RNA was tested for influenza A, B, and D,4-6 coronavirus,7 and enterovirus8 using Superscript III One-step RT-PCR with Platinum Taq Polymerase. Extracted DNA was tested for adenovirus by realtime PCR using a QuantiNova Probe PCR kit (Qiagen) (Table 1).8

For adenovirus-positive aerosol samples, 500 μL of sample was inoculated into adenocarcinomic human alveolar basal epithelial (A549) cells (ATCCR CCL 185™) with Dulbecco’s Modified Eagle Medium (DMEM) 2% (v/v) Fetal Bovine Serum (FBS), and incubated at 37°C. After 72 hours, inoculated shell vials were observed for cytopathic effect (CPE) daily for ten days. For influenza A virus-positive aerosol samples, 200 μL of sample was inoculated into Madin Darby Canine Kidney (MDCK) cells (ATCCR PTA-6500™) with DMEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, 0.2% (w/v) BSA, 25 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and 1 μg/mL Tosyl phenylalanyl chloromethyl ketone (TPCK)- treated trypsin, and incubated at 37°C for 7 days with daily observances for CPE.

Bovine clinical samples used in this study were submitted to KSVDL for routine diagnostic testing. The samples were obtained from naturally infected animals in the field by licensed veterinarians as a part of normal veterinary care and diagnostic investigations.

The positive controls were consistently positive throughout the analyses, and all negative controls were negative. BCoV RNA was detected on all boots, coats and stethoscopes, and on seven out of eight wristwatches, 24 h after exposure. The eighth watch was positive for BCoV RNA 15 min and two hours after exposure. The copy numbers of BCoV RNA 24 h after exposure are presented in Fig. 3. BRSV RNA was detected on 18 out of 19 boots sampled after two hours and 16 out of 19 boots after 24 h. For the coats, 17 out of 19 were positive two hours after exposure, and 18 out of 19 were positive after 24 h. There were minor differences in BRSV RNA copy numbers between samples collected 2 and 24 h after exposure and no tendency of reduction in copy numbers (Fig. 4).

BRSV infectivity was tested in ten swabs showing the highest level of viral RNA by RT-ddPCR; eight swabs from coats and two from human nostrils, collected 24 h and 30 min after exposure, respectively. Fetal bovine turbinate cells (courtesy of Swedish Veterinary Institute) propagated in Eagle’s minimal essential medium (BioWhittaker, Belgium) in 96 well plates were incubated with 50 μl filtered swab samples for 30 min. Medium with 2% FCS was added, the plates were incubated at 37 °C in 5% CO2 and the supernatant passaged after seven days. Samples were cultivated in duplicates with positive (cultivated BRSV from a calf in the experiment) and negative controls (cells only). The cells were observed for cytopathic effect (CPE) and infection visualized by direct immunofluorescence test using FITC Moab a-BRSV (Bio-X Diagnostics, Rochefort, Belgium). Culture supernatants were harvested and tested by the BRSV RT-ddPCR as described.

Clinical samples (nasal and pharyngeal swabs and lung tissue) from bovine respiratory disease submissions to KSVDL were screened by a BRDC PCR panel which detects BVDV, BHV-1, BRSV and bovine coronavirus (BCV). A total of 204 samples were screened. Samples were collected in years 2011–2014 from twelve states: Nebraska (n = 48), Kansas (n = 112), Colorado (n = 6), Missouri (n = 1), Mississippi (n = 7), Texas (n = 4), Oklahoma (n = 2), Idaho (n = 2), Montana (n = 6), Oregon (n = 4), Washington (n = 11) and Virginia (n = 1). Samples were also screened for influenza D virus (IDV) using quantitative real time reverse transcription PCR as previously described. A 5’-nuclease reverse transcription PCR assay was designed to detect bovine rhinitis viruses targeting the 3D polymerase gene: probe, 5’-FAM-CGG CAG TCC AGG TCC AGT GT-Iowa Black-3’; Forward: 5’-CTT TTC GGT GTG ATT GGC AG-3’; Reverse: 5’-GAA ATC TAT CAG GGC AGG TCT G-3’. Viral RNA was extracted using the MagMAX-96 viral RNA isolation kit (Life Technologies) according to the manufacturer’s instructions. Real time reverse transcription PCR was performed using Qiagen Quantitect RT-PCR with BRV primers and probe as follows: 50°C, 30 minutes; 95°C, 15 minutes; followed by 40 cycles of 94°C for 15 seconds and 60°C for 60 seconds. The PCR assay specificity was confirmed using bovine rhinitis virus positive samples as determined by metagenomic sequencing as well as with cultures of common BRDC pathogens BVDV, BHV-1, BRSV, BCV, Mannheimia haemolytica, Histophilus somni, Pasteurella multocida and Mycoplasma bovis.

