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.

Clinical signs of classical swine fever usually appear 5–10 days after infection (occasionally longer). An individual pig may show one of four types of clinical effect; Peracute (sudden death, especially at the beginning of a farm outbreak), Acute (fever, depression, weakness, anorexia, conjunctivitis, diarrhoea or vomiting, purple discoloration of abdominal skin, or necrosis of the tips of extremities, and neurological signs), Chronic (weight loss, hair loss, dermatitis, discoloration of abdomen or ears) and subclinical. Affected pigs may recover or relapse, depending on the severity of the disease. Reproductive effects is also common; abortions, stillbirths, mummifications and also congenital tremor of piglets.

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.

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.

Despite efforts to reduce the incidence of nosocomial infections by measures such as practicing good hand hygiene and the use of personal protective equipment, hospital-acquired infections are still frequent. Two of the largest hospitals in Singapore report that on average, one in seven hospitalized patients acquire a nosocomial infection,1 with immunocompromised children at greatest risk. Little is known regarding the transmission of respiratory viruses in clinical environments. Theoretically, influenza and other respiratory viruses are transmitted through contact with contagious persons and contaminated fomites. However, there is an increasing body of evidence supporting the concept of transmission through the inhalation of virus-laden particles. Specifically, airborne virus-laden particles ≤4μm are thought to play a significant role in respiratory virus transmission as they can remain in the air for prolonged periods of time and are inhaled deep into the lungs.2

Coronaviruses (CoVs) are well-known causes of severe infections, respiratory, enteric and systemic, in humans and numerous animal hosts. The CoV infections have been reported in cattle, swine, horses, camels, rodents, cats, dogs, bats, palm civets, ferrets, mink, rabbits, snake, and several other wild animals and avian species (Fehr and Perlman 2015; Kahn and McIntosh 2005). The coronaviruses of relevant veterinary species are shown in Table 1 with organ affected and clinical signs. Though human CoVs were identified for the first time in the year 1960 from respiratory infections in adults as well as children, the major scientific interest in CoVs research grew only after the emergence of Severe Acute Respiratory Syndrome CoV (SARS-CoV) in the year 2002-2003 (Drosten et al. 2003; Ksiazek et al. 2003; Peiris et al. 2003). In this SARS-CoV epidemic, around 8000 confirmed human cases with 774 deaths (around 9.5% mortality rate) occurred that was a result of its global spread (Kahn and McIntosh 2005). Initially, the virus was detected in the caged Himalayan palm civets and these were thought to be the natural host of this virus (Guan et al. 2003). Following SARS-CoV incidence in 2003, a similar CoV named HKU3-1 to HKU3-3 were identified in the horseshoe bats (non-caged) in 2005 from Hong Kong (Lau et al. 2005). Since then, bats are considered to be the natural host and potential reservoir species that could be held responsible for any future CoVs epidemics and/or pandemics (Cui et al. 2019, Li et al. 2005). After the 2003 and 2005 SARS-CoV epidemics, an analogous virus emerged in the Middle East region of the world leading to severe respiratory illness and was named the Middle East Respiratory Syndrome CoV (MERS-CoV) (Zaki et al. 2012). The mortality was higher than previous SARS-CoV pandemic claiming around 919 lives out of the total 2521 human cases (around 35% mortality) (World Health Organization 2015). Notably, dromedary camels were connected with the transmission of MERS-CoV (Alagaili et al. 2014). Further, its origin was also traced from bats (Ithete et al. 2013). All these highly pathogenic human CoVs, SARS and MERS, show emergence over wider areas of the world posing high risk of human-to-human transmission and fatal consequences thereto (Figure 1).

This decade’s first CoV emergency was from Hubei province of China, and as on February 4, 2020, 425 deaths have been reported in China only (World Health Organization 2020b). Further, the spread of this novel coronavirus, named 2019-nCoV, has been noted in 24 countries till date. Considering the global threat of the 2019-nCoV, the World Health Organization (WHO) declared it as a ‘Public Health Emergency of International Concern’ on January 30th, 2020.

This rapid communication provides an overview of the recently emerging coronavirus (2019-nCoV) with regards to its current scenario, comparative analysis with respects to previously reported CoVs, evolutionary perspective based on genome analysis while covering the recent advances on vaccines and therapeutics in brief.

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.

Border disease is an important disease in sheep, caused by infection of the foetus in early pregnancy. Surviving lambs are persistently viremic, and the virus is present in their excretions and secretions, including semen. Ruminants and possibly also pigs can be readily infected by contact with these persistent excretors or with acutely infected sheep. Acute infections in immunocompetent animals usually are transient and subclinical and result in immunity to challenge with homologous but not heterologous strains of virus. The disease is characterised by low birth weight and viability, poor conformation, tremor, and an excessively hairy birth coat in normally smooth-coated breeds. Kids may also be affected, and a similar condition occasionally occurs in calves. The disease has been recognized in most sheep-rearing areas of the world.

