In summary, we conducted a 7-week pilot study to monitor for aerosolized respiratory viruses among inpatients in a pediatric ward in Singapore. We found molecular evidence of influenza A virus and adenovirus, demonstrating the potential for airborne transmission. To comprehend this potential risk of transmission, our proof-of-concept project might be expanded in the future to specifically study patients with known positive clinical infections (e.g. through nasopharyngeal swabs) and determine how far away viable viruses can be detected from a patient’s bedside. Additionally, future studies might involve more comprehensive demographic and clinical risk factor analyses to help us better understand phenomenon such as super-spreading. Lastly, bioaerosol surveillance might be useful in monitoring clinical populations for incursions of novel respiratory viruses, especially since aerosol sampling in shared clinical spaces often requires no informed consent.

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.

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.

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.

The present study showed that calves infected with BCoV shed viral RNA for five weeks, and harbored viral RNA in intestinal tissues and lymph nodes even longer. Interestingly, contact with these calves three weeks after challenge, when the clinical condition had improved and the calves had seroconverted, did not lead to infection in sentinel calves and virus isolation was not possible from calves shedding viral RNA at this time point.

In concordance with other studies [18, 29], all EG calves became BCoV positive shortly after contact with infected calves and shed viral RNA continuously for two weeks. This supports that introduction of BCoV into a naïve population leads to a high basic reproduction number (R0). R0 depends on the duration of the infectious period, the number of exposed susceptible individuals and the probability of a susceptible individual to be infected. In herds and transportation systems where cattle from different herds are commingled, the risk of virus transmission is high.

The detection of BCoV RNA in nasal swabs from naïve calves in EG shortly after exposure might be due to passive inhalation of virus excreted by the FG, or to virus replication in the respiratory tract. Since the viral load in the nasal swabs from EG exceeded that of FG at D2, the study confirms that BCoV replicated massively in the airways of EG calves already at D2. Fecal shedding started later than nasal shedding which is in concurrence with other studies. Saif and colleagues found that when inoculating calves intranasally, BCoV was first detected in nasal epithelial cells and secondly in feces. In contrast, in calves inoculated orally, fecal detection of BCoV preceded detection in nasal swab specimens. They concluded that the infection route could determine the sequence of infection of the respiratory and intestinal tract. The present study supports that the respiratory route is the most common infection route when calves are naturally infected by direct contact. With indirect virus spread, the fecal-oral route could be more common.

Nasal swabs were more often positive for BCoV than fecal samples in this trial, most likely due to a higher limit of detection for BCoV in feces than in nasal swabs. For diagnostic purposes, nasal swab specimens therefore seem advantageous to fecal samples for virus detection in calves with suspected BCoV related disease.

Moving and commingling are associated with stress, which has been found to affect the intestinal immune system. It is possible that stress increased the BCoV RNA shedding observed in the EG calves after introduction of the sentinel calves. Buying and selling of calves often involve extended transportation and commingling with susceptible cattle. The stress response, and a possible increased fecal shedding of virus, would probably be higher under field conditions.

In the acute stage of the infection, the agreement between positive PCR results and clinical score was relatively high. Three weeks after exposure to BCoV, the clinical signs and detection of viral RNA varied more independently. In an experiment with porcine deltacoronavirus, the severity of the clinical signs did not correlate with the shedding of virus in conventionally reared piglets, only in gnotobiotic piglets. This indicates that secondary pathogens and changes in microbiota are important for disease development and clinical signs. The present study supports that after the acute stage of disease other factors than virus replication are important for clinical signs; for instance secondary bacterial infections.

Although the sentinel calves did not get infected with BCoV, they showed sporadic unspecific signs during the trial, but below the mildest category “mild disease” in the clinical scoring system. Since acclimatization was not possible, the calves changed environment including feeding routines when enrolled in the experiment, which could cause the signs observed. Other infectious agents could also have been present, and if so, most likely less virulent pathogens. Bovine virus diarrhea virus and bovine herpesvirus 1 are not present in Sweden, and the sentinel calves showed no serologic response to BRSV. Co-infection between BCoV and other agents is likewise possible in FG and EG, as is the case under field conditions.