Serum samples were obtained from livestock farmers and their family members to be used as reference samples for assay evaluation. This was done after consent was obtained from the participants. For livestock, 10 mL of whole blood was collected and for the humans 5 mL was collected. This was done by trained veterinary technicians and clinical phlebotomists, respectively. Blood samples were then transported to the laboratory where they were centrifuged to obtain serum and immediately frozen with liquid nitrogen.

We developed a whole-virus enzyme linked immunosorbent assay (ELISA) to test for HCoV-NL63 in livestock as part of a two-stage testing algorithm also involving a recombinant immunofluorescence assay. For a sample to be considered positive for HCoV-NL63, it had to be in the top 5% most reactive samples as determined with the whole-virus ELISA and also positive in a confirmatory test with a more specific recombinant immunofluorescence assay (rIFA). This was the procedure adopted for swine, sheep, and goat sera. This ELISA relied on bovine products in the form of fetal calf serum in cell culture and milk powder for blocking and dilution of sera, and as such, cattle sera were tested directly with the recombinant immunofluorescence assay that had been optimized with less bovine products in the testing process to minimize background signals. Few donkey samples were obtained, and these were also tested directly with the recombinant immunofluorescence assay. A selection of coronavirus-characterized serum samples was used for assay optimization and the study samples for evaluation. All serum samples were heat inactivated at 56 °C for 30 min before testing.

Bovine kidney cells (MDBK/NBL-1; ATCC® CCL-22™) were cultured at 37 °C with 5% CO2, in DMEM (Fisher Scientific, Loughborough, UK) supplemented with 8% horse serum. Virus isolation and determination of the median of tissue culture infective dose were performed on MDBK cells. Virus isolation was performed as follows: The three nasal swabs, positive for BPIV3, were filtrated through a 0.22 μm filter (Millipore, Milford, MA, USA) and inoculated to a monolayer culture of MDBK cells cultured in Dulbecco’s modified eagle medium (DMEM, Fisher Scientific, Loughborough, UK) supplemented with 8% horse serum (Fisher Scientific, Loughborough, UK). MDBK cells were maintained at 37 °C in an atmosphere of 5% CO2. The cytopathic effect (CPE) was examined daily. The storage solution was exposed to a ten-fold dilution and filtered with a 0.22 μm filter. Then, the filtrate was inoculated into MDBK cells for 1 h. Finally, the culture medium was replaced with DMEM containing 2% horse serum. MDBK cells inoculated with filtrate were cultured in an incubator continually for 72 h. The propagation of the virus was performed three times.

The complete genome sequences of BPIV3 available in GenBank were used for genetic and phylogenetic analysis. BLASTn was initially used to identify viral sequences through their sequence similarity to annotated viral genomes in GenBank. Based on the best hits of blastx searches, the following 24 complete genomes were choose for the next analyses: Genbank numbers: KU198929; KT071671; JX969001; KJ647287; KJ647285; LC000638; LC040886; EU277658; KJ647284; KJ647286; KP764763; KJ647289; JQ063064; KP757872; D84095; AB770485; AB770484; KJ647288; EU439428; AF178654; EU439429; AF178655; and HQ530153. These genomes were then aligned using Clustal X software. Subsequently, a phylogenetic tree was constructed by the Maximum Composite Likelihood (MCL) approach assuming the Hasegawa-Kishino-Yano model plus a discrete Gamma distribution (with five categories and an estimated alpha parameter = 3.1840) and the rate of invariable sites of 54.73%. All phylogenetic analyses and tree editions were conducted using MEGAX software.

