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.

What Patterns in Online Classified Puppy Advertisements Can Tell Us about the Current UK Puppy Trade.

The UK online puppy trade has rapidly outgrown the current legislation, aided by the anonymity provided by classified advertisement platforms. In an effort to meet increased demand, some unregulated and regulated breeders may have employed practices that negatively impact canine welfare. A paucity of up-to-date empirical data, necessary to characterise the scale and nature of this industry, makes intervention challenging. This study quantifies the online puppy trade via web-scraped online classified advertisements, providing empirical data that reveal market trends, along with spatial and temporal patterns. A total of 17,389 unique dog advertisements were collated and analysed over a 2-year period (1 June 2018 to 31 May 2020). The second year included the COVID-19 Lockdown (23 March 2020 to 31 May 2020). Statistical comparisons between dependent and independent variables were performed by linear regression. In the case of a single continuous variable, a one sample t-test was used. Of these advertisements, 57.2% were sourced from a pet-specific classified advertisement website (Pets4Homes, n = 9948), and the remaining 42.8% from two general classified advertisement websites (Gumtree, n = 7149, 41.1%; Preloved, n = 292, 1.7%, respectively). England exhibited the greatest number of advertisements (n = 10,493), followed by Wales (n = 1566), Scotland (n = 975), and Northern Ireland (NI; n = 344). Scaled for estimated human population density, Wales possessed as many advertisements per million inhabitants (489.4) as the other three combined (England = 186.4, Scotland = 177.3, and NI = 181.1). Across both years, 559 unique breeds were advertised, yet 66% of all advertisements focused on 20 breeds, and 48% advertisements focused on only 10 breeds. Regional breed popularity was suggested, with French Bulldog as the most advertised breed in England (7.3%), Scotland (6.8%), and Wales (6.8%), but Schnauzers were most popular within Northern Ireland (6.83%). Within the 559 unique breeds advertised, only 3.4% had links to conformational disorders CD); however, these breeds were among the most commonly advertised, totalling 46.9% of all ads. Across all regions, price density peaked between GBP 300 and GBP 1000, with Bulldogs presenting the greatest cost (mean = GBP 1461.38, SD = GBP 940.56), followed closely by French Bulldog (mean = GBP 1279.44, SD = GBP 664.76) and Cavapoo (mean = GBP 1064.56, SD = GBP 509.17). CD breeds were found to be GBP 208.07 more expensive, on average, than non-CD breeds. Our results represent a buoyant online market with regional and seasonal fluctuations in price, advertised breed frequency and total counts. This suggests a market influenced by consumer trends, with a particular focus on breed preference, despite links to illness/disease associated with conformation. Our findings highlight the value of utilising online classified advertisement data for long-term monitoring, in order to assist with evidence-based regulatory reform, impact measurement of targeted campaigns, and legislative enforcement.

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

Virological Investigation in Synanthropic Rodents in North Italy

Intro: Several rodents, including mice and the brown rat, are synanthropic animals usually found in rural and urban environments in contact with other animals and humans. Rodents are natural reservoirs of infectious agents and could harbour a plethora of zoonotic pathogens of public health importance. Taking advantage of a parallel study on presence and distribution of Hantaviruses, we aimed to investigate the occurrence in mice of other viruses with zoonotic or economic impact. Method(s): From May to July 2022, 41 mice (Mus domesticus) were captured and killed by using baited snap traps in 13 selected cattle, goat and poultry farms located in the Piedmont region. Gut and lung samples were homogenised and tested by PCR methods for pan-Coronavirus (CoV) and SARS-CoV-2, pan-Pestivirus, Mammalian orthoreoviruses, Canine Distemper virus (CDV), Flaviviruses, Influenza A (IAV) and D (IDV) viruses. Finding(s): All captured animals did not present at necropsy lesions related to infectious diseases. Virological investigations detected the presence of CoV in six mice. By sequencing Rodent CoVs was identified in two samples (four more pending). Mammalian orthoreovirus was detected in nine animals and typing and characterization are in progress. One mouse, captured in a bovine farm, tested slightly positive for IDV and confirmation of positivity is in progress by complete sequencing with NGS approach. All samples were negative for Flaviviruses, IAV, CDV, pan-Pestivirus and SARS-CoV-2. Conclusion(s): Rodents are well adapted to a wide range of habitats, including peri-urban and rural environments, where they benefit from human activities. These results, although preliminary, underline the importance of enhancing surveillance in rodents in anthropized areas to better assess the presence of zoonotic agents and the potential risk of transmission.Copyright © 2023

Canine Olfactory Detection of SARS-CoV-2-Infected Humans - a Systematic Review.