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.

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.

Bovine respiratory disease complex (BRDC), a multi-factorial disease, is an economically important health problem of cattle worldwide. The disease is commonly referred to as “Shipping fever” and causes an increase in morbidity mortality rates. The multiple factors that cause BRDC include stress, infectious agents, immunity, and housing conditions. The infectious agents associated with BRDC include viruses, bacteria, and mycoplasmas. While most acute infections with uncomplicated infectious agents are sub-clinical, they can cause respiratory disease characterized by a cough, fever, and nasal discharge. Mixed infections with two or more infectious agents are thought to contribute to BRDC. The primary viral infectious pathogens that cause BRDC are bovine parainfluenza virus 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BHV-1), bovine coronavirus (BCV), and so forth.

Bovine parainfluenza virus type 3 (BPIV3) was one of the most important viruses associated with BRDC in cattle. It was first isolated in 1959 and first identified in cases of BRDC. BPIV3 is an enveloped, non-segmented negative-strand RNA virus within the genus Respirovirus. BPIV3 induces respiratory tract damage and immunosuppression. More severe secondary bacterial and mycoplasma infections are caused in susceptible animals in instances of high stress, such as transportation and feedlot situations.

Up to now, based on phylogenetic analysis, BPIV3 has been divided into three genotypes: Genotype A, genotype B, and genotype C. Multiple BPIV3 genotype A strains have been isolated in USA, China, Argentina, and Japan. Genotype B was initially identified in Australia. Isolation of BPIV3 genotype C, first identified in China, has also been conducted in South Korea, Japan, Argentina, and USA. A high seropositivity rate for BPIV3 in dairy cattle indicated that a high level of BPIV3 infections occurs. Many efforts have been made focusing on the prevention and control of BRDC in order to reduce production losses in the livestock industry.

Here, we describe the cell culture isolation and genomic sequencing of a BPIV3 genotype A strain isolated from cattle in China. Although BPIV3 is endemic in cattle, little is known about the pathogenesis of this virus and information regarding antigenic variation owing to the genetic variability is rare. The phylogenetic comparison of our isolated strain with strains previously characterized in China indicated the presence of divergent strains of genotype A circulating in the country. The diversity of BPIV3 in China seems to mirror the diversity of this virus, which is observed in the USA. In addition, the full characterization of our BPIV3 genotype A strain will lend support to molecular diagnoses and to future studies aimed at developing an efficient vaccine against multiple viral lineages.

Porcine epidemic diarrhea virus (PEDV) is an enveloped, positive-stranded RNA virus which readily infects pigs, resulting in highly contagious porcine epidemic diarrhea. PEDV belongs to family Coronaviridae, subfamily Coronavirinae and genus Alphacoronavirus. Some viruses of the Coronaviridae family cause severe disease in humans such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) [3, 4]. Coronaviruses of veterinary significance include avian infectious bronchitis virus infecting chickens, transmissible gastroenteritis virus (TGEV) infecting pigs, bovine coronavirus, feline coronaviruses, canine coronavirus and turkey coronavirus.

Porcine epidemic diarrhea (PED) was first observed in Europe in the early 1970s, and PEDV was first isolated in Belgium in 1978. Subsequently, PED has become an endemic disease in Asian pig farming countries. Severe PED outbreaks were reported in China in 2010–2012 [7, 8]. From April 2013 to the present, major PEDV outbreaks have been reported in the USA, Canada, Taiwan and Europian countries [12, 13]. The PED is characterized by the presence of watery diarrhea in the infected piglets in first few weeks of their life, dehydration, vomiting and anorexia resulting in high morbidity and mortality. PEDV infection of older pigs results in considerably lower morbidity and mortality. The symptoms of the disease are similar to transmissible gastroenteritis of pigs and hence only laboratory tests can aid in differencial diagnosis. Although, some efforts have been made to create the vaccine against PEDV with varied success, no effective vaccine is available in the market to protect the newborn piglets [14, 15].

The size of PEDV genomic RNA is about 28 kb, and contains seven open reading frames (ORFs) encoding viral proteins: 1A, 1B, spike (S), ORF3, envelope (E), membrane (M) and nucleocapsid (N). The S protein is present at the outer surface of the virion and is 1386 amino acid long. The spike protein of coronaviruses forms trimers and plays an important role in the virus attachment and in virus-cell membrane fusion. Porcine aminopeptidase N has been demonstrated to be a functional receptor for the PEDV coronavirus. The S protein of PEDV is a class I membrane glycoprotein consisting of two subunits: the N-terminal S1 and the C-terminal S2. Cleavage of spike protein into S1 and S2 is an essential event in the cellular entry for wild-type PEDV virus but not for cell culture adapted PEDV. Proteolytic cleavage of spike protein in PEDV needs trypsin [19, 20]. Several neutralizing epitopes have been identified on the S protein sequence [21–23], and the recombinant S1 protein was previously shown to have protective activity in piglets.

