First Diagnosed Case of Camelpox Virus in Israel

Article First Diagnosed Case of Camelpox Virus in Israel

Oran Erster 1,†, Sharon Melamed 2,†, Nir Paran 2, Shay Weiss 2, Yevgeny Khinich 1, Boris Gelman 1, Aharon Solomony 3 and Orly Laskar-Levy 2,* 1 Division of Virology, Kimron Veterinary Institute, P.O. Box 12, Beit Dagan 50250, Israel; [email protected] (O.E.); [email protected] (Y.K.); [email protected] (B.G) 2 Department of Infectious Diseases, IIBR P.O. Box 19, Ness Ziona 74100, Israel; [email protected] (S.M.); [email protected] (N.P.); [email protected] (S.W.) 3 Negev Veterinary Bureau, Israeli Veterinary Services, Binyamin Ben Asa 1, Be0er Sheba 84102, Israel; [email protected] * Correspondence: [email protected]; Tel.: +972-8-938-16051 † These authors contributed equally to this work.

Received: 10 January 2018; Accepted: 12 February 2018; Published: 13 February 2018

Abstract: An outbreak of a disease in camels with skin lesions was reported in Israel during 2016. To identify the etiological agent of this illness, we employed a multidisciplinary diagnostic approach. Transmission electron microscopy (TEM) analysis of lesion material revealed the presence of an orthopox-like virus, based on its characteristic brick shape. The virus from the skin lesions successfully infected chorioallantoic membranes and induced cytopathic effect in Vero cells, which were subsequently positively stained by an orthopox-specific antibody. The definite identification of the virus was accomplished by two independent qPCR, one of which was developed in this study, followed by sequencing of several regions of the viral genome. The qPCR and sequencing results confirmed the presence of camelpox virus (CMLV), and indicated that it is different from the previously annotated CMLV sequence available from GenBank. This is the first reported case of CMLV in Israel, and the first description of the isolated CMLV subtype.

Keywords: orthopoxvirus; camelpox virus; transmission electron microscopy; PCR; Immunoﬂuorescence assay (IFA); chorioallantoic membrane

1. Introduction Pox and pox-like diseases belongs to a group of systemic diseases that in their classical form are manifested by skin lesions with various morphologies [1]. In camels, the diagnosis of such skin lesions involves three main suspected viruses that can cause similar skin lesions [2]. camelpox virus (CMLV), the causative agent of camelpox, belongs to the genus Orthopoxvirus (OPV), and Camel contagious ecthyma virus (CCEV), the causative agent of Auzdik disease, which belongs to the genus Parapoxvirus (PPV). The third virus is camelus dromedary papillomavirus, the causative agent of camel papillomatosis [3]. Although the clinical signs may look similar, the consequences of each viral infection are completely different in terms of possible human infection [4,5] and economic implication [6,7]. Phylogenetic analysis shows that among OPV, CMLV is the closest to variola virus (VARV), the causative agent of smallpox [8]. Despite the above, each virus exhibits a strictly narrow host range [9]; VARV exhibits exclusive human-speciﬁcity, whereas CMLV infect exclusively old world camelids [7], with very rare human infection cases [5]. Camel contagious ecthyma virus [10] (CCEV) induces acute cutaneous pustular lesions in camels that can be transmitted to humans. In humans, the lesions remain localized, and infections on the hand are relatively common among people working in close contact with animals. It was previously shown that orf virus, the prototype of PPV, has adapted to skin tissue via unique genes that were acquired from the host during evolution [11,12].

Viruses 2018, 10, 78; doi:10.3390/v10020078 www.mdpi.com/journal/viruses Viruses 2018, 10, 78 2 of 13

Camelus dromedary papillomavirus (CDPV) belongs to the genus Delta papillomavirus of the family Papillomaviridae [3], which can be clearly distinguished using Transmission Electron Microscopy (TEM), as its shape and size is completely different from that of poxviruses. CMLV is endemic in Africa and Asia, circulating in old-world camelids [13]. Camels are used as a source of wool, milk, and meat. Therefore, the emergence of such a disease is of economic importance, with implications on animal handlers’ health. Here, we describe the first diagnosed cases of CMLV in Israel using a sensitive, specific, and rapid diagnostic approach for differentiating between CMLV, CCEV, and CDPV. The first method includes imaging the infectious agent using TEM. This rapid method enables the differentiation among papillomavirus, OPV, and PPV. In case of insufficient material, an isolation of virus particles can be achieved by infecting chorioallantoic membranes or cell cultures. As supporting evidence for the presence of OPV, we performed an immunofluorescence assay on infected cells using an orthopox-specific antibody. Pock formation on chorioallantoic membranes, and the induction of typical cytopathic effects, are common accessory methods that are used in parallel. Lastly, a combination of PCR and DNA sequencing approaches were used for the definite identification of CMLV.

2. Materials and Methods

2.1. Cells and Viruses BS-C-1 (ATCC, CCL-26) and Vero (ATCC CCL-81) cells were routinely maintained in Dulbecco0s modiﬁed Eagle medium (DMEM) supplemented with 10% fetal calf serum, 2 mM of glutamine, 0.1 mg/mL of streptomycin, 100 units/mL of penicillin, 1.25 units/mL of nystatin, and non-essential amino acids (Biological Industries, Beit-Haemek, Israel). Vaccinia virus-Western Reserve (VACV-WR, ATCC VR-119) and cowpox virus (CPXV, strain Brighton, ATCC VR-302) were grown in HeLa cells, and puriﬁed as described previously [14]. CMLV titers were determined by plaque assay on Vero cells, as described elsewhere [15].

2.2. Samples and Antibodies Samples from eight animals showing clinical signs that are characteristic of a poxvirus infection, were collected from four locations in the Israel desert (Negev) and southeastern areas as follows: two samples from Hura, two from Kseifeh, one from Tel Arad, and three from the Hebron region. All of the diagnostic assays in this study were performed on skin scrapes, except qPCR, which was also performed on whole blood samples. Preparation of the anti-VACV hyperimmune serum was described previously [16,17].

2.3. TEM Imaging A small piece of skin lesion was processed by vortexing in PBS, then inactivation and ﬁxation were performed using 2% buffered formaldehyde (FA) for 30 min at 25 ◦C, followed by an additional 30 min of incubation at 25 ◦C[18]. A volume of 10 µL was applied on 1% Alcian blue treated, 300 mesh carbon-coated copper TEM grids. Samples were incubated for 10 min, and then excess liquid was blotted using Whatman paper. Grids were washed three times with distilled water, and stained with 1% phosphotungstic acid. After air-drying, samples were visualized using an Tecnai T12 TEM (Thermo Fisher, Oregon, USA) operated at 120 kV and equipped with a Gatan ES500W Erlangshen camera. Scaling was done using a standard of known size measured at different magniﬁcations. The viral length of each particle was measured by stretching a line from end to end.