Coronavirus disease 2019 (COVID-19) developed into a pandemic within months. SARS-CoV-2 testing measures and vaccines became quickly accessible. However, due to pre- or asymptomatic transmission, effective disease control remains challenging. To complement conventional testing methods, scientists around the world have investigated dogs' olfactory capability for true real-time detection. Several diseases are known to produce specific scents in affected individuals, excreted as volatile organic compounds, which can be easily detected by dogs within seconds. This systematic review evaluates the current evidence for using dogs' olfactory system as a reliable COVID-19-screening tool. Two independent procedures for study quality assessment were used: the QUADAS-2 tool for the evaluation of laboratory tests' diagnostic accuracy, designed for systematic reviews, and a second system for the general evaluation of canine scent detection studies, adapted with a focus on medical scent detection. Twenty-seven studies from thirteen countries were evaluated. Particular attention was paid to potential confounding factors, e.g., study design, patient/sample selection, dog characteristics, training protocols, and sample types/treatment. These analysis systems revealed that respectively four and six studies had low risk of bias and high quality. The four QUADAS-2 non-biased studies resulted in sensitivity and specificity ranges of 81-97% and 91-100%, whereas the six high quality studies according to the general evaluation system revealed sensitivity and specificity ranges of 82-97% and 83-100%, respectively. The other studies contained high risk of bias, concerns about the methodological applicability and/or quality concerns. Standardization and certification procedures as used for canine explosives detection should be established for medical scent detection dogs in order to use their undoubtful potential in an optimal and structured way. '.

Coinfection in the cases of feline panleukopenia: a literature review

Coinfection caused by bacteria, parasites, or viruses complicates almost all feline panleukopenia virus (FPV) infections. Pathogens that colonize the gastrointestinal tract, Clostridium perfingens, Clostridium piliforme, Cryptosporidium spp, Giardia spp, Tritrichomonas fetus, canine parvovirus type 2,Salmonella sp., feline coronavirus, feline bocavirus, and feline astrovirus were isolated in the presence of FPV infection. Complex mechanisms between viruses, bacteria, protozoa, and hosts contribute to the pathogenesis and severity of coinfection. Prompt and accurate diagnosis, vaccination precautions, and appropriate treatment play important roles in reducing morbidity and mortality. This article outlines the etiology, pathogenesis, diagnosis, prevention, and treatment that can help veterinarians and pet owners improve their knowledge of managing the diseases.

An immunoinformatics study: designing multivalent T-cell epitope vaccine against canine circovirus.

BACKGROUND: Canine circovirus is a deadly pathogen of dogs and causes vasculitis and hemorrhagic enteritis. It causes lethal gastroenteritis in pigs, fox, and dogs. Canine circovirus genome contains two main (and opposite) transcription units which encode two open reading frames (ORFs), a replicase-associated protein (Rep) and the capsid (Cap) protein. The replicase protein and capsid protein consist of 303 amino acids and 270 amino acids respectively. Several immuno-informatics methods such as epitope screening, molecular docking, and molecular-dynamics simulations were used to craft peptide-based vaccine construct against canine circovirus. RESULTS: The vaccine construct was designed by joining the selected epitopes with adjuvants by suitable linker. The cloning and expression of the vaccine construct was also performed using in silico methods. Screening of epitopes was conducted by NetMHC server that uses ANN (Artificial neural networking) algorithm. These methods are fast and cost-effective for screening epitopes that can interact with dog leukocyte antigens (DLA) and initiate an immune response. Overall, 5 epitopes, YQHLPPFRF, YIRAKWINW, ALYRRLTLI, HLQGFVNLK, and GTMNFVARR, were selected and used to design a vaccine construct. The molecular docking and molecular dynamics simulation studies show that these epitopes can bind with DLA molecules with stability. The codon adaptation and in silico cloning studies show that the vaccine can be expressed by Escherichia coli K12 strain. CONCLUSION: The results suggest that the vaccine construct can be useful in preventing the dogs from canine circovirus infections. However, the results need further validation by performing other in vitro and in vivo experiments.