Toroviruses are enveloped, positive-stranded polyadenylated RNA viruses, which belong to the family Coronaviridae and also toroviruses are potential gastroenteritis causing agents affecting humans, calves, pigs, and horses [1–6]. In 1982, bovine torovirus (BToV) was first isolated from a case of neonatal calf diarrhea in the United States and BToV was reported to be related to calf diarrhea in experimentally infected gnotobiotic calves and under field conditions. Porcine torovirus is a member of the genus Torovirus (family Coronaviridae, order Nidovirales), and its genome organization is similar to other toroviruses, consisting of ~28000 nucleotides organized into five ORFs expressing a replicase polyprotein and four structural proteins: spike (S), membrane (M), hemagglutinin-esterase (HE) and nucleocapsid (N) [6–8]. Porcine torovirus has been reported in Canada, South Africa and European countries, Italy, Hungary, and in recent years also in Spain [5, 9, 10]. However, to our knowledge, detection of PToV in China has not been reported. In 2011 winter, there were epidemic outbreaks of diarrhea that occurred with high morbidity and mortality in China, which has caused great economic losses. Diarrhea samples were collected for examination of enteric pathogens, in which PToV was included. In this study, we reported the first detection of PToV in southwest China and analyzed the phylogenetic relationships between the Chinese PToV and PToV reference strains as well as other representative toroviruses. A survey for other enteric pathogens was also conducted and statistical analysis of the epidemiological study with regard to clinical signs (diarrhea) was performed to reveal any association of PToV infection with diarrhea in piglets.

Coronaviruses (CoVs) are enveloped viruses with a positive-stranded RNA genome and classified into four genera (alpha-, beta-, gamma- and deltacoronavirus) within the subfamily Orthocoronavirinae in the family Coronaviridae of the order Nidovirales. CoVs are found in a variety of mammals and birds, in which they can cause respiratory, enteric and systemic infections. Additionally, CoVs have proven ability for cross-species transmission, exemplified by the emergence of severe acute respiratory syndrome (SARS) coronavirus in 2002/2003, and of the Middle-East respiratory syndrome (MERS) coronavirus in 2012. Both viruses belong to the Betacoronavirus genus and have an animal origin. SARS coronavirus crossed over from bats via intermediate hosts to humans, became human-adapted and quickly spread worldwide before its containment. MERS coronavirus recurrently enters the human population via its dromedary camel reservoir host, with limited, non-sustained human-to-human transmission particularly in healthcare settings. Apart from SARS- and MERS-CoV, all four globally endemic human CoVs (HCoV-OC43, HCoV-NL63, HCoV-229E and HCoV-HKU1) originate from animals. In addition, cross-species transmission potential of CoVs is also illustrated by the occurrence of chimeric coronaviruses that resulted from recombination events between feline CoVs (FCoV) and canine CoVs (CCoV).

In order to get insight into the frequency of interspecies transmission of coronaviruses within and between animal and human populations and the risk of subsequent development of a pandemic, it is useful to screen for coronavirus infections in animal species; especially those that are in close contact with humans. Serological assays that can detect virus-specific antibody responses against infection play an important role in these epidemiological studies.

Cats live in close contact with humans and often roam around freely in the environment. Hence cats are an interesting species to study for infections with coronaviruses. Infections with feline coronaviruses (FCoVs) are recognized and widespread. FCoVs are classified into two types, type 1 and type 2, based on the genetic and antigenic difference of their spike (S) protein. In the field, the majority of FCoV infections are caused by FCoV type 1, while FCoV type 2, derived from recombination events of type 1 FCoVs and CCoVs obtaining the S gene and some flanking regions of CCoVs, is less prevalent. Depending on the virulence of the FCoV strain and the immune response of the cat, the clinical presentation can range from apparently asymptomatic, through diarrhea, to full-blown feline infectious peritonitis. FCoVs are members of the genus alphacoronavirus, to which also HCoV-229E, porcine transmissible gastroenteritis virus (TGEV), and CCoV belong. The latter three viruses and FCoV type 2 have been proven to use feline aminopeptidase N (fAPN) as a functional receptor in vitro. The receptor for type 1 FCoV has still not been identified. Notably, previous studies have shown that HCoV-229E and CCoV could infect cats after experimental inoculation, causing an asymptomatic infection. Thus, cats might potentially become naturally infected with CoVs of other species which may lead to virus-host adaptation e.g., mutation or recombination, resulting in emergence of novel coronaviruses and potentially new diseases. The extent to which infections with CoVs of other species occur in the field, has not been explored in previous epidemiological studies of CoV infections in cats.

