Development of multiplex real-time PCR for simultaneous detection of SARS-CoV-2, CCoV, and FIPV.

Introduction: Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), canine coronavirus (CCoV), and feline infectious peritonitis virus (FIPV), have the potential for interspecies transmission. These viruses can be present in complex environments where humans, dogs, and cats coexist, posing a significant threat to both human and animal safety. Methods and results: In this study, we developed a novel multiplex TaqMan-probe-based real-time PCR assay for the simultaneous detection and differentiation of SARS-CoV-2, CCoV, and FIPV. Specific primers and TaqMan fluorescent probes were designed based on the N region of SARS-CoV-2 and FIPV, as well as the S region of CCoV, which demonstrated a remarkable sensitivity and specificity toward the targeted viruses, as few as 21.83, 17.25 and 9.25 copies/µL for SARS-CoV-2, CCoV and FIPV, respectively. The standard curve constructed by the optimized method in our present study showed a high amplification efficiency within or near the optimal range of 91% to 116% and R(2) values were at least 0.95 for the abovementioned coronaviruses. A total of 91 samples, including six plasmid mixed mock samples, four virus fluid mixing simulated samples, and 81 clinical samples, were analyzed using this method. Results demonstrated strong agreement with conventional approaches. Discussion: By enabling the simultaneous detection of three viruses, this method enhances testing efficiency while decreasing costs. Importantly, it provides a valuable tool for the prevalence and geographical distribution of suspected and co-infected animals, ultimately contributing to the advancement of both animal and public health.

Epidemiological investigation of enteric canine coronaviruses in domestic dogs: A systematic review and meta-analysis.

Canine enteric coronavirus (CeCoV) is a globally distributed enteric pathogen that causes significant harm to canines. The objective of this systematic review was to examine the global dissemination of CeCoV and assess the potential for infected canines to be exposed to various CeCoV genotypes and subtypes. With an aggregated prevalence of 18.8%, the study predicted regional variations, indicating that CeCoV is an exceptionally prevalent disease. The increased likelihood that infected canines will be asymptomatic is a significant cause for concern, as undetected cases of CeCoV infection could persist and spread the disease. This underscores the significance of ongoing surveillance of CeCoV in order to avert its transmission. Nevertheless, further investigation is necessary in order to ascertain the moderators that significantly impact the prevalence and distribution of distinct subtypes and genotypes of CeCoV. Hence, it is imperative to undertake randomized clinical trials in order to acquire a more accurate understanding of the variables that influence the prevalence of CeCoV. By conducting ongoing surveillance, regional variations in the prevalence of CeCoV in canines can be accounted for, thereby enhancing our comprehension of the illness and ultimately impeding its transmission.

Emerging Variants of Canine Enteric Coronavirus Associated with Outbreaks of Gastroenteric Disease.

A 2022 canine gastroenteritis outbreak in the United Kingdom was associated with circulation of a new canine enteric coronavirus closely related to a 2020 variant with an additional spike gene recombination. The variants are unrelated to canine enteric coronavirus-like viruses associated with human disease but represent a model for coronavirus population adaptation.

A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody.

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

Discovery and identification of a novel canine coronavirus causing a diarrhea outbreak in Vulpes.

Cross-species transmission of viruses from wildlife animal reservoirs, such as bats, poses a threat to human and domestic animal health. Previous studies have shown that domestic animals have important roles as intermediate hosts, enabling the transmission of genetically diverse coronaviruses from natural hosts to humans. Here, we report the identification and characterization of a novel canine coronavirus (VuCCoV), which caused an epidemic of acute diarrhea in Vulpes (foxes) in Shenyang, China. The epidemic started on November 8, 2019, and caused more than 39,600 deaths by January 1, 2022. Full-length viral genomic sequences were obtained from 15 foxes with diarrhea at the early stage of this outbreak. The VuCCoV genome shared more than 90% nucleotide identity with canine coronavirus (CCoV) for three of the four structural genes, with the S gene showing a larger amount of divergence. In addition, 67% (10/15) of the VuCCoV genomes contained an open reading frame (ORF3) gene, which was previously only detected in CCoV-I genomes. Notably, VuCCoV had only two to three amino acid differences at the partial RNA-dependent RNA polymerase (RdRp) level to bat CoV, suggesting a close genetic relationship. Therefore, these novel VuCCoV genomes represent a previously unsampled lineage of CCoVs. We also show that the VuCCoV spike protein binds to canine and fox aminopeptidase N (APN), which may allow this protein to serve as an entry receptor. In addition, cell lines were identified that are sensitive to VuCCoV using a pseudovirus system. These data highlight the importance of identifying the diversity and distribution of coronaviruses in domestic animals, which could mitigate future outbreaks that could threaten livestock, public health, and economic growth.

