50% PFU of the viral control.

#### IgG ELISA

Flat-bottom 96 well plates (Nunc MaxiSorp R ) were coated with inactivated and purified VACV- WR viral particles; these plates were treated with a solution of PBS plus tween 20 (0.05%) and 5% casein. Sera were diluted 100x in a solution of PBS plus tween 20 (0.05%) and 1% casein.

To detect antibodies, anti-horse IgG conjugated to horse radish peroxidase (HRP) (SIGMA-ALDRICH R ) and tetramethylbenzidine (TMB, BD R ) were used for colorimetric detection at the concentration of 1:20000. All samples were tested in duplicates, and the results were read at 450 nm. Five known negative equid control sera were used. The average of all five negative controls minus three times their standard deviation determined the cutoff value for each plate.

Additionally, a known positive equid serum was tested for each plate as a positive control.

#### Statistical Analysis

Epidemiologic and demographic variables as well as qualitative laboratory findings were analyzed using parametric and non-parametric statistical tests. Pearson Chi-square test was employed for analyzing multiple-category non-parametric data. The Mantel-Haenszel common odds ratio (OR) and Fisher's exact tests (2-tailed) were used for categorical variables. A p-value < 0.050 was used to assign significance of association. All analyses were performed using IBM R SPSS Statistics version 19.

#### RESULTS

A total of 128 equids (20.6%) were seropositive for OPV by either the ELISA or PRNT assay (**Table 1**). Seropositive animals were found in all mesoregions of MG state. There was a lower frequency of seropositive equines in RI (4.8%, western MG) and RVII (13.9%, northern MG) and a higher frequency in RIV (29.4%, southeast) compared to the other regions of MG. The results from RI and RIV significantly deviate (−16% and +9%, respectively) from that which would be expected by chance alone (Chi-square 20.5, df =6, 2-sided p = 0.002). Moreover, a statistically significant difference was observed between these two regions (**Figure 2A**).

∗positive by either PRNT or ELISA. † > 8% deviation from that expected by chance alone.

The location of the equids was significantly associated with seropositivity (Chi-square 20.5, df = 6, 2-sided p = 0.002). No significant relationship was observed for species, age or sex. The existence of seropositive equids throughout MG State, represents a unique result, since the possibility of VACV natural circulation among horses had been hypothesized, but not demonstrated.

Except in RII and RIV, a history of prior BV or EV occurrence was determined by property owner report only; such reports could not be independently confirmed. In cases where the property owner was not aware of the disease, pictoral representations of typical VACV lesions of humans, bovines and equids were provided. Regarding animals from properties whose owners confirmed BV or EV history – specific cases where samples were sent by IMA to official laboratories and results confirmed VACV infection – the frequency of seropositive equids from BV areas was almost two times higher (52.9%) than the frequency of seropositive equids in EV areas (27%). Furthermore, seropositive equids were found to be three times more likely to be from a property which formerly experiences a BV episode than those from areas where EV has occurred (OR = 3.0, 95% CI, 1.03–8.96, p = 0.044) (**Table 2** and **Figure 2B**).

#### DISCUSSION

These results demonstrate OPV exposure among equids in MG state, Brazil. As no OPV other than VACV has been proven to circulate in the country, results are hypothesized to be most likely related to the circulation of VACV. Moreover, the presence of serologically positive equids from 2003 to 2004 demonstrates that exposures, and perhaps unrecognized infections, occurred prior to the first described outbreak in Brazilian equids in 2008 (Brum et al., 2010). Results regarding the lower seropositivity observed in RI and higher seropositivity in RIV corroborate several studies in bovines, which associate intense VACV circulation with small property size (Kroon et al., 2011). Also, this result would be expected if there is a relationship between VACV circulation among equids and bovines. RIV is an important dairy region where several BV outbreaks have been reported (Lobato et al., 2005; Trindade et al., 2006; De Souza Trindade et al., 2007). The states that share a boundary with RIV – Espirito Santo and Rio de Janeiro – are also known for the frequent occurrence of BV

FIGURE 2 | Regional differences in seropositivity and a history of EV and BV outbreaks. (A) Two mesoregions of MG presented a discrepancy in equid seropositivity (RI and RIV). Both are highlighted; their absolute number and correspondent percentage of seropositive equids are indicated by arrows [n(%)]. (B) A difference of OPV seropositivity among equids derived from areas with EV or BV history was also observed. The white target indicates an area with a history of EV; the black target indicates an area with a history of BV. The absolute number and correspondent percentage of seropositive are indicated by arrows [n(%)].