Being the main envelope protein of coronaviruses, the spike (S) protein mediates cell attachment and membrane fusion to allow viral entry. S functions as the main determinant of cell-, organ- and host-tropism. Additionally, it is also the major target of neutralizing antibodies. Spike comprises two functionally interdependent subunits, S1 and S2, with S1 responsible for receptor binding and S2 for membrane fusion. The S1 subunit is the least conserved and the most variable immunogenic antigen between coronavirus species. Therefore, the S1 subunit is well suited as an antigen to screen for coronavirus type specific antibodies.

In this study, CoVs infection in cats were detected through profiling antibody presence in serum samples from cats. Recombinant CoV spike S1 subunits of different animal and human CoVs were expressed in a mammalian expression system and used for screening of cat sera for the presence of antibodies against the respective proteins. Positive samples were also tested by virus neutralization assays to support the specificity of the reaction. This investigation intends to extend our knowledge of CoV epidemiology, potential reservoirs, and cross-species transmission.

During the first week of December 2019, a few cases of pneumonia appeared in the city of Wuhan, Hubei province of China. The patients exhibited a history of visiting the local nearby Huanan seafood market which deals in the sale of different live animals, where zoonotic (animal-to-human) transmission suspected as the main route of disease origin (Hui et al. 2020). Firstly, the affected patients presented with pneumonia-like symptoms, followed by a severe acute respiratory infection. Some cases showed rapid development of acute respiratory distress syndrome (ARDS) followed by serious complications in the respiratory tract. On Jan 7th, 2020, it was confirmed by the Chinese Center for Disease Control and Prevention (CDC) that a new coronavirus has emerged and was named 2019-nCoV. As on February 4th 2020, China has confirmed 20471 cases with 425 deaths and 2788 severe cases of 2019-nCoV. In addition to China, 24 different countries from Europe, Northern America, Southeast Asia, Eastern Mediterranean, and Western Pacific Asia have reported the confirmed cases of this disease making the total tally of confirmed cases to 20630 worldwide (Figure 2). Although the mortality rate due to 2019-nCoV is comparatively lesser than the earlier outbreaks of SARS and MERS-CoVs, as well as this virus presents relatively mild manifestations, the total number of cases are increasing speedily and are crossing the old census. There is a high risk of human-to-human transmission which has also been reported in family clusters and medical workers. The infected patients with nCoV exhibit high fever and dyspnea with chest radiographs showing acute invasive lesions in both lungs.

Coronaviruses (CoVs) are enveloped, positive single-stranded RNA viruses that belong to the subfamily Orthocoronavirinae in the family Coronaviridae of the order Nidovirales. They are classified into four genera (alpha-, beta-, gamma- and deltacoronavirus) and infect both mammalian and avian hosts. Equine coronavirus (ECoV) belongs to Betacoronavirus 1 species, within the Embecovirus subgenus of the Betacoronavirus genus, as does human coronavirus OC43, HKU1 and bovine coronavirus. ECoV was isolated for the first time from a two-week-old diarrheic foal in North Carolina (USA) in 1999, suggesting the role of ECoV in causing enteric disease. Since 2010, several cases of ECoV infections have also been reported in adult horses from the United States, Europe and Japan. Equine coronavirus has been detected in fecal samples from horses with clinical signs that included anorexia, lethargy, fever and, less frequently, diarrhea, colic and neurologic deficits. The morbidity rate varies from 10% to 83% during outbreaks. Mortality is low and has been related to endotoxemia, septicemia or hyperammonemia-associated encephalopathy. The outbreaks in adult horses demand further studies on the pathogenesis and epidemiology of ECoV infections. For this, diagnostic assays with high sensitivity and specificity are crucial.

ECoV is known to be associated with enteric infections but can also be detected in a small percentage of horses with respiratory signs. Virus shedding can be observed in fecal samples or nasal swabs from sick horses as well as healthy horses, but with a strong association between clinical signs assumed to be related to ECoV infection and virus detection in fecal samples suggesting a possible etiological role of ECoV. Recently, real-time quantitative PCR (qPCR) methods have been established and were shown to be able to detect ECoV in feces efficiently. However, ECoV viral nucleic acid is generally only detectable by qPCR within a limited timeframe of 3–9 days post infection, as reported from both field and experimental studies. On the other hand, serological assays can be used to support the diagnosis of a clinical ECoV infection by showing seroconversion or a significant increase in antibody titer in paired serum samples. Serological assays are also needed to gain more insight into the transmission rate of infection within animal populations. Antibodies induced by betacoronaviruses persist in blood for a longer period after infection. The virus neutralization (VN) assay has long been used as a gold standard to confirm serological responses to coronavirus infections. Although the VN assay is highly specific for the detection of antibodies, it is also time-consuming and laborious to perform. Alternative high-throughput serologic assays that correlate well with neutralizing antibodies are therefore needed. Severe infections of ECoV have been shown to be associated with high viral load, but mild or asymptomatic infections may occur with low levels of virus replication being negative in PCR and with variable immune responses. Consequently, specific, sensitive and high-throughput serodiagnostic methods are necessary to avoid the underestimation of prevalence in surveillance studies.