In Vitro Evaluation of Antiviral Activities of Funicone-like Compounds Vermistatin and Penisimplicissin against Canine Coronavirus Infection.

Recent studies have demonstrated that 3-O-methylfunicone (OMF), a fungal secondary metabolite from Talaromyces pinophilus belonging to the class of funicone-like compounds, has antiviral activity against canine coronaviruses (CCoV), which causes enteritis in dogs. Herein, we selected two additional funicone-like compounds named vermistatin (VER) and penisimplicissin (PS) and investigated their inhibitory activity towards CCoV infection. Thus, both compounds have been tested for their cytotoxicity and for antiviral activity against CCoV in A72 cells, a fibrosarcoma cell line suitable for investigating CCoV. Our findings showed an increase in cell viability, with an improvement of morphological features in CCoV-infected cells at the non-toxic doses of 1 µM for VER and 0.5 µM for PS. In addition, we observed that these compounds caused a strong inhibition in the expression of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which is activated during CCoV infection. Our results also showed the alkalinization of lysosomes in the presence of VER or PS, which may be involved in the observed antiviral activities.

Engineered Cross-Linked Silane with Urea Polymer Thin Durable Coatings onto Polymeric Films for Controlled Antiviral Release of Activated Chlorine and Essential Oils.

In March 2020, the World Health Organization announced a pandemic attributed to SARS-CoV-2, a novel beta-coronavirus, which spread widely from China. As a result, the need for antiviral surfaces has increased significantly. Here, the preparation and characterization of new antiviral coatings on polycarbonate (PC) for controlled release of activated chlorine (Cl+) and thymol separately and combined are described. Thin coatings were prepared by polymerization of 1-[3-(trimethoxysilyl)propyl] urea (TMSPU) in ethanol/water basic solution by modified Stöber polymerization, followed by spreading the formed dispersion onto surface-oxidized PC film using a Mayer rod with appropriate thickness. Activated Cl-releasing coating was prepared by chlorination of the PC/SiO2-urea film with NaOCl through the urea amide groups to form a Cl-amine derivatized coating. Thymol releasing coating was prepared by linking thymol to TMSPU or its polymer via hydrogen bonds between thymol hydroxyl and urea amide groups. The activity towards T4 bacteriophage and canine coronavirus (CCV) was measured. PC/SiO2-urea-thymol enhanced bacteriophage persistence, while PC/SiO2-urea-Cl reduced its amount by 84%. Temperature-dependent release is presented. Surprisingly, the combination of thymol and chlorine had an improved antiviral activity, reducing the amount of both viruses by four orders of magnitude, indicating synergistic activity. For CCV, coating with only thymol was inactive, while SiO2-urea-Cl reduced it below a detectable level.

Prevalence and genetic diversity of canine coronavirus in northeastern China during 2019-2021.