Unlike most enteric viruses, BCoV is enveloped and therefore susceptible to environmental inactivation. One might expect that the conditions in the forestomaches and abomasum would inactivate BCoV and one possibility is that BCoV is transported from the oronasal cavity to the small intestines through the bloodstream. However, viremia was not detected in the present study, and transport of the virus to the intestines appears to have been through the digestive tract. Park and colleagues detected BCoV RNA in serum samples from calves infected with a winter dysentery strain between day three and eight post inoculation. They used nested PCR for detection, which is generally a more sensitive method than RT-qPCR, but also more vulnerable for contamination. Short viremic period or intake of a lower virus dose in naturally infected calves could also explain the negative results in the present study. Inhibition of the RT-qPCR by plasma components was tested and ruled out. Despite the absence of detectable viremia in the present study, BCoV RNA was found in mesenteric lymph nodes at late stages of the infection. Viral RNA must have been transferred in low concentrations in blood or lymph to the draining lymph node, by antigen presenting cells or as free virus particles.

The finding of BCoV RNA in lymph nodes, ileum and colon six weeks after infection indicates coronavirus persistence in calves, however, the importance of this persistence for virus transmission is uncertain. Other coronaviruses are known to create persistent or chronic infections in mice and cats [35, 36]. MERS-CoV is shown to be excreted for more than a month in humans and human coronavirus 229E creates persistent infections in vitro. Although fecal shedding of BCoV RNA was detected five weeks post infection in the present study, the transmission potential at this stage is most likely negligible, as at three weeks post infection.

BCoV VRC were quantified by RT-qPCR, which does not give information on the number of infective particles. The ratio of total to infective particles (T/I) is challenging to establish for BCoV due to difficulties in cultivating virus from clinical samples. In the present study, virus titration showed a T/I ratio of approximately 5 log10. With this high T/I ratio it is not surprising that virus isolation was unsuccessful after D13, when the VRC numbers are decreasing. It also agrees with the sentinel calves not getting infected D21. In contrast, roughly 8.8 log10 VRC were detected per nasal swab and gram feces from the seronegative FG calves that infected the EG calves. With a T/I ratio of 5 log10, each nasal swab and gram of feces contained more than 3.8 log10 infective virus particles.

The high T/I ratio and the failure of virus isolation after D13 could be due to either few infective particles or low sensitivity of the isolation method. Low levels of infective particles could be caused either by high production of defective particles or by neutralizing effect of antibodies. Low sensitivity could be caused by suboptimal conditions in cell culture compared to in vivo (particularly for virus from clinical samples not adapted to cell culture growth), dilution of viral content in the swab, and freezing and thawing of the material. For feline enteric coronavirus, the T/I increased from 3–4 log10 during the first week after infection, to up to 8 log10 28 days post infection, the increase possibly caused by the antibody response.

Few methods are available for studying transmission potential apart from live animal experiments, although ethically challenging and resource demanding. Existing literature is based on experimental studies examining BCoV shedding for 14 [20, 22, 40] to 21 [19, 41] days. To the authors’ knowledge, the present study is the first to study the shedding for as long as six weeks under experimental conditions. In addition, it is also the first to study the impact of this shedding using sentinel calves. Although a low number of calves were used, the results indicate that calves are not infectious three weeks after exposure to BCoV. This information is important and relevant in order to produce scientific based advices on how to avoid introduction of BCoV into herds. Further investigation of calves at different stages of disease is recommended to verify and corroborate these findings. The effect of stress related to transport on viral shedding and infectivity should also be considered.

In the present study, the virus that caused winter dysentery in adult cattle primarily gave respiratory disease in calves. Niskanen et al. also found that BCoV derived from an outbreak of winter dysentery caused mainly respiratory disease in weaned calves, supporting that BCoV is an important cause of respiratory disease in calves [42, 43] and winter dysentery in adults. The economic and welfare consequences of BCoV therefore include the combined effects of neonatal enteritis, respiratory disease in young cattle and winter dysentery in adults. Also considering the high prevalence worldwide, BCoV is an important loss-inflicting factor in the cattle industry.

Previously, virus discovery in animals has focused on pathogenic infections or on animals that on the basis of relevance to (re-)emerging viruses are thought to represent a key risk host for emerging virus-associated disease in humans. With the recent advances in the metagenomics field, a substantial increase in studies looking at virus epidemiological baseline levels in different (wildlife) animals has been observed,,,–[68]. Even though ferrets are a very important animal model for a number of human viral infections, not much is known about viruses that naturally occur in ferrets–[21]. This study describes the viral communities in fecal material of ferrets (Mustela putorius furo).

