Antibody responses to equine parapoxvirus reveal a re-emerging pattern

Antibody responses to equine parapoxvirus reveal a re-emerging pattern | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antibody responses to equine parapoxvirus reveal a re-emerging pattern Jenni Virtanen, Lev Levanov, Sanna Tervo, Katja Hautala, Kirsi Aaltonen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5528230/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Parapoxviruses (PPV) cause skin and mucous membrane signs to several animal species and humans worldwide. Equine parapoxvirus (EqPPV) was first detected in a sick horse in Finland in 2013. It is potentially zoonotic, and a similar virus has been detected in humans in the USA. In winter 2021–2022, EqPPV caused a large-scale pastern dermatitis epidemic in racehorses all over Finland. Field reports suggest that similar epidemics of unverified cause have also occurred in 2015 and 2019. The aim of this study was to develop a serological test and study the immune response, seroprevalence, and history of the virus utilizing serum samples from clinical cases and archived horse samples (2012–2022). A recombinant protein-based immunofluorescent assay was established using envelope proteins B2L and F1L. EqPPV induced a fast immune response within a few days from the onset of the clinical signs. Two horses that were additionally tested a year after the disease still had similar IgG titers as a year prior. Seroprevalences in archived horse samples varied between 1.8–14.6% and peaks were observed in 2015 and 2022. The results suggest that EqPPV is a re-emerging pathogen that has a potential to cause large epidemics, bringing a need for more studies and preparedness. Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Microbiology/Virology/Pox virus Biological sciences/Microbiology/Virology/Viral epidemiology Biological sciences/Microbiology/Virology/Virus host interactions EqPPV parapoxviruses serology immunofluorescence assay western blot Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Parapoxviruses (PPV) are known to cause skin and mucous membrane lesions in several animal species worldwide, most notably in domestic and wild ruminants 1 . Five virus species are officially classified: orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV), red deer parapoxvirus (RDPV), and grey sealpox virus (GSEPV) 2 . Additionally, equine parapoxvirus (EqPPV) has recently been identified and characterized 3 . ORFV, BPSV, and PCPV are known to be zoonotic, and others are potentially zoonotic, causing painful skin lesions in humans 4,5 . In Finland, ORFV, BPSV, PCPV, and EqPPV are detected in sheep, cattle, reindeer, horses, and, occasionally, humans. EqPPV infection was first described in a severely sick horse in Finland in 2013 3,6 . Around the same time, a similar virus was identified in two humans that had been in contact with horses and donkeys in the United States 7 . The two viruses showed 99–100% nucleotide identity (100% amino acid similarity) based on a partial RNA polymerase sequence (ORF147, ORF numbers according to Delhon et al. 8 ), but the short length of the sequence prevents definite conclusions on whether findings from humans in United States and horses in Finland represent the same virus species 6 . In winter 2021–2022, a pastern dermatitis epidemic occurred in racehorses in Finland. Hundreds of horses got sick with painful skin lesions in the pastern area 9 . Clinical signs lasted for a few weeks and many sick horses had to be taken out of training, leading to financial losses. PCR screening of affected horses identified EqPPV in 23/26 tested samples from different regions of the country, making it the most likely cause of the widespread epidemic. Based on a questionnaire for case and control stables, around one third of the horses in affected stables got the disease and the median time between the onset of the signs of the first and second case was three days. Interestingly, skin symptoms in humans in contact with infected horses were reported significantly more frequently by case stables than control stables 9 . Unfortunately, lack of human samples prevented the confirmation of zoonotic transmission. The exact case number, especially before the 2021–2022 epidemic, remains unclear and the lack of serological test has prevented following the immune response of individuals and prevalence in horse population. Based on the information from the racing community and veterinarians, similar pastern dermatitis epidemics were observed in winters 2014–2015 and 2018–2019. As no samples were taken until 2021, the causes of the previous epidemics have never been confirmed in the laboratory. However, knowledge about the immune response, disease history, and population immunity would be important in predicting, diagnosing, and controlling future outbreaks. Most of the EqPPV genome has been sequenced along with a preliminary annotation 3,9 . Based on other PPVs, envelope proteins F1L (ORF059) and B2L (ORF011) are highly immunogenic and have been used for recombinant protein-based serological assays for detecting ORFV and GSEPV antibodies 10–13 . F1L (36 kDa) is a homologue to Vaccinia virus (VACV) envelope protein H3L that is localized in intracellular mature virus (IMV) membrane and involved in virus entry into host cell 14,15 . B2L (41 kDa) is a homologue to VACV F13L protein that is localized on the inner surface of the outer membrane and needed for formation of enveloped virus particles before they are released from the host cell 16,17 . EqPPV F1L gene is 60–64% identical and B2L 77–79% identical to ORFV proteins in amino acid level. The aim of this study was to establish a serological test detecting antibodies against EqPPV and study the immune response of affected horses during and after the 2021–2022 epidemic. Additionally, we aimed at acquiring information about the seroprevalence in horse population during and before the latest epidemic to understand temporal patterns and numbers of unreported infections in the disease outbreaks. Methods Serum samples Serum samples were collected from 26 horses from 12 stables during the most recent pastern dermatitis epidemic between December 2021 and March 2022. All the horses showed signs of pastern dermatitis. In twenty of the horses, EqPPV DNA had been detected by PCR, one had provided borderline results, and one had been PCR-negative 9 (Table S1 ). Four had not been tested with PCR but the clinical signs were consistent with PCR-verified cases, and two of these horses lived at stables with PCR-verified EqPPV infection (Table S1 ). In total, 24/26 tested horses, from which we received sera, lived with at least one PCR-confirmed stablemate (Table S1 ). Three horses were sampled within the first day after the clinical signs were noticed, twelve horses were sampled at some other point during the first week, and the rest were sampled at some point during the first few weeks (Table S1 , supplementary file). Additional samples were acquired from two horses (C3 and I1) a year after the disease (February 2023). Three serum samples and one whole blood sample from horses sampled for diagnostic purposes before the epidemic were used as negative controls. Three of these had been tested for parapoxviruses with PCR due to showing signs of dermatitis and had turned out negative. One had earlier been tested to be positive for orthopoxvirus (OPV) IgG antibodies as part of routine diagnostics. One had been collected for diagnostics of another viral disease. A total of 266 archived horse serum samples collected between January and March in 2012, 2015, 2019, 2021, and 2022 (Table S2, supplementary file and Fig. 1 ) were acquired from Finnish Food Authority. Most horses had been sampled for breeding purposes (180/272) or for diagnosing a disease (48/272). Additionally, 11 had been sampled for export and 25 for import reasons. Nine out of 48 horses that were sampled for disease diagnostics were suspected of having a respiratory infection, four had suspected equine viral arteritis and one a spontaneous abortion. The suspected disease was unknown for the rest (35) of these horses. Sample set included both racehorses and riding horses. Virus isolation attempt Earlier, unsuccessful EqPPV isolation attempts have been done in baby hamster kidney cells, African green monkey kidney cells, and primary bovine oesophagus cells 3,6,9 . Here, equine dermis cells and aneuploid human keratinocyte cells (HaCat) were grown in DMEM High Glucose media (Sigma-Aldrich) supplemented with 10% (for maintenance) or 2% (for infection) heat inactivated fetal bovine serum (Gibco), 100 IU/ml of penicillin (Sigma-Aldrich), 100 µg/ml of streptomycin (Sigma-Aldrich), and 2 mM of L-glutamine (Sigma-Aldrich). Hacat and NBL-6 cells were seeded to 6-well plates in density 100,000 cells/well day before the infection. Before inoculating the cells, dry swabs taken from skin lesions of horses suffering from pastern dermatitis 9 were incubated in 1 ml of Dulbecco’s phosphate buffered saline containing 0.2% of bovine serum albumin (DPBS + BSA) overnight at 4°C. Samples were filtered with 0,45 µM polyethersulfone membrane filters and inoculated into the cells for 1 h at 37 o C, 5% CO 2 . Fresh culture medium was added. Cells were observed daily for cytopathic effect and culture supernatants from each five passages were tested with EqPPV-specific PCR described earlier 9 . DNA was extracted with Nucleospin tissue kit (Macherey-Nagel). Preparing the antigen and immunofluorescence assay slides An immunofluorescence assay (IFA) was set up with a recombinant protein technique based on B2L and F1L sequences from the first detected equine case (GenBank no. OQ110635.1, OQ248663.1) 3 . The B2L and F1L genes with 6×Histidine (His) and Hemagglutinin (HA) tags on C-terminus were amplified by PCR from synthetic genes (Thermo Fisher Scientific), using appropriate primers with incorporated initiation and termination codons and flanked by KpnI and SgsI sites. Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) was used for PCR amplification according to manufacturer’s instructions. The amplified fragments were digested with KpnI/SgsI restriction enzymes (Thermo Fisher Scientific) and cloned into the pCAGGS expression vector 18 . The constructed plasmids pCAGGS-B2L and pCAGGS-F1L were transfected into Vero E6 cells in a 100-mm plate using FuGENE HD transection reagent (Promega). The transfected cells were grown in Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 10% fetal calf serum (Gibco), 100 IU/ml of penicillin (Sigma-Aldrich), 100 µg/ml of streptomycin (Sigma-Aldrich), and 2 mM of L-glutamine (Sigma-Aldrich). Seventy-two hours post-transfection, the cells were harvested, pelleted at 4000 × g for 5 min at 4°C, and washed twice with 1×PBS. The washed transfected cells were placed on microscope slides, fixed with cold acetone for 10 min, and stored at -20°C until use. The washed pelleted cells were also used as the antigen in immunoblotting. Immunofluorescence assay All the serum samples were screened for IgG antibodies against both antigens, F1L and B2L. First, the slides were blocked with DPBS + BSA (25 µl/well) for 30 min in a 37°C moist chamber and washed 3x5 min with PBS and once with ultrapure water (Milli-Q). Next, they were incubated in 37°C moist chamber for another 30 min with serum samples diluted 1:20 in DPBS + BSA (25 µl/well). First, anti-HA antibodies (BioLegend) diluted 1:1000 and later, positive serum sample from a PCR-verified patient horse were used as positive controls. After washing the slides another 3x5 min with PBS and 1x with water, they were incubated in 37°C moist chamber for additional 30 min with 25 µl/well of Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Horse IgG (H + L) (horse samples, Jackson ImmunoResearch; diluted 1:100 or 1:150 in DPBS depending on slide batch) or Anti-Mouse IgG (H + L) (HA control, diluted 1:100). Finally, slides were washed 3x5 min with PBS and once with water, dried, covered with mounting media (Epredia Immu-Mount, Thermo Scientific) and a cover glass, and checked with a fluorescent microscope (Zeiss Axioplan 2). All the IFA results