Canine coronavirus (CCoV) is associated with diarrhea in dogs, with a high incidence and sometimes even death. However, there is currently limited information about its prevalence and molecular characterization in northeastern China. Therefore, in this study, we examined 325 canine fecal specimens in four provinces in northeastern China from 2019 to 2021. PCR results revealed that 57 out of 325 (17.5%) samples were found to be positive for CCoV, and the positive rate varies obviously with city, season, age and so on. High incidence (65%) of viral co-infection was detected in the diarrhea samples and mixed infection of distinct CCoV genotypes occurs extensively. More importantly, sequence analysis showed that the S gene has a strong mutation. Phylogenetic analysis demonstrated that CCoV-I and CCoV-II strains has different origins. In particular, we found the CCoV-IIa strains of S gene sequenced and the reference strain B906_ZJ_2019 were highly clustered, and the reference strain was a recombinant strain of CCoV-I and CCoV-II. Our findings provide useful orienting clues for evaluating the pathogenic potential of CCoV in canines, and point out more details on characterization in northeastern China. Further work is required to determine the significance and continuous genetic evolution of CCoV.

Effectiveness of the Fungal Metabolite 3-O-Methylfunicone towards Canine Coronavirus in a Canine Fibrosarcoma Cell Line (A72).

Canine coronavirus (CCoV), an alphacoronavirus, may cause self-limiting enteric disease in dogs, especially in puppies. The noteworthy plasticity of coronaviruses (CoVs) occurs through mutation and recombination processes, which sometimes generate new dangerous variants. The ongoing SARS-CoV-2 pandemic and the isolation of a novel canine-feline recombinant alphacoronavirus from humans emphasizes the cross-species transmission ability of CoVs. In this context, exploring antiviral compounds is essential to find new tools for fighting against CoVs infections. Fungi produce secondary metabolites, which are often developed as antibiotics, fungicides, hormones, and plant growth regulators. Previous examinations of benzo-γ-pyrone 3-O-methylfunicone (OMF), obtained from Talaromyces pinophilus, showed that it reduces the infectivity of hepatitis C virus and bovine herpesvirus 1. Based on this evidence, this study evaluated the antiviral ability of OMF against CCoV infection in a canine fibrosarcoma (A72) cell line. During CCoV infection, a non-toxic dose of OMF markedly increased features of cell viability. Moreover, OMF induced a significant reduction in virus yield in the presence of an intense downregulation of the viral nucleocapsid protein (NP). These findings occurred in the presence of a marked reduction in the aryl hydrocarbon receptor (AhR) expression. Taken together, preliminary findings suggest that OMF inhibiting AhR shows promising activity against CCoV infection.

Epidemiological investigation of canine coronavirus infection in Chinese domestic dogs: A systematic review and data synthesis.

Canine enteric coronavirus (CCoV) is a pathogenic virus that infects dogs worldwide, causing enteric issues and causing harm to the dog industry and dogs. Although CCoV is not recognized as a highly lethal canine intestinal pathogen, it has been reported that CCoV is significantly associated with canine diarrhea in dogs. CCoV is a common health problem in dogs, attracting major concern from veterinarians and dog owners across China. In this study, we summarized the prevalence and epidemiological characteristics of CCoV in dogs in mainland China. The study revealed that the pooled prevalence of CCoV infection was 33%, and which associated with age, but not with sex, season and immunization status. In addition, the study also further suggested that CCoV-II was the predominant CCoV subtype in Chinese dogs. This study will provide valuable information for CCoV infections across China and other countries. Furthermore, this study also suggested that continuous surveillance and epidemiological studies of CCoV are necessary.

Antiviral activity of canine interferon lambda 3 expressed using a recombinant adenovirus against canine coronavirus, canine parvovirus, and canine distemper virus.

Canine coronavirus (CCoV), canine parvovirus (CPV), and canine distemper virus (CDV) are highly contagious canine pathogens; dogs with these diseases are difficult to treat. In a previous study, we developed a recombinant adenovirus expressing canine interferon lambda 3 (Ad-caIFNλ3) in canine epithelial cells. In this study, we aimed to investigate the antiviral activity of Ad-caIFNλ3 against CCoV, CPV, and CDV in two canine cell lines, A72 and MDCK. Ad-caIFNλ3 transduction suppressed replication of these viruses without cytotoxicity. Our results suggest that Ad-caIFNλ3 may be a therapeutic candidate for canine viral diseases.

Phylogeny, Evolution, and Transmission Dynamics of Canine and Feline Coronaviruses: A Retro-Prospective Study.