All calves tested negative for antibodies to BCoV at the beginning of the trial. At D14 all calves in FG and EG had seroconverted (Additional file 1: Table S1). The SG was still seronegative to BCoV D42 and did not show an increase in titer for antibodies to BRSV.

BCoV RNA was not detected in any of the blood samples analyzed.

Proper specimen collection and delivery to a diagnostic lab is commonly neglected, and significantly impacts the diagnostic outcome. Antemortem samples for diagnostic testing should minimally include feces from acutely diarrheic animals prior to therapy with optional blood samples. Necropsy specimens from freshly sacrificed, moribund, or euthanized calves are of great value for diagnosis during severe outbreaks. Fresh and formalin-fixed gastrointestinal tissues (abomasum, small intestine, or colon) including ones from regional lymph nodes and liver should be collected along with colonic contents. Fresh fecal samples should be directly recovered from diarrheic animal into a specimen container with either rectal swabs or by rectal stimulation while avoiding environmental contamination (by soil, urine, or other feces). Once collected, the sample should be stored in a transporting medium or special stool container with refrigeration to maintain pathogen viability and sample integrity (e.g., reduced overgrowth of undesired bacteria and prevention of nucleic acid degradation). Samples of anaerobic bacteria (e.g., C. perfringens) should be kept in an oxygen-free transport medium during shipping if possible.

A total of 1138 equine serum samples were included in this study. The details of serum panels A–H (n = 1084) are shown in Table 1. They were retrieved from the serum bank at GD Animal health Deventer, the Netherlands. All of them were collected for the monitoring of other diseases independent to this study, and their ECoV exposure status was unknown. With the exception of panel H (collected from Iceland), all serum samples from panel A to G were collected from horses in the Netherlands. Additionally, panel I included 27 paired (acute- and convalescent-phase) serum samples that were collected during an ECoV outbreak in the USA (2014). All samples were stored at −20 °C until tested.

Several attempts have been made to develop vaccines against human coronavirus infection in the past decades. But the degree of cross-protection provided by such vaccines is greatly limited due to the extensive diversity in antigenic variants even within the strains of a phylogenetic sub-cluster (Graham et al. 2013). As for MERS and SARS coronaviruses, there is no licensed specific antiviral treatment or vaccine available till now. However, few of the advances made in developing vaccines and therapeutics for SARS-CoV and MERS-CoV could be exploited for the countering 2019-nCoV. But since the efforts to design and develop any vaccine or antiviral agent to tackle the presently emerging coronavirus pathogen would take some time, therefore till then we need to rely extensively on enforcing highly effective prevention and control measures to minimize the risk of 2019-nCoV transmission and spread to the best feasible extent (Cheng et al. 2020). Majority of the vaccines that are being developed for coronaviruses targets the Spike glycoprotein or S protein (Graham et al. 2013). This is mainly because of the fact that S protein is the major inducer of neutralizing antibodies (Jiang et al. 2005). Several kinds of vaccines and antiviral drugs that are based on S protein have been previously evaluated. Among them, the S protein-based vaccines include full-length S protein vaccines, viral vector-based vaccine, DNA-based vaccine, recombinant S protein-based and recombinant RBD protein-based vaccines. Whereas S protein based antiviral therapies include RBD–ACE2 blockers, S cleavage inhibitors, fusion core blockers, neutralizing antibodies, protease inhibitors, S protein inhibitors, and small interfering RNAs (Du et al. 2009). Even though such therapeutic options have proven efficacy in the in vitro studies, however most of these haven’t undergone randomized animal or human trials and hence are of limited use in our present 2019-nCoV scenario. Remdesivir is a novel nucleotide analog prodrug that was intended to be used for the treatment of Ebola virus disease. It also has anti-coronavirus activity due to its inhibitory action on the SARS-CoV and MERS-CoV replication (Sheahan et al. 2017). At present, efforts are being made to identify and develop monoclonal antibodies that are specific and effective against 2019-nCoV. Combination therapy with 2019-nCoV specific monoclonal antibodies and remdesivir can be considered as the ideal therapeutic option for 2019-nCoV (Cohen 2020). Further evaluation is required before confirming the efficacy of such combination therapy. A variety of different therapeutic and vaccine designing approaches against coronaviruses are being explored and yet to be evaluated in terms of their potency, efficacy and safety, but hopefully the process of evaluation will be accelerated in the coming days (Cyranoski 2020; Lu 2020; Pillaiyar et al. 2020; Zaher et al. 2020).