2.4. Infection of Chorioalanthoic Membrane & Vero Cells In order to isolate the virus, lysates from skin samples were used to inoculate speciﬁc pathogen free embryonated chicken eggs (SPF-ECE) and Vero cells. Skin samples from suspected camels were ground in sterile PBS supplemented with 0.1 mg/mL of streptomycin, 100 units/mL of penicillin, Viruses 2018, 10, 78 3 of 13 and 1.25 units/mL of nystatin antibiotic mix. The extract was layered on chorioallantoic membranes (CAM) of 10-days old SPF-ECE using a 23 G sterile needle. Following egg death, the chorioallantoic membrane was excised and used to generate an infectious suspension for Vero cells infection. Vero cells were infected with the SPF-ECE CAM resuspension, and grown under standard conditions.

2.5. Infection, Immunofluorescence Assay (IFA) and Cells Staining For immunofluorescence labeling, Vero cells were plated on LabTek (Thermo Scientific Nunc, Waltham, MA, USA). Following the different infections and treatments, cells were fixed with 3% paraformaldehyde (PFA) in PBS for 20 min, and permeabilized with 0.5% Triton X-100 for 2 min. The fixed cells were rinsed with PBS and blocked with PBS supplemented with 2% Bovine Serum Albumin (Sigma, St. Louis, MI, USA), and the LabTek were incubated for 45 min with anti-VACV hyperimmune serum. Following the PBS washes, the cells were incubated with the appropriate fluorescently conjugated secondary antibodies for 30 min. For nuclei visualization, cells were stained with 5 µg/mL (40,6-diamidino-2-phenylindole) DAPI [17]. For cytopathic effect and syncytial formation, cells were infected (Figure 4) and fixed with 3.7% formalin for 20 min. Hematoxylin-eosin staining was performed using the EnVision kit (Dakocytomation, Carpinteria, CA, USA). Light and fluorescence microscopy were carried out using the Nikon TE2000 fluorescent microscope (Tokyo, Japan). The images were acquired by a Nikon DXM-1200F camera, and processed with Adobe Photoshop software (5.0, San Jose, CA, USA).

2.6. Primer Design and Multiple Alignment Analysis Primers were designed and tested in silico using the Geneious software (Biomatters, version 8.0, Auckland, New Zealand). A multiple alignment analysis of partial genomic regions was performed using the MUSCLE algorithm [19]. Alignments of complete Poxvirus genomes were performed using the BLASTZ algorithm [20]. The alignment ﬁle was then used to construct a phylogenetic tree for the tested region, using the Mega6 software [21], according to the maximum likelihood method.

2.7. DNA Extraction and PCR Procedures DNA was extracted from blood and skin samples using the iNTRON viral extraction kit (http://intronbio.com/eng/viral-gene-spin.html). Two quantitative PCR (qPCR) assays were used. The first one was based on the C18L gene described by Balamurugan [22], using the following primers: C18L_F50-GCGTTAACGCGACGTCGTG-30 and C18L_R50-GATCGGAGATATC ATACTTT ACTTTAG-30. The reaction mix was prepared as follows: 2 µL of DNA template, 5 µL of PerfeCTa SYBR® Green SuperMix (http://www.quantabio.com/perfecta-syber-green-supermix), and 0.4 µM ◦ 0 each of primer and ddH2O, to a final reaction volume of 10 µL. Reaction conditions were: 95 C 5 , [95 ◦C 100, 62–64.4 ◦C 300 (plate read)] × 40, melt curve: 70.0 ◦C to 84.4 ◦C, increment of 0.4 ◦C for 1 min. A second PCR, termed 137637, was designed during the course of this study, and directed to amplify a different region in the CMLV genome. Two primers were designed to target a 192 bp region located downstream to the gene encoding RNA polymerase subunit RPO132 (acc. No. NC_003391). This region is approximately 61Kb upstream to the C18L region. The primers sequence was as follows: CMLV_137637_FWD 50-AGTTGATCTCAAGCCGTC-30, and CMLV_137829_REV 50-GAGGTGTAACAGCCACTTG-30. The reaction mix and conditions were as described for the C18L reaction. The sensitivity of the test was assayed using decimal dilutions of a purified control PCR product that contained the reaction target sequence. The primers that were used to generate the control PCR product were as follows: CMLV_137401F 50-GTTTTAAAAGACTCAGAAGAGG-30, and CMLV_138164R 50-AACGTGAATTGGAATCTGAACG-30. The conditions for the control product amplification were as described for the standard PCR run above. In order to characterize the infecting virus, three regions of the genomes were amplified and sequenced. The primer sets designed for the characterization procedure are listed in Table1. Viruses 2018, 10, x FOR PEER REVIEW 4 of 13

In order to characterize the infecting virus, three regions of the genomes were amplified and Viruses 2018, 10, 78 4 of 13 sequenced. The primer sets designed for the characterization procedure are listed in Table 1.