Canine coronavirus (CCoV) and feline coronavirus (FCoV) are endemic in companion animals. Due to their high mutation rates and tendencies of genome recombination, they pose potential threats to public health. The molecular characteristics and genetic variation of both CCoV and FCoV have been thoroughly studied, but their origin and evolutionary dynamics still require further assessment. In the present study, we applied a comprehensive approach and analyzed the S, M, and N genes of different CCoV/FCoV isolates. Discriminant analysis of principal components (DAPC) and phylogenetic analysis showed that the FCoV sequences from Chinese isolates were closely related to the FCoV clusters in Netherlands, while recombination analysis indicated that of S N-terminal domain (NTD) was the most susceptible region of mutation, and recombination of this region is an important cause of the emergence of new lineages. Natural selection showed that CCoV and FCoV subgenotypes were in selection constraints, and CCoV-IIb was in strong positive selection. Phylodynamics showed that the mean evolution rate of S1 genes of CCoV and FCoV was 1.281 × 10-3 and 1.244 × 10-3 subs/site/year, respectively, and the tMRCA of CCoV and FCoV was about 1901 and 1822, respectively. Taken together, our study centered on tracing the origin of CCoV/FCoV and provided ample insights into the phylogeny and evolution of canine and feline coronaviruses.

Recent Zoonotic Spillover and Tropism Shift of a Canine Coronavirus Is Associated with Relaxed Selection and Putative Loss of Function in NTD Subdomain of Spike Protein.

A canine coronavirus (CCoV) has now been reported from two independent human samples from Malaysia (respiratory, collected in 2017-2018; CCoV-HuPn-2018) and Haiti (urine, collected in 2017); these two viruses were nearly genetically identical. In an effort to identify any novel adaptations associated with this apparent shift in tropism we carried out detailed evolutionary analyses of the spike gene of this virus in the context of related Alphacoronavirus 1 species. The spike 0-domain retains homology to CCoV2b (enteric infections) and Transmissible Gastroenteritis Virus (TGEV; enteric and respiratory). This domain is subject to relaxed selection pressure and an increased rate of molecular evolution. It contains unique amino acid substitutions, including within a region important for sialic acid binding and pathogenesis in TGEV. Overall, the spike gene is extensively recombinant, with a feline coronavirus type II strain serving a prominent role in the recombinant history of the virus. Molecular divergence time for a segment of the gene where temporal signal could be determined, was estimated at around 60 years ago. We hypothesize that the virus had an enteric origin, but that it may be losing that particular tropism, possibly because of mutations in the sialic acid binding region of the spike 0-domain.

Detection and molecular characteristics of canine coronavirus in Chengdu city, Southwest China from 2020 to 2021.

Canine coronavirus (CCoV) is generally thought of as a mild, but highly contagious, enteritis of young dogs. This study was to investigate the molecular detection and characteristics of CCoV in Chengdu city, Southwest China. 218 canine fecal samples were collected from four animal hospitals and one animal shelter from 2020 to 2021. Fifty-nine CCoV-positive samples were detected by RT-PCR, including 40 CCoV-I, 25 CCoV-IIa, one CCoV-IIb and 10 untyped. To further analyze the genetic diversity of CCoV, we amplified ten complete spike (S) genes, including four CCoV-I and six CCoV-II strains. The amino acid sequence obtained in this study revealed 85.95% ± 12.55% homology with the reference strains. Moreover, in the N-terminal structural domain, there were two amino acid insertions (17QQ18) in two strains of CCoV-I and four amino acid insertions (95IGTN98) in CCoV-IIb strain. Interestingly, we identified that the S1/S2 cleavage site of the S protein of CCoV strains (SWU-SSX3 and SWU-SSX10) were consistent with feline coronavirus (FCoV). In the evolutionary tree, a strain of CCoV-I (SWU-SSX10) was found to be more closely related to FCoV, while SWU-SSX7 of CCoV-IIb was more closely related to coronavirus from the Chinese ferret badger. In addition, for the first time, recombination in a CCoV-IIb strain was found to occur between two subtypes occurring in the C domain of the S1 subunit, with a breakpoint starting at 2141 nt. The results enriched the epidemiological information of CCoV and provided an important reference for the prevention of CCoV in Chengdu city, Southwest China.