were classified as positive, unclear, or negative. Clinical samples that were positive with both antigens were also titrated with a two-fold dilution series (1:20 − 1:320) against the B2L antigen. Samples from horses with dermatitis and other samples from 2022 were further tested for IgM antibodies with B2L antigen. For IgM IFA, IgG was first removed with GullSORB IgG Inactivation Reagent (Thermo Scientific): one drop was mixed with 5 µl of serum, incubated for 10 min at room temperature (RT), centrifuged for 8 sec in 11 000 g, and diluted 1:2 to acquire 1:20 dilution. The IgM IFA was performed similarly to the IgG IFA except for that instead of 30 min, serum samples were incubated for 2,5 h, and FITC-conjugated Goat anti-Equine IgM Heavy Chain secondary antibody (Novus) with a 1:100 dilution was used as secondary antibody. Samples showing high background fluorescence in IFA were repeated by first preadsorbing the sera with cells used in the transfection. Nontransfected Vero E6 cells were detached from confluent T75-bottles, washed with DPBS + BSA and split into 24 tubes. Cells were then fixed with 50 µl of + 4°C acetone for 7 min after which the acetone was removed by centrifugation and pellets were airdried. Before the IFA, serum dilutions were mixed with the cell pellets and incubated 5–10 min at RT. Cells were pelleted again by centrifugation and the sera were used in IFA as described above. All the centrifugations were done at 500 g for 5 min. Western blot The antigens used in the IFA protocols were further assessed by testing three IFA-IgG-positive and one negative serum with Western blot (WB). First, Vero E6 cell pellets containing B2L and F1L antigens and pelleted antigen-free cells were suspended in 4x Laemmli buffer and denatured for 5 min at 95°C. Five µl of cell suspension was run in SDS-PAGE gel (Mini-PROTEAN TGX Stain-Free Gels, Bio-Rad) along with 3 µl of PrecisionPlus dual color protein standard (Bio-Rad) and transferred into Amersham Prothan 0.45 µm NC nitrocellulose blotting membrane (Cytiva). After blocking the membrane in 4% milk in tris-buffered saline (TBS, Medicago) overnight at 4°C, membranes were incubated with diluted serum samples for 1 h at RT with shaking (PSU-10i Orbital Shaker, Biosan, 100 rpm). One of the positive samples was tested with 1:100 and 1:200 serum dilutions to estimate the correct dilution and the rest only with 1:200 dilution. Membrane was then washed 3x5 min in TBS + 0.05% Tween20 (TBST) and incubated with peroxidase-conjugated AffiniPure Goat Anti-Horse IgG (H + L) secondary antibody (Jackson ImmunoResearch) diluted 1:40 000 for another 1 h after which it was washed 3x5 min in TBST and 1x5 min in ultrapure water. The membrane was visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific) according to manufacturer’s instructions and imaged on X-ray film (Fujifilm Super RX Medical X-ray film). Data analysis For calculation purposes, animals were considered seropositive for EqPPV if they showed a positive reaction with both antigens and unclear if they were positive with one antigen only. Samples that were negative with both antigens were considered seronegative. Similarity of seroprevalence between groups was tested with Fisher exact test with a significance level of 0.05. All the statistical tests were done with IBM SPSS Statistics 29.0.2.0. Pictures were drawn in Jupyter Notebook utilizing Pandas, SciPy, Matplotlib, Seaborn, and NumPy packages 19–23 . Confidence intervals were calculated with Wilson score method. Ethics declaration This study relied on samples collected during routine diagnostics within a non-experimental clinical veterinary setting. Hence, no ethical approval was required (European Union directive 2010/63/EU and the national act 497/2013). Results Virus isolation We attempted to cultivate five samples positive for EqPPV RNA in equine dermis cells and aneuploid human keratinocyte cells (HaCat). Cytopathic effect (CPE), characterized by rounded cells, was observed with both cell lines in 1 of the samples 7 days after infection (passage 1). However, based on a PCR performed on culturing media (Table S3, supplementary file), CPE could not be confirmed to have been caused by EqPPV. The CPE was not seen in any further passages and Ct-values increased during each passage. Expression of EqPPV recombinant proteins and evaluating an immunofluorescence assay As the culturing attempts of EqPPV were repeatedly unsuccessful, and with almost full-length EqPPV genome sequence available 3 , we concentrated our efforts on establishing serological assays based on antigenic recombinant proteins. As previous literature showed antigenicity of homologues B2L and F1L proteins of other PPVs, we selected these for further studies. Both proteins were successfully expressed with HA and His tags from the related ORFs from pCAGGS plasmids transfected in VeroE6 cells. In IFA, a control anti-HA antibody detected the C-terminal HA tag in B2L but not in F1L protein construct suggesting that this epitope was potentially hidden in F1L (Fig. 2 ). However, positive serum samples were identified with both antigens (detailed in the following sections). The four negative control horses sampled before the epidemic were negative, including the OPV-positive horse. Based on the amino acid sequences, sizes of the proteins are 41 kDa (B2L) and 36 kDa (F1L) 9 . Bands of these sizes were detected in Western blot (WB) when using two IFA-positive serum samples against cell suspension containing these antigens whereas bands of these sizes were not detected when testing these sera against cell suspension without the antigens (Fig. 3 ). No bands were detected with an IFA-negative control serum and in one IFA-positive sample of the three tested. Due to the novelty of the virus and the lack of a golden standard test, specificity and sensitivity could not be calculated. All samples combined, IgG positivity and negativity to B2L and F1L were in 97.6% agreement in IFA. Seven samples out of 294 gave conflicting results. In all the seven cases, antibodies to F1L were positive and to B2L either negative or unclear. Antibody response of horses with pastern dermatitis In total, 79% (22/28) of samples collected from horses with pastern dermatitis between December 2021 and March 2022 were IgG positive with B2L, 86% (24/28) with FL1, and 79% (22/28) with both (Table 1 ). Seventy-three percent (73%; 11/15) of the horses sampled within a week from onset and 82% (9/11) sampled between a few weeks and few months were positive with both F1L and B2L. The two horses (C3 and I1) that were resampled a year after a PCR-verified infection were still IgG positive in both assays. Sera from two horses only were negative with both antigens, D2 [sampled 1day post-onset, borderline case in PCR] and F4 [sampled 4 days post-onset, positive in PCR] (Table S1 ). Serum from one horse, F1, [sampled between a few days and a few weeks, positive in PCR]) provided unclear result with both antigens. The results of sera from three horses (F3, G1 and H1) differed between antigens: all were positive with F1L but negative or unclear with B2L. The only sample that was negative in PCR 9 , J1, [sampled within a few days post-onset] was IgG positive with both antigens. Geometric mean titer of the positive samples was 39.8 (titer range from 20 to 160, Table S1 ) and no variation was detected between sampling groups. No clear positive samples were identified with IgM IFA. Four samples gave unclear results that could not be clearly classified as positive or negative. Three of these were from horses sampled within a week from the first detection of the clinical signs and one was sampled over 3 weeks after the onset. Table 1 The proportion of IgG positive cases among horses with pastern dermatitis, December 2021- March 2022. Sampling time after first detection of signs Seroprevalence (F1L) a , % Seroprevalence (B2L) a , % Seroprevalence (Both) a ,% A few days 86.6 (13/15) 73.3 (11/15) 73.3 (11/15) A few days-few weeks 90.9 (10/11) 81.8 (9/11) 81.8 (9/11) 1 year c 100.0 (2/2) 100.0 (2/2) 100.0 (2/2) Total 85.7 (24/28) 78.6 (22/28) 78.6 (22/28) a Reported as % (n/N) b Reported as geometric mean titer c Both horses were also sampled earlier EqPPV seroprevalence in Finland between 2012–2022 Seroprevalence of horses to EqPPV in Finland varied between 3.6 and 16.7% (F1L, p = 0.096, Table S4, supplementary file) and 1.8–14.6% (B2L, p = 0.101) between five time-points. Highest seroprevalences were detected in 2015 and 2022 with both antigens and the lowest in 2021 (Table 2 and Fig. 4 ). Seroprevalence was highest in horses sampled for breeding or disease diagnostics. None of the exported or imported horses were seropositive. No significant differences were detected between racehorses and riding horses (F1L, p = 0.073; B2L, p = 0.224). No antibodies were detected in ponies, whereas total seroprevalence in horses was 8.5% (F1L, p = 0.232; B2L, p = 0.382). Out of the positive horses, one lived in Uusimaa region (Southern Finland), five in Ostrobothnia (Western Finland), and one in Savo (Eastern Finland). Three of the positive horses had been brought to Finland from North America (#71, #94, and #137) and one from Germany (#211). Due to the frequent travelling of many of the horses, no detailed geographical analysis was done. One of the horses (#237) sampled in 2022 was positive for IgM (Fig. 5 , Table S2) and one (#231) could not be clearly defined as positive or negative. Both horses were sampled for breeding purposes in February. Table 2 Seroprevalences and Wilson score intervals (95% confidence level) grouped by sampling year, use of the horse, reason for sampling, and horse type. Numbers of positive samples and total samples are reported in brackets. Class F1L B2L Both Prevalence (%) 95% CI Prevalence (%) 95% CI Prevalence (%) 95% CI 2012 5.0 (2/40) 1.4–16.5 5.0 (2/40) 1.4–16.5 5.0 (2/40) 1.4–16.5 2015 13.6 (8/59) 7.0-24.5 10.2 (6/59) 4.7–20.5 9.8 (6/61) 4.6–19.9 2019 6.4 (4/63) 2.5–15.2 6.4 (4/63) 2.5–15.2 6.4 (4/63) 2.5–15.2 2021 3.6 (2/56) 1.0-12.1 1.8 (1/56) 0.3–9.5 1.8 (1/56) 0.3–9.5 2022 16.7 (8/48) 8.7–29.6 14.6 (7/48) 7.3–27.2 14.6 (7/48) 7.3–27.2 Racehorses 11.4 (19/167) 7.4–17.1 9.0 (15/167) 5.5–14.3 9.0 (15/167) 5.5–14.3 Riding horses 5.9 (5/85) 2.5–13.0 5.9 (5/85) 2.5–13.0 5.9 (5/85) 2.5–13.0 Others 0.0 (0/2) 0.0-65.8 0.0 (0/2) 0.0-65.8 0.0 (0/2) 0.0-65.8 Breeding 10.4 (19/182) 6.8–15.7 8.9 (16/182) 5.5–13.8 8.9 (16/182) 5.5–13.8 Import 0.0 (0/25) 0.0-13.3 0.0 (0/25) 0.0-13.3 0.0 (0/25) 0.0-13.3 Export 0.0 (0/11) 0.0-25.9 0.0 (0/11) 0.0-25.9 0.0 (0/11) 0.0-25.9 Disease 10.4 (5/48) 4.5–22.2 8.3 (4/48) 3.3–19.6 8.3 (4/48) 3.3–19.6 Finnhorses 8.5 (8/94) 4.4–15.9 6.4 (6/94) 3.0-13.2 6.4 (6/94) 3.0–13.2 Standardbreds 14.4 (13/90) 8.6–23.2 12.2 (11/90) 7.0-20.6 12.2 (11/90) 7.0–20.6 Warmbloods 6.3 (2/32) 1.7–20.1 6.3 (2/32) 1.7–20.1 6.3 (2/32) 1.7–20.1 Ponies/Icelandic horses 0.0 (0/20) 0.00-16.1 0.0 (0/20) 0.0-16.1 0.0 (0/20) 0.0–16.1 Others 0.0 (0/2) 0.00-65.8 0.0 (0/2) 0.0-65.8 0.0 (0/2) 0.0–65.8 Discussion Here, we developed a serological method to detect antibodies raised by a novel parapoxvirus, EqPPV, which caused a widespread pastern dermatitis epidemic among horses in Finland during winter 2021–2022. With this method, we analyzed serum samples from affected horses as well as archived samples to acquire information on immune response, prevalence, and history of EqPPV. Due to its novelty, pathogenesis and virulence factors of EqPPV are poorly known. Hence, the exact mechanisms of infection and host immune response can mainly be guessed based on what is known about other PPVs, especially the most extensively studied ORFV. ORFV causes both cell-mediated and humoral immune response in humans and sheep, first of which seems to play a bigger role 24 . Antibodies do not seem to give protective immunity against ORFV or BPSV and reinfections are possible, although the clinical picture may be milder in secondary infections 24–26 . Based on a preliminary annotation of EqPPV, many of the anti-inflammatory ORFV genes (including chemokine binding protein (ORF112), and at least two out of three inhibitors of the NF-kB signaling pathway (ORF024 ja