The spike protein (S) of coronaviruses is the key mediator in virus cell entry and therefore the major target for neutralizing antibodies. The S ectodomain consists of two functionally interdependent subunits, S1 and S2. The N-terminal S1 subunit is responsible for receptor binding, while the C-terminal S2 subunit mediates membrane fusion. The S1 subunit is the most variable immunogenic antigen among coronaviruses, and therefore it is an ideal candidate for the detection of CoV species-specific antibodies. The objective of the study was to develop and validate an ELISA method for the detection of specific antibodies to ECoV and provide a tool for the diagnosis and the future estimation of ECoV prevalence and incidence in various equine (sub) populations.

Porcine deltacoronavirus (PDCoV), a member of the Deltacoronavirus genus, causes diarrhea and vomiting in pigs of any age. The clinical symptoms of PDCoV are similar to those of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), but the mortality rates are lower in PDCoV-affected nursing pigs. PDCoV was first identified in pigs in Hong Kong in 2012, while the first PDCoV outbreak in swine herds was reported in the United States in 2014. Subsequently, PDCoV was detected in Canada, South Korea, mainland China, Thailand, Laos, and Vietnam, indicating that it is widespread among the world's pig populations; moreover, PDCoV can have devastating effects on the swine industry. However, no treatments or vaccines are available for PDCoV.

Hemagglutinin, an envelope glycoprotein of some enveloped viruses, can adsorb to erythrocytes. In the presence of virus particles, erythrocytes clump together as a result of the interaction between hemagglutinin and erythrocytes, leading to a lattice formation. Hemagglutination (HA) assessment has been widely used for detecting and quantifying viruses that cause red blood agglutination, such as orthomyxoviruses, paramyxoviruses, and encephalomyocarditis virus. Some viruses of the family Coronaviridae, such as bovine coronavirus (BCoV), human coronavirus OC43 (HCoV-OC43), hemagglutinating encephalomyelitis virus (HEV), avian infectious bronchitis virus (IBV), and PEDV, have been shown to possess the ability to hemagglutinate erythrocytes.

As PDCoV is a newly emerging swine enteropathogenic coronavirus, it is unclear whether PDCoV has HA activity. We, therefore, investigated the hemagglutinating property of PDCoV in the present study. The following describes our observations on the HA and HA-inhibition (HI) characteristics of PDCoV. Our results will provide important information for the investigation of PDCoV biological characteristics.

Influenza viruses are enveloped, segmented, single-stranded, negative-sense RNA viruses and belong to the family Orthomyxoviridae. The genomes of influenza A virus (IAV) and influenza B virus (IBV) consist of eight RNA segments, whereas influenza C viruses (ICV) only have seven segments. Both IAV and IBV contain two major surface glycoproteins: the hemagglutinin (HA), which binds to sialylated host cell receptors and mediates membrane fusion; and the neuraminidase (NA), which destroys the receptor by cleaving sialic acid from host cell membranes, thereby releasing newly assembled virus particles, and likely assisting initial invasion by destroying sialylated mucin decoys. ICV, however, has only one major surface glycoprotein, the hemagglutinin-esterase-fusion (HEF) protein, which possesses all-in-one of receptor binding, receptor destroying and membrane fusion activities [3, 4]. While IAV infects avian, human, swine, and many other mammalian species including dogs, horses, tigers and seals, IBV and ICV are found principally in humans and rarely infect other species.

ICV usually causes mild upper respiratory tract infections in children with cough, rhinitis and rhinorrhea as clinical symptoms [5, 6]. The virus only occasionally spreads to the lower respiratory tract and causes bronchitis, bronchiectasie and broncho-pneumonia. Encephalopathy has also been occasionally reported. Seroepidemiological studies have revealed that ICV is widely distributed globally and that the majority of humans acquire antibodies against the virus early in life [9, 10]. Aside from humans, there is evidence that ICV possesses the ability to infect animals. Serological studies showed that antibodies against ICV are present in pigs [11–13]. In 1981, fifteen strains of ICV were isolated from domestic pigs in China, which showed characters highly related to viruses isolated from humans in Japan [15, 16]. Furthermore, pigs have been shown to be susceptible to experimental infection with both pig and human ICVs, and the virus is able to be transmitted from the infected to uninfected contact pigs, suggesting that interspecies transmission of ICV between humans and pigs might occur in nature. Dogs may also serve as a natural reservoir for human ICV due to the presence of viral replication and clinical symptoms in experimental infections and the prevalence of antibody to ICV among dogs [12, 17–19].