TableTable 1. 1. PrimerPrimer sets sets used used for for characterization characterization of of the the infecting infecting camelpox camelpox virus (CMLV). Amplicon Length in Primer Name Primer Sequence Amplicon Length in Sequence Primer Name Primer Sequence Sequence No. NC-003391 No. NC-003391 15458 Fwd 5′-CTATATCTATATGAGATGAC-3′ 0 0 420 bp 15877 15458Rev Fwd 5 5-CTATATCTATATGAGATGAC-3′-GTTGGTAGTAGGGTACTCGTG-3′ 0 0 420 bp CMLV 3359015877 F Rev 5 5′-GTTGGTAGTAGGGTACTCGTG-3-TCTGGAAGTGGATATACATAG-3′ 902 bp CMLVCMLV 34492 R 33590 F 5′-GGGATAATCCAGAATTGATAATAGTGG-350-TCTGGAAGTGGATATACATAG-30 ′ 902 bp CMLV CMLV188946 34492F R 5′0-GTATAATGTATGTAACCCGTAC-3-GGGATAATCCAGAATTGATAATAGTGG-3′ 0 1094 bp CMLVCMLV 190040 188946 R F 55′-TACATACCATTAATAATGCAAGC-30-GTATAATGTATGTAACCCGTAC-30 ′ 1094 bp CMLV 190040 R 50-TACATACCATTAATAATGCAAGC-30 The mix for each reaction was assembled as follows: 10 μL of Takara EmeraldAmp mix (http://ww.clontech.com/), 2–4 μL of the DNA extraction, 0.4 μM of the final concentration of each The mix for each reaction was assembled as follows: 10 µL of Takara EmeraldAmp mix primer, and ddH2O to a final reaction volume of 20 μL. The following conditions were used in a (http://ww.clontech.com/), 2–4 µL of the DNA extraction, 0.4 µM of the final concentration of ProFlex thermocycler (Thermo Fisher, Waltham, MA, USA): 98 °C 1′, (98 °C 20″, 51 °C 50″, 72 °C 20″) each primer, and ddH2O to a final reaction volume of 20 µL. The following conditions were used in x 35, 72 °C 5′. PCR products were resolved on agarose 1.2% gel, and visualized using Olerup SSP a ProFlex thermocycler (Thermo Fisher, Waltham, MA, USA): 98 ◦C 10, (98 ◦C 20”, 51 ◦C 50”, 72 ◦C GelReddye (http://www.olerup.com/). For the identification of CPXV and VACV DNA, the extension 20”) x 35, 72 ◦C 50. PCR products were resolved on agarose 1.2% gel, and visualized using Olerup SSP time was extended to 1′25″. The PCR products were purified either following gel separation or GelReddye (http://www.olerup.com/). For the identification of CPXV and VACV DNA, the extension directly from the reaction tube, and were sequenced. time was extended to 1025”. The PCR products were purified either following gel separation or directly from the reaction tube, and were sequenced. 3. Results 3. Results 3.1. Case Description and Clinical Signs 3.1. Case Description and Clinical Signs In the summer of 2016, a disease with skin manifestation was reported in camels in the southern part ofIn Israel. the summer Infected of animals 2016, a disease were reported with skin in manifestationtwo herds in the was Be reported′er-Sheba in district, camels inone the in southern the Tel- Aradpart ofregion, Israel. and Infected one in animals the Hebron were reportedregion. Insp in twoected herds herds in of the mainly Be0er-Sheba adult district,females oneexhibited in the symptomsTel-Arad region, including and weakness, one in the loss Hebron of appetite, region. fever, Inspected abortions, herds and of mainly multifoc adultal lesions females on exhibited the skin (Figuresymptoms 1A– includingD). During weakness, the initial loss stages of appetite, of the disease, fever, abortions, stiff papules and multifocalwere observed, lesions while on the at skinthe advanced(Figure1). stage During they the become initial stages uncerated of the with disease, purule stiffnt papules secretion. were No observed, mortalities while were at reported, the advanced and thestage inspected they become animals uncerated recovered with after purulent a few secretion. weeks. NoThere mortalities were no were subsequent reported, reports and the of inspected further spreadinganimals recovered of the disease. after a No few infections weeks. There in husban weredry no subsequentpersonnel were reports reported. of further In order spreading to identify of the thedisease. etiologic No infections agent, skin in husbandry samples from personnel eight were representative reported. In camels order to were identify subjected the etiologic to various agent, diagnosticskin samples tests. from eight representative camels were subjected to various diagnostic tests.

FigureFigure 1. 1. SkinSkin lesions lesions observed observed in in infected infected camels. camels. (A () ASkin) Skin lesions lesions on onthe theneck neck and and (B) hip (B) hipof the of left the rearleft rearleg of leg an ofinfected an infected camel. camel. Enlargement Enlargement of (C) stiff of ( papulesC) stiff papulesand (D) ulcerated and (D) ulcerated papules with papules purulent with secretion.purulent secretion.

Viruses 2018, 10, 78 5 of 13 Viruses 2018, 10, x FOR PEER REVIEW 5 of 13

3.2. Diagnostic Diagnostic Transmission Transmission Electron Microscopy Samples from camel skin lesions lesions were were prepared prepared for for diagnostic TEM. TEM. Out Out of of the the eight eight samples, samples, fourfour were analyzed by TEM. All four negatively-stainednegatively-stained samples clearly showed the characteristic brick-shaped morphology ofof OPVOPV (Figure(Figure2 A–E),2A–E), in in contrast contrast to to the the oval oval shape shape of of PPV PPV with with tubules tubules in in a acrisscross crisscross pattern pattern (Figure (Figure2F). 2F). At At least least 20 20 particles particles could could be be identiﬁed identified in onein one grid. grid. The The dimensions dimensions of ± × ± ofthe the viral viral diameter diameter were were 217.6 217.6 ±18.7 18.7 ×293.15 293.15 ± 18.8 nm. These values are in accordance with those reported in the literature [23]. [23]. Thus, Thus, diagnostic diagnostic TE TEMM indicated indicated the the existence existence of of OPV-like OPV-like virus in the skin of camels. No No papillomavirus papillomavirus particles were observed.

Figure 2. (A–E) Transmission electron microscopy (TEM) images of viral particles extracted from Figure 2. (A–E) Transmission electron microscopy (TEM) images of viral particles extracted from lesions taken from four individual camels and negatively stained with 1% Phosphotungstic acid lesions taken from four individual camels and negatively stained with 1% Phosphotungstic acid (PTA). (PTA). Note that the morphology and dimensions of the virus particles corresponds with the Note that the morphology and dimensions of the virus particles corresponds with the morphology and morphologydimensions ofand the dimensionsOrthopoxvirus of the(OPV) Orthopoxvirus family. (F) ( ForOPV) comparison, family. (F) aFor TEM comparison, image of an a TEM orf virus image after of annegative orf virus staining. after negative Note the staining. profound Note morphology the profound differences morphology between differences the orf virus between (F) that the belongs orf virus to (theF) that PPV belongs family, andto the the PPV OPV family, family and (A –theE). OPV family (A–E).

3.3. Pock Formation on CAM 3.3. Pock Formation on CAM The chorioallantoic membranes (CAM) of 10-day old embryonic chicken eggs were infected with The chorioallantoic membranes (CAM) of 10-day old embryonic chicken eggs were infected with skin extracts, as described above. Egg death occurred four days post-injection, at which time white skin extracts, as described above. Egg death occurred four days post-injection, at which time white pocks were observed on the membrane (Figure 3A). The pocks were excised from the membrane, and pocks were observed on the membrane (Figure3A). The pocks were excised from the membrane, and resuspended in sterile PBS. resuspended in sterile PBS.

3.4. CPE CPE on on Vero Vero Cells The resuspended pock material was used to infect Vero cells.cells. Cytopathic effects (CPE) were 5 observed four days post-infection (Figure3 3C).C). VirusVirus titer titer was was determined determined as as TCID50 TCID50 10 105/mL.