ORF121)) are also found in EqPPV genome, even though confirming that they share a same function would need further experimentation. In ORFV, both contribute to the ability of the virus to evade host immune response 24,27 . Due to a few gaps in the EqPPV sequence, we cannot rule out that we are still missing some genes. Based on current data, it is also not possible to tell if and how fast horses can be reinfected by EqPPV and if the second infection is milder. Even though the clinical signs associated with EqPPV are mainly restricted to the skin of pastern area, our serological data demonstrate that the infection is severe and deep enough to stimulate a quick and long-lasting immune response. IgG antibodies could be detected in most affected horses during the first few days after onset and still prevailed a year after the disease. This is faster than expected based on knowledge on other PPVs. For example, IgG response was observed around two weeks after the primary infection in experimental infections of sheep with ORFV and cows with BPSV 24,28 . Some of the difference of EqPPV to other PPVs could be explained by the fact that the IgG response in experimental infections has been measured from the infection whereas we measured it from the time when the clinical signs were first detected, not taking incubation period or potential delay between the onset and its detection into account. However, since reports from the field suggest that the incubation time likely is no more than a few days (median time between 1st and 2nd case of affected stables being 3 days), IgG response can still be considered fast 9 . Another study detected a more rapid increase in IgG titer against ORFV in sheep after secondary infection four weeks after the primary infection 25 . In this case, it is unlikely that all the horses had been exposed to EqPPV before. Based on reports and photos from the field 9 , clinical signs were relatively severe in comparison with other PPVs, potentially explaining the fast IgG response. Despite other PPVs generally causing only a short-lived protective immunity, a detectable antibody response has been observed in a cow experimentally infected with BPSV even three years after the infection even though it should be noted that subcutaneous experimental infection does not fully resemble natural infection 28 . In association with EqPPV infection, a follow-up longer than one year would be needed to determine the duration of IgG response. There is a slight chance that some of the antibodies of the clinical cases are a result of a previous infection. However, this would likely be a minor proportion of the cases based on our data on seroprevalence of the archived samples (1.79% in 2021). Also, most of the horses were PCR-positive at the time of the sampling and were hence known to be infected. Whether detectable IgG antibodies are protective and how big a role T cell responses may play in EqPPV infection remains to be clarified in further studies. Since the culturing attempts were unsuccessful- which is not uncommon for parapoxviruses 29 - serological assay was set up with a recombinant-protein based technique relying on two known immunogenic proteins, B2L and F1L, instead of the whole virus. Based on the results, IFA was able to detect IgG with both the antigens with good agreement. Detection by HA antibody failed with F1L, possibly due to the short length of the HA tag which may be folded so that is inaccessible for the antibodies. The WB analysis confirmed the existence of proteins that were close to the expected sizes and were not detectable in antigen-free cell suspension in two out of three tested positive samples. The negative result of the third IFA positive sample in WB can be explained by a possibly lower sensitivity of WB: when one of the IFA positive samples was tested with two different serum dilutions (1:100 and 1:200), the result was positive with 1:100 dilution only. When relatively low mean titer of 40 is also considered, it is probable that the antibody titer in some of the samples is too low for WB to detect with standard 1:200 dilution. On the other hand, high background may make smaller serum dilutions more difficult to read. Due to the lack of a golden standard test, specificity and sensitivity of the IFA could not be calculated. One option for the future would be a virus neutralization test for which, however, a virus isolate is needed first. Our EqPPV isolation attempts have been unsuccessful so far. Another option for validation would be a larger sample panel from PCR-verified cases 2–6 weeks from infection. When it comes to specificity of the assay, a panel of certainly negative samples is challenging to find. For that, a panel of healthy horses born after the previous outbreak would be the most potential solution. Cross-reaction with other poxviruses that can infect horses cannot be fully excluded. However, considering that the OPV-positive serum sample was negative with this assay, cross-reaction is unlikely. Still, cross-reaction with other PPVs remains possible, but those have not been reported in horses as far as we know. To maximize sensitivity, F1L antigen should be used in screening, because a few samples were clearly positive only with this one and negative or unclear with B2L. This implies that F1L is either slightly more sensitive or less specific than B2L. No clear IgM antibodies were detected in horses that had pastern dermatitis. However, possible positive samples were detected in archived samples from early 2022. Detecting IgM in general horse population at that time is possible as that was the peak of the epidemic, and the epidemic was suspected to affect hundreds of horses 9 . An IgM-positive horse sampled for breeding in 2022 may have been infected at the time of the sampling but not displaying clear clinical signs yet. We also don’t know if breeding horses were clinically healthy at the time of the sampling or if asymptomatic infections are possible. When it comes to clinical cases, it is possible that IgM response is so short that it had gone down by the time the clinical signs were spotted and samples were taken. However, these IgM assays could lack sensitivity and the results should be considered uncertain due to the lack of a positive control for protocol optimization, and any alternative test for confirming the results. In comparison, ORFV has been shown to induce a detectable IgM response in most animals that show clinical signs 30 . Data on archived samples indicate that EqPPV has been circulating in the Finnish horse population for several years. Based on one equine serum sample from 2012 that was confirmed to be IgG-antibody-positive with both antigens, the virus has been circulating even before the first report in 2013. Peaking seroprevalences were detected in 2015 and 2022, well in line with the field reports claiming that epidemics have been raging before 2021–2022, most notable year being 2015. However, the differences between years were not statistically significant, possibly impacted by our limited sample size. Peak in 2019, another claimed epidemic year, was less clear, which may be either due to the sample size, smaller scale of the epidemic, or a different causative agent. This is the first step towards a serological assay for this new pathogen. In the future, larger sample sets, dating further back and potentially focusing on racehorses should be tested. As testing of hundreds of samples with IFA is resource intensive and the test is subjective, setting up a test that is more quantitative and more suitable for large sample panels (e.g. enzyme-linked immune sorbent assay) would be beneficial. The 2021–2022 epidemic was only reported in racehorses. In archived samples, antibodies were also found in riding horses, although with a slightly lower seroprevalence (nonsignificant difference). Still, it is important to raise awareness among riders that riding horses can also be infected and competition events should be avoided if the horse shows signs of EqPPV infection. It is unknown if the horses are infectious before the onset of the clinical signs, making it difficult to give clear instructions on whether healthy horses from stables that have the disease can pose a transmission risk. However, frequent movement of the horses and suspicions that most introductions into stables came from racing events 9 , make competitions and other events a potential platform for the large-scale spread of the virus. Antibodies were detected in all three horse types that were tested. No antibodies were detected in ponies but the number of sampled ponies was insufficient and most likely they are also at risk for infection if they are exposed to EqPPV. It should also be noted that some sampling biases may be caused by different distribution of horse uses, types, and reasons for sampling between groups (Fig. 1 ). For example, it may be that some groups (racehorses vs riding horses and horses vs ponies) attend less commonly events where the virus spreads. After the epidemic in 2022, no cases have been detected despite ongoing diagnostic surveillance of dermatitis cases. One factor that may explain the clearly cyclic nature of the disease would be a protective herd immunity of the horse population. When the herd immunity decreases, a new epidemic arises. The seroprevalence of less than 20% detected in this study is not high enough to fully support this hypothesis. However, the sensitivity of this pilot detection is unknown, and if we only look at the high-risk horses that actively participate in racing events, seroprevalence may be higher and contribute to some level of herd immunity among racehorses. As many poxviruses are known to jump from one species into another, the role of other reservoir or amplification animals (e.g. rodents) should also be considered. To our knowledge, this is the 1st serological study on this novel virus and provides new information for predicting and controlling future epidemics. Based on the reports from the field and the results of this study, further epidemics are likely within the next few years. No cases have been reported in horses outside Finland so far either because the virus has not spread to other countries, or it has not been recognized and reported. Considering the findings in humans in the USA and racehorses travelling between countries, the latter explanation is more likely. For example, some of the seropositive horses in this study had been imported to Finland from other countries, including the USA, and it is not possible to conclude weather they were infected in Finland or not. In the future, serological screening should be broadened to other countries to understand the global distribution of the virus. Declarations Acknowledgements We thank DVM, PhD Heli Koskinen for her support in the beginning of the project, Mikko Virta for assistance with collecting the archived samples, and Jonas Wensman et al. (Swedish Veterinary Agency) for providing the equine dermis cells. We also thank the veterinarians and owners who contributed to getting the serum samples. Author’s contributions Conceptualization: JV, PMK, KH, KA. Data curation: PMK, KH, TG, LK. Formal Analysis: JV. Funding acquisition: JV, PMK, KH. Investigation: ST, LL, MU. Methodology: JV, PMK, ST, LL, MU, KA. Project administration: JV, PMK. Resources: OV, TS. Supervision: JV, PMK. Validation: JV, LL, OV, MU. Visualization: JV. Writing – original draft: JV. Writing – review & editing: JV, PMK, MU, OV. All authors read and approved the final manuscript. Data availability The datasets supporting the conclusions of this article are included within the article and its supplementary file. Funding This study was funded by Finnish Veterinary Foundation (JV), Finnish Foundation of Veterinary Research (JV), Niemi Foundation (PMK), Erkki Rajakoski foundation (PMK), Finnish Foundation for Research on Viral Diseases (KH), Orion research foundation (JV), and Sakari Alhopuro Foundation (JV). Conflicts of interests Besides holding the title of Associate Professor (Docent) of University of Helsinki, PMK is an employee of MSD Animal Health. The EqPPV studies were initiated before her joining the company. References Essbauer, S., Pfeffer, M. & Meyer, H. Zoonotic poxviruses. Vet Microbiol 140 , 229–236 (2010). https://doi.org/10.1016/j.vetmic.2009.08.026 McInnes, C. J. et al. ICTV Virus Taxonomy Profile: Poxviridae 2023. J Gen Virol 104 (2023). https://doi.org/10.1099/jgv.0.001849 Virtanen, J. et al. Partial Genome Characterization of Novel Parapoxvirus in Horse, Finland. Emerg Infect Dis 29 , 1941–1944 (2023). https://doi.org/10.3201/eid2909.230049 Gaspari, V. et al. Are Parapoxvirus zoonotic diseases doomed to remain neglected? New Microbiol 45 , 358–362 (2022). Khmaladze, E. et al. Geographic distribution and genetic characterization of poxviruses from human infections in Georgia, 2009–2014. Arch Virol 166 , 1729–1733 (2021). https://doi.org/10.1007/s00705-020-04922-x Airas, N. et al. Infection with Possible Novel Parapoxvirus in Horse, Finland, 2013. Emerg Infect Dis 22 , 1242–1245 (2016). https://doi.org/10.3201/eid2207.151636 Osadebe, L. U. et al. Novel poxvirus infection in 2 patients from the United States. Clin Infect Dis 60 , 195–202 (2015). https://doi.org/10.1093/cid/ciu790 Delhon, G. et al. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus. J Virol 78 , 168–177 (2004). https://doi.org/10.1128/jvi.78.1.168-177.2004 Virtanen, J. et al. Equine dermatitis outbreak associated with parapoxvirus. J Gen Virol 104 (2023). https://doi.org/10.1099/jgv.0.001940 Housawi, F. M. et al. The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein. Arch Virol 143 , 2289–2303 (1998). https://doi.org/10.1007/s007050050461 Badr, Y. et al. A New Enzyme-linked Immunosorbent Assay for Serological Diagnosis of Seal Parapoxvirus Infection in Marine Mammals. J Vet Res 66 , 43–52 (2022). https://doi.org/10.2478/jvetres-2022-0005 Yogisharadhya, R., Kumar, A., Bhanuprakash, V. & Shivachandra, S. B. Evaluation of a recombinant major envelope protein (F1L) based indirect- ELISA for sero-diagnosis of orf in sheep and goats. J Virol Methods 261 , 112–120 (2018). https://doi.org/10.1016/j.jviromet.2018.08.015 Yogisharadhya, R. et al. Functional characterization of recombinant major envelope protein (rB2L) of orf virus. Arch Virol 162 , 953–962 (2017). https://doi.org/10.1007/s00705-016-3178-z Scagliarini, A. et al. Heparin binding activity of orf virus F1L protein. Virus Res 105 , 107–112 (2004). https://doi.org/10.1016/j.virusres.2004.04.018 Jensen, O. N. et al. Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis. J Virol 70 , 7485–7497 (1996). https://doi.org/10.1128/JVI.70.11.7485-7497.1996 Sullivan, J. T., Mercer, A. A., Fleming, S. B. & Robinson, A. J. Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K. Virology 202 , 968–973 (1994). https://doi.org/10.1006/viro.1994.1420 Schmutz, C., Rindisbacher, L., Galmiche, M. C. & Wittek, R. Biochemical analysis of the major vaccinia virus envelope antigen. Virology 213 , 19–27 (1995). https://doi.org/10.1006/viro.1995.1542 Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108 , 193–199 (1991). https://doi.org/10.1016/0378-1119(91)90434-d Harris, C. R. et al. Array programming with NumPy. Nature 585 , 357–362 (2020). https://doi.org/10.1038/s41586-020-2649-2 Hunter, J. D. Matplotlib: A 2D graphics environment. Computing in Science & Engineering 9 , 90–95 (2007). McKinney, W. Data structures for statistical computing in python. Proceedings of the 9th Python in Science Conference , 51–56 (2010). Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods 17 , 261–272 (2020). https://doi.org/10.1038/s41592-019-0686-2 Waskom, M. et al. mwaskom/seaborn: v0.8.1 (September 2017). Zenodo (2017). https://doi.org/doi.org/10.5281/zenodo.883859 Bergqvist, C., Kurban, M. & Abbas, O. Orf virus infection. Rev Med Virol 27 (2017). https://doi.org/10.1002/rmv.1932 Yirrell, D. L., Reid, H. W., Norval, M. & Howie, S. E. Immune response of lambs to experimental infection with Orf virus. Vet Immunol Immunopathol 22 , 321–332 (1989). https://doi.org/10.1016/0165-2427(89)90168-2 Huang, T. et al. Coinfection with multiple strains of bovine papular stomatitis virus. Arch Virol 160 , 1527–1532 (2015). https://doi.org/10.1007/s00705-015-2394-2 Haig, D. M. Orf virus infection and host immunity. Curr Opin Infect Dis 19 , 127–131 (2006). https://doi.org/10.1097/01.qco.0000216622.75326.ef Iketani, Y. et al. Persistent parapoxvirus infection in cattle. Microbiol Immunol 46 , 285–291 (2002). https://doi.org/10.1111/j.1348-0421.2002.tb02697.x Suzuki, T. et al. Isolation and antibody prevalence of a parapoxvirus in wild Japanese serows (Capricornis crispus). J Wildl Dis 29 , 384–389 (1993). https://doi.org/10.7589/0090-3558-29.3.384 Azwai, S. M., Carter, S. D. & Woldehiwet, Z. Immune responses of the camel (Camelus dromedarius) to contagious ecthyma (Orf) virus infection. Vet Microbiol 47 , 119–131 (1995). https://doi.org/10.1016/0378-1135(95)00055-f Additional Declarations Competing interest reported. Besides holding the title of Associate Professor (Docent) of University of Helsinki, PMK is an employee of MSD Animal Health. The EqPPV studies were initiated before her joining the company. Other authors have nothing to declare. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5528230","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":410944582,"identity":"a06eb588-553b-4958-be4b-5585f4cc3702","order_by":0,"name":"Jenni Virtanen","email":"data:image/png;base64,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","orcid":"","institution":"University of Helsinki","correspondingAuthor":true,"prefix":"","firstName":"Jenni","middleName":"","lastName":"Virtanen","suffix":""},{"id":410944583,"identity":"c47a7025-ac52-4207-8351-b1d5062c3168","order_by":1,"name":"Lev Levanov","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Lev","middleName":"","lastName":"Levanov","suffix":""},{"id":410944584,"identity":"b863f625-62d4-4a84-a8c5-f8fe9e264932","order_by":2,"name":"Sanna Tervo","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Sanna","middleName":"","lastName":"Tervo","suffix":""},{"id":410944585,"identity":"89dbeb9f-6a38-431a-aab2-64406c0abfa1","order_by":3,"name":"Katja Hautala","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Katja","middleName":"","lastName":"Hautala","suffix":""},{"id":410944586,"identity":"11a8026c-ce9d-4c6c-ab9a-a017e3122b57","order_by":4,"name":"Kirsi Aaltonen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Kirsi","middleName":"","lastName":"Aaltonen","suffix":""},{"id":410944587,"identity":"73f9d3a6-6ba4-4697-9df3-1214227aab76","order_by":5,"name":"Mira Utriainen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Mira","middleName":"","lastName":"Utriainen","suffix":""},{"id":410944588,"identity":"8cc0cd84-f4c5-43f4-8de1-6418e293964d","order_by":6,"name":"Lauri Kareinen","email":"","orcid":"","institution":"Finnish Food Authority","correspondingAuthor":false,"prefix":"","firstName":"Lauri","middleName":"","lastName":"Kareinen","suffix":""},{"id":410944590,"identity":"154e2b42-cd64-4bbd-94cc-65cb7a3eeccc","order_by":7,"name":"Tuija Gadd","email":"","orcid":"","institution":"Finnish Food Authority","correspondingAuthor":false,"prefix":"","firstName":"Tuija","middleName":"","lastName":"Gadd","suffix":""},{"id":410944592,"identity":"7e6351a7-265f-4833-bc4c-c81f69572528","order_by":8,"name":"Tarja Sironen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Tarja","middleName":"","lastName":"Sironen","suffix":""},{"id":410944593,"identity":"d19ddc40-9cec-42bf-bc85-1dc59c80846f","order_by":9,"name":"Olli Vapalahti","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Olli","middleName":"","lastName":"Vapalahti","suffix":""},{"id":410944594,"identity":"8e1d78f2-22d4-4507-a2fc-7b174914ebf8","order_by":10,"name":"Paula Kinnunen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Kinnunen","suffix":""}],"badges":[],"createdAt":"2024-11-26 13:23:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5528230/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5528230/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75704862,"identity":"caa69a8b-f7dd-451b-9e92-3b4a79b2858c","added_by":"auto","created_at":"2025-02-07 10:06:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40844,"visible":true,"origin":"","legend":"\u003cp\u003eMetadata on archived samples. Distribution of reason for sampling (\u003cstrong\u003eA\u003c/strong\u003e), use (\u003cstrong\u003eB\u003c/strong\u003e), and type (\u003cstrong\u003eC\u003c/strong\u003eand \u003cstrong\u003eD\u003c/strong\u003e) grouped by collection year (\u003cstrong\u003eA-C\u003c/strong\u003e) and type (\u003cstrong\u003eD\u003c/strong\u003e). Sample numbers are shown above the pictures.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/5b59a1fcb864c87d1f0b070c.png"},{"id":75704867,"identity":"9b091308-6cc2-4bdc-960e-d5ea6dc3bd9f","added_by":"auto","created_at":"2025-02-07 10:06:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":210119,"visible":true,"origin":"","legend":"\u003cp\u003eEquine parapoxvirus immunofluorescence assay. Positive serum samples (\u003cstrong\u003eA, B\u003c/strong\u003e), negative serum samples (\u003cstrong\u003eC, D\u003c/strong\u003e) and HA-control (\u003cstrong\u003eE, F\u003c/strong\u003e) with F1L (\u003cstrong\u003eA, C, \u003c/strong\u003eand \u003cstrong\u003eE\u003c/strong\u003e) and B2L (\u003cstrong\u003eB, D,\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e) antigens (x20).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/4313dff49343b8ce47fc8a1f.jpeg"},{"id":75705682,"identity":"1471559c-3ee8-4349-a462-104e18214c8b","added_by":"auto","created_at":"2025-02-07 10:14:34","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118904,"visible":true,"origin":"","legend":"\u003cp\u003eEquine parapoxvirus Western blot assay based on F1L and B2L antigens, used with horse sera. Serum from a EqPPV-PCR and IFA-negative horse (A), sera from PCR and IFA-positive horses collected \u0026lt;1 week (B, horse B1) and 1-2 months (C-D, horse K1) after the onset of the disease, and serum from a horse that had a PCR and IFA-confirmed EqPPV infection 1 year earlier (E, horse C3). 1st lane of each subpicture has cell suspension with the antigen, and the 2nd lane has cell suspension without the antigen. Sample from horse K1 was tested with two serum dilutions (C: 1:100 and D: 1:200). Other samples were tested with 1:200 dilution only. Expected protein sizes (36 kDa for F1L and 41 kDa for B2L) are marked with arrows. Each subplot represents an individual sample on a separate membrane and size markers (Precision Plus Protein Dual Color Standard) were included in each membrane (not shown separately for each subplot). Sample lanes are uncropped.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/b41a924420520c5c9c8e0d82.jpeg"},{"id":75704863,"identity":"e7b3ffad-9c1f-4f94-89e6-637f812c7131","added_by":"auto","created_at":"2025-02-07 10:06:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49743,"visible":true,"origin":"","legend":"\u003cp\u003eEquine parapoxvirus IgG seroprevalences in Finnish horses. Samples have been classified by collection year (\u003cstrong\u003eA\u003c/strong\u003e), the reason for collection (\u003cstrong\u003eB\u003c/strong\u003e), use of the horse (\u003cstrong\u003eC\u003c/strong\u003e), and horse type (\u003cstrong\u003eD\u003c/strong\u003e). Error bars represent 95 % confidence intervals (Wilson score method).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/00a9a0a3993dda2580157fd3.png"},{"id":75704520,"identity":"d0656618-b5e7-48af-8972-70ab3313ff6c","added_by":"auto","created_at":"2025-02-07 09:58:34","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154334,"visible":true,"origin":"","legend":"\u003cp\u003eIgM IFA. A possible IgM positive serum sample (\u003cstrong\u003eA\u003c/strong\u003e) and IFA negative sample (\u003cstrong\u003eB\u003c/strong\u003e) from early 2022 (x20).