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).

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

Taken together, the antiviral effect of the AMC/DCBA and HR containing lozenges varies widely, depending on the specific formulation of the lozenges and the the virus which causes the sore throat. This questions their suitability to be used as causative therapy in a clinical setting, where the disease causing virus mostly remains unknown. In contrast, carrageenan containing lozenges were highly active against all viruses tested, with a minimum superiority toward all other lozenges of 2- to 1368-fold for HRV1a and CoV OC43, respectively. Therefore, iota-carrageenan containing lozenges are an appropriate measure to effectively reduce the number of viral particles in the mouth. They can be used as a causative therapy against viral infections of the throat.

The assay was performed as described elsewhere13 with small adaptations to account for the high viscosity of the lozenges solutions. In short, the virus was preincubated with a semilogarithmic dilution series of lozenges or control solutions for 30 minutes at RT (prophylactic treatment) before it was added to a monolayer of MDCK cells for infection. After an infection period of 45 minutes at RT the inoculum was removed; cells were overlaid with semiliquid carboxymethylcellulose medium and then cultured at 37°C, hereby maintaining the same concentrations of active agent as in the prophylactic treatment. Staining was performed on fixed cells using an antibody directed against the influenza virus A nucleoprotein with a horse radish peroxidase labeled detection antibody and TMB as substrate. To enable direct comparison of the antiviral effectiveness of the lozenges, the 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 lozenge #1.

Aerosol samples were collected in a general pediatric ward at KK Women’s and Children’s Hospital, Singapore, using three National Institute for Occupational Safety and Health (NIOSH) two-stage cyclone samplers and one SKC filter cassette preloaded with a 37mm polytetrafluoroethylene (PTFE) filter (0.3μm pore size) designed to sample for severe acute respiratory syndromeassociated coronavirus (SARS-CoV). Aerosol samples were collected once per week for seven weeks in May and June 2017. The NIOSH samplers were stationed on tripods and placed along the corridor outside the open patient bedding area, and one SKC filter cassette was attached to a mobile computer on wheels (COW) used by doctors and nurses during ward rounds. Each sampler was connected to an AirChekR TOUCH Sample Pump (SKC, Eighty- Four, USA) with Tygon tubing (61 cm length, 0.635 cm diameter) for air collection at a rate of 3.5 L/min. A total of 840 L of air was collected during each four-hour sampling period. Each NIOSH sampler separates collected particles into three aerodynamic diameters: >4μm, 1-4μm, and <1μm.3 Filter cassettes and sample tubes from the NIOSH samplers were stored at -80˚C before processing. Prior to nucleic acid extraction, sample material collected in the 1-4 μm and <1μm size fractions were combined (described below).

Bovine coronavirus (BCoV) and bovine respiratory syncytial virus (BRSV) are contagious pathogens detrimentally affecting production and animal welfare in the cattle industry. The viruses are part of the bovine respiratory disease complex and are endemic worldwide. BRSV and BCoV can cause epidemics of respiratory disease and additionally BCoV cause diarrhea in calves and adult cattle (winter dysentery) [1–4]. The traditional way of handling and preventing these diseases is through metaphylactic antibiotic treatment, use of vaccines, or changes in management to improve calf health in herds. The within-herd prevalence and morbidity of BCoV and BRSV infections are high [6, 7] and once the virus enters a herd, circulation is difficult to mitigate. An additional preventive strategy is therefore to reduce inter-herd transmission of virus. Movement of live animals between herds is an important transmission route. If this risk is under control, the next question concerns the contribution of indirect spread of virus between herds. Indirect spread can occur via e.g. personnel travelling between herds, their clothes or equipment.

Important risk factors for indirect spread are the level of virus contamination of relevant surfaces and the infectivity of the viruses. Enveloped respiratory viruses like BCoV and BRSV are generally fragile outside the host. However, as related viruses like human respiratory syncytial virus (HRSV) and human coronavirus 229E remain infective for several hours on contaminated surfaces like countertops and surgical gloves [10, 11], there is a potential for indirect transmission. Epidemiological studies also point out the importance of indirect transmission; Ohlson et al. found that lack of boot provision for visitors was a risk factor for infections with both viruses and Toftaker et al. found that a herd’s BCoV and BRSV antibody status was influenced by the status of its neighboring herds.