Infectious disease Roundup 2022

This paper describes the epidemiology, prevalence, transmission, prevention and control of some infectious diseases in companion animals, livestock, wild animals and humans in Ontario, Canada, in 2022, including SARS-CoV-2;Echinococcus multilocularis, Leishmania spp. and SARS-CoV-2;antimicrobial stewardship resources;2 cases of rabid dogs imported from Iran (July 2021 and January 2022);prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriacea, Dirofilaria immitis, Brucella canis, canine parainfluenza and adeno- and herpes viruses in dogs recently imported from Asia;Paragonimus kellicotti lung flukes and Streptococcus equi subsp. zooepidemicus in dogs;African swine fever in pet pigs, backyard pigs and wild pigs and blastomycosis in dogs and humans.

Disease threats to tigers and their prey

The contraction of the global tiger population over the last 100 years into small, often isolated subpopulations has made them increasingly vulnerable to the impact of disease. Despite this, the health of wild tigers continues to be insufficiently funded and explored. For example, canine distemper virus (CDV), has been associated with localized declines and increased risk of extinction, and yet has received little research attention in most tiger range countries. The emergence of new pathogenic threats has posed fresh challenges, including African swine fever virus (ASFV), which has the potential to devastate wild boar populations, and severe acute respiratory syndrome coronavirus (SARS-CoV2) with implications for tiger conservation that remain unknown. The objective of this review is to synthesize current research on the health of tigers and their prey that impacts the conservation of tigers in the wild. Published sources are interpreted based on three mechanisms through which disease can affect the viability of tiger populations: (1) by reducing the survival of adult tigers, (2) by reducing breeding productivity, and (3) by reducing the carrying capacity of tiger habitat through decreased prey abundance. Examples of CDV, SARS-CoV2, carnivore protoparvovirus 1 and ASFV are used to illustrate these processes and inform discussion of research and mitigation priorities. Copyright © 2023 Gilbert, Dvornicky-Raymond and Bodgener.

Understanding the performance of truffle dogs

Trained dogs can detect many biochemical traces, from the most hostile, such as COVID-19, to the most expensive, such as truffles. However, it is still unclear how the interplay of genetic disposition, education, experience, personality, and environmental conditions influences the performance of dogs. Here, we combine published evidence with the knowledge of truffle hunters to assess direct and indirect factors that affect the hunting performance of truffle dogs. Since the transport of truffle aroma into the canine nasal cavity is most puzzling, we propose 5 experiments to further investigate the role of weather conditions, soil parameters, and bacterial composition on the dispersal of truffle volatiles perceived by trained dogs. We also suggest exploring whether the application of multiple dogs increases the success of truffle hunting. Moreover, we argue that interdisciplinary research on dog behavior and performance, including insights from veterinary science and comparative psychology as well as the experience of truffle hunters and farmers, is needed to improve the quantity and quality of truffle harvests. (PsycInfo Database Record (c) 2023 APA, all rights reserved)

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.

Pathogenesis of Anemia in Canine Babesiosis: Possible Contribution of Pro-Inflammatory Cytokines and Chemokines-A Review.

Canine babesiosis is a tick-borne protozoan disease caused by intraerythrocytic parasites of the genus Babesia. The infection may lead to anemia in infected dogs. However, anemia is not directly caused by the pathogen. The parasite's developmental stages only have a marginal role in contributing to a decreased red blood cell (RBC) count. The main cause of anemia in affected dogs is the immune response to the infection. This response includes antibody production, erythrophagocytosis, oxidative damage of RBCs, complement activation, and antibody-dependent cellular cytotoxicity. Moreover, both infected and uninfected erythrocytes are retained in the spleen and sequestered in micro-vessels. All these actions are driven by pro-inflammatory cytokines and chemokines, especially IFN-γ, TNF-α, IL-6, and IL-8. Additionally, imbalance between the actions of pro- and anti-inflammatory cytokines plays a role in patho-mechanisms leading to anemia in canine babesiosis. This article is a review of the studies on the pathogenesis of anemia in canine babesiosis and related diseases, such as bovine or murine babesiosis and human or murine malaria, and the role of pro-inflammatory cytokines and chemokines in the mechanisms leading to anemia in infected dogs.