Clinical (e.g., age, vaccination record, and clinical signs) and farm history should be provided to clinicians for determining the cause of diarrhea. Once the specimens are submitted to a veterinary diagnostic laboratory, the diagnostician sorts the samples to ensure proper delivery to testing laboratories based on the history and sample type. Generally, fecal sample are examined by microscopy (for C. parvum and Coccidia), bacterial culturing (for Salmonella spp., E. coli, and C. perfringens), and PCR (for BRV and BCoV). In contrast, intestinal tissues are subjected to immunohistochemistry or bacterial culturing. More recently, nucleic acid-based techniques such as PCR and an antigen-capturing enzyme-linked immunosorbent assay (Ag-ELISA) have been more commonly used for the rapid detection of various bacterial and viral pathogens in clinical specimens from diarrheic calves. When the laboratory test results are available, clinicians should consider the overall farm and clinical history in conjunction with lab results before identifying the causative pathogen.

Serum anti-BCV IgG antibodies were measured by ELISA in 195 samples collected from birth through weaning for 39 calves from Herd 2 that were involved in the mass treatment for BRD on August 12, 2016 in order to determine the mean antibody abundance and range at each sample acquisition time (Fig. 4). The mean (± standard deviation) anti-BCV antibody abundance declined from a maximum of 1186 ± 699 at birth to a low of 138 ± 88 at the time of mass treatment. Mean antibody abundance increased slightly following mass treatment, with mean antibody abundances of 176 ± 83 and 182 ± 95 at preconditioning and weaning, respectively.

Neutralizing antibody titers were measured in 60 of these samples from 12 randomly selected calves to determine the relationship between total anti-BCV reactive antibodies measured by ELISA and neutralizing antibody titers measured by a virus neutralization test (VNT). The effect of altering the strain of the test virus used in the VNT was also evaluated. A high positive mean correlation was observed between the ELISA and VNT assays regardless of the test virus used (Pearson’s rank correlation, ρ = 0.81 with BRCV_2014 strain and ρ = 0.91 with Mebus strain), indicating good to excellent agreement between the two tests under these conditions (Additional file 2). Thus, the ELISA was used for subsequent measurements of anti-BCV antibodies.

A total of 416 bovine serum samples and two positive control sera were tested with the IFA to distinguish negative serum samples from SFTSV. The results showed that 97.4% (407/418) of the bovine serum samples were negative, while 2.6% (11/418) of the bovine serum samples were positive according to the IFA. cELISA was performed using the SFTS-negative group samples, and the mean inhibition percent was 11.3% with a standard deviation of 12.7%. Therefore, we determined the cut-off value to be 49.5% (mean ± 3SD) (Fig. 5). The cELISA and IFA were compared using this cut-off value, and 98.1% (410/418) consistency was observed between results (Table 1). The accuracy of cELISA was demonstrated with a plot of the distribution of PI values obtained from the IFA-negative or positive cattle group (Fig. 6).

Because official standard methods for the antibody detection of SFTSV have not been determined, the cut-off criteria determination for IFA requires further optimization. Therefore, ROC curve was employed to determine the sensitivity and specificity and define the optimal cut-off value for IFA. The ROC curves of four different IFA cut-off values were compared, and the results showed that a 1/80 dilution of the sample strongly supports the result of cELISA. For this dilution, the area under the curve (AUC) was 0.91. While a 1/128 dilution rate as an IFA cut-off point maximizes the specificity and sensitivity compared with other dilution rates, this condition is too strict to detect positive samples above the strict threshold in cELISA. The other IFA cut-off conditions (1/16 and 1/64) were also excluded because they showed moderate AUC values (Fig. 7).