∗positive by either PRNT or ELISA.

(Kroon et al., 2011). RI, on the other hand, is known for its large herds of beef cattle2 and fewer BV reports (Kroon et al., 2011).

When equids and bovines are maintained together on a single property, their degree of contact is often dictated by the size of the operation and type of production that occurs on the property. In small dairy herds, because of the nature of the work required, equid contact with bovines is considerably more intense than on larger, more sophisticated dairy farms. During dry seasons, these semi-intensive small properties require daily feed dispersal to support dairy production. The transportation of feed on these properties is usually accomplished by equids. Cattle are herded towards the corral and later back to the pastures, often by a human riding an equid. It is important to point out that despite its use in several other activities as sports, leisure and even therapy, one of its main functions, however, remains the daily work in agricultural activities, where about five million animals in Brazil are used primarily for the MAPA. In these situations, it is common for equids to freely access corral areas, share pastures, water, and feeding devices with bovines; therefore, equids housed on small properties with dairy cattle may indeed be more exposed to any pathogen circulating among the dairy cattle as compared to equids involved with beef cattle production or that are found on more technologically sophisticated farms.

The pure breed beef cattle that predominate in RI are highly valued economically3 . This additional capital value allows for broader investments in a given property's dairy production. Therefore, the lower seropositivity observed among equids from this region may be a reflection of the investment-based technological innovations that have proliferated throughout the region. Increasing levels of sophistication on dairy properties may lead to the substitution of horses for tractors and crawlers thereby reducing bovine and equid contact. When equids from areas with a previous occurrence of BV versus a previous occurrence of EV were evaluated, a higher number of seropositive equids were found to be from BV properties.

Evaluating the typical systems in which horses are kept, opportunities for disease acquisition and transmission are somewhat different from those typical for small dairy cattle properties. Properties which dedicate part of their labor force to breeding horses tend to have individual stalls for their stallions; pastures present a density of equids considerably lower than those used with dairy cattle. Even if VACV replication and shedding is naturally efficient among horses and bovines, equids exposure would be significantly lower considering typical management practices for horses – those that are not working animals on a dairy farm.

2http://www.abcz.org.br/

3http://www.abcz.org.br/

The demonstration of VACV shedding on feces from deliberately infected cows (Rivetti et al., 2013) and the seroconversion of naïve animals after direct or indirect contact with this material (D'Anunciação et al., 2012) corroborate the possible co-dependency of VACV circulation observed among equids and bovines, as equids exposed to an OPV positive herd and environment may induce these equids seroconversion. It has been suggested that VACV is more successful in replicating and being shed by bovines as compared to equids; the experimental infection in equids with Brazilian samples of VACV indicated a low level of replication at the inoculation site with mild cutaneous lesions when compared with the course of infection of other hosts. The authors hypothesized equids have a low potential for viral maintenance and transmission to other species, albeit being susceptible to VACV infection (Barbosa et al., 2016).

Damaso, Esparza, Schrick, and collegues have revisited Edward Jenner' Inquiry in 2017 and vaccines tested by the doctor with specimens obtained from horses have come to all attention. Jenner attested "horse material" would most likely protect humans against smallpox only if previously inoculated in cows, as it rarely produced the "take" (exanthema developed at the site of inoculation) when directly sampled from horses and promptly inoculated in humans (reviewed by Baxby, 1999). Considering the possibility of VACV-like samples circulation in 19th Europe and their unknown use by Jenner, these observations may relate to what Barbosa called "a low level of replication" of Brazilian VACV samples in deliberately infected equids. The absence of human cases associated to all EV outbreaks reported in Brazil so far, also follows the pattern previously proposed and instigate the conclusion of a probable route of VACV from bovines to equids, as these last apparently are less effective in shedding the virus. Many characteristics of the infection of equids by VACV, however, remain to be investigated and assumptions should not be made based only at these studies.

Most importantly the evidence of silent (or possibly unreported) VACV exposure and disease in Brazilian equids in southeast Brazil has been demonstrated. VACV multiplication in equids occurs and even if it is less effective than in bovines, virus shedding into the environment constitutes a potential source of infection to other animals, domestic or wild, and to humans. Little is known about possible VACV reservoirs in Brazil as well as the exact importance of equids for VACV maintenance and circulation. Care must be taken in order to avoid VACV dissemination; veterinarians, horse and cattle caretakers, and all others involved in the equid industry should be informed of the risks related to EV.