Human nasal mucosa might also be a vector for inter-herd virus transmission, as traffic of personnel between herds is common. Carriage of BCoV and BRSV in human nostrils has not been studied. Generally, there are few studies on indirect transmission of these viruses, and no experimental studies have been performed. Molecular methods and virus isolation in cell culture can be used to study the level of virus carriage and infectivity, which are determinants for virus transmission. Combined, these methods provide sensitive quantification of viral genomes and assessment of virus infectivity.

Consequently, the aim of the present study was to investigate whether personnel (nostrils) and fomites carry viral RNA and infective viruses after exposure to BCoV or BRSV infected animals.

Alpaca (Vicugna pacos, also known as Lama guanicoe pacos) are domesticated members of the New World camelid species (Lamini), which also include guanaco (Lama guanicoe), vicuna (Vicugna vicugna), and llama (Lama glama). The natural habitat for alpaca is at high altitude (3500–5000 m) in South America (Peru, Ecuador, Bolivia, and Chile) where they are kept as livestock in herds and their fiber is used much like wool. Approximately 300,000 animals are in the U.S. Compared to other livestock, e.g., about 96 million cattle, their number is still relatively small.

Previously reported viral infections in domestic alpaca include adenovirus, equine viral arteritis virus, rabies, bluetongue virus, foot-and-mouth disease virus, bovine respiratory syncytial virus, influenza A virus, rotavirus, orf virus, bovine papillomavirus, vesicular stomatitis virus, coronavirus, bovine parainfluenza-3 virus, West Nile virus, equine herpesvirus-1,, and bovine viral diarrhea virus–[13]. Bovine enteroviruses (BEV) have not previously been reported to infect alpaca. The bovine enterovirus species previously contained two types, BEV-A and BEV-B, although a new classification structure was ratified recently, redesignating these as species Enterovirus E (EV-E) and Enterovirus F (EV-F), respectively,. Each of the new BEV species includes multiple serotypes, with EV-E comprising four described serotypes (previously A1–4, renamed E1–E4), and EV-F containing six reported serotypes (previously B1–6, renamed F1–F6).

Recently developed approaches to virus detection have the potential to further expand understanding of viral disease in animals, including alpaca. Many of these approaches are based on non-specific PCR amplification used in conjunction with standard or high-throughput sequencing to identify PCR products.

We utilized such a method–[19] to investigate an outbreak of a respiratory infection in alpaca, identifying a bovine enterovirus (EV-F), named Enterovirus F, strain IL/Alpaca, after other techniques had failed to detect any pathogen.

Infectious viral diseases, both emerging and re-emerging, pose a continuous health threat and disease burden to humans. Many important human pathogens are zoonotic or originated as zoonoses before adapting to humans–[4]. This is exemplified by recently emerged diseases in which mortality ranged from a few hundred people due to infection with H5N1 avian influenza A virus to millions of HIV-infected people from acquired immunodeficiency syndrome–[8]. Severe acute respiratory syndrome (SARS) coronavirus and the pandemic influenza A/H1N1(2009) virus in humans were linked to transmission from animal to human hosts as well and have highlighted this problem–[11]. An ongoing systematic global effort to monitor for emerging and re-emerging pathogens in animals, especially those in key reservoir species that have previously shown to represent an imminent health threat to humans, is crucial in countering the potential public health threat caused by these viruses.

Relatively few studies have been conducted on diseases of non-domestic carnivores, especially regarding diseases of small carnivores (e.g. mustelids). Ferrets (Mustela putorius furo) can carry bacteria and parasites such as Campylobacter, Giardia, and Cryptosporidium in their intestinal tract and potentially spread them to people,. In addition, they can transmit influenza A virus to humans and possibly on rare occasions rabies virus as well–[16]. Because of their susceptibility to several human respiratory viruses, including human and avian influenza viruses, SARS coronavirus, nipah virus, and morbilliviruses–[21], ferrets have been used as small animal model for these viruses. To further characterize this important animal model and to obtain epidemiological baseline information about pathogens in ferrets, the fecal viral flora of ferrets was studied using a metagenomics approach. Both known and new viruses were identified.