Using a 1/80 dilution as cut-off for the IFA and a PI of 49.5% as a cut-off for cELISA, 0.95% (4/418) of the bovine serum samples were identified as positive, while 97.1% (406/418) of the serum samples were negative.

Tick-mediated SFTSV is an emerging infectious virus identified in 2009 that shows 12% lethality in humans. However, the range of host species or virus circulation routes in the environment is difficult to determine because a standard diagnosis method is not available. Therefore, a simple and effective method of detecting SFTSV antibody is needed to identify potential hosts and develop anti-viral strategies. In an effort to detect SFTSV-specific antibodies, in-house ELISA and double-antigen sandwich ELISA (DAS-ELISA) assays were developed. Specifically, DAS-ELISA was used to determine the seroprevalence of humans and animals in China. A multiplexed Luminex-based immunoassay method was also established for high-throughput detection. These newly developed techniques are highly specific and sensitive, but are not commercially available, and some of these methods require special equipment for diagnosis.

Here, we report the development of cELISA using mAb to detect antibodies against SFTSV. The cELISA format was selected as a serodiagnostic test that has value in handling field serum samples because it requires only small volumes of serum for diagnosis and shows species flexibility. Immunostaining and Western blotting were used to detect mAbs with high affinity to SFTSV-infected Vero cells. The results indicated that the generated mAbs were appropriate for use in the development of a cELISA.

A previous study showed that the involvement of domestic animal husbandry is a significant factor in SFTSV infection when compared with outdoor activities or tick exposure in China. Therefore, we collected 416 field bovine sera from farms in Gyeongsang Province in Korea, where 30.6% (11/36) of human SFTSV infections were reported in 2013. First, we tested the field bovine serum samples using IFA. The bovine serum samples that yielded negative results in the IFA were used as a negative group to determine the cut-off value for the cELISA. All field serum samples and two experimentally immunized bovine sera were examined for an immune response to SFTSV using both IFA and cELISA.

The results showed that 97.4% of the total serum samples yielded negative results in the IFA; therefore, this group was regarded as negative to SFTSV. The cELISA cutoff value was subsequently calculated using the mean inhibition rate of the IFA-negative population ± 3S.D. (cutoff PI = 49.5%). Using this cut-off, 99.8% of the IFA-negative bovine serum samples were also considered to be negative in the cELISA. Although the newly developed cELISA is highly specific, more field animals infected with SFTSV would need to be measured to improve the sensitivity.

SFTSV was first isolated in goats, after which antibodies against SFTSV were detected in the sera obtained from goats at one goat farm in the southern part of Korea (Gyeongsang province) in September 2014 (personal communication with Dr. Lee, Yoon-Hee and Mr. Choi, Jeong-Soo, Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Korea). Serological diagnostic methods are necessary to test large numbers of animals. While the cELISA developed in the present study requires additional tests to validate its performance, the results observed to date are promising. Further studies to adapt this diagnostic technique to a broad spectrum of animal species are currently in progress.

A cell-based assay in a 96-well format was used to test the antiviral effectiveness of the different lozenges solutions against HRV1a/8 and Coxsackievirus A10. Here, the virus was preincubated with a semilogarithmic dilution series of the respective test samples or control for 30 minutes at room temperature (RT; prophylactic treatment) before it was added to HeLa/RD cells for infection. After an infection period of 30 minutes at RT, cells were washed with medium and then cultured at 33°C (HRV) or 37°C (Coxsackievirus), hereby maintaining the same concentrations of active agent as in the prophylactic treatment. The antiviral effectiveness of the test samples was assessed by determining cell viability using resazurin fluorescent dye when >90% cells of a control infection in the absence of any active agent have died. An incubation of cells with the same dilution series in the absence of viral infection was performed to monitor a potential toxicity of the treatment. To enable direct comparison of the antiviral effectiveness of the lozenges, the half maximal inhibitory dilution (ID50) value of each sample was calculated for a sigmoidal dose–response model with XLfit Excel add-in version 5.3.1. From this value, corresponding iota-carrageenan concentrations were calculated for the lozenge #1.