Little investment has been made so far to prevent and control VACV infections in Brazil, either associated to BV or to the relatively recent EV. Disease prevention efforts are

#### REFERENCES

Abrahão, J. S., Drumond, B. P., Trindade Gde, S., da Silva-Fernandes, A. T., Ferreira, J. M., Alves, P. A., et al. (2010a). Rapid detection of Orthopoxvirus by semi-nested PCR directly from clinical specimens: a useful alternative for routine laboratories. J. Med. Virol. 82, 692–699. doi: 10.1002/jmv. 21617

typically intensified only after a county suffers a significant outbreak. Municipalities that are potentially at risk remain uninformed about the disease until the impacts are apparent, including reduced milk production and painful human infections during BV outbreaks, bovine or equine morbidity during BV and EV outbreaks, respectively. Livestock disease surveillance programs have a role in outbreaks with suspected VACV cases, concomitant notifications to human public health authorities would speed the rapid inception of control measures, but research constitutes still the major investment related to VACV in Brazil. The continuation of serological monitoring and complementary research of VACV in equids is therefore fundamental to assess the spectrum of VACV impacts throughout the country, as well as to the development of prevention and control methods for its circulation nationwide.

#### AUTHOR CONTRIBUTIONS

IB did the standardization of the laboratorial techniques, field and laboratory work, data compilation, and paper writing. MR and AMM were responsible for guidance, statistical analysis, and paper writing. POF and FV did the field and laboratory work. LA, GC, ACM, VAA, ZL, and JR did the field work. PCF, ZL, JR, and EK provided guidance. GT contributed to the idealization of the project, guidance, and paper writing.

### FUNDING

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Pro-Reitoria de Pesquisa da UFMG (PRPq-UFMG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Ministério da Agricultura, Pecuária e Abastecimento (MAPA). IB was supported by a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. GT, EK, ZL, and JR are researchers of Conselho Nacional de Desenvolvimento Científico e Tecnológico.

### ACKNOWLEDGMENTS

We thank all colleagues from Laboratorio de Virus for excellent technical support. We also would like to thank Fernanda Gonçalves de Oliveira for sera preparation, Valdísio for driving and helping us during the expeditions, and the Instituto Mineiro de Agropecuária (IMA) for technical support.

volepox virus pathogenesis in California mice (Peromyscus californicus). PLoS One 7:e43881. doi: 10.1371/journal.pone.0043881

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Borges, Reynolds, McCollum, Figueiredo, Ambrosio, Vieira, Costa, Matos, de Andrade Almeida, Ferreira, Lobato, dos Reis, Kroon and Trindade. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US

Bazla Ali 1 , Maxim I. Desmond1 , Sara A. Mallory 1 , Andrea D. Benítez 1†, Larry J. Buckley 1 , Susan T. Weintraub2 , Michael V. Osier 1 , Lindsay W. Black 3 and Julie A. Thomas 1 \*

1 Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY, United States, 2 Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States, 3 University of Maryland School of Medicine, Baltimore, MD, United States

#### Edited by:

Jonatas Abrahao, Universidade Federal de Minas Gerais, Brazil

#### Reviewed by:

Juliana Cortines, Universidade Federal do Rio de Janeiro (UFRJ), Brazil Gabriel Almeida, University of Jyväskylä, Finland

> \*Correspondence: Julie A. Thomas<EMAIL_ADDRESS>
#### † Present Address:

Andrea D. Benítez, Instituto Nacional de Investigación en Salud Pública, Centro Nacional de Referencia e Investigación en Vectores, Quito, Ecuador

#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 18 August 2017 Accepted: 31 October 2017 Published: 15 November 2017

#### Citation:

Ali B, Desmond MI, Mallory SA, Benítez AD, Buckley LJ, Weintraub ST, Osier MV, Black LW and Thomas JA (2017) To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US. Front. Microbiol. 8:2251. doi: 10.3389/fmicb.2017.02251 Giant Salmonella phage SPN3US has a 240-kb dsDNA genome and a large complex virion composed of many proteins for which the functions of most are undefined. We recently determined that SPN3US shares a core set of genes with related giant phages and sequenced and characterized 18 amber mutants to facilitate its use as a genetic model system. Notably, SPN3US and related giant phages contain a bolus of ejection proteins within their heads, including a multi-subunit virion RNA polymerase (vRNAP), that enter the host cell with the DNA during infection. In this study, we characterized the SPN3US virion using mass spectrometry to gain insight into its head composition and the features that its head shares with those of related giant phages and with T4 phage. SPN3US has only homologs to the T4 proteins critical for prohead shell formation, the portal and major capsid proteins, as well as to the major enzymes essential for head maturation, the prohead protease and large terminase subunit. Eight of ∼50 SPN3US head proteins were found to undergo proteolytic processing at a cleavage motif by the prohead protease gp245. Gp245 undergoes auto-cleavage of its C-terminus, suggesting this is a conserved activation and/or maturation feature of related phage proteases. Analyses of essential head gene mutants showed that the five subunits of the vRNAP must be assembled for any subunit to be incorporated into the prohead, although the assembled vRNAP must then undergo subsequent major conformational rearrangements in the DNA packed capsid to allow ejection through the ∼30 Å diameter tail tube for transcription from the injected DNA. In addition, ejection protein candidate gp243 was found to play a critical role in head assembly. Our analyses of the vRNAP and gp243 mutants highlighted an unexpected dichotomy in giant phage head maturation: while all analyzed giant phages have a homologous protease that processes major capsid and portal proteins, processing of ejection proteins is not always a stable/defining feature. Our identification in SPN3US, and related phages, of a diverged paralog to the prohead protease further hints toward a complicated evolutionary pathway for giant phage head structure and assembly.