Could veterinarians be immune to contracting SARS-CoV-2?

Introduction: The aim of the study was to assess the epidemic situation among veterinarians of the Swietokrzyskie Voivodeship, Poland, in relation to the control group. Material and methods: The research was divided into 3 stages. Stage I involved the selection of subjects. In stage II, flow cytometry for immunophenotyping was performed and the percentage of the sub-population of CD4 cells and CD8 cells was assessed. Stage III involved collection of nasopharyngeal swab samples in order to determine the canine coronavirus CR-CoV mRNA with the rT-PCR method. Results: The percentage of the CD4 and CD8 lymphocyte subpopulation in relation to the total lymphocyte population in veterinarians did not differ statistically from the percentage in the control group. The CD4/CD8 ratio in the group of veterinarians was on average 1.93, and 2.04 in the control group. There was no statistically significant difference between the groups, p = 0.591. Canine CR-CoV mRNA was not detected in any of the veterinarians or in the control group. Conclusions: None of the veterinarians had a significant increase in T lymphocytes, which could be an effective defense against SARS-CoV-2.

The knotty biology of canine coronavirus: A worrying model of coronaviruses' danger.

Severe clinical diseases associated to αCoronavirus (αCoV) infections were recently demonstrated for the first time in humans and a closely related but distinct canine CoV (CCoV) variant was identified in the nasopharyngeal swabs of children with pneumonia hospitalized in Malaysia, in 2017-2018. The complete genome sequence analysis demonstrated that the isolated strain, CCoV-HuPn-2018, was a novel canine-feline-like recombinant virus with a unique nucleoprotein. The occurrence of three human epidemics/pandemic caused by CoVs in the recent years and the detection of CCoV-HuPn-2018, raises questions about the ability of these viruses to overcome species barriers from their reservoirs jumping to humans. Interestingly, in this perspective, it is interesting to consider the report concerning new CCoV strains with a potential dual recombinant origin through partial S-gene exchange with porcine transmissible gastroenteritis virus (TGEV) identified in pups died with acute gastroenteritis in 2009. The significance of the ability of CCoVs to evolve is still unclear, but several questions arisen on the biology of these viruses, focusing important epidemiological outcomes in the field, in terms of both virus evolution and prophylaxis. The new CCoV-Hupn-2018 should lead researchers to pay more attention to the mechanisms of recombination among CoVs, rather than to the onset of variants as a result of mutations, suggesting a continuous monitoring of these viruses and in particular of SARS-CoV-2.

Identification of a canine coronavirus in Australian racing Greyhounds.

Coronavirus infection can cause a range of syndromes, which in dogs can include mild-to-severe enteritis that generally resolves rapidly. Fatalities can occur from coinfection with other pathogens, including canine parvovirus. Between late December 2019 and April 2020, canine coronavirus (CCoV) was detected in Australian racing Greyhounds that displayed signs of gastrointestinal disease. The CCoV was genotyped using high-throughput sequencing, recovering 98.3% of a type IIb CCoV, generally thought to cause a mild but highly contagious enteric disease. The Australian CCoV was almost identical (99.9%, whole-genome sequence) to another CCoV associated with an outbreak of severe vomiting in dogs in the United Kingdom at the same time (December 2019-March 2020).

Novel Canine Coronavirus Isolated from a Hospitalized Patient With Pneumonia in East Malaysia.