Lozenges were dissolved with the respective assay medium. The toxicity testing was performed under the assay conditions of the respective antiviral assays. Cell survival was assessed with resazurin fluorescent dye and crystal violet (HeLa and RD cells) or crystal violet only (MDCK cells). The highest noncytotoxic concentration was used as starting point in the subsequent antiviral activity assays.

Seeing the possible role of animals in 2019-nCoV infection, WHO in its advice for public recommended to avoid the unprotected contact with both farm and wild animals (World Health Organization 2020a). The live-animal markets such as in China could provide chances to animal CoVs to get transmitted to humans and these markets may act as critical places for the origin of novel zoonotic pathogens and pose high public health risks during an outbreak.

The emergency pathogens could be counteracted by opting immediate and timely international collaborative efforts, cooperative efforts between human and animal health sectors. Other effective measures include One health approach, implementation of effective prevention and control strategies, rapid communication and networking, and exploring advances in science and technology for developing rapid and confirmatory diagnostics, enhancing disease surveillance and monitoring, implementation of strict biosecurity measures, and timely efforts toward designing appropriate and effective vaccines and therapeutics (Cheng et al. 2020; Cohen 2020; Cyranoski 2020; Lu 2020; Munjal et al. 2017; Singh et al. 2017).

In the present scenario of not having any direct acting anti-viral agent and vaccines, strict implementation of high vigilance for 2019-nCoV and appropriate prevention and control measures are of utmost importance to check the further spread and control of this virus (Cheng et al. 2020). Researchers and Authorities (WHO, CDC Atlanta and others) across the globe are working to combat the current ongoing 2019-nCoV outbreaks, identifying the possible origin of this novel virus, and to design and develop effective vaccines and therapeutics (Cohen 2020, Cyranoski 2020, Lu 2020, Mahase 2020). Studying the virus in details, its molecular biology and immunology, adaptive genetic changes, mutations and recombination events, elucidating clinical pathology and pathogenesis, identifying the route of origin, role of any mixing vessels (like birds, pigs, and mammals), jumping the species barrier, zoonotic potential, human-to-human transmission events, altogether would pave ways for designing effective prevention and control measures to counter 2019-nCoV

To confirm the specificity of the fragments obtained by RT-PCR, PCR products were purified and used for sequencing. The purposed band, about 450 bp, was excised and then purified using Biomed gel extraction kit (BEIJING BILOMED CO., LTD) according to the manufacturer's instructions. The resulting products were cloned into pMDT-19 simple vector (Takara) for sequencing.

Equine coronavirus (ECoV) belongs to the species Betacoronavirus 1 in the genus Betacoronavirus which includes bovine coronavirus (BCoV) and dromedary camel coronavirus HKU23 [1, 8, 17]. Clinical symptoms of fever, anorexia, lethargy, leucopenia and digestive problems were seen in horses affected by ECoV in

several outbreaks in the United States [12, 13] and Japan [7, 10, 11], and in an experimental challenge study. About 20 to 30% of draft horses kept at a racecourse in Japan were affected in one ECoV outbreak [10, 11]. Those results indicate that ECoV is a highly contagious virus. Although most infected horses recovered, ECoV occasionally led to fatal symptoms like

necrotizing enteritis and hyperammonemic encephalopathy in the United States [2, 3]. Vaccination is one of the most important ways of minimizing the symptoms

of infectious viral diseases, but a vaccine against ECoV is so far not available anywhere in the world.

BCoV belongs to the same species as ECoV, and it has been reported that bovine and rabbit anti-sera against BCoV cross-react with ECoV to some extent [4, 11]. These results indicate that BCoV is related to ECoV both genetically and antigenically. An inactivated BCoV vaccine is available in Japan [6, 14] and it might also induce antibodies against not only BCoV but also ECoV in horses. This means that the BCoV vaccine could possibly become a surrogate ECoV vaccine. In this study, we investigated the antibody response to

ECoV in horses inoculated with the BCoV vaccine.