Keywords: Salmonella, myovirus, giant phage, mass spectrometry, prohead protease, CTS (capsid targeting sequence), ejection protein, virion RNA polymerase (vRNAP)

## INTRODUCTION

In recent years there has been a remarkable realization that standard phage isolation techniques were biased against larger phages and that "giant" or "jumbo" dsDNA tailed phages with genomes >200 kb can be readily isolated from a diversity of environmental samples and locales (Serwer et al., 2004, 2007, 2009; Krylov et al., 2007). The first giant phage genome published was that of Pseudomonas aeruginosa phage φKZ (280 kb) in 2002 (Mesyanzhinov et al., 2002). Since then even longer phage genomes have been reported, up to 480 kb (Bacillus megaterium phageG, Hendrix, 2009). Both φKZ and phageG were isolated decades prior to genome sequencing (Donelli, 1968; Krylov et al., 1984) and were considered rare oddities with fascinatingly complex virions but of little general relevance. However, φKZlike phages have been incorporated into therapeutic mixtures of phages for phage therapy of P. aeruginosa infections (Krylov et al., 2007, 2012). There has been a surge in interest in phage therapy due to the problems of multi-drug resistant bacteria (Harper and Morales, 2012). Consequently, more than thirty phages related to φKZ infecting a range of hosts including Salmonella (e.g., SPN3US), Erwinia amylovora (e.g., Ea35-70), Cronobacter sakazakii (e.g., CR5) and Vibrio spp. (e.g., JM-2012) have now been isolated with the goal of developing novel phagebased therapeutics (Lee et al., 2011, 2016; Jang et al., 2013; Meczker et al., 2014; Yagubi et al., 2014; Bhunchoth et al., 2016; Danis-Wlodarczyk et al., 2016). For example, PhiEaH2 is one of two phages in "Erwiphage," the first marketed bacteriophagebased pesticide against E. amylovora in Hungary (Meczker et al., 2014).

There is also great interest in giant phages related to φKZ as their long genomes, which range in length from 211 to 316 kb, have many genes without counterparts in other tailed phage taxa. Not surprisingly, these phages have numbers of highly unusual traits relative to other phage types, including virions amongst the most complex of known phages, and atypical mechanisms for replication within the bacterial cell, as highlighted in recent studies (Kraemer James et al., 2012; Ceyssens et al., 2014; Zehr Elena et al., 2014; De Smet et al., 2016; Leskinen et al., 2016; Van den Bossche et al., 2016; Chaikeeratisak et al., 2017). Studies on φKZ determined that its virion is unusually large with a T = 27 capsid (Fokine et al., 2005a, 2007) and an odd structure within its head—a large proteinaceous inner body (IB), around which the DNA is tightly wrapped (Krylov et al., 1984). We determined the three-dimensional reconstruction of the φKZ IB and estimate it is comprised composed of 15–20 MDa of protein (Wu et al., 2012). That is, essentially the IB represents a large bolus or mass of many different proteins that are ejected into the host cell along with the genome (Wu et al., 2012). As observed for φKZ, other relatives of φKZ also show "bubblegrams" under the cryoelectron microscope indicative of an IB/ejection protein mass (Thomas et al., 2008; Sokolova et al., 2014).