Viruses 2018, 10, 78 6 of 13 Viruses 2018, 10, x FOR PEER REVIEW 6 of 13

Figure 3. Virus isolation. (A) Infected skin samples extracts were used to infect chorioallantoic Figure 3. Virus isolation. (A) Infected skin samples extracts were used to infect chorioallantoic membrane of embryonated chicken eggs, and the onset of pock lesions was observed four days post- membrane of embryonated chicken eggs, and the onset of pock lesions was observed four days injectionpost-injection (marked (marked with witharrows). arrows). The pocks The pocks were were excised excised from from the themembrane, membrane, and and used used to generate to generate a suspendeda suspended inoculum inoculum for for the the infection infection of ofVero Vero cells. cells. Cytopathic Cytopathic effect effect was was observed observed four four days days after after μ inoculationinoculation ( (CC),), while while the the uninfected uninfected cells cells maintained maintained normal normal morphology morphology ( (BB),), Bar Bar =100 =100 µm.m.

3.5. Immunofluorescence Assay 3.5. Immunofluorescence Assay Members of the OPV family share common antigens [23]; thus, the use of OPV-specific Members of the OPV family share common antigens [23]; thus, the use of OPV-specific antibodies antibodies generated against VACV, the prototype of OPV, can recognize other members of the OPV generated against VACV, the prototype of OPV, can recognize other members of the OPV family. family. We performed an immunofluorescence assay using anti-OPV hyperimmune serum on Vero We performed an immunofluorescence assay using anti-OPV hyperimmune serum on Vero cells cells infected with the suspended pock material, as described in Section 2.4. Our results show infected infected with the suspended pock material, as described in Section 2.4. Our results show infected cells cells that were positively labeled by the anti OPV hyperimmune serum (Figure 4A). that were positively labeled by the anti OPV hyperimmune serum (Figure4A). Moreover, viral factories stained by DAPI and anti-OPV antibodies, which are characteristic of Moreover, viral factories stained by DAPI and anti-OPV antibodies, which are characteristic of OPV-infected cells, can be seen in the infected cells (Figure 4A-3). These results support the OPV-infected cells, can be seen in the infected cells (Figure4A-3). These results support the hypothesis hypothesis that the infectious agent belongs to the OPV family. As this sample was obtained from a that the infectious agent belongs to the OPV family. As this sample was obtained from a camel, and camel, and taking into consideration that CMLV is a species-specific OPV, these findings support our taking into consideration that CMLV is a species-specific OPV, these findings support our assumption assumption that the OPV isolated from the camels dermal lesions contains CMLV. that the OPV isolated from the camels dermal lesions contains CMLV.

3.6.3.6. Syncytia Syncytia Formation Formation in in Cultured Cultured Cells Cells SyncytiaSyncytia (cell–cell (cell–cell fusion) fusion) formation formation is is charac characteristicteristic of of several several pathogenic pathogenic OPV OPV members, members, includingincluding CMLV, CMLV, ectromelia ectromelia virus virus (ECTV), (ECTV), and and monkey monkeypoxpox (MPXV) (MPXV) [24]. [In24 order]. In to order verify to the verify ability the ofability the isolated of the isolated virus virusto induce to induce cell–cell cell–cell fusion, fusion, BS-C-1 BS-C-1 cells cells were were infected infected with with the the sample sample at at multiplicitiesmultiplicities of of infection infection (MOI) (MOI) of of0.005 0.005 and and 0.05 0.05 (Figure (Figure 4B,4 panelsB, panels 5 and 5 and6, respectively). 6, respectively). Cells Cellswere incubatedwere incubated for 24 forh, and 24 h, cytopathic and cytopathic effects effects were evaluated. were evaluated. The most The prominent most prominent cytopathic cytopathic effecteffect was thewas formation the formation of syncytia, of syncytia, which which was was manifested manifested by bypolykaryocytosis polykaryocytosis (Figure (Figure 4B).4B). These These giant multinucleatedmultinucleated cells cells comprised comprised few few to to dozens dozens of of nuclei, nuclei, which which were were positioned positioned in in many many cases cases in in a a ring-shapering-shape form. form. These These results results further further supported supported the the assumption assumption that that the the infectious infectious agent agent is is CMLV. CMLV.

Viruses 2018, 10, 78 7 of 13 Viruses 2018, 10, x FOR PEER REVIEW 7 of 13

FigureFigure 4. 4. ImmunofluorescenceImmunoﬂuorescence and and sync syncytiaytia formation formation assays. assays. (A (A) Vero) Vero cells cells were were infected infected with with (2 (2) ) VacciniaVaccinia virus-Western virus-Western Reserve Reserve (VACV-WR) (VACV-WR) at at a a mu multiplicityltiplicity of of infection infection (MOI) (MOI) = = 0.01, 0.01, or or (3 (3) )skin skin lesionlesion suspensions suspensions or or (1 (1) )left left uninfected. uninfected. At At 24 24 hours hours post-infection, post-infection, cells cells were were fixed ﬁxed and and stained stained for for NucleiNuclei (DAPI: (DAPI: blue) blue) and and OrthopovirusOrthopovirus antigensantigens (anti-VACV: (anti-VACV: green). green). Arrows Arrows indicate indicate viral viral factories. factories. (B(B) )Induction Induction of of cell–cell cell–cell fusion (syncytia)(syncytia) byby camelpoxviruscamelpoxvirus CMLV CMLV is is infection infection titer-dependent. titer-dependent. BS-C-1 BS- C-1cells cells were were infected infected at different at different dilutions. dilutions. (4) Control (4) Control uninfected uninfected BS-C-1 cellsBS-C-1 (5) BS-C-1cells (5 cells) BS-C-1 infected cells at infectedMOI = 0.005at MOI and = 0.005 (6) at and MOI (6 =) 0.05.at MOI Bar = =0.05. 100 Barµm. = 100 μm.