The BCoV vaccine used in this study was CattleWin BC (Kyoto Biken Laboratories, Kyoto, Japan). This vaccine contains aluminum hydroxide gel as an adjuvant and formalin-inactivated BCoV strain No. 66/HL. Original strain No. 66 was

isolated in Japan in 1977 from the feces of a naturally infected calf. Strain No. 66/H is the strain that sequentially cultured the original strain in bovine kidney cell cultures, BEK-l

cells and HAL cells. Additionally, vaccine strain No. 66/HL is strain No. 66/H that has been propagated in HmLu-1 cells. The manufacturer’s instructions indicate that 1 ml

of the vaccine is to be intramuscularly administered to cattle twice, about 1 month apart.

Six 1-year-old Thoroughbred horses were randomly divided into two groups of three, each group receiving either 1 or 2 ml of the vaccine. Horses were vaccinated intramuscularly twice, 28 days apart. Clinical

examinations were performed daily for 3 days after each vaccination, and rectal temperatures were measured once daily during this study. Horses with rectal temperatures exceeding 38.5°C were defined as having significant pyrexia. The

experimental protocol and all animal procedures were approved by the Animal Care Committee of the Equine Research Institute of the Japan Racing Association.

The virus neutralization tests for BCoV No. 66/H and ECoV NC99 were performed on serum samples collected at 0, 7, 14, 28, 42 and 56 days post first inoculation (dpi) as described previously. ECoV strain NC99 is a reference strain that was first isolated in the United States in 1999 [4, 17]. Two-fold serial dilutions of serum were

mixed with an equal volume of viral suspensions containing two hundred 50% tissue culture infective doses per 0.1 ml and incubated for 60 min at 37°C. Then, 0.1 ml of each mixture was applied to

HRT-18G cells on 96-well plates and incubated for 6 to 7 days. Virus-neutralizing antibody titers were expressed as the reciprocal of the highest serum dilution that inhibited viral cytopathic effects.

Statistical analysis was carried out using Ekuseru-Toukei 2012 (SSRI, Tokyo, Japan). Logarithmic transformations of the reciprocal antibody titers were made to stabilize variances. Antibody titers after logarithmic transformation

were analyzed by one-way ANOVA with Dunnett’s multiple comparison post hoc test using the antibody titers at 0 dpi as control. A P-value of <0.05 was considered statistically significant.

The virus-neutralizing antibody titers of horses inoculated with 1 or 2 ml of the BCoV vaccine are shown in Table 1. In horses inoculated with 1 ml of vaccine, the geometric mean antibody titers against BCoV at 0, 7, 14, 28, 42 and 56 dpi were 4, 5, 32, 102, 645 and 323, respectively, and the geometric mean antibody

titers against ECoV were 4, 6, 20, 25, 40 and 51 (Table 1). Compared with the antibody titers at 0 dpi, the antibody titers against both BCoV and ECoV significantly increased at 14, 28, 42

and 56 dpi. In horses inoculated with 2 ml of vaccine, the geometric mean titers against BCoV were 8, 161, 323, 203, 406 and 512, respectively, and the geometric mean titers against ECoV were 4, 16, 32, 25, 64 and 64

(Table 1). The antibody titers against BCoV significantly increased at 7, 14, 28, 42 and 56 dpi, and the antibody titers against ECoV significantly increased at 14, 28, 42 and 56 dpi in

comparison with the antibody titers at 0 dpi. This study showed that in all horses inoculated with the BCoV vaccine antibody titers against ECoV increased from 14 dpi, although the antibody titers against ECoV were lower than those

against BCoV. Maximum antibody titers against ECoV in each horse ranged from 32 to 128. An experimental inoculation study conducted earlier also showed that neutralizing antibody titers against ECoV strain NC99, which is the same

strain as used in this study, were 32 to 128 in three horses at 14 days after their inoculation with ECoV-positive feces. However, in horses naturally infected by ECoVs in the 2009 and 2012

outbreaks in Japan [10, 11], the geometric means of neutralizing antibody titers were 304.4 (6 horses) and 348.4 (9 horses), respectively. Thus, the

antibody titers of horses inoculated with the BCoV vaccine were similar to the titers of the experimentally infected horses but were lower than the titers of horses naturally infected in actual outbreaks. An experimental challenge