Mass spectral analyses of phages φKZ and relatives P. aeruginosa phage EL and P. chlororaphis phage 201φ2-1 revealed that the virions of these phages are comprised of 60–70 different proteins—twice the number of different proteins found in the virion of the model phage T4 (Fokine et al., 2005a; Thomas et al., 2008, 2012; Lecoutere et al., 2009; Sycheva et al., 2012). The heads alone of phages related to φKZ contain ∼50 different proteins and the IB/ejection protein bolus is likely the locale for many of these proteins (Thomas et al., 2012). We hypothesize that these structures/proteins are likely multi-functional having roles in assembly, stability and host-takeover (Black and Thomas, 2012; Thomas et al., 2012). Support for the latter is that all related giant phages have a multi-subunit virion RNA polymerase (vRNAP) which is packaged within their heads for ejection into the host cell for production of early gene transcripts (Thomas et al., 2008; Ceyssens et al., 2014). The vRNAP and a second multi-subunit non-virion RNAP (nvRNAP) are a hallmark feature of all giant phages related to φKZ and are highly unusual as they are comprised of β and β' subunits which themselves are split into 2–3 subunits. This is very different from the single subunit RNAPs of phages T7 and N4 (Kazmierczak et al., 2002; Sousa and Mukherjee, 2003; Gleghorn et al., 2008) and likely enables transcription of giant phage proteins to be completely, or almost completely, independent of the host transcriptional machinery (Ceyssens et al., 2014; Leskinen et al., 2016).

We determined that head proteins in 201φ2-1 and φKZ, including the major capsid protein (MCP) and ejection protein candidates, undergo proteolysis by a protease (Thomas et al., 2012; Thomas and Black, 2013). We identified the φKZ enzyme, gp175, using bioinformatics analyses and then demonstrated that it cleaved two inner body proteins, making it the first phage protease to be expressed, highly purified and shown to be active in vitro (Thomas and Black, 2013). This enzyme is now the type protease for the MEROPs family S80 (Rawlings et al., 2012). Importantly, we identified that φKZ gp175 and its homologs in related giant phages, such as SPN3US gp245, share diverged sequence similarity and predicted structural elements with the prohead protease of T4 phage, gp21 (Thomas et al., 2012; Thomas and Black, 2013). Remarkably, the T4 prohead protease has been shown to conserve the structural elements and catalytic residues of Herpesvirus protease (Cheng et al., 2004; Liu and Mushegian, 2004; Rossmann et al., 2007). This finding, in conjunction with shared folds in MCP, portal and large terminase proteins, support a shared ancestor for Herpesviruses and tailed phages (Baker et al., 2005; Fokine et al., 2005b; Rixon and Schmid, 2014).

In T4, proteolytic processing by gp21 is an essential step in head assembly and results in a vast remodeling of the capsid architecture, setting the stage for genome packaging (Black et al., 1994; Miller et al., 2003). Briefly, T4 heads assemble via nucleation of the portal ring on the inner host membrane. Upon this ring a spherical mass of protein assembles, referred to as the core structure, which consists of the scaffold protein, gp22 (576 copies), internal proteins (∼1,000 copies), Alt (40 copies), gp67 (341 copies), gp68 (240 copies), and gp21 (100 copies) (Black et al., 1994). All of the T4 internal head proteins associate with the core structure via a capsid-targeting-sequence or CTS (Mullaney and Black, 1996). Upon completion of the core complex the shell then assembles around. The shell is composed of two proteins, the MCP, gp23 (960 copies) and gp24 (55 copies), which is located at the capsid vertices and is a paralog of gp24 (Fokine et al., 2006). At this point, the protease gp21 cleaves the core proteins gp22, gp67, and gp68 into short fragments, as well as N-terminal propeptides from gp23, gp24, Alt and the IPs (Showe et al., 1976a,b). Fragments of the scaffold and core proteins, as well as the propeptides of the internal proteins exit the capsid and the capsid shell expands, increasing the internal volume of the capsid by 30–50% (Black and Rao, 2012). The prohead is then released from the host membrane and undergoes the final step of head assembly—DNA packaging where the terminase proteins packages the 170 kb genome into the head to remarkably high density (∼500 mg/ml) (Black and Rao, 2012; Black, 2015). The addition of the contractile tail assembled through a separate pathway and two decoration proteins, HOC and SOC, to the exterior of the capsid completes virion assembly.