3.7.3.7. CMLV CMLV Identification Identification Using Using PCR PCR InIn order order to to enable enable a a definitive definitive identification identification of of the the etiological etiological agent agent of of the the disease, disease, PCR PCR was was employed,employed, with with both both rapid rapid SYBR-based SYBR-based qPCR, qPCR, and and the the amplification amplification of of several several genomically genomically distant distant regionsregions for for sequencing. sequencing. For For preliminary preliminary detection detection and and identification, identification, two two separate separate qPCR qPCR were were used, used, identifyingidentifying different different regions regions in in the the CMLV CMLV genome. genome. The The C18L C18L test test [22] [22 ]and and the the “137,637” “137,637” test test were were developeddeveloped in in this this study. study. The The rationale rationale for for developin developingg an an additional additional test test was was to to confirm confirm the the results results obtainedobtained using using the the C18L C18L test, test, by by targeting targeting a adifferen differentt region region in in the the CMLV CMLV genome. genome. The The selected selected target target regionregion is is located located downstream downstream to the gene, encodingencoding thethe RNA-dependentRNA-dependent DNADNA polymerasepolymerase subunit subunit B B(reference (reference accession accession No. No. NC_003391).NC_003391). ThisThis region is non-coding, and and its its variability variability among among different different OPVOPV species species is is relatively relatively high high (data (data not not shown), shown), thus thus enabling enabling the the design design of of a CMLV-specific a CMLV-specific reaction. reaction. ByBy using using this this region region as as the the target, target, the the specificity specificity of of the the sample sample analysis analysis was was further further increased, increased, thereby thereby furtherfurther minimizing minimizing the the chance chance of of erroneous erroneous identification. identification. Additionally, Additionally, in in all all of of the the annotated annotated CMLV CMLV sequences,sequences, there there were were two two mismatches mismatches in in the the revers reversee primer primer of of the the C18L C18L reaction reaction [22]. [22]. In In order order to to maximizemaximize the the chances chances of of a a successful successful amplification, amplification, we we developed developed the the “137,637” “137,637” test, test, consisting consisting of of primersprimers with with 100% 100% complementarity complementarity to to the the annotated annotated sequences. sequences. InIn order order toto determinedetermine the the sensitivity sensitivity of theof the new new qPCR, qPCR, we performed we performed a calibration a calibration test to comparetest to compareits sensitivity its sensitivity with that with of the that C18L of testthe (FigureC18L test S1). (Figure The two S1). tests The showed two tests similar showed performances, similar performances,detecting less detecting than 10 targetless than copies. 10 target The copies new test. The showed new test a slightlyshowed highera slightly efficiency higher efficiency compared comparedwith the C18Lwith the test: C18L 90.9% test: versus 90.9% 82.7%, versus respectively 82.7%, respectively (Figure (Figure S1). The S1). specificity The specificity of the of two the qPCR two qPCRwas examined was examined by using by using different different poxvirus poxvirus samples samples as template. as template. DNA DNA extracted extracted from from the orf the virus orf virus(PPV) (PPV) and lumpyand lumpy skin skin disease disease virus virus (capripoxvirus) (capripoxvirus) did notdid yieldnot yield any any product product (data (data not not shown). shown). The The primers designed for the “137,637” reaction were identical to the CMLV-corresponding genomic region (accession NC_003391), but contained two and three mismatches with respect to the

Viruses 2018, 10, 78 8 of 13

primersViruses 2018 designed, 10, x FOR for PEER the REVIEW “137,637” reaction were identical to the CMLV-corresponding genomic region8 of 13 (accession NC_003391), but contained two and three mismatches with respect to the corresponding VACVcorresponding and CPXV VACV virus regionsand CPXV (accession virus numbersregions LN864565(accession andnumbers AY313848, LN864565 respectively). and AY313848, However, underrespectively). permissive However, conditions, under i.e., permissive 60 ◦C annealing condit temperature,ions, i.e., 60 DNA °C samplesannealing of thesetemperature, two OPV DNA were positivesamples (Figureof these5 A).two OPV were positive (Figure 5A).

Figure 5.5. Development of Orthopoxvirus-specific-specific qPCRqPCR andand samplessamples examination.examination. ( A) qPCR ofof ◦ Camelpoxvirus (CMLV), cowpox virus (CPXV) andand Vaccinia virusvirus (VACV)(VACV) atat 6060 °CC annealingannealing temperature.temperature. LeftLeft panel:panel: TheThe “137,637”“137,637” reaction.reaction. RightRight panel:panel: The C18L reaction.reaction. (B) Similar reactions ◦ asas inin ((AA),), butbut withwith anan annealingannealing temperaturetemperature ofof 64.564.5 °C.C. (C)) MeltMelt profilesprofiles ofof thethe “137,637”“137,637” (left)(left) andand ◦ C18L (right)(right) reactionsreactions at at 60 60C °C annealing annealing temperature. temperature. (D )(D Representative) Representative results results of the of “137,367” the “137,367” (left) and(left) C18L and (right)C18L (right) tests that tests were that used were to used detect to anddetect confirm and confirm the presence the presen of thece virus of the in eightvirus samplesin eight fromsamples four from locations, four aslocations, detailed as in Sectiondetailed 3.1 in. BloodSection and 3.1. skin Blood DNA and extracts skin wereDNA both extracts positive. were The both Ct

valuespositive. for The each Ct sample values arefor detailedeach sample in Table are detailed S1. in Table S1.

The specificity of the reaction increased significantly following an increase of the annealing temperature to 64.5 °C, at which point the CMLV signal remained robust, while the CPXV and VACV signals decreased markedly (Figure 5B). Moreover, the melt profile, determined by the post- amplification melt analysis, was different for each sample, thus enabling specific identification of the