In 2011, an influenza C-like virus was isolated from swine in Oklahoma (D/swine/Oklahoma/1334/ 2011 [D/OK]) exhibiting influenza-like symptoms. The genome of this virus also contains seven segments, and sequence analysis showed approximately 50% overall amino acid homology to either human or previous swine ICVs. D/OK did not cross-react with antibodies against human ICVs and, importantly, was unable to reassort with human ICVs or generate viable progeny [20–22]. However, the low seroprevalence rate observed in both swine and humans to D/OK (9.5% and 1.3%, respectively) suggested that swine and humans are not likely to be a major reservoir of this novel virus. Subsequent serological studies have showed that antibodies against D/OK are almost ubiquitously present in cattle, and several novel D/OK-like virus strains have been isolated from cattle with respiratory disease which could be divided into two distinct lineages represented by D/OK and D/bovine/Oklahoma/660/2013 (D/660) [21, 23]. These two genetic and antigenic distinct clades have been shown to reassort with each other. In addition, D/OK has a broader cell tropism than human ICV and is capable of infecting ferrets, pigs and guinea pigs and transmit to naive animals by direct contact [20, 24]. Based on these differences to ICV it was suggested that this virus warrants classification as a new genus of influenza virus, named influenza D virus (IDV) with cattle as the potential reservoir. Subsequently, more IDVs or viral genomic segments were identified from China and France in cattle, suggesting the wide geographic distribution of IDV [25, 26]. Interestingly, IDV is common in clinical samples of bovine respiratory disease complex (BRDC), which is the leading cause of morbidity and mortality in feedlot cattle [23, 27]. BRDC is a challenging multi-factorial disease caused by viral, bacterial pathogens and environmental factors, leading to severe clinical signs and deaths. IDV was detected in clinical BRDC samples, co-infected with bovine coronavirus (BCV), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BHV-1), and Pasteurella multocida, Mannheimia haemolytica, Histophilus somni et al, suggesting IDV has the pathogenic potential in BRDC. In addition, a latest serological study showed that antibodies to IDV were present in sheep and goats in United States, suggesting that small ruminants are also susceptible to IDV infection.

To further evaluate the infectivity and transmissibility of IDV, we expressed and purified the ectodomain of D/OK HEF, and determined that it also uses 9-O-Acetyl-Sia as its receptor by glycan microarray. We also solved the crystal structure of D/OK HEF, both in its native state (resolution of 2.4 Å) and in complex with its receptor analogue (resolution of 3.1 Å), and the structure of the enzymatically inactive HEF (resolution of 2.4 Å) alone and in complex with two receptor analogs respectively (both resolutions of 2.2 Å). Indeed, our results show that IDV HEF is functionally and structurally similar to ICV HEF, but with some distinct characteristics.

Coronaviruses (CoV, family Coronaviridae) are large enveloped viral particles containing a positive sense single stranded RNA genome (26–30 kb), coding for several structural proteins, including polymerase (Pol), nucleocapsid (N), membrane (M), hemagglutinin-esterase (HE), spike (S) proteins and several non-structural proteins (NSPs). Coronaviruses have been associated with respiratory and enteric infections in humans and ruminants.

Enteric Bovine coronavirus (BCoV) replicates in the epithelial cells of the gut, destroying villi, resulting in severe, often bloody diarrhea in calves, which can be life threatening, due to loss of electrolytes and malnutrition. Disease in calves usually occurs within the first month of life, with respiratory and enteric infections being the most common conditions diagnosed. In adult cows, as a result of close confinement during transport or housing, BCoV is associated with winter dysentery and shipping fever. The spike proteins of BCoV play an important role in immune response, eliciting both cellular immune responses and neutralizing antibodies.

BCoV has been detected in Ireland using molecular or immunological techniques [4–6], but it has not been characterized or compared to other global BCoV strains. Enteric pathogens frequently isolated from neonatal calves with enteritis in Ireland are rotavirus, cryptosporidium and much less frequently, coronavirus. Currently in Ireland, a trivalent vaccine is licensed for the immunization of pregnant cows against rotavirus, coronavirus and Escherichia coli, confering passive immunity to calves, the coronavirus aspect of the vaccine is based on an inactivated Mebus strain. In this study we aimed to: (i) characterize bovine coronavirus in the South of Ireland via analysis of the Spike gene, and (ii) compare Irish BCoV to global and vaccine isolates to identify variations in the hyper-variable region of the spike gene.