These steps in T4 assembly, and many other features of this model phage, were elucidated with the aid of an elegant genetic system (Epstein et al., 1963, 2012). Not surprisingly, this system showed that the T4 genes associated with formation and maturation of the head, gps 17, 20, 21, 22, 23, gp24, 67, and 68, are essential (Miller et al., 2003). The proteins packaged within the dsDNA and ejected into the host cell, the IPs and the ADP-ribosyltransferase Alt, are not essential for assembly; however IPI is essential for the inhibition of a Type IV restriction endonuclease in E. coli strains containing the GmrSD enzyme gene (Black and Abremski, 1974; Abremski and Black, 1979; Bair et al., 2007; Rifat et al., 2008). Noting the power of such a genetic system and that in all phages related to φKZ there is a core set of conserved genes (including genes for major virion proteins and the vRNAPS), we sought to establish Salmonella phage SPN3US as a model giant phage genetic system (Thomas et al., 2016). We isolated, sequenced, and characterized 18 amber mutants of SPN3US, identifying 13 essential genes, only two of which, a SbcC and vRNAP β subunit, had been assigned putative functions previously. Illustrating the potential for this system, analyses of a putative neck gene mutant determined that ∼50 gene products are present in the mature SPN3US head. Additionally, analyses of the vRNAP β mutant facilitated identification of the previously unidentified C-terminal domain of the giant phage vRNAP β ′ subunit and suggested a new phenomenon in phage head assembly—the five-subunit vRNAP enzyme complex assembles prior to incorporation into the prohead.

We continue to develop our novel genetic resource with the goal of implementing it to resolve questions regarding giant phage biology. To assist this process, in this study we sought to characterize the wild-type SPN3US virion using mass spectrometry and determine if proteolysis of head proteins occurs, as observed in related phages, and if so, define which proteins are processed and to what degree. In doing so, we aimed to gain insight into the head features and maturation characteristics that are shared in related giant phages and those that are shared between giant phages and T4 phage. Our results show that while SPN3US shares a head maturation protease with diverged sequence similarity to that of T4 and other major head features of T4, there are substantial variations in the proteolysis step in head maturation in SPN3US relative to T4 and also to other giant phages. Unexpectedly, we only identified approximately half the number of different proteins that undergo proteolytic processing in SPN3US vs. the number found in previously analyzed giant phage heads, despite all phages having approximately the same number of overall head proteins and sharing homologous proteins. In addition, our analyses of four SPN3US head gene mutants identified one of the processed head proteins as a novel head ejection protein (gp47), and another (gp243) that is essential for the assembly of the two most abundant, processed head ejection proteins into the prohead. The analyses of two vRNAP subunit mutants provided further evidence to support that the vRNAP is incorporated into the prohead as a multimer.

The identification of a diverged paralog to the prohead protease in SPN3US, and other giant phages, that is truncated and presumably no longer active (i.e., a cryptic protease) suggests a complex evolutionary pathway for the head proteolysis maturation step.

#### MATERIALS AND METHODS

#### Propagation and Purification of Phages

Bacterial stocks of E. coli and Salmonella enterica Typhimurium strains TT9079 and TT6675 and phages SPN3US and T4 were propagated using LB media. SPN3US wild-type and mutant phages were propagated in overlays containing 0.34% agar at 30◦C. Phage dilutions were prepared in SM buffer. SPN3US amber mutants were isolated via hydroxylamine mutagenesis as described previously (Thomas et al., 2016).

Amber mutant particles were purified after propagation on the non-permissive host (TT9079) in LB broth supplemented with 1 mM CaCl2 and 1 mM MgCl2 at 30◦C. Typically, an overnight culture was diluted 1:100 and grown to an OD600 of 0.3–0.5 at which point phage was added (MOI of 10). Phages were allowed to adsorb for 15 min then cells were pelleted twice at low speed (5,000 rpm, 3 min, RT) and resuspended in fresh media. Infected cells were then incubated with shaking for 3 h 30◦C, and then treated with lysozyme (1 mg/ml) at room temperature with gentle shaking for 30 min. Mutant particles were concentrated by differential centrifugation then purified by sequential CsCl step then overnight buoyant density gradient ultracentrifugation as described previously (Thomas et al., 2016).

### SPN3US Amber Mutant Genome Sequencing

SPN3US wild-type and mutant phage DNAs were extracted from high titer stocks that had undergone differential centrifugation (titers typically 5 × 1011 pfu/ml) and purified using a phage DNA extraction kit (Norgen). Mutant phage genomes underwent genome sequencing at the University of Rochester Genomics Research Center on an Illumina MiSeq machine (2 × 250). Genomes were assembled and SNP reports generated using SeqMan NGen and SeqMan Pro, respectively (DNASTAR). The reference sequence used for alignments was the wild-type SPN3US genome GenBank accession JN641803 (Lee et al., 2011).