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/770e8925181c67f77a9adfae.jpeg"},{"id":78872878,"identity":"11e696cd-d558-4a4d-b5ca-ef63541ef884","added_by":"auto","created_at":"2025-03-20 06:32:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1249522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/b08e7641-9593-4470-8a4b-950ccfb5c454.pdf"},{"id":75704518,"identity":"3918a8c9-0f68-4d32-8ce6-e0bbe8b52c96","added_by":"auto","created_at":"2025-02-07 09:58:34","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37629,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5528230/v1/86b044a4cbff4eb5426a8509.xlsx"}],"financialInterests":"Competing interest reported. Besides holding the title of Associate Professor (Docent) of University of Helsinki, PMK is an employee of MSD Animal Health. The EqPPV studies were initiated before her joining the company. Other authors have nothing to declare.","formattedTitle":"Antibody responses to equine parapoxvirus reveal a re-emerging pattern","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParapoxviruses (PPV) are known to cause skin and mucous membrane lesions in several animal species worldwide, most notably in domestic and wild ruminants \u003csup\u003e1\u003c/sup\u003e. Five virus species are officially classified: orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV), red deer parapoxvirus (RDPV), and grey sealpox virus (GSEPV) \u003csup\u003e2\u003c/sup\u003e. Additionally, equine parapoxvirus (EqPPV) has recently been identified and characterized \u003csup\u003e3\u003c/sup\u003e. ORFV, BPSV, and PCPV are known to be zoonotic, and others are potentially zoonotic, causing painful skin lesions in humans \u003csup\u003e4,5\u003c/sup\u003e. In Finland, ORFV, BPSV, PCPV, and EqPPV are detected in sheep, cattle, reindeer, horses, and, occasionally, humans.\u003c/p\u003e \u003cp\u003eEqPPV infection was first described in a severely sick horse in Finland in 2013 \u003csup\u003e3,6\u003c/sup\u003e. Around the same time, a similar virus was identified in two humans that had been in contact with horses and donkeys in the United States \u003csup\u003e7\u003c/sup\u003e. The two viruses showed 99–100% nucleotide identity (100% amino acid similarity) based on a partial RNA polymerase sequence (ORF147, ORF numbers according to Delhon et al. \u003csup\u003e8\u003c/sup\u003e), but the short length of the sequence prevents definite conclusions on whether findings from humans in United States and horses in Finland represent the same virus species \u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn winter 2021–2022, a pastern dermatitis epidemic occurred in racehorses in Finland. Hundreds of horses got sick with painful skin lesions in the pastern area \u003csup\u003e9\u003c/sup\u003e. Clinical signs lasted for a few weeks and many sick horses had to be taken out of training, leading to financial losses. PCR screening of affected horses identified EqPPV in 23/26 tested samples from different regions of the country, making it the most likely cause of the widespread epidemic. Based on a questionnaire for case and control stables, around one third of the horses in affected stables got the disease and the median time between the onset of the signs of the first and second case was three days. Interestingly, skin symptoms in humans in contact with infected horses were reported significantly more frequently by case stables than control stables \u003csup\u003e9\u003c/sup\u003e. Unfortunately, lack of human samples prevented the confirmation of zoonotic transmission.\u003c/p\u003e \u003cp\u003eThe exact case number, especially before the 2021–2022 epidemic, remains unclear and the lack of serological test has prevented following the immune response of individuals and prevalence in horse population. Based on the information from the racing community and veterinarians, similar pastern dermatitis epidemics were observed in winters 2014–2015 and 2018–2019. As no samples were taken until 2021, the causes of the previous epidemics have never been confirmed in the laboratory. However, knowledge about the immune response, disease history, and population immunity would be important in predicting, diagnosing, and controlling future outbreaks.\u003c/p\u003e \u003cp\u003eMost of the EqPPV genome has been sequenced along with a preliminary annotation \u003csup\u003e3,9\u003c/sup\u003e. Based on other PPVs, envelope proteins F1L (ORF059) and B2L (ORF011) are highly immunogenic and have been used for recombinant protein-based serological assays for detecting ORFV and GSEPV antibodies \u003csup\u003e10–13\u003c/sup\u003e. F1L (36 kDa) is a homologue to Vaccinia virus (VACV) envelope protein H3L that is localized in intracellular mature virus (IMV) membrane and involved in virus entry into host cell \u003csup\u003e14,15\u003c/sup\u003e. B2L (41 kDa) is a homologue to VACV F13L protein that is localized on the inner surface of the outer membrane and needed for formation of enveloped virus particles before they are released from the host cell \u003csup\u003e16,17\u003c/sup\u003e. EqPPV F1L gene is 60–64% identical and B2L 77–79% identical to ORFV proteins in amino acid level.\u003c/p\u003e \u003cp\u003eThe aim of this study was to establish a serological test detecting antibodies against EqPPV and study the immune response of affected horses during and after the 2021–2022 epidemic. Additionally, we aimed at acquiring information about the seroprevalence in horse population during and before the latest epidemic to understand temporal patterns and numbers of unreported infections in the disease outbreaks.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eSerum samples\u003c/p\u003e\u003cp\u003eSerum samples were collected from 26 horses from 12 stables during the most recent pastern dermatitis epidemic between December 2021 and March 2022. All the horses showed signs of pastern dermatitis. In twenty of the horses, EqPPV DNA had been detected by PCR, one had provided borderline results, and one had been PCR-negative \u003csup\u003e9\u003c/sup\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Four had not been tested with PCR but the clinical signs were consistent with PCR-verified cases, and two of these horses lived at stables with PCR-verified EqPPV infection (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In total, 24/26 tested horses, from which we received sera, lived with at least one PCR-confirmed stablemate (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThree horses were sampled within the first day after the clinical signs were noticed, twelve horses were sampled at some other point during the first week, and the rest were sampled at some point during the first few weeks (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, supplementary file). Additional samples were acquired from two horses (C3 and I1) a year after the disease (February 2023).\u003c/p\u003e\u003cp\u003eThree serum samples and one whole blood sample from horses sampled for diagnostic purposes before the epidemic were used as negative controls. Three of these had been tested for parapoxviruses with PCR due to showing signs of dermatitis and had turned out negative. One had earlier been tested to be positive for orthopoxvirus (OPV) IgG antibodies as part of routine diagnostics. One had been collected for diagnostics of another viral disease.\u003c/p\u003e\u003cp\u003eA total of 266 archived horse serum samples collected between January and March in 2012, 2015, 2019, 2021, and 2022 (Table S2, supplementary file and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were acquired from Finnish Food Authority. Most horses had been sampled for breeding purposes (180/272) or for diagnosing a disease (48/272). Additionally, 11 had been sampled for export and 25 for import reasons. Nine out of 48 horses that were sampled for disease diagnostics were suspected of having a respiratory infection, four had suspected equine viral arteritis and one a spontaneous abortion. The suspected disease was unknown for the rest (35) of these horses. Sample set included both racehorses and riding horses.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eVirus isolation attempt\u003c/p\u003e\u003cp\u003eEarlier, unsuccessful EqPPV isolation attempts have been done in baby hamster kidney cells, African green monkey kidney cells, and primary bovine oesophagus cells \u003csup\u003e3,6,9\u003c/sup\u003e. Here, equine dermis cells and aneuploid human keratinocyte cells (HaCat) were grown in DMEM High Glucose media (Sigma-Aldrich) supplemented with 10% (for maintenance) or 2% (for infection) heat inactivated fetal bovine serum (Gibco), 100 IU/ml of penicillin (Sigma-Aldrich), 100 µg/ml of streptomycin (Sigma-Aldrich), and 2 mM of L-glutamine (Sigma-Aldrich). Hacat and NBL-6 cells were seeded to 6-well plates in density 100,000 cells/well day before the infection. Before inoculating the cells, dry swabs taken from skin lesions of horses suffering from pastern dermatitis \u003csup\u003e9\u003c/sup\u003e were incubated in 1 ml of Dulbecco’s phosphate buffered saline containing 0.2% of bovine serum albumin (DPBS + BSA) overnight at 4°C. Samples were filtered with 0,45 µM polyethersulfone membrane filters and inoculated into the cells for 1 h at 37\u003csup\u003eo\u003c/sup\u003eC, 5% CO\u003csub\u003e2\u003c/sub\u003e. Fresh culture medium was added. Cells were observed daily for cytopathic effect and culture supernatants from each five passages were tested with EqPPV-specific PCR described earlier \u003csup\u003e9\u003c/sup\u003e. DNA was extracted with Nucleospin tissue kit (Macherey-Nagel).\u003c/p\u003e\u003cp\u003ePreparing the antigen and immunofluorescence assay slides\u003c/p\u003e\u003cp\u003eAn immunofluorescence assay (IFA) was set up with a recombinant protein technique based on B2L and F1L sequences from the first detected equine case (GenBank no. OQ110635.1, OQ248663.1)\u003csup\u003e3\u003c/sup\u003e. The B2L and F1L genes with 6×Histidine (His) and Hemagglutinin (HA) tags on C-terminus were amplified by PCR from synthetic genes (Thermo Fisher Scientific), using appropriate primers with incorporated initiation and termination codons and flanked by KpnI and SgsI sites. Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) was used for PCR amplification according to manufacturer’s instructions. The amplified fragments were digested with KpnI/SgsI restriction enzymes (Thermo Fisher Scientific) and cloned into the pCAGGS expression vector \u003csup\u003e18\u003c/sup\u003e. The constructed plasmids pCAGGS-B2L and pCAGGS-F1L were transfected into Vero E6 cells in a 100-mm plate using FuGENE HD transection reagent (Promega). The transfected cells were grown in Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 10% fetal calf serum (Gibco), 100 IU/ml of penicillin (Sigma-Aldrich), 100 µg/ml of streptomycin (Sigma-Aldrich), and 2 mM of L-glutamine (Sigma-Aldrich). Seventy-two hours post-transfection, the cells were harvested, pelleted at 4000 × g for 5 min at 4°C, and washed twice with 1×PBS. The washed transfected cells were placed on microscope slides, fixed with cold acetone for 10 min, and stored at -20°C until use. The washed pelleted cells were also used as the antigen in immunoblotting.\u003c/p\u003e\u003cp\u003eImmunofluorescence assay\u003c/p\u003e\u003cp\u003eAll the serum samples were screened for IgG antibodies against both antigens, F1L and B2L. First, the slides were blocked with DPBS + BSA (25 µl/well) for 30 min in a 37°C moist chamber and washed 3x5 min with PBS and once with ultrapure water (Milli-Q). Next, they were incubated in 37°C moist chamber for another 30 min with serum samples diluted 1:20 in DPBS + BSA (25 µl/well). First, anti-HA antibodies (BioLegend) diluted 1:1000 and later, positive serum sample from a PCR-verified patient horse were used as positive controls. After washing the slides another 3x5 min with PBS and 1x with water, they were incubated in 37°C moist chamber for additional 30 min with 25 µl/well of Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Horse IgG (H + L) (horse samples, Jackson ImmunoResearch; diluted 1:100 or 1:150 in DPBS depending on slide batch) or Anti-Mouse IgG (H + L) (HA control, diluted 1:100). Finally, slides were washed 3x5 min with PBS and once with water, dried, covered with mounting media (Epredia Immu-Mount, Thermo Scientific) and a cover glass, and checked with a fluorescent microscope (Zeiss Axioplan 2). All the IFA results were classified as positive, unclear, or negative. Clinical samples that were positive with both antigens were also titrated with a two-fold dilution series (1:20 − 1:320) against the B2L antigen.