PTFE filters were removed from the cassettes attached to the NIOSH samplers, transferred to 50 mL falcon tubes and vortexed for 15 s. One mL of 0.5% bovine serum albumin (BSA) fraction V in molecular grade water was then added to each 50mL falcon tube and vortexed again for 15 s. One mL of 0.5% BSA was added to each 1.5mL conical tube from the NIOSH samplers and vortexed for 15 s. These BSA solutions were then pooled into a 2mL cryovial tube. Two mL of 0.5% BSA fraction V solution was added to each 15mL falcon tube from the NIOSH samplers, vortexed for 15 s, and transferred to a cryovial tube and stored at - 80˚C until further used. Styrene filters from SKC cassettes were swabbed with FLOQSwabs soaked in 0.5% BSA fraction V solution. Swabs were then placed in 50mL falcon tubes, vortexed for 15 s, and transferred to cryovials. QIAamp viral RNA kit and QIAamp DNA Blood kit (Qiagen) were then used to extract RNA and DNA, respectively, from the sample solutions following the manufacturer’s protocol.

No CPE was seen in cells incubated with swab material or with passaged material and RT-ddPCR results did not indicate any virus replication after two passages in the cells. Positive control wells were positive, and negative control wells were negative.

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 importance of cronaviruses as emerging zoonotic viruses became evident after the international public health threat caused by severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002/2003. Thereafter, there have been several studies that looked for novel coronaviruses aimed at assessing their zoonotic potential. Coronaviruses are members of the order Nidovirales and family Coronaviridae which are made up of single-stranded positive sense RNA genomes and infect both mammalian and avian hosts. They are divided into four genera namely Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In 2003, a coronavirus belonging to the Alphacoronavirus genus was discovered in an infant in the Netherlands and was designated human coronavirus NL63 (HCoV-NL63). This, among other coronaviruses, namely human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV predominantly cause respiratory disease. Human coronavirus NL63 has a worldwide distribution and is known to be associated with both upper and lower respiratory tract infections in both adults and children with seroconversion occurring at a very early age. Most of the known human coronaviruses are believed to have originated from mammalian reservoirs such as bats and used other mammalian hosts as intermediate hosts before ending up in the human population. Some of these, like HCoV-229E and MERS-CoV, used camelid species, while SARS-CoV went through Himalayan palm civets as intermediate hosts. Further, HCoV-OC43 is reported to have originated directly from cattle. Unlike these groups of coronaviruses, HCoV-NL63 and HCoV-HKU1 have no known intermediate mammalian hosts. Human coronavirus NL63 is known to use the same receptor as SARS-CoV, and may therefore, like some SARS-CoV-related viruses, be capable of infecting swine. This assertion is, however, yet to be explored through surveillance data. Different serological studies have mainly employed enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) approaches for investigating HCoV-NL63. Most of these assays are designed for specific purposes ranging from seroprevalence studies to studies of the HCoV-NL63 genome, and would therefore vary in parameters like sensitivity and specificity. There is no single assay that is widely accepted as the standard for serological detection of HCoV-NL63, and this presents a challenge in the general validation of new assays. Coronaviruses have the potential to recombine to produce new viruses, and as such, knowledge of potential hosts other than humans that can be infected by two human coronaviruses is important to provide information on potential sources of novel human coronaviruses that may later spillover into human populations and cause disease. Knowledge of potential intermediate hosts of human coronaviruses will also provide information on the evolution of coronaviruses in general and interspecies transmission events that lead to emergence. The purpose of this study was therefore to assess the potential of domestic livestock species as intermediate hosts for HCoV-NL63.

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.