#### Mass Spectrometry

Samples were boiled for 10 min in SDS sample buffer prior to electrophoresis on Criterion XT MOPS 12% SDS-PAGE reducing gels (Bio-Rad) and subsequent protein visualization by staining with Coomassie blue. Gel lanes were divided into slices (10 for the wild-type phage, six for the mutants). Efforts were made to avoid transecting visible stained bands. No replicates of samples were analyzed. After de-staining, proteins in the gel slices were reduced with TCEP [tris(2 carboxyethyl)phosphine hydrochloride] and then alkylated with iodoacetamide before digestion with trypsin (Promega). HPLCelectrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) was accomplished on a Thermo Fisher LTQ Orbitrap Velos Pro mass spectrometer or a Thermo Fisher Orbitrap Fusion Lumos mass spectrometer. Mascot (Matrix Science; London, UK) was used to search the MS files against a locally generated SPN3US protein database that had been concatenated with the SwissProt database (2012\_11\_170320; version 51.6). Subset searching of the Mascot output by X! Tandem, determination of probabilities of peptide assignments and protein identifications, and cross correlation of the Mascot and X! Tandem identifications were accomplished by Scaffold (Proteome Software). MS data files were either processed individually or the files for an entire gel lane were combined via the "MudPIT" option.

Peptides generated by cleavage by a prohead protease were identified through use of database searching (Mascot and X! Tandem) using an enzyme specificity of "semi-trypsin" followed by visual inspection of the results in Scaffold (Proteome software). This process was necessary because of unknown cleavage specificity of the SPN3US protease. Our previous studies have found variations in protease specificity even between the homologous proteases of φKZ and 201φ2-1 (Thomas et al., 2010, 2012; Thomas and Black, 2013). The results for identified proteins, numbers of unique peptides, total spectra, and sequence coverage for each experiment were exported from Scaffold with the following quality filters: peptide, 95%; protein, 99.9%; minimum number of peptides, 3. Microsoft Excel was then used to generate spectrum count profiles, as described previously (Thomas et al., 2010). An estimate of the relative abundance of SPN3US virion proteins was calculated by dividing the total number of spectra assigned for each protein (spectral count, SC) in the MudPIT analyses by its molecular mass (SC/ M) as performed for phages 0305φ8-36 (Thomas et al., 2007), 201φ2- 1 (Thomas et al., 2007, 2010), RIO-1 (Hardies et al., 2016), and φKZ (Thomas et al., 2012). Our results from other phages have demonstrated that SC/M provides a useful indicator of relative abundance of different virion proteins. That is, proteins with similar SC/M values are typically present in similar relative abundances in the virion. In addition, proteins with SC/M ≤ 1 are likely to be present in only few copies, or even less than one copy, per virion. The copy numbers of several SPN3US virion proteins are known; major capsid protein (gp75), 1560 copies; tail sheath (gp256), 264 copies (Alasdair Steven and Weimin Wu, personal communication); and portal protein (gp81), 12 copies.

There are limitations to estimations of protein abundance using SC/M; therefore we also used densitometry, as conducted previously for the complex myoviruses 0305φ8-36 (Thomas et al., 2007) and φKZ (Thomas et al., 2012) and the podovirus RIO-1 (Hardies et al., 2016), for additional confirmation of head proteins assigned by SC/molecular mass as highly abundant. For these analyses, as two stoichiometric controls, the major sheath and portal proteins, migrate to the same region of the gel as the broad band of the highly abundant MCP (**Figure 1**) we used results from SPN3US mutant 64\_112(am27) propagated under non-permissive conditions (it has a tailless phenotype) to assess the abundance of high copy number head proteins (Thomas et al., 2016).