The Middle East Respiratory Syndrome (MERS) is caused by a beta-coronavirus (CoV) first detected in a Saudi male in 2012. Since then, the World Health Organization (WHO) has recorded 2260 laboratory-confirmed cases, at least 803 of these being fatal. Surveys of camels in the Middle East and Africa showed that they are a natural host of MERS-CoV and they are a source of human infection.

Host specificity of MERS-CoV is determined by the presence of the dipeptidyl peptidase-4 (DPP4) receptors expressed on cell surfaces. DPP4 was found to be a functional receptor for the receptor-binding S1 domain (RBD) of the MERS-CoV spike protein. There is limited data on the susceptibility of other livestock in close contact with camels to MERS-CoV infection. A survey of goats, camels, and sheep from Jordan showed no evidence of infection. Surveys on horses in the United Arab Emirates, Saudi Arabia, and Oman did not provide conclusive evidence of infection.

We expanded our MERS-CoV surveillance programme in Egypt, Tunisia, and Senegal to include other domestic livestock in contact with camels.

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.

Porcine hemagglutinating encephalomyelitis coronavirus (PHEV) is a member of the Coronaviridae family, which causes porcine encephalomyelitis. PHEV predominantly affects 1-3 week-old piglets[1], with clinical piglets vomiting, exhaustion and obvious neurological symptoms as the main feature. The mortality rate is up to 20-100%[2]. Since 1958, when the disease broke out in the Canadian province of Ontario for the first time[3], many countries have reported about it. Serological test results proved that it is common for the pigs to be infected by PHEV[4,5], and the disease may have spread worldwide. In August 2006, the disease broke out in part of the pig farms in Argentina, resulting to 1226 deaths, with the morbidity rate up to 52.6%[1]. In China, an PHEV infection has been reported occurring in a pig farm in Beijing as early as in 1985, followed with reports from Jilin, Liaoning, Shandong, Taiwan, etc. The large-scale epidemics of HEV occurred in Taiwan in 1994 had a fatality rate of almost 100%[6], resulting to serious economic losses. Serological survey conducted by foreign scholars revealed that PHEV infection in pigs is very common, with a worldwide distribution[4,5].

Coronaviruses are usually divided into three groups based on genetic and serological relationship. HEV, togather with murine hepatitis virus (MHV), bovine coronavirus (BCoV), human coronavirus OC43 (HcoV-OC43), rat coronavirus (RCoV), belongs to group 2[8].

In this report, the clinical and neuropathologic feathers of spontaneous PHEV infection had been reported. Microscopically, coronavirus-like particles were detected in the supernatant of the brain samples by electron microscopy. One coronavirus strain (isolate PHEV-JLsp09) was isolated from the piglets. The Hemagglutinin- esterase (HE) gene of PHEV strains was amplified by reverse transcription- polymerase chain reaction (RT-PCR) and sequenced. The homology and phylogenetic analyses were done between the group 2 coronaviruses and influenza C virus strains downloaded from Genbank, based on the sequence of HE gene.

Three quarters of the recently discovered human pathogens are viral, and most of those are RNA viruses. Some of these emergent viruses, such as HIV and SARS coronavirus (SARS-CoV), are capable of causing epidemics of human disease. RNA virus populations sustain high genetic diversity due to the low fidelity of their polymerase, short genome, high replication rates and large population size. For this reason a single RNA virus population can consist of a multiplicity of slightly different genomes, sometimes referred to as a mutant spectra. The high mutation rate of RNA viruses increases the ability of these viruses to adapt to diverse hosts (interspecies transmission events) and the potential cause new human and zoonotic diseases, however, very little is known about the particular mutations that enable interspecies transmission events to occur.

Coronaviruses are particularly adept at adapting to new hosts due in part to their amazing capacity for genome recombination. Coronaviruses have the largest genome of RNA viruses, consisting of 27–30 kb positive sense single-stranded RNA. Although recombination can lead to an interspecies transmission event, as was believed to be the case with SARS-CoV, accumulation of point mutations may also enable the coronaviruses to adapt to new host species–[7].