\u003c/p\u003e\u003cp\u003eSamples from horses with dermatitis and other samples from 2022 were further tested for IgM antibodies with B2L antigen. For IgM IFA, IgG was first removed with GullSORB IgG Inactivation Reagent (Thermo Scientific): one drop was mixed with 5 µl of serum, incubated for 10 min at room temperature (RT), centrifuged for 8 sec in 11 000 g, and diluted 1:2 to acquire 1:20 dilution. The IgM IFA was performed similarly to the IgG IFA except for that instead of 30 min, serum samples were incubated for 2,5 h, and FITC-conjugated Goat anti-Equine IgM Heavy Chain secondary antibody (Novus) with a 1:100 dilution was used as secondary antibody.\u003c/p\u003e\u003cp\u003eSamples showing high background fluorescence in IFA were repeated by first preadsorbing the sera with cells used in the transfection. Nontransfected Vero E6 cells were detached from confluent T75-bottles, washed with DPBS + BSA and split into 24 tubes. Cells were then fixed with 50 µl of + 4°C acetone for 7 min after which the acetone was removed by centrifugation and pellets were airdried. Before the IFA, serum dilutions were mixed with the cell pellets and incubated 5–10 min at RT. Cells were pelleted again by centrifugation and the sera were used in IFA as described above. All the centrifugations were done at 500 g for 5 min.\u003c/p\u003e\u003cp\u003eWestern blot\u003c/p\u003e\u003cp\u003eThe antigens used in the IFA protocols were further assessed by testing three IFA-IgG-positive and one negative serum with Western blot (WB). First, Vero E6 cell pellets containing B2L and F1L antigens and pelleted antigen-free cells were suspended in 4x Laemmli buffer and denatured for 5 min at 95°C. Five µl of cell suspension was run in SDS-PAGE gel (Mini-PROTEAN TGX Stain-Free Gels, Bio-Rad) along with 3 µl of PrecisionPlus dual color protein standard (Bio-Rad) and transferred into Amersham Prothan 0.45 µm NC nitrocellulose blotting membrane (Cytiva). After blocking the membrane in 4% milk in tris-buffered saline (TBS, Medicago) overnight at 4°C, membranes were incubated with diluted serum samples for 1 h at RT with shaking (PSU-10i Orbital Shaker, Biosan, 100 rpm). One of the positive samples was tested with 1:100 and 1:200 serum dilutions to estimate the correct dilution and the rest only with 1:200 dilution. Membrane was then washed 3x5 min in TBS + 0.05% Tween20 (TBST) and incubated with peroxidase-conjugated AffiniPure Goat Anti-Horse IgG (H + L) secondary antibody (Jackson ImmunoResearch) diluted 1:40 000 for another 1 h after which it was washed 3x5 min in TBST and 1x5 min in ultrapure water. The membrane was visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific) according to manufacturer’s instructions and imaged on X-ray film (Fujifilm Super RX Medical X-ray film).\u003c/p\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eFor calculation purposes, animals were considered seropositive for EqPPV if they showed a positive reaction with both antigens and unclear if they were positive with one antigen only. Samples that were negative with both antigens were considered seronegative. Similarity of seroprevalence between groups was tested with Fisher exact test with a significance level of 0.05. All the statistical tests were done with IBM SPSS Statistics 29.0.2.0. Pictures were drawn in Jupyter Notebook utilizing Pandas, SciPy, Matplotlib, Seaborn, and NumPy packages \u003csup\u003e19–23\u003c/sup\u003e. Confidence intervals were calculated with Wilson score method.\u003c/p\u003e\u003cp\u003eEthics declaration\u003c/p\u003e\u003cp\u003eThis study relied on samples collected during routine diagnostics within a non-experimental clinical veterinary setting. Hence, no ethical approval was required (European Union directive 2010/63/EU and the national act 497/2013).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eVirus isolation\u003c/p\u003e \u003cp\u003eWe attempted to cultivate five samples positive for EqPPV RNA in equine dermis cells and aneuploid human keratinocyte cells (HaCat). Cytopathic effect (CPE), characterized by rounded cells, was observed with both cell lines in 1 of the samples 7 days after infection (passage 1). However, based on a PCR performed on culturing media (Table S3, supplementary file), CPE could not be confirmed to have been caused by EqPPV. The CPE was not seen in any further passages and Ct-values increased during each passage.\u003c/p\u003e \u003cp\u003eExpression of EqPPV recombinant proteins and evaluating an immunofluorescence assay\u003c/p\u003e \u003cp\u003eAs the culturing attempts of EqPPV were repeatedly unsuccessful, and with almost full-length EqPPV genome sequence available \u003csup\u003e3\u003c/sup\u003e, we concentrated our efforts on establishing serological assays based on antigenic recombinant proteins. As previous literature showed antigenicity of homologues B2L and F1L proteins of other PPVs, we selected these for further studies. Both proteins were successfully expressed with HA and His tags from the related ORFs from pCAGGS plasmids transfected in VeroE6 cells. In IFA, a control anti-HA antibody detected the C-terminal HA tag in B2L but not in F1L protein construct suggesting that this epitope was potentially hidden in F1L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, positive serum samples were identified with both antigens (detailed in the following sections). The four negative control horses sampled before the epidemic were negative, including the OPV-positive horse.\u003c/p\u003e \u003cp\u003eBased on the amino acid sequences, sizes of the proteins are 41 kDa (B2L) and 36 kDa (F1L) \u003csup\u003e9\u003c/sup\u003e. Bands of these sizes were detected in Western blot (WB) when using two IFA-positive serum samples against cell suspension containing these antigens whereas bands of these sizes were not detected when testing these sera against cell suspension without the antigens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No bands were detected with an IFA-negative control serum and in one IFA-positive sample of the three tested. Due to the novelty of the virus and the lack of a golden standard test, specificity and sensitivity could not be calculated.\u003c/p\u003e \u003cp\u003eAll samples combined, IgG positivity and negativity to B2L and F1L were in 97.6% agreement in IFA. Seven samples out of 294 gave conflicting results. In all the seven cases, antibodies to F1L were positive and to B2L either negative or unclear.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAntibody response of horses with pastern dermatitis\u003c/p\u003e \u003cp\u003eIn total, 79% (22/28) of samples collected from horses with pastern dermatitis between December 2021 and March 2022 were IgG positive with B2L, 86% (24/28) with FL1, and 79% (22/28) with both (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seventy-three percent (73%; 11/15) of the horses sampled within a week from onset and 82% (9/11) sampled between a few weeks and few months were positive with both F1L and B2L. The two horses (C3 and I1) that were resampled a year after a PCR-verified infection were still IgG positive in both assays. Sera from two horses only were negative with both antigens, D2 [sampled 1day post-onset, borderline case in PCR] and F4 [sampled 4 days post-onset, positive in PCR] (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Serum from one horse, F1, [sampled between a few days and a few weeks, positive in PCR]) provided unclear result with both antigens. The results of sera from three horses (F3, G1 and H1) differed between antigens: all were positive with F1L but negative or unclear with B2L. The only sample that was negative in PCR \u003csup\u003e9\u003c/sup\u003e, J1, [sampled within a few days post-onset] was IgG positive with both antigens. Geometric mean titer of the positive samples was 39.8 (titer range from 20 to 160, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and no variation was detected between sampling groups.\u003c/p\u003e \u003cp\u003eNo clear positive samples were identified with IgM IFA. Four samples gave unclear results that could not be clearly classified as positive or negative. Three of these were from horses sampled within a week from the first detection of the clinical signs and one was sampled over 3 weeks after the onset.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe proportion of IgG positive cases among horses with pastern dermatitis, December 2021- March 2022.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSampling time after first detection of signs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeroprevalence (F1L)\u003csup\u003ea\u003c/sup\u003e, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSeroprevalence (B2L)\u003csup\u003ea\u003c/sup\u003e, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSeroprevalence (Both)\u003csup\u003ea\u003c/sup\u003e ,%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA few days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e86.6 (13/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73.3 (11/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73.3 (11/15)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA few days-few weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90.9 (10/11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e81.8 (9/11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81.8 (9/11)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 year\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.0 (2/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.0 (2/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.0 (2/2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e85.7 (24/28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78.6 (22/28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78.6 (22/28)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e Reported as % (n/N)\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003eb\u003c/sup\u003e Reported as geometric mean titer\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ec\u003c/sup\u003e Both horses were also sampled earlier\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003ch2\u003e\u003ctd colspan=\"4\"\u003eEqPPV seroprevalence in Finland between 2012\u0026ndash;2022\u003c/td\u003e\u003c/h2\u003e\u003cp\u003eSeroprevalence of horses to EqPPV in Finland varied between 3.6 and 16.7% (F1L, p\u0026thinsp;=\u0026thinsp;0.096, Table S4, supplementary file) and 1.8\u0026ndash;14.6% (B2L, p\u0026thinsp;=\u0026thinsp;0.101) between five time-points. Highest seroprevalences were detected in 2015 and 2022 with both antigens and the lowest in 2021 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Seroprevalence was highest in horses sampled for breeding or disease diagnostics. None of the exported or imported horses were seropositive. No significant differences were detected between racehorses and riding horses (F1L, p\u0026thinsp;=\u0026thinsp;0.073; B2L, p\u0026thinsp;=\u0026thinsp;0.224). No antibodies were detected in ponies, whereas total seroprevalence in horses was 8.5% (F1L, p\u0026thinsp;=\u0026thinsp;0.232; B2L, p\u0026thinsp;=\u0026thinsp;0.382).\u003c/p\u003e \u003cp\u003eOut of the positive horses, one lived in Uusimaa region (Southern Finland), five in Ostrobothnia (Western Finland), and one in Savo (Eastern Finland). Three of the positive horses had been brought to Finland from North America (#71, #94, and #137) and one from Germany (#211). Due to the frequent travelling of many of the horses, no detailed geographical analysis was done. One of the horses (#237) sampled in 2022 was positive for IgM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table S2) and one (#231) could not be clearly defined as positive or negative. Both horses were sampled for breeding purposes in February.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSeroprevalences and Wilson score intervals (95% confidence level) grouped by sampling year, use of the horse, reason for sampling, and horse type. Numbers of positive samples and total samples are reported in brackets.