BACKGROUND: During the validation of a highly sensitive panspecies coronavirus (CoV) seminested reverse-transcription polymerase chain reaction (RT-PCR) assay, we found canine CoV (CCoV) RNA in nasopharyngeal swab samples from 8 of 301 patients (2.5%) hospitalized with pneumonia during 2017-2018 in Sarawak, Malaysia. Most patients were children living in rural areas with frequent exposure to domesticated animals and wildlife. METHODS: Specimens were further studied with universal and species-specific CoV and CCoV 1-step RT-PCR assays, and viral isolation was performed in A72 canine cells. Complete genome sequencing was conducted using the Sanger method. RESULTS: Two of 8 specimens contained sufficient amounts of CCoVs as confirmed by less-sensitive single-step RT-PCR assays, and 1 specimen demonstrated cytopathic effects in A72 cells. Complete genome sequencing of the virus causing cytopathic effects identified it as a novel canine-feline recombinant alphacoronavirus (genotype II) that we named CCoV-human pneumonia (HuPn)-2018. Most of the CCoV-HuPn-2018 genome is more closely related to a CCoV TN-449, while its S gene shared significantly higher sequence identity with CCoV-UCD-1 (S1 domain) and a feline CoV WSU 79-1683 (S2 domain). CCoV-HuPn-2018 is unique for a 36-nucleotide (12-amino acid) deletion in the N protein and the presence of full-length and truncated 7b nonstructural protein, which may have clinical relevance. CONCLUSIONS: This is the first report of a novel canine-feline recombinant alphacoronavirus isolated from a human patient with pneumonia. If confirmed as a pathogen, it may represent the eighth unique coronavirus known to cause disease in humans. Our findings underscore the public health threat of animal CoVs and a need to conduct better surveillance for them.

Identification and phylogenetic analysis of two canine coronavirus strains.

Canine coronavirus (CCoV), a member of the genus Alphacoronavirus, is an enveloped, single-stranded positive-sense RNA virus that responsible for gastroenteritis in dogs. In this study, two CCoV isolates were successfully propagated from 53 CCoV-positive clinical specimens by serial passaging in A-72 cells. These two strains, CCoV JS1706 and CCoV JS1712, caused cytopathic effects in A-72 cells. The sizes of virus plaque formed by them differed in early passages. Electron microscopy revealed a large quantity of typical coronavirus particles with 80-120 nm in diameter in cell culture media and cytoplasm of infected cells, in which they appeared as inclusion bodies. RT-PCR analysis of S gene indicated that these two isolates were belonged to CCoV IIa subtype. Homology of RdRp, S, M and N proteins between the two strains were 100, 99.6, 99.2 and 100.0%, respectively, whereas they were 99.4-100%, 83.1-95.2%, 88.5-99.2% and 91.9-99.7% identity compared to CCoV II reference strains. Phylogenetic analysis of RdRp, S, M and N protein showed that they were closely related to CCoV II strains. These two subtype IIa isolates will be useful for evaluating the pathogenesis and evolution of CCoV and for developing diagnostic reagents and vaccines.

Molecular characterization of the FCoV-like canine coronavirus HLJ-071 in China.

BACKGROUND: According to the differences of antigen and genetic composition, canine coronavirus (CCoV) consists of two genotypes, CCoV-I and CCoV-II. Since 2004, CCoVs with point mutations or deletions of NSPs are contributing to the changes in tropism and virulence in dogs. RESULTS: In this study, we isolated a CCoV, designated HLJ-071, from a dead 5-week-old female Welsh Corgi with severe diarrhea and vomit. Sequence analysis suggested that HLJ-071 bearing a complete ORF3abc compared with classic CCoV isolates (1-71, K378 and S378). In addition, a variable region was located between S gene and ORF 3a gene, in which a deletion with 104 nts for HLJ-071 when compared with classic CCoV strains 1-71, S378 and K378. Phylogenetic analysis based on the S gene and complete sequences showed that HLJ-071 was closely related to FCoV II. Recombination analysis suggested that HLJ-071 originated from the recombination of FCoV 79-1683, FCoV DF2 and CCoV A76. Finally, according to cell tropism experiments, it suggested that HLJ-071 could replicate in canine macrophages/monocytes cells. CONCLUSION: The present study involved the isolation and genetic characterization of a variant CCoV strain and spike protein and ORF3abc of CCoV might play a key role in viral tropism, which could affect the replication in monocyte/macrophage cells. It will provide essential information for further understanding the evolution in China.