Viruses 2018, 10, x FOR PEER REVIEW 9 of 13

CMLV virus even under permissive reaction conditions (Figure 5C). The C18L test detected both CMLV and CPXV, but not VACV, in the 60 °C annealing reaction, with different melt profiles (Figure 5A,C), and only CMLV in the 64.5 °C annealing reaction (Figure 5B). A total of eight samples (skin and blood) obtained from four locations (Section 2.2) were identified (Figure 5D). Both the C18L and “137,637” tests gave similar results, with Ct values ranging from 9.87 to 31 (Table S1). VirusesIn2018 order, 10, 78to further establish the identity of the isolated virus, we developed three standard 9PCR of 13 to amplify three regions within the CMLV genomes. The first reaction was designed to amplify the regionThe between specificity positions of the 15,458 reaction and increased 15,877 in significantly the CMLV followinggenome (accession an increase NC_003391). of the annealing This reactiontemperature specifically to 64.5 identified◦C, at which CMLV point and the distinguished CMLV signal it remained from other robust, OPV, while namely the CPXV CPXV and and VACV.VACV This signals was decreased accomplished markedly by amplifying (Figure5 aB). region Moreover, whose thelength melt is 420 profile, bp in determinedthe CMLV genome, by the 1140post-amplification bp in the CPXV melt genome analysis, (accession was different LN864565) for, each and sample,1048 bp thusin the enabling VACV (accession specific identification AY313848). Byof thekeeping CMLV the virus extension even time under of permissivethe reaction reaction below 25 conditions s, only the (Figure CMLV5 C).presence The C18L could test be detecteddetected (Figureboth CMLV 6A). and CPXV, but not VACV, in the 60 ◦C annealing reaction, with different melt profiles (FigureHowever,5A,C), andby extending only CMLV this in stage the 64.5 to one◦C annealingminute and reaction 25 s, two (Figure longer5B). pr Aoducts total ofwere eight generated, samples demonstrating(skin and blood) that obtained under permissive from four locations conditions, (Section this test 2.2 could) were differentially identified (Figure detect5D). CMLV Both and the C18Lother related viruses (Figure 6B). This reaction successfully amplified the target sequence in all of the and “137,637” tests gave similar results, with Ct values ranging from 9.87 to 31 (Table S1). samples,In order except to furtherone sample, establish which the could identity not of be the am isolatedplified virus,despite we repeated developed attempts three standard (Figure PCR6C). Twoto amplify other regions three regions within withinthe CMLV the CMLV genome genomes. were amplified The first and reaction sequenced, was designed one between to amplify positions the 33,590region and between 34,492 positions in sequence 15,458 number and 15,877 NC_003391 in the CMLV (902 bp), genome and (accessionthe other between NC_003391). positions This reaction188,946 andspecifically 190,040 identified in sequence CMLV number and distinguished NC_003391 it( from1094 otherbp). The OPV, sequences namely CPXV were and deposited VACV. This in wasthe GeneBankaccomplished with by the amplifying following a region accession whose numbers: length is KX770279–KX770292. 420 bp in the CMLV genome, Multiple 1140 alignments bp in the followedCPXV genome by phylogenetic (accession LN864565),analyses showed and 1048 that bp al inl of the the VACV examined (accession samples AY313848). clustered By to keeping the same the clade,extension thereby time generating of the reaction a distinct below clade, 25 s, separa only theted CMLV from the presence other couldannotated be detected CMLV isolates (Figure6 (FigureA). 7).

FigureFigure 6. DevelopmentDevelopment of of standard standard PCR PCR for for differential differential detection detection of of camelpoxvirus camelpoxvirus (CMLV), (CMLV), cowpoxviruscowpoxvirus (CPXV), (CPXV), andvaccinia andvaccinia virus virus (VACV) (VACV) (A (,AB,)B and) and samples samples examination examination (C). ( C(A).,B (A) Lane,B) Lane 1 = CMLV,1 = CMLV, lane lane 2 = CPXV, 2 = CPXV, lane lane3 = VACV 3 = VACV.. As negative As negative controls, controls, lumpy lumpy skin disease skin disease virus virus(capripox, (capripox, lane 4),lane and 4), orf and virus orf virus (parapox, (parapox, lane lane 5) were 5) were used. used. (A) ( APCR) PCR for for the the amplification amplification of of the the region region spanning spanning positionspositions 15,458 15,458 to to 15,877 15,877 with with an an extension extension time time of of 20 20 s s allowed allowed the the amplification amplification of of a a 420-bp 420-bp length length productproduct from from CMLV CMLV only (lane 1). ( (BB)) Increased Increased extension extension time time of of one one minute minute and and 20 20 s s enabled enabled the the amplificationamplification of of CMLV, CPXV,CPXV, andand VACV VACV DNA DNA samples samples with with different different product product lengths. lengths. (C ()C A) A 420-bp 420- bpproduct product was was detected detected in in seven seven out out of of the the eight eight samples, samples, and and waswas usedused asas anan additional test for for the the quantitativequantitative analysis analysis an andd sequencing sequencing of of a CMLV-specific CMLV-specific amplifiedamplified region.region.

However, by extending this stage to one minute and 25 s, two longer products were generated, demonstrating that under permissive conditions, this test could differentially detect CMLV and other

related viruses (Figure6B). This reaction successfully amplified the target sequence in all of the samples, except one sample, which could not be amplified despite repeated attempts (Figure6C). Two other regions within the CMLV genome were amplified and sequenced, one between positions 33,590 and 34,492 in sequence number NC_003391 (902 bp), and the other between positions 188,946 and 190,040 Viruses 2018, 10, 78 10 of 13 in sequence number NC_003391 (1094 bp). The sequences were deposited in the GeneBank with the following accession numbers: KX770279–KX770292. Multiple alignments followed by phylogenetic analyses showed that all of the examined samples clustered to the same clade, thereby generating a distinctViruses 2018 clade,, 10, x separatedFOR PEER REVIEW from the other annotated CMLV isolates (Figure7). 10 of 13

FigureFigure 7.7. PhylogeneticPhylogenetic analysisanalysis ofof isolatedisolated camelpoxviruscamelpoxvirus (CMLV)(CMLV) samples.samples. The sequencessequences ofof threethree differentdifferent (not(not consecutive)consecutive) regions in thethe CMLVCMLV genomegenome werewere comparedcompared toto annotatedannotated sequencessequences ofof CMLVCMLV andand relatedrelated OrthopoxvirusesOrthopoxviruses (OPV).(OPV). AnalysisAnalysis waswas performedperformed followingfollowing multiplemultiple alignments,alignments, asas describeddescribed inin SectionSection 2.62.6..( (A) AnalysisAnalysis ofof thethe regionregion spanningspanning positionspositions 15,45815,458 toto 15,87715,877 inin sequencesequence numbernumber NC_003391NC_003391 (420 bpbp size).size). (B) AnalysisAnalysis of thethe regionregion spanningspanning positionspositions 33,59033,590 toto 34,49234,492 inin sequencesequence number NC_003391 (902 (902 bp bp size). size). (C (C) )Analysis Analysis of of the the region region spanning spanning positions positions 188,946 188,946 to to190,040 190,040 in insequence sequence number number NC_003391 NC_003391 (1094 (1094 bp bp size size).). The The CMLV CMLV clade clade is is shaded, shaded, and the KVIKVI (Kimron(Kimron VeterinaryVeterinary Institute)-isolated Institute)-isolated samples samples are are marked marked with with a a rectangle. rectangle.

TheThe sequencedsequenced samples shared a high degree of identity, andand werewere differentdifferent fromfrom thethe annotatedannotated CMLVCMLV isolatesisolates availableavailable fromfrom GenBankGenBank (Table(Table S1).S1). Collectively, thesethese resultsresults suggestsuggest thatthat allall ofof thethe CMLVCMLV samplessamples identiﬁedidentified inin thisthis studystudy belongbelong toto thethe samesame subtype,subtype, whichwhich is differentdifferent fromfrom thethe annotatedannotated CMLVCMLV isolatesisolates currentlycurrently availableavailable fromfrom thethe NCBINCBI GenBank.GenBank.