In 2011, a new influenza virus was isolated from pigs with influenza-like symptoms and shared only 50% overall homology to human influenza C virus. This virus was considered as a new genus and named thereafter influenza D virus (IDV). IDV circulates widely and has been detected in America, Europe, Asia and Africa. Several studies demonstrated that IDV has a large host range and a higher prevalence in cattle than in swine and other species, suggesting that bovine could be a main host for IDV. The virus or its specific antibodies were also detected in horses, small ruminants, camels or feral swine. However, the zoonotic potential of IDV is still unclear. The circulation of IDV in Europe is not fully understood but data is available in Luxembourg and Italy with small cohorts tested: 80% and 93% of the tested cattle sera were positive in Luxembourg and Italy, respectively (n = 480 and 420 sera tested in each country).

Here, we performed a large scale seroprevalence study of IDV in large and small domestic ruminants at a country level. As we aimed to detect IDV antibodies with an individual prevalence limit of 0.1% for cattle and 0.5% for small ruminants with 95% confidence, at least 3000 and 600 sera were needed, respectively.

Along with equine rhinitis virus (ERV) and foot and mouth disease virus (FMDV), bovine rhinitis A and B viruses (BRAV and BRBV, respectively) are species in the genus Aphthovirus, family Picornaviridae. Two serotypes of BRAV have been identified, BRAV1 and BRAV2, while BRBV consists of a single serotype. The BRAV1 strain SD-1 was isolated in Germany in 1962 from nasal secretions from a calf with rhinitis. Additional BRAV1 strains were subsequently isolated from both healthy and diseased bovines in England, Japan, Italy and the U.S. and shown to cross react in serum neutralization assays [3–6]. The sole BRBV isolate EC-11 was isolated in England in 1964 by Reed from the lung of a specific pathogen free calf with respiratory disease. Likewise, BRAV2 consists of a single specimen, strain H-1, isolated from an outbreak of respiratory disease in cattle in 1984. Despite numerous studies on bovine rhinitis viruses (BRV) in the 1960’s through mid-1980’s, little work has been published on their epidemiology and ecology the past several decades.

Bovine respiratory disease complex (BRDC) is the most economically significant disease of the cattle industry, leading to losses due to mortality, morbidity, treatment costs and feed inefficiency in excess of $750 million dollars per year in the U.S. alone. BRDC has a multifactorial etiology involving a variety of bacteria and viruses in addition to host and environmental factors. Numerous commercial vaccines including both killed and attenuated live bacteria are available. Viruses commonly included in commercial vaccine include bovine viral diarrhea virus (BVDV), bovine herpes virus 1 (BHV1), parainfluenza virus 3 (PI3) and bovine respiratory syncytial virus (BRSV). Despite their widespread use, BRDC incidence has increased over the past 20 years. BRDC pathogenesis often involves a primary viral infection which damages respiratory mucosa and alters host immune responses leading to secondary bacterial pneumonia caused by commensal bacteria already present in the respiratory tract.

Both BRAV and BRBV are established but rarely studied etiologic agents of BRDC. Experimental inoculation of calves with BRAV1 via intranasal (IN) or intratracheal (IT) routes, either singly or in combination, resulted in variable clinical signs of respiratory disease and histologic lesions consistent with pneumonia. BRAV1 was also recovered from nasal swabs of IN inoculated animals and all animals inoculated or exposed by contact seroconverted to BRAV1 by day seven post inoculation. A similar experiment using a different BRAV1 strain (RS 3x) and colostrum deprived calves failed to reproduce clinical disease but was successful in isolating BRAV1 from nasal swabs post inoculation and found histological lesions of focal rhinitis and a neutralizing antibody response in all inoculated calves. BRBV pathogenesis was investigated using intranasal inoculation of gnotobiotic calves. Clinical signs including fever, nasal discharge and increased respiration rate were observed. Foci of epithelial necrosis were observed histologically in the turbinates and trachea and interstitial pneumonia was evident in the lungs. Virus was isolated from multiple tissues and was neutralized by convalescent antiserum. In addition to controlled studies, numerous investigations of acute respiratory disease in cattle resulted in the isolation of bovine rhinitis viruses where paired acute and convalescent sera suggested a causative role for bovine rhinitis virus.