Sequences closely related to known viral sequences were identified, with homology to ferret coronavirus, ferret hepatitis E virus, Aleutian mink disease virus, different avian viruses and murine astrovirus STL1,,. The avian viruses may be a reflection of the diet of these ferrets, which were fed chicken. It is of note that both the viral screening with random amplification and next-generation sequencing and a specific ferret hepatitis E virus taqman assay indicated that household ferrets that are kept as pets are significantly more likely to excrete ferret hepatitis E virus in their fecal material than farm animals in this study. Such a correlation was not observed for ferret coronavirus excretion for which prevalence in both cohorts seemed similar, but not as high as previously reported. Sporadic cases of human hepatitis E virus infections seem to be increasing and several observations suggest that these cases are caused by zoonotic spread of infection from wild or domestic animals–[71]. Follow-up studies into seroprevalence and/or virus prevalence in household ferrets, their owners, and possibly other pets may provide indications for cross species transmission.

We report on the first identification of a kobuvirus, parechovirus, papillomavirus and anellovirus in ferrets. The kobu- and parechovirus belong to the family Picornaviridae that currently comprises 17 genera and many more different virus species. They infect a wide range of mammals and are well-known for their involvement in gastroenteritis. Our phylogenetic analysis revealed that the ferret kobuvirus clustered with bovine and ovine kobuvirus in species Aichivirus B. The close relationship between bovine, ovine, and ferret kobuviruses may indicate past cross species transmission events and subsequent evolution in the separate host species resulting in the distinct types seen today. As was observed for ferret hepatitis E virus but not ferret coronavirus, ferret kobuvirus is significantly more often detected in household ferrets than in farm ferrets by a specific ferret kobuvirus taqman assay, although random amplification and next-generation sequencing suggested the presence of kobuvirus in two additional samples from farm ferrets, which could reflect the presence of another divergent kobuvirus species. Also here follow-up studies into seroprevalence and/or virus prevalence in household ferrets, their owners, and possibly other pets should be performed. An overall kobuvirus prevalence of ∼15% was observed in this study. Two out of 3 animals (67%) with diarrhea and 4 animals out of 36 (11%) without diarrhea were positive by taqman for ferret kobuvirus. Because of the small sample size and the fact that other causes for diarrhea were not excluded, further in depth studies are needed to show that these viruses can cause diarrhea in ferrets.

The ferret parechovirus is the third parechovirus species identified to date. It is highly divergent from human parechovirus and Ljungan virus and may even constitute a new genus in the family Picornaviridae based on the observed sequence diversity. Interestingly, the P3 genome region of MpPeV1, encoding the nonstructural proteins NS3A–3D, is not more conserved than the P1 genome region, encoding the structural capsid proteins, when compared to the corresponding regions of human parechoviruses 1 and 2. This has been observed for Ljungan virus as well by us and others and requires more in-depth studies. Only 1 out of 39 ferrets was positive for MpPeV1, suggesting that the ferret parechovirus prevalence may be relatively low (<2.5%). In humans, parechovirus infections are most common in young children and the prevalence in adults may be low as well. It is of note that the parechovirus-positive animal did not show any signs of gastroenteritis. Low virus prevalence was observed for the identified ferret papillomavirus as well. Papillomaviruses are, however, not typically detected in fecal material or associated with gastroenteritis like parechoviruses. Although recently a full-length papillomavirus genome was characterized from fecal material of a deer mouse as well. Not much knowledge exists on papillomatosis in ferrets as a disease, or the virus(es) causing them. The impact of papillomavirus infections on ferrets in general and the specific role of MpPV1 thus has to be further addressed.

Like many other mammals, it seems that ferrets also harbor anelloviruses. The ferret anellovirus MpfTTV1 is most closely related to the recently identified anellovirus from pine marten. Based on phylogenetic analysis, we propose that MpfTTV1 should be placed in the proposed new anellovirus genus, Xitorquevirus, in analogy to the classification of torque teno viruses in nine genera named Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Eta-, Iota-, Theta-, and Zetatorquevirus, and the proposed five genera Kappa-, Lambda-, Mu-, Nu-, and Xitorquevirus

[25],,. A closely related virus to MpfTTV1 seemed to be present in one other ferret (Table 1) besides ferret 21, resulting in a virus prevalence of ∼5% in this study. Anelloviruses do not generally seem to be as abundantly present in fecal material as in serum, as evidenced by fecal virome studies in different host species,,,,,,,.