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClass\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eF1L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eB2L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eBoth\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.0 (2/40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u0026ndash;16.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0 (2/40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.4\u0026ndash;16.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.0 (2/40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.4\u0026ndash;16.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.6 (8/59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.0-24.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.2 (6/59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.7\u0026ndash;20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.8 (6/61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.6\u0026ndash;19.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.4 (4/63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026ndash;15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.4 (4/63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5\u0026ndash;15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.4 (4/63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.5\u0026ndash;15.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.6 (2/56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0-12.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8 (1/56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3\u0026ndash;9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.8 (1/56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.3\u0026ndash;9.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.7 (8/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.7\u0026ndash;29.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.6 (7/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.3\u0026ndash;27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.6 (7/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.3\u0026ndash;27.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRacehorses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.4 (19/167)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.4\u0026ndash;17.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.0 (15/167)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.5\u0026ndash;14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.0 (15/167)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.5\u0026ndash;14.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRiding horses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.9 (5/85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026ndash;13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.9 (5/85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5\u0026ndash;13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.9 (5/85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.5\u0026ndash;13.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0 (0/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0-65.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0 (0/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0-65.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0 (0/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0-65.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.4 (19/182)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.8\u0026ndash;15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.9 (16/182)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.5\u0026ndash;13.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.9 (16/182)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.5\u0026ndash;13.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImport\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0 (0/25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0-13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0 (0/25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0-13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0 (0/25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0-13.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExport\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0 (0/11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0-25.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0 (0/11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0-25.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0 (0/11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0-25.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.4 (5/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u0026ndash;22.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.3 (4/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.3\u0026ndash;19.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.3 (4/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.3\u0026ndash;19.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinnhorses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.5 (8/94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.4\u0026ndash;15.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.4 (6/94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.0-13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.4 (6/94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.0\u0026ndash;13.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandardbreds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.4 (13/90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.6\u0026ndash;23.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.2 (11/90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.0-20.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.2 (11/90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.0\u0026ndash;20.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWarmbloods\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.3 (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7\u0026ndash;20.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.3 (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.7\u0026ndash;20.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.3 (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.7\u0026ndash;20.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePonies/Icelandic horses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00-16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0-16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0 (0/20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0\u0026ndash;16.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0 (0/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00-65.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0 (0/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0-65.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0 (0/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0\u0026ndash;65.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we developed a serological method to detect antibodies raised by a novel parapoxvirus, EqPPV, which caused a widespread pastern dermatitis epidemic among horses in Finland during winter 2021\u0026ndash;2022. With this method, we analyzed serum samples from affected horses as well as archived samples to acquire information on immune response, prevalence, and history of EqPPV.\u003c/p\u003e \u003cp\u003eDue to its novelty, pathogenesis and virulence factors of EqPPV are poorly known. Hence, the exact mechanisms of infection and host immune response can mainly be guessed based on what is known about other PPVs, especially the most extensively studied ORFV. ORFV causes both cell-mediated and humoral immune response in humans and sheep, first of which seems to play a bigger role \u003csup\u003e24\u003c/sup\u003e. Antibodies do not seem to give protective immunity against ORFV or BPSV and reinfections are possible, although the clinical picture may be milder in secondary infections \u003csup\u003e24\u0026ndash;26\u003c/sup\u003e. Based on a preliminary annotation of EqPPV, many of the anti-inflammatory ORFV genes (including chemokine binding protein (ORF112), and at least two out of three inhibitors of the NF-kB signaling pathway (ORF024 ja ORF121)) are also found in EqPPV genome, even though confirming that they share a same function would need further experimentation. In ORFV, both contribute to the ability of the virus to evade host immune response \u003csup\u003e24,27\u003c/sup\u003e. Due to a few gaps in the EqPPV sequence, we cannot rule out that we are still missing some genes. Based on current data, it is also not possible to tell if and how fast horses can be reinfected by EqPPV and if the second infection is milder.\u003c/p\u003e \u003cp\u003eEven though the clinical signs associated with EqPPV are mainly restricted to the skin of pastern area, our serological data demonstrate that the infection is severe and deep enough to stimulate a quick and long-lasting immune response. IgG antibodies could be detected in most affected horses during the first few days after onset and still prevailed a year after the disease. This is faster than expected based on knowledge on other PPVs. For example, IgG response was observed around two weeks after the primary infection in experimental infections of sheep with ORFV and cows with BPSV \u003csup\u003e24,28\u003c/sup\u003e. Some of the difference of EqPPV to other PPVs could be explained by the fact that the IgG response in experimental infections has been measured from the infection whereas we measured it from the time when the clinical signs were first detected, not taking incubation period or potential delay between the onset and its detection into account. However, since reports from the field suggest that the incubation time likely is no more than a few days (median time between 1st and 2nd case of affected stables being 3 days), IgG response can still be considered fast \u003csup\u003e9\u003c/sup\u003e. Another study detected a more rapid increase in IgG titer against ORFV in sheep after secondary infection four weeks after the primary infection \u003csup\u003e25\u003c/sup\u003e. In this case, it is unlikely that all the horses had been exposed to EqPPV before. Based on reports and photos from the field \u003csup\u003e9\u003c/sup\u003e, clinical signs were relatively severe in comparison with other PPVs, potentially explaining the fast IgG response.\u003c/p\u003e \u003cp\u003eDespite other PPVs generally causing only a short-lived protective immunity, a detectable antibody response has been observed in a cow experimentally infected with BPSV even three years after the infection even though it should be noted that subcutaneous experimental infection does not fully resemble natural infection \u003csup\u003e28\u003c/sup\u003e. In association with EqPPV infection, a follow-up longer than one year would be needed to determine the duration of IgG response. There is a slight chance that some of the antibodies of the clinical cases are a result of a previous infection. However, this would likely be a minor proportion of the cases based on our data on seroprevalence of the archived samples (1.79% in 2021). Also, most of the horses were PCR-positive at the time of the sampling and were hence known to be infected. Whether detectable IgG antibodies are protective and how big a role T cell responses may play in EqPPV infection remains to be clarified in further studies.