Establishment and application of triple PCR method for CPV, CCoV and CRV

Canine parvovirus (CPV), canine coronavirus (CCoV) and canine rotavirus (CRV) are the three main causative viruses of diarrhea in dogs with similar clinical symptoms;thereby it is necessary to establish a high effective molecular detection method for differentiating the above pathogens. By optimizing the primer concentration and annealing temperature, a triple PCR method was established for simultaneous detection of CPV, CCoV and CRV, and then the specificity, sensitivity and repeatability of the method were tested. The results showed that the target fragments of CPV VP2 gene (253 bp), CCoV ORF-1b gene (379 bp) and CRV VP6 gene (852 bp) could be accurately amplified by the triple PCR method with high specificity, the detection limits of CPV, CCOV and CRV were 6.44x10-1 pg/L, 8.72x10-1 pg/L and 8.35x10-1 pg/L respectively with high sensitivity, and the method had good stability. Using this triple PCR method, 135 canine diarrhea fecal samples collected in Chengdu region from 2019 to 2020 were detected, and compared with those of single PCR method. The detection rates of CPV, CCoV and CRV were 16.30%, 20.74% and 4.44%, respectively, and the total infection rate was 51.11% (65/135) with 20.00% (13/65) co-infection rate. The detection results were consistent with three single PCR methods. In conclusion, CPV/CCoV/CRV triple PCR method successfully established in this paper can be applied as an effective molecular method to detection of related pathogens and to the epidemiological investigation.

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.

Molecular Detection of Canine Parvovirus-2c (Cpv-2c) in Diseased Coyote Pups (Canis Latrans) at Northeastern Mexico

Intro: Canine parvovirus type 2 (PVC-2), Protoparvovirus genus of the Parvoviridae family, is a worldwide distribution virus that affects the Canidae family. In free-living coyotes (Canis latrans), the presence of the PCV-2a, PCV-2b and PCV-2c subtypes of PVC-2 has been reported, but there are no reports of their presence as a cause of clinical damage. The objective of this study is to report the presence of PVC-2c in an outbreak of mild gastroenteritis in three coyote pups detected in northeastern Mexico Methods: During the fall of 2019, in the suburban area of Monterrey, N.L., 3 affected coyote pups were detected with a mild gastroenteric condition consisting of mild diarrhea with loose stools, vomiting, dehydration, loss of appetite, pale mucous membranes, and low weight. Stool samples were tested for Canine Parvovirus (CPV-2), Canine Coronavirus (CCV) or Giardia antigens with a commercial kit. All samples were positive for CPV-2 and these were subsequently analyzed by PCR and sequencing of the CPV-2 VP2 gene. Using bioinformatics, the VP2 gene sequence data obtained were used to establish phylogenetic relationships with homologous sequences reported in coyotes and CPV-2 vaccines. Finding(s): The genetic sequence of VP2 obtained showed a high homology (98.1 to 100%) with CPV-2c. The sequences obtained from the pups showed 100% homology to each other. The phylogenetic tree showed that the sequences reported in coyotes are grouped in different clades and that the sequence of the VP2 gene of CPV-2c from coyote pups is grouped in a different monophyletic group. Conclusion(s): Information suggests that wild coyotes may not only act as asymptomatic reservoir hosts but may also be clinically affected by PVC-2c. It is necessary to carry out studies to know the effects of the genetic subtypes of PVC-2 in the population of coyotes and other wild canids of northeastern Mexico.Copyright © 2023

Differentiating infectious from non-infectious respiratory disease in the cat and dog

This paper describes the clinical signs and use of differential laboratory diagnostic techniques (computed tomography, cytology, histopathology, antigen/antibody detection and polymerase chain reaction) for infectious (viral, bacterial, fungal and parasitic) and non-infectious (inflammatory/immune mediated, neoplastic, cardiac, malformation, foreign body, smoke inhalation, aspiration of caustic material, non-cardiogenic, pulmonary oedema, traumativ, pneumothorax, pulmonary contusions and idiopathic) causes of respiratory diseases in cats and dogs in Ontario, Canada.