Ferrets are carnivores that are significantly affected by a number of human pathogens and hence are widely used as a small animal model for viral infections–[21]. In addition, they are capable of spreading viral infections to humans, among which influenza A virus and on rare occasions rabies virus–[16]. The characterization of the fecal virome of ferrets provides epidemiological baseline information about pathogens which allows the swift identification of possible sources of future zoonotic infections and their subsequent control. Especially the seemingly interesting correlation between certain viral infections in ferrets and their presence in human households as pets needs to be further addressed. In addition, this study shows that ferrets can be infected with a wide range of different viruses, some of which may influence the outcome in experimental virus infection studies, thus delivering a strong argument in favor of development of specific pathogen-free ferret colonies.

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.

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).

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).

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.

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.

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.

In this report, we describe an enterovirus that was isolated on subpassage from pulmonary tissue of an alpaca that died with evidence of respiratory and systemic infection. Using a universal virus detection assay, we identified a significant portion of the genome of this picornavirus with a single PCR. This finding is consistent with the EM data that visualized non-enveloped viral particles of approximately 25–30 nm in diameter. This is the first report of a BEV isolation from alpaca.

All four of the diseased animals had similar clinical symptomatology and had similar pulmonary histology on autopsy. Because EV-F was the only potential pathogen isolated from any of these animals, the alpaca-adapted EV is a potential cause of this syndrome, although these experiments clearly did not fulfill Koch's postulates and limitations in sensitivity of the other tests that were performed do not exclude the potential for other causes. Attempts to identify EV-F by PCR of paraffin-embedded pulmonary tissue samples obtained from these animals failed. This could be due to low copy number of EV-F RNA in the sections of the paraffin blocks that were examined or low stability of the EV-F RNA under the conditions of paraffin block storage.

Enteroviruses comprise one of the nine genera of picornaviruses; all of which include members that infect vertebrates. Picornaviridae members are small, non-enveloped viruses with a single-stranded RNA genome of positive polarity. Members of the Enterovirus genus include human pathogenic poliovirus, coxsackieviruses, enteroviruses, and echoviruses. Other mammalian enteroviruses, including those infecting bovine, simian and porcine species, also have been described. The only picornavirus previously reported to infect alpaca is the foot-and-mouth disease virus (FMDV), which belongs to the Aphthovirus genus. However, FMDV does not usually cause severe disease in alpaca,.

EV-E and EV-F are globally prevalent infections in cattle, and while virus can be shed in high titers in the feces, such infections are usually subclinical and their ability to cause disease in any animal is unclear. Earlier studies described enteroviruses isolated from calves suffering from respiratory disease–[27]. However, in these studies, respiratory disease could not be reproduced using viral isolates from the infected calves. Subsequent studies in cattle have not been reported.

We hoped to be able to identify sequences that could account for the alpaca infection. While there are insufficient data to determine whether or not the virus adapted to alpaca, the frequent housing of alpaca with cattle without other such reports suggests that these infections are unusual. The alpaca-sourced virus has the interesting characteristic of possessing sequences that are most similar to serotype 1 (including the capsid region that is used to determine picornavirus serotype), but in at least one gene is closest to serotype 3, suggesting that this virus could have arisen by recombination of other EV-F serotypes. It is thus possible that recombination of viruses from two EV-F serotypes led to this unusual infection. The isolate has approximately 80–85% homology in its protein sequence to previously described EV-F strains, which is similar to the degree of homology shared among protein sequences from previously sequenced EV-F strains isolated from cattle, which ranges from 79–99% for EV-F strains, and 50 to 95% when EV-E strains are also considered. The sequence of the alpaca-infecting virus isolate is divergent enough from previously reported strains that it does not provide clear evidence for the basis of its pathogenicity. While it is possible that this virus was transmitted directly from cattle to alpaca, it also is possible that there were one or more intermediate hosts. Besides cattle, EV-F has been reported as an infection of possum and of capped langur,. We suspect that the absence of previously reported bovine enterovirus infections in U.S. alpaca is related to the relative isolation of alpaca herds, making it less likely that an alpaca-adapted virus would be further transmitted among alpaca.