4.4. Discussion ThisThis studystudy reports reports the ﬁrstthe documentedfirst documented outbreak outb ofreak camelpox of camelpox disease in Israel,disease and in demonstrates Israel, and thedemonstrates diagnostic proceduresthe diagnostic used procedures for its diagnosis, used for which its diagnosis, include morphological, which include morphological, cellular, immunological, cellular, andimmunological, molecular methods. and molecu Thelar employment methods. The of employment several approaches of seve basedral approaches on different based features on different of the causativefeatures of agent the causative signiﬁcantly agent improved significantly the reliability improved of the the reliability diagnosis of [25 the]. Since diagnosis then, [25]. no other Since CMLV then, casesno other in this CMLV area werecases reported in this (Internationalarea were reported Society (International for Infectious Society Diseases–ProMED). for Infectious Diseases– ProMED).The disease occurs mainly in adult females camels, and is manifested by skin lesions on hairless skin regionsThe disease such occurs as the mainly face and in neck, adult whereas females incamels, several and cases, is manifested lesions appear by skin throughout lesions on the hairless body. Symptomsskin regions such such as as fever, the face weakness, and neck, lack whereas of appetite, in several and abortioncases, lesions were appear observed throughout with no mortality.the body. Symptoms such as fever, weakness, lack of appetite, and abortion were observed with no mortality. Since camels serve as transport and milk-producing animals, such a disease that is infectious and easily transmitted will have profound economic implications on societies that rely on camels. Eight skin and blood samples from sick camels were examined in this study. A disease involving exanthematous skin conditions in camels raises several possibilities regarding the causative agent: camelus dromedary papillomavirus, CCEV, CMLV, or co-infection with CCEV and CMLV. This necessitated the use of a differential diagnosis using several methods, the first of which was

Viruses 2018, 10, 78 11 of 13

Since camels serve as transport and milk-producing animals, such a disease that is infectious and easily transmitted will have profound economic implications on societies that rely on camels. Eight skin and blood samples from sick camels were examined in this study. A disease involving exanthematous skin conditions in camels raises several possibilities regarding the causative agent: camelus dromedary papillomavirus, CCEV, CMLV, or co-infection with CCEV and CMLV. This necessitated the use of a differential diagnosis using several methods, the first of which was diagnostic TEM [26]. It should be noted that this approach, which was performed on negatively stained samples, is fast and relatively simple, but requires expensive and complicated equipment, as well as professional expertise. Indeed, in the current study, using TEM, the viral particles could be observed easily with their characteristic brick-shape morphology, assigning them to the OPV family. For comparison, the CCEV, which causes a disease with similar symptoms, belongs to the PPV with a different morphology, which is characterized by an oval shape and a tubules’ texture with crisscross patterns. Hence, TEM is a powerful technique for differentiation between OPV and PPV [27]. Further examination and characterization was carried by the infection of CAM and Vero cells. While the infection of SPF-ECE resulted in typical pocks formation, the infected Vero cells were positive by immunofluorescence using an OPV-specific antibody. In order to determine the virus identity at the species level, PCR followed by sequence analysis was used. Of the various OPV, the primary candidate was CMLV. To establish this, we employed the C18L test [22], and due to sequence variation in the region amplified in the C18L test, as evident from CMLV genomes deposited recently in GenBank, an additional test was developed. The new PCR developed in this study was based on a non-coding genome sequence that shows significant variation among the OPV family, thus allowing the specific identification of CMLV. Using this test in parallel with the C18L PCR, we clearly demonstrated that the infecting virus is CMLV. The recent CMLV-annotated genomes suggested the presence of a different CMLV. The three endpoint PCR tests aimed to establish the subspecies identity of the isolated virus, and showed that all of the tested samples were of the same subspecies, which was distinct from the other four annotated subtypes currently available from GenBank. The regions selected for the analysis contained variations and insertions that enabled a clear distinction between CMLV and other OPV species, such as monkeypox, cowpox, and rabbitpox viruses (Figure7, Table S2). Recent studies published after the completion of this manuscript compared a large number of samples using the HA and B2L genes [28] and the C18L gene [13], which were not studied here. The completion of the full genome sequence of the newly isolated subtype (work in progress) will allow for a better comparison, and will also determine its similarity to other studied isolates. Nonetheless, this report describes the first identification of CMLV in Israel, and highlights the significance of a combined approach for the quick and definitive identification of a pathogen that had been previously unreported in this country. Such a prompt response is of major importance in order to launch effective treatment and prevention processes. Moreover, we describe new PCR tests that can (1) distinguish between CMLV and related OPV species (VACV, CPXV) that exhibit broad species tropism, and (2) determine that the new, CMLV isolate is genetically distinct from the currently annotated camelpox isolates.

5. Conclusions This study describes the ﬁrst diagnosed outbreak of camelpox virus in Israel. Although camelpox virus infects camels, it is genetically the closest relative of the variola virus, the causative agent of smallpox. Although rare, human infections of camelpox have been previously described. The ﬁndings in this study adds to the recent emergence of orthopoxvirus human infections, raising the need for adequate diagnostic approaches, as outlined in this study.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/10/2/78/s1. Acknowledgments: We are thankful to the staff of the regional Beer-Sheba veterinary bureau for their valuable help in the ﬁeld work. Viruses 2018, 10, 78 12 of 13

Author Contributions: Orly Laskar-Levy, Sharon Melamed, Oran Erster, Nir Paran, Shay Weiss, Yevgeny Khinich and Boris Gelman conceived and designed the experiments; Orly Laskar-Levy, Sharon Melamed, Oran Erster, Nir Paran, Yevgeny Khinich, Boris Gelman performed the experiments; Orly Laskar-Levy, Sharon Melamed, Oran Erster and Nir Paran analyzed the data; Aharon Solomony and Shay Weiss contributed reagents/materials/analysis tools; Orly Laskar-Levy, Sharon Melamed, Oran Erster and Nir Paran wrote the paper. Oran Erster and Sharon Melamed equal contribution. Conﬂicts of Interest: The authors declare no conﬂict of interest. Founding was based on institutional support.