Metagenomic sequencing on nasal swabs obtained from BRDC diagnostic submissions were performed to survey viruses present. Contigs with high identity to BRAV2 and BRBV were identified in one swab. To further our understanding of the epidemiology and ecology of bovine rhinitis viruses in BRDC, a more comprehensive survey was performed.

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.

An overview of clinical signs in all groups is presented in Table 2. Five out of six FG calves showed mild clinical disease. EG’s daily clinical scores are shown in Fig. 2. Three out of four EG calves showed mild disease, and one calf moderate clinical disease. SG did not develop clinical signs that were categorized as disease in the clinical scoring system. However, both calves had some days with intermittent nasal discharge and sporadic cough and S1 had a few days with intermittently runny feces. Blood-tinged diarrhea or nasal discharge was not observed in any of the groups.

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.

Prophylactic administration of low doses of antibiotics has been historically used to promote the growth and avoid infectious diseases in livestock animals. However, due to the emergence of antibiotic resistant microbes, several governments in countries around the world have prohibited the use of antibiotics as growth promoters for animals.

One of the most important challenges of agricultural immunology therefore is to find alternatives for developing drug-independent safe food production systems by modulating the immune system of animals. The work reviewed here encourages the research of probiotics to beneficially modulate the immune system of the bovine host. This review provides comprehensive information on the innate antiviral immune response of bovine IECs against virus, which can be further studied for the development of strategies aimed to improve antiviral defenses. The analyzed data also suggest that beneficial microbes have a great potential to be used as antiviral alternatives able to reduce severity of infections in the bovine host.

The development of specific in vitro study systems for cattle such as BIE cells as well as the selection and characterization of microbes that exert beneficial functions specifically and efficiently in the bovine host are key points for the successful development of immunomodulatory feeds aimed to protect against infections and reduce or avoid the use of antibiotics.

The family of Coronaviridae is composed of group 1–3 coronaviruses (CoVs). These viruses are able to infect human, canine, feline, murine, bovine, porcine, rat, and avian species. The etiological importance and zoonotic characteristics of coronaviruses have received much attention since the discovery of the newly emerged severe acute respiratory syndrome associated coronavirus (SARS-CoV) in 2003. Coronaviruses have a high frequency of viral genome recombination and polymerase infidelity, which may have contributed to the increase of viral pathogenesis, inter-species transmission, and tissue tropism. In the case of SARS-CoV, its ancestral origin remains undetermined, but some evidence suggests that Chinese horseshoe bats may be the natural reservoirs, while Himalayan palm civets harbor and support inter-species transmission to humans. Other examples of extended tissue tropisms can also be found in some group 2 CoVs. It is speculated that the acquisition of hemagglutinin esterase (HE) activity from influenza C virus gives rise to the ability of sialic acid recognition and the extended tissue tropism and pathogenesis for some group 2 CoVs. Furthermore, bovine coronavirus (BCV) is thought to have jumped to human hosts, possibly by recombining with influenza C virus, thus giving rise to human coronavirus-OC43 (HCoV-OC43) around 1890.

Receptor interaction between the virus and its host is the first step leading to a successful entry and productive replication. Viruses increase fitness by adapting to environmental pressure through mutation and recombination. In contrast to other families of viruses that utilize a universal receptor to gain entry into host cells, members in the coronavirus family use a variety of cellular proteins and/or co-factors. Group 1 CoVs – including human coronavirus-229E (HCoV-229E), feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCV) – utilize human, feline, porcine, and canine aminopeptidase N (APN) as functional receptors during virus entry. The only notable exception is HCoV-NL63, which utilizes angiotensin-converting enzyme 2 (ACE2). In group 2 CoV, mouse hepatitis virus (MHV) of group 2a and SARS-CoV of group 2b independently utilize carcinoembryonic antigen-cell adhesion molecule (CEACAM1) and ACE2 to mediate infection. However, other group 2a CoVs, including HCoV-OC43 and BCoV recognize N-acetyl-9-O-acetylneuraminic acid as a functional receptor. While the cellular receptors for both groups 1 and 2 CoVs have been identified and independently confirmed, group 3 CoV receptors remains undetermined.