Introducing new species (as livestock or as pets) to a habitat potentially increases the risk of an indigenous pathogen causing infections in new species. While most pathogens do not cross the species barrier due to adaptive constraints, those that succeed often cause more severe disease in the new host. Notable human examples of this phenomenon are yellow fever virus, HIV and more recently Nipah virus, Hendra virus and SARS virus. Animal examples include the devastating infections of canine distemper virus in raccoons and African lions–[38]. U.S. alpaca are outside their native South American habitat and are exposed to viruses endemic to the U.S., especially those from U.S. domestic farm animals to which U.S. alpaca herds often have close proximity. Thus, recently described alpaca infections include bovine viral diarrhea virus, equine herpesvirus 1, and bluetongue virus. Newly introduced animals also can potentially carry pathogens that are relatively benign to them, but not to the indigenous fauna. The risk obviously increases if newly introduced and indigenous livestock are kept in close proximity and if their pathogens are able to remain stable in the environment or persist in the host species. Since picornaviruses are non-enveloped viruses, they often are very stable under environmental conditions, increasing the opportunity for infection of different hosts over a prolonged period of time.

While the enterovirus infection described in this report was temporally associated with illness in three other alpaca in the affected herd that may have represented limited spread of the virus, the sparse distribution of alpaca, together with the severe and rapid course of disease likely prevented further dissemination of the virus, as evidenced by the absence of other reports of similar illnesses in the herd or other alpaca in the region. However, even though it appears that this outbreak was controlled, bovine enteroviruses should be added to the list of viruses that can infect alpaca, and that could potentially be associated with severe respiratory and systemic infections in alpaca. Furthermore, considering the relative stability of enteroviruses, the ubiquity of cattle and likely frequent co-location of domestic cattle with alpaca, it is quite plausible that similar outbreaks may occur in the future. Therefore, this alpaca virus infection serves to remind us that viral species are constantly evolving and that the opportunity to infect new hosts may hasten that process.

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.

Co-detection of BCV and H. somni at the time of the disease outbreak suggests that these two pathogens contributed to disease pathogenesis. Characterizing the factors that may have contributed to the outbreak described here can help veterinarians, researchers, and producers better understand the risk factors associated with pre-weaning BRD. This information can be used to guide prevention and control strategies to decrease the incidence of BRD and associated adverse animal health issues and production losses.

Our results confirm that if French ovine and caprine are susceptible to IDV, as previously shown in the USA, China and Togo, bovines are the main host for IDV, as previously observed in Luxembourg or in the United States. Whether virological factors (differences in susceptibility of small and large ruminants to IDV) and/or epidemiological factors (breeding systems, decreasing number of mixed breeding farms in France with time, etc.) are responsible for the differences in prevalence is still not known and further studies are needed to understand the mechanism. Further epidemiological and serological studies including a higher number of mixed breeding farms will also be required to understand the potential transmission of IDV between ruminant species.

We also observed differences of seroprevalence between regions only for cattle. This may be partly explained by the breeding systems with high numbers of fattening units of young bulls or veal calves in Pays de la Loire (highest seroprevalence of 70% with GMT of 86) inducing more exchanges and introductions of young animals between farms from several origins. In contrast, regions such as Hauts-de-France (lowest seroprevalence of 31% with GMT of 45) consist mainly of classical breeding dairy or beef farms. In addition, the seroprevalence reported in Luxembourgish cattle (76%–88%) was higher than the seroprevalence observed in France (31%–70%). Differences in breeding systems and in number of animals per farm may account for the different seroprevalences observed between the two countries.

We previously showed that IDV was detected in France in 2011. The high seroprevalence we found between 2014 and 2018 either suggests that once IDV is introduced, it seems to spread very efficiently throughout the country, or that the virus may have emerged well before 2011 in France. Archive sera should be screened to figure out when the virus may have really emerged in the country.