Seroepidemiological and clinicopathological investigation of Canine Coronavirus infection in dogs, in t..rkiye

Domestic and wild dogs of all ages and breeds are susceptible to Canine Coronavirus(CCoV) infections and be seen in T..rkiyeand amongst world. CCoV has recently been declared a zoonotic disease agent and the eighth pathogenic human coronavirus. This study was conducted on 143 naturally infected dogs with gastroenteritis which were not vaccinated against CCoV in T..rkiye in 2015-2020. The data of dogs were analyzed seroepidemiologically, clinicopathologically and statistically. CCOV antibodies in serum and CCOV antigens in stool were detected by ELISA and lateral immunochromatography. The rising CCoV IgG antibody titers were detected at all dogs and were as follows;<10 ng/L in 3 (2%), 10-20 ng/L in 18 (13%), 20-30 ng/L in 16 (11%), 30-40 ng/L in 14 (%10), 40-64 ng/L in 11 (8%) and >64 ng/L in 81 (81%) dogs. CCOV and Canine Parvovirus (CPV) antigen were detected together in the stool of the 41 (28.7%) dogs. As a result, it was concluded that the CCOV agent is in circulation among dogs living in T..rkiye. CCOV and CPV can cause co-infections and increased mortality. Although infection can be seen in dogs of all ages, it can be seen more frequently in dogs younger than 1 year of age, and especially in dogs younger than 6 months, and can cause enteritis, low hemoglobin, erythropenia, lymphopenia, leukopenia, thrombocytopenia, and hypoproteinemia.

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.

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.

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 Canine Coronavirus

Canine coronavirus (CCoV) is usually the cause of mild gastroenteritis in dogs and is known to have spread worldwide. In the last decade, as a consequence of the extraordinary large RNA genome, novel recombinant variants of CCoV have been found that are closely related to feline and porcine strains. Moreover highly virulent pantropic CCoV strains were recently identified in dogs. The molecular characterization of the CCoV circulating in canine population is essential for understanding viral evolution. Copyright © Springer Science+Business Media New York 2016.

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.

Efficacy of Probiotic on Duodenal TNF-α Expression and the Histological Findings in the Liver and Lung in Animal Model Canine Coronavirus

Currently, Canine coronavirus (CCoV) is an enteric pathogen of the Alphacoronavirus-1 species that causes mild to severe diarrhea in puppies. The pathogenesis of this infection will cause severe lymphopenia and lead to death in puppies. This study aimed to determine the administration of probiotics on TNF-α expression, histological findings of the liver and lung in mice infected with CCoV. A total of 28 mice were randomly assigned into seven treatment groups, i.e. (C-) placebo;(C+) active CCoV vaccine induction;(T1) CCov + Isopronosin;(T2) CCoV + Lactobacillus acidophilus probiotic;(T3) CCoV + Lactobacillus Acidophylus and Bifidobacterium probiotics;(T4) CCoV + colustrum fermentation probiotic;(T5) CCoV + ginger, turmeric and ginger probiotics. Thereafter, the expression of TNF-α in the duodenum was stained using immunohistochemistry, liver and lung were stained using hematoxylin eosin. The data were analyzed using the ANOVA test followed by the Tukey test with a significance level (p<0.05). TNF-α expression on T4 and T5 decreased significantly (p<0.05) compared to C+, T1, T2 and T3. Histologic findings of the liver in the C- and T4 groups showed normal features in the central vein. On the other hand, glycogen accumulation was found in hepatocyte cells, hemorrhage with sinusoid dilation, lymphocyte infiltration in centro lobular area in group C+. Lung histology showed normal features of sinusoids and alveolar septa in groups C- and T4. Meanwhile, intra-alveolar hemorrhage was found with neutrophil cell infiltration and fibrin plasma accumulation in group C+. In conclusion, colostrum fermentation probiotics can reduce TNF-α expression in the duodenum and improve the liver and lung physiology in mice infected with CCoV.

Detection and molecular characterization of canine Coronavirus in dogs with lower respiratory tract infection