The avian CoVs, such as turkey CoV and infectious bronchitis viruses (IBV), have been classified in group 3, with IBV the most extensively studied. Recently, Winter and colleagues suggested that sialic acids are responsible for IBV strain Massachusetts 41 entry. However, group 3 CoVs lack HE as a key viral protein regulating sialic acid binding, and the use of sialic acid would not explain the dependence on chicken cells for infection. Therefore IBV is unlikely to use sialic acids as a functional entry receptor, but rather as a non-specific attachment factor. Heparan sulfate may also serve as an attachment factor for the IBV strain Beaudette (IBV_Bdtt). IBV_Bdtt is a highly chicken embryo-adapted strain, which has an extensive tropism in cell culture and efficiently infects various cell types, including BHK-21 cells. In contrast, clinical isolates and field strains of IBV typically only infect chicken cells.

In the effort to identify the receptor for group 3 CoVs, feline APN (fAPN) was reported to allow entry of the IBV strain Arkansas 99 (IBV_Ark99). This could therefore be the first indication of a more universal receptor for the CoV family. APN belongs to a family of metalloproteases. It is a type II membrane-bound glycoprotein, and it is expressed on a variety of cell types, including granulocytes, monocytes, and fibroblasts. APN can also be found on the synaptic membranes of the central nervous system neurons, and on epithelial cells in the proximal convoluted tubules, intestinal brush border, and respiratory tract. For coronaviruses in general, there is a cross-species restriction that permits cells of a certain species to be infected only by its own complimentary CoV. However, several studies on FIPV and HCoV-229E, CCV and TGEV have identified fAPN as a universal entry receptor for group 1 coronaviruses. The demonstration that fAPN can allow infection by the IBV strain Ark_99 prompted us to examine both prototype and field isolates of IBV and test them for the potential use of fAPN as a receptor.

In this study, we first verified the use of the expressed fAPN as a receptor for FIPV and TGEV by transient and constitutive expression of fAPN in non-permissive BHK-21 cells. We also cultured seven strains of IBV, including Arkansas 99, Arkansas_DPI, California 99, Connecticut 46, Holland 52, Iowa 97, and Massachusetts 41 (designated as Ark99, Ark_DPI, CA99, Conn46, H52, Iowa97, and Mass41) as candidates to test for fAPN utilization by group 3 avian CoVs. Surprisingly, expression of fAPN did not increase viral infection in any of the strains tested. As a consequence, we conclude that fAPN is not a functional receptor during IBV entry. The authentic receptor is still under investigation.

The third-generation virus sub-cultured in MDBK was used for complete genome amplification. Twelve pairs of primers (described in Table S2 in the supplementary material) were used for the whole genome amplification. PCR products were purified, cloned into pMD18-T, and sequenced. Sequences data were compiled to generate the complete genome sequence of BPIV3. Sequences were assembled using SeqMan (DNASTAR, Madison, WI, USA).

Microneutralization: The methods used have been described before. MERS-CoV (strains: EMC and camel/Egypt/163/14) and bovine coronavirus (BCoV) (ATCC BRCV-OK-0514-2) were used. Vero cells (ATCC CCL-81) were used for MERS-CoV and HRT-18G cells (obtained from ATCC) for BCoV. Serum dilutions were mixed with equal volumes of 200 tissue culture infective dose 50 of virus and incubated for one hour at 37°C. The virus-serum mixture was then added in quadruplicate to cell monolayers in 96-well microtitre plates. After one hour of adsorption, the virus-serum mixture was removed and 150 μl of fresh culture medium was added to each well and the plates incubated at 37°C in 5% CO2 in a humidified incubator. A virus back-titration was performed without immune serum to assess input virus dose. Cytopathic effect (CPE) was read at three days post-infection for MERS-CoV and four days post-infection for BCoV. The highest serum dilution that completely protected the cells from CPE in half of the wells was defined as the neutralizing antibody.