study using cattle inoculated with inactivated strain No. 66/H showed that inoculated cattle possessing neutralizing antibody titers of more than 640 showed no clinical signs after challenge with a virulent BCoV, whereas in contrast,

inoculated cattle possessing neutralizing antibody titers of less than 160 developed watery diarrhea and fever. Needless to say, the animal species, strain of challenge virus, and method of

virus neutralization test in that study are different from our present study. Nevertheless, the antibody titers of all vaccinated horses in the present study were no more than 128, and we therefore consider that the BCoV vaccine will

have limited efficacy against ECoV infection in horses. To clarify this, ECoV challenge studies in horses inoculated with the BCoV vaccine will be needed to evaluate the efficacy the vaccine.

The three horses inoculated with 1 ml of the vaccine did not exhibit any adverse reaction during this study. In contrast, two out of the three horses inoculated with 2 ml of the vaccine exhibited

swelling at the inoculation site after the second vaccination. None of the horses developed a fever after the vaccinations. Administration of more than 2 ml of the vaccine to horses would likely increase the risk of

adverse reactions. As described above, a significant increase in antibody titers against ECoV was observed from 14 dpi irrespective of whether 1 or 2 ml was administered. Additionally, the differences in antibody

titers against ECoV at each dpi from 14 dpi were less than twofold between horses inoculated with 1 and 2 ml. These results suggest that inoculation of 1 ml is suitable for horses as well as for

cattle.

Although horse No. 5 had no detectable antibodies against ECoV before vaccination, the horse already had antibodies against BCoV (Table 1). That horse was born and had been kept at a farm

that reared cows before coming to our facility. In Saudi Arabia, dromedary camel coronavirus HKU23 was detected in apparently healthy horses kept at facilities that reared camels, sheep, goats, and chickens. HKU23, which is closely related to BCoV, is endemic in camels of the Middle East and the HKU23-positive horses frequently came into contact with camels and other

animals. HKU23 may have been transmitted from infected camels to those horses. Horse No. 5 in the present study may have also been in contact with infected cows, and BCoV may have been

transmitted from infected cows to the horse. However, because there is no epidemiological information, it is unknown whether there were in fact BCoV-infected cows at the farm or whether horse No. 5 had shown any clinical signs.

This study showed that a BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether the antibodies provided by the BCoV vaccine are sufficient to be effective against ECoV, and therefore ECoV

challenge studies in horses are needed to evaluate the efficacy of the vaccine in the future.

Five thousand three hundred and seventy-three animal sera (n = 33,181,430 and 625 sera coming from n = 92, 45, and 13 herds for cattle, sheep and goat respectively, Table S1) were collected in official veterinary laboratories and at the Veterinary School of Toulouse from five French regions. Most of these sera were initially collected for infectious bovine rhinotracheitis monitoring. The sampling plan was representative of the population taking into account the major cattle-rearing areas including Bretagne, Pays de la Loire, Bourgogne-Franche-Comté, Hauts-de-France and Occitanie. In addition, sera from Occitanie were retrieved from the Veterinary School of Toulouse large animal clinics (n = 509). No data was available on history of respiratory diseases in the farms of each region. All the tested animals were older than 1-year-of age and the detection of maternally derived antibodies can therefore be ruled out. The type of sera, localization and years of collection are described in Table S1. Three controls sera were used: an in-house polyclonal rabbit anti-IDV serum generated by inoculating rabbits with D/Bovine/Nebraska/9-2/2012 subcutaneously (as described in); IDV negative and positive French cattle sera generated during an experimental infection. All sera were treated with receptor destroying enzyme (RDE, Seika) following the manufacturer’s instructions and hemadsorbed on packed horse red blood cells. Hemagglutination Inhibition (HI) assays were performed as previously described, with four hemagglutination units of D/bovine/France/5920/2014 and 1% horse red blood cells. Samples with antibody titers ≥1:20 were considered positive. Statistical analyses were carried out using Graph Pad Prism 5.0. A p value ≤0.05 was considered significant. A χ2 test was used to compare IDV seroprevalences between species and between French provinces.