References

1. Breman, J.G.; Henderson, D.A. Diagnosis and management of smallpox. N. Engl. J. Med. 2002, 346, 1300–1308. [CrossRef][PubMed] 2. Khalafalla, A.I.; Al-Busada, K.A.; El-Sabagh, I.M. Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary camels. Virol. J. 2015, 12, 102. [CrossRef][PubMed] 3. Bernard, H.U.; Burk, R.D.; Chen, Z.; van Doorslaer, K.; zur Hausen, H.; de Villiers, E.M. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 2010, 401, 70–79. [CrossRef][PubMed] 4. Bera, B.C.; Shanmugasundaram, K.; Barua, S.; Venkatesan, G.; Virmani, N.; Riyesh, T.; Gulati, B.R.; Bhanuprakash, V.; Vaid, R.K.; Kakker, N.K.; et al. Zoonotic cases of camelpox infection in India. Vet. Microbiol. 2011, 152, 29–38. [CrossRef][PubMed] 5. Khalafalla, A.I.; Abdelazim, F. Human and dromedary camel infection with camelpox virus in Eastern Sudan. Vector Borne Zoonotic Dis. 2017, 17, 281–284. [CrossRef][PubMed] 6. Dahiya, S.S.; Kumar, S.; Mehta, S.C.; Narnaware, S.D.; Singh, R.; Tuteja, F.C. Camelpox: A brief review on its epidemiology, current status and challenges. Acta Trop. 2016, 158, 32–38. [CrossRef][PubMed] 7. Duraffour, S.; Meyer, H.; Andrei, G.; Snoeck, R. Camelpox virus. Antivir. Res. 2011, 92, 167–186. [CrossRef] [PubMed] 8. Gubser, C.; Smith, G.L. The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox. J. Gen. Virol. 2002, 83, 855–872. [CrossRef][PubMed] 9. Haller, S.L.; Peng, C.; McFadden, G.; Rothenburg, S. Poxviruses and the evolution of host range and virulence. Infect. Genet. Evol. 2014, 21, 15–40. [CrossRef][PubMed] 10. Dashtseren, T.; Solovyev, B.V.; Varejka, F.; Khokhoo, A. Camel contagious ecthyma (pustular dermatitis). Acta Virol. 1984, 28, 122–127. [PubMed] 11. Fleming, S.B.; Wise, L.M.; Mercer, A.A. Molecular genetic analysis of orf virus: A poxvirus that has adapted to skin. Viruses 2015, 7, 1505–1539. [CrossRef][PubMed] 12. Friederichs, S.; Krebs, S.; Blum, H.; Wolf, E.; Lang, H.; von Buttlar, H.; Buttner, M. Comparative and retrospective molecular analysis of parapoxvirus (PPV) isolates. Virus Res. 2014, 181, 11–21. [CrossRef] [PubMed] 13. Dahiya, S.S.; Kumar, S.; Mehta, S.C.; Singh, R.; Nath, K.; Narnaware, S.D.; Tuteja, F.C. Molecular characterization of camelpox virus isolates from bikaner, India: Evidence of its endemicity. Acta Trop. 2017, 171, 1–5. [CrossRef][PubMed] 14. Melamed, S.; Paran, N.; Katz, L.; Ben-Nathan, D.; Israely, T.; Schneider, P.; Levin, R.; Lustig, S. Tail scarification with vaccinia virus lister as a model for evaluation of smallpox vaccine potency in mice. Vaccine 2007, 25, 7743–7753. [PubMed] 15. Paran, N.; Suezer, Y.; Lustig, S.; Israely, T.; Schwantes, A.; Melamed, S.; Katz, L.; Preuss, T.; Hanschmann, K.M.; Kalinke, U.; et al. Postexposure immunization with modified vaccinia virus ankara or conventional lister vaccine provides solid protection in a murine model of human smallpox. J. Infect. Dis. 2009, 199, 39–48. [CrossRef][PubMed] 16. Achdout, H.; Lustig, S.; Israely, T.; Erez, N.; Politi, B.; Tamir, H.; Israeli, O.; Waner, T.; Melamed, S.; Paran, N. Induction, treatment and prevention of eczema vaccinatum in atopic dermatitis mouse models. Vaccine 2017, 35, 4245–4254. [CrossRef][PubMed] 17. Erez, N.; Paran, N.; Maik-Rachline, G.; Politi, B.; Israely, T.; Schnider, P.; Fuchs, P.; Melamed, S.; Lustig, S. Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex. Virol. J. 2009, 6, 151. [CrossRef][PubMed] Viruses 2018, 10, 78 13 of 13

18. Moller, L.; Schunadel, L.; Nitsche, A.; Schwebke, I.; Hanisch, M.; Laue, M. Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy. Viruses 2015, 7, 666–679. [CrossRef] [PubMed] 19. Edgar, R.C. Muscle: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [CrossRef][PubMed] 20. Schwartz, S.; Kent, W.J.; Smit, A.; Zhang, Z.; Baertsch, R.; Hardison, R.C.; Haussler, D.; Miller, W. Human-mouse alignments with blastz. Genome Res. 2003, 13, 103–107. [CrossRef][PubMed] 21. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. Mega6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [CrossRef][PubMed] 22. Balamurugan, V.; Bhanuprakash, V.; Hosamani, M.; Jayappa, K.D.; Venkatesan, G.; Chauhan, B.; Singh, R.K. A polymerase chain reaction strategy for the diagnosis of camelpox. J. Vet. Diagn. Investig. 2009, 21, 231–237. [CrossRef][PubMed] 23. Moss, B. Poxvirus DNA replication. Cold Spring Harb. Perspect. Biol. 2013, 5, a010199. [CrossRef][PubMed] 24. Balamurugan, V.; Venkatesan, G.; Bhanuprakash, V.; Singh, R.K. Camelpox, an emerging orthopox viral disease. Indian J. Virol. 2013, 24, 295–305. [CrossRef][PubMed] 25. Levy, O.; Oron, C.; Paran, N.; Keysary, A.; Israeli, O.; Yitzhaki, S.; Olshevsky, U. Establishment of cell-based reporter system for diagnosis of poxvirus infection. J. Virol. Methods 2010, 167, 23–30. [CrossRef][PubMed] 26. Hazelton, P.R.; Gelderblom, H.R. Electron microscopy for rapid diagnosis of infectious agents in emergent situations. Emerg. Infect. Dis. 2003, 9, 294–303. [CrossRef][PubMed] 27. Wibbelt, G.; Tausch, S.H.; Dabrowski, P.W.; Kershaw, O.; Nitsche, A.; Schrick, L. Berlin squirrelpox virus, a new poxvirus in red squirrels, Berlin, Germany. Emerg. Infect. Dis. 2017, 23, 1726–1729. [CrossRef][PubMed] 28. Gelaye, E.; Achenbach, J.E.; Ayelet, G.; Jenberie, S.; Yami, M.; Grabherr, R.; Loitsch, A.; Diallo, A.; Lamien, C.E. Genetic characterization of poxviruses in Camelus dromedarius in Ethiopia, 2011–2014. Antivir. Res. 2016, 134, 17–25. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).