Plaque reduction neutralization (PRNT): The PRNT assays were performed on heat inactivated sera in 24-well tissue culture plates in duplicate for each serum dilution as previously described. Briefly, two-fold serum dilutions were incubated with 40–60 plaque-forming units of MERS-CoV (strain EMC) virus for 1 h at 37°C. Then the virus and serum mixture were added to a pre-formed Vero cell monolayer and incubated for 1 hr at 37°C in a 5% CO2 incubator. Then, the supernatant was removed and the cells were overlaid with 1% agarose (SeaKem LE Agarose, Lonza, Switzerland) in cell culture medium (Minimum Essential Medium with 2% foetal bovine serum). After three days incubation the plates were fixed and stained. Endpoint PRNT antibody titres were defined as the highest serum dilutions that resulted at ≥50% (PRNT50) and ≥90% (PRNT90) inhibition of the number of plaques, respectively. Only the PRNT90 data is shown because this is the more stringent end point for virus neutralization.

The genomes of identified viruses described in detail here were deposited in GenBank under the following accession numbers: ferret kobuviruses MpKoV38, KF006985; MpKoV32, KF006987; MpKoV39, KF006986; ferret parechovirus (MpPeV1), KF006989; ferret papillomavirus (MpPV1), KF006988; ferret anellovirus (MpfTTV1), KF006990.

There were no adverse reactions noticed at the injection site or overall health of sow post-vaccination. Each day post-challenge, the piglets were evaluated by animal services veterinarian for the clinical scores. The clinical score was assigned as 0 (healthy) to 4 (dead or moribund) for each piglet based on parameters such as animal demeanor, degree of depression and willingness to nurse. The average clinical scores for the vaccinated sow’s piglets was close to zero, whereas the control sow’s piglets had average scores reaching on day 2 and 3 post-challenge (Fig. 8a). Similarly, fecal scores were recorded from 0 (normal pasty faces) to 2 (watery diarrhea) for all the piglets each day post-challenge. Diarrhea increased until day 4 post-challenge and then started to reduce for both groups of piglets (Fig. 8b).

The weight of each piglet was monitored every day post-farrowing for 10 days, and the analysis of the average weight of piglets showed no appreciable differences between two groups of piglets (Fig. 8c).

Survival of the piglets was monitored for 10 days after challenge. At 6 days post-challenge, 7 out of 8 piglets (87.5 %) of vaccinated sow survived whereas only 3 out of 7 piglets (42.9 %) of control sow survived the challenge (Fig. 8d). Statistically significant (P = 0.0002) better survival rate in the litter of vaccinated sow can be explained by the fact that these piglets were less depressed and showed more willingness to nurse than piglets of a control sow. However, more experiments with large number of sows are needed to confirm these data.

In summary, our vaccine had negligible effect in either preventing diarrhea or preventing PEDV-mediated weight loss, but did partially protect piglets in terms of severity of clinical disease and significantly reduced mortality. Future work to improve the protective efficacy of the subunit vaccine for PEDV may include testing new adjuvants. For instance, in the recently published report, oil-in-water adjuvant was used to formulate recombinant S1 protein. Another approach is use of oral immunization instead of intramuscular (IM) route. A field study demonstrated that orally vaccinated sows with live attenuated PEDV vaccine exhibited higher IgA and virus neutralizing antibody levels in the colostrum or sera compared to those of the counterparts administered the IM vaccine with the same dose. To deliver a recombinant protein orally, a live vector such as adenoviral vector might be used.

Many different infectious agents, viruses, bacteria, parasites and fungi, can lead to perinatal diseases in animals. Some infectious agents have the genital system as their primary target; they will mainly lead to infertility, abortion, stillbirth or weak newborns. Other agents give a more generalised infection and reproductive problems are only a small part of the picture. This lecture will focus on some important infectious agents that infect the animals during late gestation leading to abortion, stillbirth or foetus anomalies. Infections of the newborn animals the first few days after birth will also be considered.