#### Cloning, Expression, and Purification of the SPN3US Prohead Protease

The full-length form of the SPN3US protease (gp245) gene was amplified from SPN3US DNA by PCR using the primers pHS245F (5′ -GCGCCATGGAAAACTTGTCACTACGTTATA ACTGCGTGGC-3′ ) and pHS245R (5′ - GCGTCTAGATTA CCAGCTCCTTACACCCATGCCCATTACC-3′ ). The gp245 gene was then cloned into the vector pHERD20T (Qiu et al., 2008) using the NcoI and XbaI sites and transformed into E. coli DH10B. Codons for a six-histidine tag were subsequently added to the 5' end of gp245 gene via site-directed mutagenesis (SDM) as described in Thomas and Black (2013). PfuUltra Hotstart DNA polymerase (Agilent) was used for the amplifications, and subsequent digestion by DpnI (NEB) was undertaken to remove any remaining template DNA. Plasmid DNA was purified using the QIAprep spin miniprep kit (Qiagen) and the construct verified by DNA sequencing using the pHERD sequencing primers (Qiu et al., 2008). The gp245 construct was propagated in LB broth containing 150µg/ml of ampicillin until mid-log phase, and protein expression was induced by the addition of arabinose (1% final concentration) for 1 h at 37◦C. Cells were pelleted at 6,000 rpm (Sorvall rotor SS34) for 10 min, then resuspended in lysis buffer (20 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1 mM EDTA, and egg white lysozyme [0.3 mg/ml; Sigma]), for ∼1 h, 4◦C. The lysate was then treated with DNase (40 U/ml; Roche) at 37◦C for 20 min, centrifuged (10,000 g, 10 min), and the supernatant was mixed with HisPur nickel resin (Thermo Scientific) overnight at 4◦C. Purification of gp245 was performed according to the nickel column manufacturer's instructions at room temperature. Washes were performed in buffer containing 20 mM Tris-Cl (pH 7.5) and 300 mM NaCl with increasing imidazole concentrations (final elution buffer contained 250 mM imidazole). The eluted gp245 was dialyzed against wash buffer containing no imidazole and electrophoresed on a 12% Bis-Tris SDS-PAGE gel (Novagen). The major gel band was excised and digested separately with trypsin and chymotrypsin prior to mass spectrometric analyses as described above.

## Transmission Electron Microscopy

Purified SPN3US mutant particles were adsorbed to 400 mesh carbon-coated grids and negatively stained with uranyl acetate (1%). Samples were examined at 80.0 kV using a FEI Tecnai T12 transmission electron microscope at the University of Maryland Electron Microscopy Core Imaging Facility.

### Bioinformatics Analyses

PSI-BLAST searches were performed on a local implementations of the NCBI BLAST suite (Altschul et al., 1997), as were analyses using the Sequence Analysis and Modeling System (SAM)

(Hughey and Krogh, 1996; Karplus et al., 1998) and HHpred (Söding, 2005). Phylogenetic trees were created from alignments created by MUSCLE (Edgar, 2004) using PAUP version 4.0 (Swofford, 2002) with Maximum Likelihood (50% majority rule) and the CDMut model. The majority rule consensus tree was created from 1,000 bootstrap runs.

#### RESULTS

#### Identification of SPN3US Virion Proteins by Mass Spectrometry

Eighty-six different SPN3US proteins were detected by mass spectrometry in CsCl step purified SPN3US, with a protein identification probability of 100% (**Figure 1**, Supplementary Table 1). This indicates that ∼33% of all SPN3US genes encode virion proteins; together, these proteins represent ∼46% because there are a number of long virion genes (e.g., gp168 is 5.2 kb). These SPN3US proteins detected in the virion ranged in molecular mass from 7 kDa for gp172 (a protein of unknown function) to 259 kDa for gp239 (the tail tape measure protein) (**Table 1**, Supplementary Table 1). The proteins were detected across a wide dynamic range of total spectra and sequence coverage, from four total spectra assigned for gp38 (a protein of unknown function) to 1,592 total spectra for gp75 (the MCP), and sequence coverage from 17% for gp49 to 94% for gp141 (Supplementary Table 1). Overall, fifty of the SPN3US proteins identified had sequence coverage of 50% or higher (Supplementary Table 1).

Spectral counting (SC) is an accepted semi-quantitative approach for estimation of relative protein abundance (Zybailov et al., 2005) and was used for this purpose for the SPN3US virion proteins. For each protein we calculated a SC/M value by dividing the total number of spectra assigned to each protein by its predicted molecular mass (**Table 1**, Supplementary Table 1). Several proteins were found to have undergone proteolytic processing (see below), and in these instances, an accordingly adjusted molecular mass was used to estimate relative abundance. After these adjustments, the protein in the virion with the highest SC/M value (22.6) was gp75, consistent with the expectation that the major capsid protein would be the most abundant protein in the virion. There are 1,560 copies of gp75 per virion based on the SPN3US capsid having the same triangulation number (T = 27) as φKZ (Alasdair Steven, Weimin Wu, personal communication). The SC/M values for gp53 and gp54 were 21.0 and 20.2, respectively, more than 2-fold the SC/M values obtained for the tail sheath and tube proteins. Two other SPN3US proteins, gp141 and gp160, had slightly higher SC/M values than the tail sheath SC/M value. The SPN3US tail sheath and tube proteins are expected to be present in ∼260 copies per particle based on the following considerations: 1. The SPN3US tail is of similar length as the φKZ tail, 2. SPN3US has sheath and tube proteins that are homologous to those of φKZ, 3. The φKZ tail is known to have 264 copies of each protein in its tail (Fokine et al., 2007).