\u003c/p\u003e \u003cp\u003eSince the culturing attempts were unsuccessful- which is not uncommon for parapoxviruses \u003csup\u003e29\u003c/sup\u003e- serological assay was set up with a recombinant-protein based technique relying on two known immunogenic proteins, B2L and F1L, instead of the whole virus. Based on the results, IFA was able to detect IgG with both the antigens with good agreement. Detection by HA antibody failed with F1L, possibly due to the short length of the HA tag which may be folded so that is inaccessible for the antibodies. The WB analysis confirmed the existence of proteins that were close to the expected sizes and were not detectable in antigen-free cell suspension in two out of three tested positive samples. The negative result of the third IFA positive sample in WB can be explained by a possibly lower sensitivity of WB: when one of the IFA positive samples was tested with two different serum dilutions (1:100 and 1:200), the result was positive with 1:100 dilution only. When relatively low mean titer of 40 is also considered, it is probable that the antibody titer in some of the samples is too low for WB to detect with standard 1:200 dilution. On the other hand, high background may make smaller serum dilutions more difficult to read.\u003c/p\u003e \u003cp\u003eDue to the lack of a golden standard test, specificity and sensitivity of the IFA could not be calculated. One option for the future would be a virus neutralization test for which, however, a virus isolate is needed first. Our EqPPV isolation attempts have been unsuccessful so far. Another option for validation would be a larger sample panel from PCR-verified cases 2\u0026ndash;6 weeks from infection. When it comes to specificity of the assay, a panel of certainly negative samples is challenging to find. For that, a panel of healthy horses born after the previous outbreak would be the most potential solution. Cross-reaction with other poxviruses that can infect horses cannot be fully excluded. However, considering that the OPV-positive serum sample was negative with this assay, cross-reaction is unlikely. Still, cross-reaction with other PPVs remains possible, but those have not been reported in horses as far as we know. To maximize sensitivity, F1L antigen should be used in screening, because a few samples were clearly positive only with this one and negative or unclear with B2L. This implies that F1L is either slightly more sensitive or less specific than B2L.\u003c/p\u003e \u003cp\u003eNo clear IgM antibodies were detected in horses that had pastern dermatitis. However, possible positive samples were detected in archived samples from early 2022. Detecting IgM in general horse population at that time is possible as that was the peak of the epidemic, and the epidemic was suspected to affect hundreds of horses \u003csup\u003e9\u003c/sup\u003e. An IgM-positive horse sampled for breeding in 2022 may have been infected at the time of the sampling but not displaying clear clinical signs yet. We also don\u0026rsquo;t know if breeding horses were clinically healthy at the time of the sampling or if asymptomatic infections are possible. When it comes to clinical cases, it is possible that IgM response is so short that it had gone down by the time the clinical signs were spotted and samples were taken. However, these IgM assays could lack sensitivity and the results should be considered uncertain due to the lack of a positive control for protocol optimization, and any alternative test for confirming the results. In comparison, ORFV has been shown to induce a detectable IgM response in most animals that show clinical signs \u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eData on archived samples indicate that EqPPV has been circulating in the Finnish horse population for several years. Based on one equine serum sample from 2012 that was confirmed to be IgG-antibody-positive with both antigens, the virus has been circulating even before the first report in 2013. Peaking seroprevalences were detected in 2015 and 2022, well in line with the field reports claiming that epidemics have been raging before 2021\u0026ndash;2022, most notable year being 2015. However, the differences between years were not statistically significant, possibly impacted by our limited sample size. Peak in 2019, another claimed epidemic year, was less clear, which may be either due to the sample size, smaller scale of the epidemic, or a different causative agent. This is the first step towards a serological assay for this new pathogen. In the future, larger sample sets, dating further back and potentially focusing on racehorses should be tested. As testing of hundreds of samples with IFA is resource intensive and the test is subjective, setting up a test that is more quantitative and more suitable for large sample panels (e.g. enzyme-linked immune sorbent assay) would be beneficial.\u003c/p\u003e \u003cp\u003eThe 2021\u0026ndash;2022 epidemic was only reported in racehorses. In archived samples, antibodies were also found in riding horses, although with a slightly lower seroprevalence (nonsignificant difference). Still, it is important to raise awareness among riders that riding horses can also be infected and competition events should be avoided if the horse shows signs of EqPPV infection. It is unknown if the horses are infectious before the onset of the clinical signs, making it difficult to give clear instructions on whether healthy horses from stables that have the disease can pose a transmission risk. However, frequent movement of the horses and suspicions that most introductions into stables came from racing events \u003csup\u003e9\u003c/sup\u003e, make competitions and other events a potential platform for the large-scale spread of the virus. Antibodies were detected in all three horse types that were tested. No antibodies were detected in ponies but the number of sampled ponies was insufficient and most likely they are also at risk for infection if they are exposed to EqPPV. It should also be noted that some sampling biases may be caused by different distribution of horse uses, types, and reasons for sampling between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For example, it may be that some groups (racehorses vs riding horses and horses vs ponies) attend less commonly events where the virus spreads.\u003c/p\u003e \u003cp\u003eAfter the epidemic in 2022, no cases have been detected despite ongoing diagnostic surveillance of dermatitis cases. One factor that may explain the clearly cyclic nature of the disease would be a protective herd immunity of the horse population. When the herd immunity decreases, a new epidemic arises. The seroprevalence of less than 20% detected in this study is not high enough to fully support this hypothesis. However, the sensitivity of this pilot detection is unknown, and if we only look at the high-risk horses that actively participate in racing events, seroprevalence may be higher and contribute to some level of herd immunity among racehorses. As many poxviruses are known to jump from one species into another, the role of other reservoir or amplification animals (e.g. rodents) should also be considered.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the 1st serological study on this novel virus and provides new information for predicting and controlling future epidemics. Based on the reports from the field and the results of this study, further epidemics are likely within the next few years. No cases have been reported in horses outside Finland so far either because the virus has not spread to other countries, or it has not been recognized and reported. Considering the findings in humans in the USA and racehorses travelling between countries, the latter explanation is more likely. For example, some of the seropositive horses in this study had been imported to Finland from other countries, including the USA, and it is not possible to conclude weather they were infected in Finland or not. In the future, serological screening should be broadened to other countries to understand the global distribution of the virus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank DVM, PhD Heli Koskinen for her support in the beginning of the project, Mikko Virta for assistance with collecting the archived samples, and Jonas Wensman et al. (Swedish Veterinary Agency) for providing the equine dermis cells. We also thank the veterinarians and owners who contributed to getting the serum samples.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization: JV, PMK, KH, KA. Data curation: PMK, KH, TG, LK. Formal Analysis: JV. Funding acquisition: JV, PMK, KH. Investigation: ST, LL, MU. Methodology: JV, PMK, ST, LL, MU, KA. Project administration: JV, PMK. Resources: OV, TS. Supervision: JV, PMK. Validation: JV, LL, OV, MU. Visualization: JV. Writing \u0026ndash; original draft: JV. Writing \u0026ndash; review \u0026amp; editing: JV, PMK, MU, OV. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its supplementary file.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was funded by Finnish Veterinary Foundation (JV), Finnish Foundation of Veterinary Research (JV), Niemi Foundation (PMK), Erkki Rajakoski foundation (PMK), Finnish Foundation for Research on Viral Diseases (KH), Orion research foundation (JV), and Sakari Alhopuro Foundation (JV).\u003c/p\u003e\n\u003ch2\u003eConflicts of interests\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eBesides holding the title of Associate Professor (Docent) of University of Helsinki, PMK is an employee of MSD Animal Health. The EqPPV studies were initiated before her joining the company.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eEssbauer, S., Pfeffer, M. \u0026amp; Meyer, H. Zoonotic poxviruses. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 229\u0026ndash;236 (2010). https://doi.org/10.1016/j.vetmic.2009.08.026\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMcInnes, C. 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D. \u0026amp; Woldehiwet, Z. Immune responses of the camel (Camelus dromedarius) to contagious ecthyma (Orf) virus infection. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 119\u0026ndash;131 (1995). https://doi.org/10.1016/0378-1135(95)00055-f\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"EqPPV, parapoxviruses, serology, immunofluorescence assay, western blot","lastPublishedDoi":"10.21203/rs.3.rs-5528230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5528230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParapoxviruses (PPV) cause skin and mucous membrane signs to several animal species and humans worldwide. Equine parapoxvirus (EqPPV) was first detected in a sick horse in Finland in 2013. It is potentially zoonotic, and a similar virus has been detected in humans in the USA. In winter 2021\u0026ndash;2022, EqPPV caused a large-scale pastern dermatitis epidemic in racehorses all over Finland. Field reports suggest that similar epidemics of unverified cause have also occurred in 2015 and 2019. The aim of this study was to develop a serological test and study the immune response, seroprevalence, and history of the virus utilizing serum samples from clinical cases and archived horse samples (2012\u0026ndash;2022). A recombinant protein-based immunofluorescent assay was established using envelope proteins B2L and F1L. EqPPV induced a fast immune response within a few days from the onset of the clinical signs. Two horses that were additionally tested a year after the disease still had similar IgG titers as a year prior. Seroprevalences in archived horse samples varied between 1.8\u0026ndash;14.6% and peaks were observed in 2015 and 2022. The results suggest that EqPPV is a re-emerging pathogen that has a potential to cause large epidemics, bringing a need for more studies and preparedness.\u003c/p\u003e","manuscriptTitle":"Antibody responses to equine parapoxvirus reveal a re-emerging pattern","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 09:58:29","doi":"10.21203/rs.3.rs-5528230/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5938b23-e93b-4043-9781-c5b2376ff67d","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43812040,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":43812041,"name":"Biological sciences/Microbiology/Virology/Pox virus"},{"id":43812042,"name":"Biological sciences/Microbiology/Virology/Viral epidemiology"},{"id":43812043,"name":"Biological sciences/Microbiology/Virology/Virus host interactions"}],"tags":[],"updatedAt":"2025-03-20T06:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 09:58:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5528230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5528230","identity":"rs-5528230","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}