Finally, the high seroprevalence of IDV in French cattle, suggesting that most animals seem to have been infected by IDV without the farmers noticing it, and the low frequency of IDV detection in lungs of calves with severe respiratory disease suggest a moderate respiratory pathogenicity of IDV. This is coherent with the recent results of limited pathogenicity of IDV in calves by experimental infections. On the other hand, the high seroprevalence of IDV in adults may explain a partial clinical protection of calves in the first weeks of life by the maternally derived antibodies. The role of IDV in bovine respiratory disease is still unclear, but current data indicate that IDV may act as an initiating pathogen as suggested for bovine parainfluenza virus type 3 (BPI-3) and bovine coronavirus (BCoV), both highly prevalent in Europe and inducing only mild clinical signs by experimental infections. IDV, BCoV and BPI-3 were mainly detected in association with other respiratory pathogens during bronchopneumonia in calves. Studies using next generation sequencing showed that IDV was more frequently detected in cattle with respiratory signs than in healthy animals. It has also become clear that many respiratory bovine pathogens (including IDV) with high prevalence are more frequently detected together than by themselves. Regular and global viro- and sero-surveillance, combined with epidemiological studies, are thus warranted to better understand the influenza D virus epidemiology and its role in bovine respiratory disease. In addition, co-infection studies should be performed to understand mechanisms behind pathogens interactions (synergies and antagonisms) in relation to the host immune response and disease severity.

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.

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.

This study represents the first prospective longitudinal study of the relationship between anti-BCV antibody levels and BCV infections and respiratory disease in nursing beef calves. Two outbreaks of respiratory disease in one research herd resulted in two-thirds of the calves being mass treated for BRD. BCV shedding was found to occur in conjunction with the pre-weaning BRD outbreak. However, when levels of passively or actively acquired immunity to BCV were measured, they were not found to associate with respiratory disease incidence between the three study herds, nor with the development of BRD in individual calves within that herd. Rather, BCV infections were likely common, with many subclinical and some persistent or recurrent infections detected.

The results from this study agree with the results from previous studies in young dairy calves [16, 17]. In these studies, dairy calves shed virus, sometimes repeatedly, in spite of relatively high anti-BCV antibody titers. Furthermore, they observed no statistically significant correlation between maternal BCV specific IgG serum antibody titers and clinical disease or infection by BCV in that population. Similarly, in the study reported here, we found that BCV specific serum IgG did not correlate with respiratory disease; and 8/45 calves (18%) from Herd 2 that were involved in mass treatment were found to be shedding BCV at two or more sample acquisition times. This result supports the idea that persistent or recurrent shedding episodes can occur in the same animal with or without signs of disease.

BCV infection (as measured by virus shedding) was similarly not found to be associated with anti-BCV antibody levels; however, this measurement was hampered by the infrequency of sample collection in our study design (Fig. 1). For example, nearly all of the animals in the Herd 2 mass treatment groups shed BCV between our routine sample collection times. While we did not see differences in anti-BCV antibody abundances in Herd 2 between those calves mass treated for BRD and those from the same herd that remained untreated, it is possible, and even likely, that many untreated calves also shed BCV between sample acquisition dates. Lack of BCV detection in nasal swabs at routine collection times may lead to “false negatives” for BCV infection that mask any influence antibodies may have on virus shedding. Furthermore, the small number of BCV positive individuals found in Herds 1 and 3 (two calves from each herd were shedding BCV at preconditioning processing) hinder our ability to detect with any confidence differences that may exist. More intensive sampling would be required to better determine the association between anti-BCV antibody titers and BCV infection. Other hypothetical reasons why serum anti-BCV IgG abundance has not been found to associate with BCV infection or treatment for disease has been discussed previously [15, 16]. Among these is the lack of knowledge related to the immune correlates of protection for respiratory BCV infections. How cell mediated immunity and other antibody isotypes (such as IgA) contribute to protection from infection and disease are areas that require additional research.

Given that BCV infection is common, it remains unknown why some animals display signs of respiratory disease while others remain sub-clinically infected. To determine whether there were differences in the BCV strains circulating in the three herds, a 1102 nucleotide fragment of spike gene was analyzed. The spike gene encodes the surface glycoprotein that is responsible for attachment to the host cell and is a major neutralizing antigen targeted by the host immune system [34, 35]. The region of the spike gene spanning the hypervariable region and antigenic domain II was selected because it is variable between coronavirus strains and isolates, and variations in this region have been associated with altered antigenicity and/or pathogenicity in other species. No polymorphisms were found in this region between Herd 1 and Herd 2 or between the isolates from subclinical shedding episodes compared to the isolates circulating at the time of the BRD outbreak. Furthermore, only one or two SNPs were found between the isolates circulating in Herd 3 compared to the isolates in Herds 1 and 2. In contrast, up to 35 SNPs and 11 amino acid differences were detected in this same region when these isolates were compared to 14 isolates collected between 2014 and 2017 from USMARC and the University of Nebraska-Lincoln Veterinary Diagnostic Center (unpublished data). Thus, differences in the BCV strains circulating in the three herds are unlikely to account for the major differences in treatment rates observed in this study.