Coronaviruses in the family Coronaviridae cause digestive and respiratory system infections in humans and animals. There are two subtypes of canine coronaviruses (CCoV), which are included in the alfacoronavirus, as CCoV I and CCoV II. CCoV-II is divided into two genotypes, CCoV-IIa and IIb. Although CCoV affects dogs of all ages and all diets, newborn puppies can be particularly susceptible and severely affected. According to the literature research, no molecular studies have been found in our country for the detection of canine coronavirus, especially in lower respiratory tract infections. In this study, it was aimed to detect and molecular characterization of CCoV un shelter dogs with lower respiratory tract infection. For this purpose, Bronchoalveolar Lavage (BAL) fluids taken from 40 shelter dogs with lower respiratory tract infections were examined. CCoV was detected in 3 of the BAL fluids of 40 dogs tested. A phylogenetic tree was constructed with the sequences obtained after the sequence analysis. It was determined that 2 of the 3 positive samples in the phylogenetic tree were CCoV-I and one sample was CCoV-II. In conclusion, this study revealed that CCoV-I and CCoV-II may play a role in lower respiratory system disorders of shelter dogs. In addition, the detection of two different CCoVs in different animals in the same shelter has been considered as an important data, and the detection of both types in dogs housed in crowded environments such as shelter conditions shows that the possibility of new variants or subtypes that may occur in the future should not be ignored.

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.

Serum Calprotectin Levels in Dogs with Diarrhea

Background: Diarrhea induced by infectious factors may lead to significant health problems in dogs. Canine parvovirus (CPV), canine coronavirus (CCV), canine distemper virus (CDV), Giardia spp., Escherichia coli (E. coli), and Salmonella spp. are the important infectious agents that may induce diarrhea in dogs. The present study aimed to investigate the effect of CPV, CCV, CDV, Giardia spp., E. coli, and Salmonella spp. infections on the change in serum calprotectin (Calp) concentration. Materials, Methods & Results: A total of 30 dogs were enrolled in the study. The study dogs were divided into 3 groups. Healthy animals as confirmed by clinical examination and animals negative for the specified pathogens were placed in Group 1. Animals infected by one or more agents, including CPV, CCV, CDV, and Giardia spp., but negative for E. coli or Salmonella spp. were placed in Group 2. Finally, animals positive for E. coli or Salmonella spp. and infected or not infected by one or more agents, including CPV, CCV, CDV, and Giardia spp., were placed in Group 3. Stool samples and rectal and conjunctival swab samples were collected to investigate the etiologic agents that induced diarrhea. Blood samples were collected through vena cephalica antebrachii for hematological and biochemical examinations. The samples were obtained via routine clinical examinations at the Prof. Dr. Servet Sekin outpatient clinic at Dicle University Veterinary Faculty. CPV, CCV, CDV, and Giardia spp. diagnoses were made based on immunochromatographic test kits. The bacteriological analysis of stool samples was used to diagnose E. coli and Salmonella spp. infection. Serum Calp concentrations were measured by Enzyme-Linked Immunosorbent Assay (ELISA). The analysis of swab and stool samples by immunochromatographic rapid diagnosis kits and microbiological methods showed that 5 animals were infected with CPV, 10 with CCV, 6 with CDV, 3 with Giardia spp., 12 with E. coli, and 2 with none of the specified agents. Total leukocyte count (WBC), lymphocyte (Lym - %), and granulocyte (Gra - %) values were higher in the diarrheal dogs when compared with the control group. In the biochemical examination of serum samples, total bilirubin (TBIL) and phosphorus (P) levels were higher and sodium (Na) levels were lower in Group 3 when compared to the control group (P = 0.025, 0.024, and 0.018, respectively). Total protein (TP) and albumin (Alb) values were lower in Group 2 compared to Groups 1 and 3 [P = 0.001 and 0.019 for TP, P = 0.000 and 0.01 for Alb, respectively]. There was a statistically significant difference in creatine kinase (CK) levels between Group 1 and Group 2 (P = 0.013). Serum Calp level was higher in the E. coli infected group (Group 3) compared to the other groups, no significant differences were noted between the groups (P > 0.05). Discussion: In conclusion, to the best of authors knowledge, this study is the first to evaluate serum Calp levels in dogs with diarrhea induced by viral, bacterial, and protozoan infections. The Calp level was higher in the sick dogs that were infected by at least one agent, including CPV, CCV, CDV, and Giardia spp., and were at the same time E. coli positive when compared with the control group and the group without E. coli infections. It was concluded that new studies could be useful to reveal the diagnostic importance of serum Calp concentration in dogs with diarrhea and that these results may contribute to future studies in this area.

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.

Animal Coronaviruses

Coronaviruses (CoVs) infect diverse animal species and cause respiratory, enteric, hepatic, renal, neurologic, and even systemic diseases. The majority of CoVs have a narrow host specificity, but a few CoVs have a broad range of host specificity. This chapter provides a brief review of animal CoVs, including SARS-CoV-2 of animals for their receptors, host tropism, and pathogenesis in target animals.