The Coronaviridae subfamily Coronovirinae is composed of three genera based on serologic and genetic characteristics: Alphacoronavirus (formerly Group 1) includes viruses that infect pigs, dogs, cats and humans; Betacoronavirus (formerly Group 2) includes bovine, bat, human, horse, pig, rodent, and bat viruses; and Gammacoronavirus (formerly Group 3) which consists of viruses adapted to birds,. Bovine coronavirus (BCoV) is a betacoronavirus which is related to SARS-CoV and has caused disease in humans on at least one occasion. BCoV is known to use 9-O-acetylated sialic acid to bind to host cells, although a second receptor may be involved. The spike protein which is present on the surface of the virion, determines host range and tissue tropism of coronvaviruses. The receptor binding domain of BCoV has not been determined, but a recent study by Peng at al. (2011) indicates that it falls within the N terminal domain. BCoV infection may cause acute and severe diarrhea and respiratory symptoms in cattle especially under stressful conditions such as transport, but subclinical infections may occur in healthy cattle. BCoV isolates are generally obtained by inoculating nasal or fecal samples from infected cattle in human rectal tumor cells (HRT-18 cells), however the genetic changes in the viral population that allow this bovine virus to adapt to human cell lines have yet to be defined.

We are interested in understanding the role of natural viral population diversity in the adaptation of BCoV to new host environments; in particular, cell types that may enable the virus to spread via the respiratory route or to cause systemic disease. The mutational dynamics of BCoV was determined by serial passage of BCoV nasal samples in human and bovine lung and macrophage cell lines as well as human enteric cells (A549, EBL, THP-1, Bomac, and HRT-18, respectively). The consensus and subconsensus (variant) nucleotide sequences of cell culture passaged samples were compared to that of the natural “unpassaged” viral populations. The genome regions analyzed included nsp1and nsp3 (genes involved with evasion of the host innate immune response and other functions such viral replication), nsp14 (designated nsp11 in NCBI BCoV Reference Sequence: NC_003045.1; involved with polymerase fidelity), and the spike protein gene (determinant of host range),–[19]. Deep sequencing was used to obtain an accurate picture of viral population diversity before and after passage in cell culture.

Bovine coronavirus (BCoV) is an important livestock pathogen with a high prevalence worldwide. The virus causes respiratory disease and diarrhea in calves and winter dysentery in adult cattle. These diseases result in substantial economic losses and reduced animal welfare. One way of reducing the negative consequences of this virus is to prevent virus transmission between herds. Inter-herd transmission is possible either directly via transfer of live animals [2, 3], or indirectly via contaminated personnel or equipment. Measures to prevent virus spread between herds must be based upon knowledge of viral shedding, the potential for transmission to susceptible animals and the role of protective immunity. Several observational studies have been published on BCoV shedding in feces of diarrheic calves and after transportation to feedlots [3, 5–10]. However, relatively few studies on BCoV pathogenesis with emphasis on transmission potential under controlled conditions have been published.

BCoV belongs to the genus Betacoronavirus within the family Coronaviridae, also including the closely related HCoV-OC43, which causes respiratory infections in humans, and the human pathogens SARS-CoV and MERS-CoV [11–13].

BCoV consists of one serotype with some antigenic variation between different strains [14, 15]. Acutely infected animals develop antibodies that persist for a long period, possibly for several years [16–18]. However, the protective immunity is shorter and incomplete. In two experimental studies, infected calves were not protected against reinfection with a different BCoV strain three weeks after the first challenge, but did not develop clinical signs [19, 20].

BCoV is transmitted via the fecal-oral or respiratory route. It infects epithelial cells in the respiratory tract and the intestines; the nasal turbinates, trachea and lungs and the villi and crypts of the small and large intestine, respectively [21, 22]. Replication leads to shedding of virus in nasal secretions and in feces. Important factors for the pathogenesis are still not fully explored, such as how the virus infects enterocytes shortly after introduction to an animal. Viremia has been detected in one study by Park et al.. Clinical signs range from none to severe, and include fever, respiratory signs and diarrhea with or without blood [1, 15]. As the time of infection is usually unknown and laboratory diagnostics are usually not performed, occurrence of clinical signs is the most relevant parameter to relate to viral shedding. The majority of experimental studies have used BCoV inoculation as challenge procedure, which may influence clinical signs and viral shedding, and thereby the transmission potential compared to natural infection. It has been hypothesized that BCoV can cause chronic subclinical infections which could be an important virus source. Kapil et al. documented viral antigen in the small and large intestines of infected calves three weeks post inoculation. Crouch et al. found that ten cows were shedding BCoV-immune complexes in the feces for 12 weeks. It is, however, difficult to establish whether there is true persistence of virus, or reinfection of partially immune animals and whether these animals represent a risk to other animals. There is a lack of experimental studies investigating viral shedding pattern for longer periods than two weeks, with sensitive detection methods. Viral load and infectivity also needs to be determined. This is of high practical relevance, since the farmers need guidance on biosecurity in trade and transport of live animals.

The current study was conducted to fill prevailing gaps in the knowledge on fundamental aspects of BCoV infection. The specific aims were to:study the duration and quantity of BCoV shedding in feces and nasal secretions, related to clinical signs in calves.study the presence of viremia and persistence of virus in lymphatic, intestinal and lung tissue.test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after BCoV infection.

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.