One side effect of respiratory viral infection is the increased risk for bacterial superinfection. While BCV may occasionally produce a clinical syndrome consistent with BRD in the absence of bacterial infection, its involvement, like other respiratory viruses, is generally considered to be a precursor to a bacterial infection, which exacerbates disease [2, 4, 36]. Thus, the presence of co/secondary bacterial infections may help explain the differences in disease severity in BCV-infected calves observed in this study. Using qPCR to look at common bacterial pathogens associated with respiratory disease in cattle, we observed H. somni in high frequency and abundance in the upper respiratory tract of sick cattle but not clinically normal cattle from the same herd collected 2–3 weeks after the outbreak. Furthermore, H. somni was not detected in these calves at initial vaccination, prior to the disease outbreak. Thus, we hypothesize that a potential secondary bacterial infection with H. somni may explain why the cattle in Herd 2 displayed more signs of respiratory disease and subsequently required treatment. Of note, however, within treatment groups there was no difference in the relative abundance of H. somni in the nasal cavity of animals when categorized by rectal temperature taken at the time of sample collection and treatment. This suggests that infection was widespread in the herd, though only 15–30% of the animals were displaying clinical signs of disease at that particular time. Frequency differences of various strains of these bacteria that have different propensities to cause disease may have been present between these populations, but were not differentiated by the assays used. It is also possible that additional pathogens that were not measured in this study could have contributed to the disease outbreak. Thus, unbiased metagenomic approaches are currently underway to determine whether any additional viral or bacterial pathogens were associated with the disease outbreak. How the URT microbiome may have influenced respiratory health is also being examined in these populations.

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.

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.

Ten nasal swabs from cattle with a slight cough and nasal discharge in an auction market were collected from Shandong Province, China, in 2010. Nasal swabs were placed in virus collection tubes (Yocon Bio. Co. Ltd., Beijing, China). RNA extraction was conducted following the manufacturer’s instructions accompanying the Viral RNA Rapid Extraction Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). Potential infectious pathogens of BRDC, including BPIV3, BVDV, BHV-1, and BRSV, were detected by PCR. Nasal swabs were placed in virus collection tubes (Yocon Bio. Co. Ltd., Beijing, China). RNA extraction was conducted following the manufacturer’s instructions accompanying the Viral RNA Rapid Extraction Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). Potential infectious pathogens of BRDC, including BPIV3, BVDV, BHV-1, and BRSV, were detected by PCR. The conditions of PCRs for the detection of BVDV, BPIV3, and BRSV were as follows: Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s, and 35 cycles for this stage; extension at 72 °C for 10 min; and storing at 4 °C for forever. The conditions of PCRs for the detection of BHV-1 were as follows: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 50 s, annealing at 60 °C for 40s, extension at 72 °C for 30 s, and 35 cycles for this stage; extension at 72 °C for 10 min; and storing at 4 °C for forever. Nasal samples, detected as BPIV3-positive, were used for virus isolation on Madin-Darby bovine kidney (MDBK) cells. Primers for PCR detection are shown in Table S1 (supplementary material).

Here, we detected a high prevalence of bovine astrovirus by RT-PCR from rectal swabs

collected from young calves of cattle and water buffalo with diarrhea. However, more studies

are needed to determine whether the persistent diarrhea observed in these calves was mainly

associated with the high prevalence of astroviruses, as previous studies reported that

bovine astroviruses were not directly associated with severe diarrhea in calves under

natural conditions [4, 24]. However, other reports [14, 26] indicated that bovine astrovirus may evolve to severe

diarrhea in co-infections with other gastrointestinal viruses, as in the case of BAstV

co-infection with BRV or BToV (Breda virus).

Nonetheless, the epidemiology of bovine astrovirus remains unclear, especially considering

the limited number of studies of cattle and water buffalo. While excretion of BAstV may

occur in up to 60–100% of calves on farms, only 5

(2.4%) of 209 rectal swabs collected from asymptomatic adult cattle were positive for BAstV

[21]. In the present study, sampling at different

time points demonstrated a high prevalence of up to 56.52% among calves with diarrhea.

Moreover, in our complementary study, astrovirus was detected alongside other

gastrointestinal viruses, including BEV, BCoV, BRV and BVDV, in 87.5% of cases.

Surprisingly, 12.5% of these cases were positive for bovine astrovirus, but yet negative for

other tested gastrointestinal viruses. In contrast, titers of BRV and BToV, which have been

previously reported as principal gastrointestinal co-infecting viruses with bovine

astrovirus, were minimal or undetectable in this study. From these results, it is clear that

astrovirus may be directly associated with diarrhea or possibly linked to other factors,

such as poor hygienic conditions or associated with other non-viral pathogens, such as

bacteria or parasites, which are known causative agents of diarrhea in calves. Similar

results were reported in Korea, where co-infection of bovine astrovirus with

gastrointestinal viruses, other than BRV and BToV, was associated with clinical symptoms of

diarrhea in 20- and 14-day-old calves.

Our phylogenetic analysis showed that all isolates in this study were closely related to

BAstV-B76-2/HK, BAstV-B18/HK, CcAstV-1/DNK/2010 and CcAstV-2/DNK/2010, but highly divergent

from a BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the

U.S.A.. Nonetheless, our results support that

proposal that BAstV and CcAstV may be different

strains of the same virus, and water buffalo may be a new host of the BAstV variant of this

virus. Moreover, most sequences derived from one farm belonged to one subgroup, although

sequence analysis clearly demonstrated origins from different strains. In addition, no

significant genetic differences were observed between the bovine and water buffalo

astrovirus strains investigated in this study (Table

4). These results further support previous evidence that one farm could serve as

a reservoir of different bovine astrovirus strains, suggesting possible outbreaks of novel

astroviruses due to mutation or recombination events. Notably, considering the history of

astroviruses, recombination events have been described in cattle, swine, humans and poultry

[19, 21,

22], and co-infection of two different astroviruses

in one head of cattle has been previously reported as well. Consequently, it is important to screen for and control astrovirus infection

in order to prevent eventual outbreaks of highly pathogenic astroviruses resulting in

mutation or recombination events on farms. Furthermore, the astrovirus isolates from cattle

and water buffalo isolated in this study displayed relatively close relationships,

indicating that these animals were infected with diverse astroviruses, which probably

evolved from the same viral ancestor. Consequently, given these close relationships,

evidence exists of possible cross-infection between the two hosts; therefore, control

measures against bovine astrovirus should also be taken into consideration when screening of

viruses in water buffalo populations.

Appreciation of the genetic diversity and evolution of astroviruses among wild and domestic

animal populations is important to fully understand this challenging gastrointestinal virus

[21]. However, the organization of the BAstV genome

has not yet been fully described. In this study, a sequence motif upstream of ORF2 was

predicted to be the signal of the putative promoter for subgenomic RNA (sgRNA) synthesis in

bovine astrovirus as well as that for the ORF1b/ORF2 overlap sequence and the most 3′-end of

ORF2. It has been suggested that in astroviruses [13,

23], a conserved sequence acts as a putative

promoter for sgRNA synthesis upstream of ORF2. In human astroviruses, the ORF1b/ORF2 overlap

is 8 nt in length, while in duck astroviruses, the ORF1b/ORF2 junctions are not overlapped

[6, 13, 23]. In contrast, the overlap in the ORF1b/ ORF2 junction

of bovine astroviruses may be longer (56 nt) than that of the other known astroviruses.

Moreover, compared with a previous report, the

3′-end of ORF2 of bovine astroviruses was highly conserved, although our results suggested

further studies and analysis of more sequences from several novel bovine astroviruses to

fully characterize ORF2 of bovine astrovirus.

In summary, this study is of interest at both the epidemiological and genetic levels. These

results will certainly contribute to the understanding of the evolution and pathology of

bovine astrovirus in cattle and water buffalo, as not only did we provide useful reference

material for further studies, but also isolated bovine astrovirus in water buffalo for the

first time. The present study is thus far among the largest epidemiological investigations

of bovine astrovirus conducted at the farm level in the dairy industry in China.

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.