Endovenous radiofrequency ablation using a new bipolar electrode in a canine model: a new endovenous radiofrequency electrode.

Purpose: This study aimed to determine the effectiveness and safety of a newly developed endovenous radiofrequency (RF) catheter compared with that of the existing RF catheter in a canine model. Methods: Seven dogs underwent ablation using 1 control catheter (ClosureFAST, CF; Covidien) and 1 experimental catheter (VENISTAR, VS; STARmed Co., Ltd.) in the femoral and cephalic veins. The ablated vein was evaluated macroscopically (2,3,5-triphenyltetrazolium chloride staining, TTC), microscopically (hematoxylin and eosin staining), and ultrasonographically. Vessel injury score was used to evaluate the ablating effect objectively. Veins from 1 dog were evaluated on the day of ablation, while in the remaining 6 dogs, the ablated veins were evaluated 2 weeks later. Results: A total of 23 veins (CF, 11 veins; VS, 12 veins) were ablated in 7 dogs. Non-TTC-stained vein wall areas were identified in all ablated veins. No significant difference was observed in the mean vessel injury score (2.54 ± 1.16 vs. 2.42 ± 1.13, P = 0.656) and the mean vessel wall thickness (0.32 ± 0.03 mm vs. 0.31 ± 0.05 mm, P = 0.212) between CF and VS. There was no blood flow in all veins ablated with VS, whereas there was remaining blood flow in 1 vein ablated with CF. Perivenous complication was not observed. Conclusion: Endovenous RF ablation using a newly developed VS RF catheter seems to provide comparable occlusion rate and degree of vein wall injury without perivenous adverse events compared to the most commonly used RF catheter (CF).

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.

Low Prevalence of SARS-CoV-2 Antibodies in Canine and Feline Serum Samples Collected during the COVID-19 Pandemic in Hong Kong and Korea.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide since its emergence in 2019. Knowing the potential capacity of the virus to adapt to other species, the serological surveillance of SARS-CoV-2 infection in susceptible animals is important. Hong Kong and Seoul are two of Asia's most densely populated urban cities, where companion animals often live in close contact with humans. Sera collected from 1040 cats and 855 dogs during the early phase of the pandemic in Hong Kong and Seoul were tested for SARS-CoV-2 antibodies using an ELISA that detects antibodies against the receptor binding domain of the viral spike protein. Positive sera were also tested for virus neutralizing antibodies using a surrogate virus neutralization (sVNT) and plaque reduction neutralization test (PRNT). Among feline sera, 4.51% and 2.54% of the samples from Korea and Hong Kong, respectively, tested ELISA positive. However, only 1.64% of the samples from Korea and 0.18% from Hong Kong tested positive by sVNT, while only 0.41% of samples from Korea tested positive by PRNT. Among canine samples, 4.94% and 6.46% from Korea and Hong Kong, respectively, tested positive by ELISA, while only 0.29% of sera from Korea were positive on sVNT and no canine sera tested positive by PRNT. These results confirm a low seroprevalence of SARS-CoV-2 exposure in companion animals in Korea and Hong Kong. The discordance between the RBD-ELISA and neutralization tests may indicate possible ELISA cross-reactivity with other coronaviruses, especially in canine sera.

Secondary zoonotic dog-to-human transmission of SARS-CoV-2 suggested by timeline but refuted by viral genome sequencing.

PURPOSE: The risk of secondary zoonotic transmission of SARS-CoV-2 from pet animals remains unclear. Here, we report on a 44 year old Caucasian male presenting to our clinic with COVID-19 pneumonia, who reported that his dog displayed respiratory signs shortly prior to his infection. The dog tested real-time-PCR (RT-PCR) positive for SARS-CoV-2 RNA and the timeline of events suggested a transmission from the dog to the patient. METHODS: RT-PCR and serological assays were used to confirm SARS-CoV-2 infection in the nasopharyngeal tract in the dog and the patient. We performed SARS-CoV-2-targeted amplicon-based next generation sequencing of respiratory samples from the dog and patient for sequence comparisons. RESULTS: SARS-CoV-2 infection of the dog was confirmed by three independent PCR-positive pharyngeal swabs and subsequent seroconversion. Sequence analysis identified two separate SARS-CoV-2 lineages in the canine and the patient's respiratory samples. The timeline strongly suggested dog-to-human transmission, yet due to the genetic distance of the canine and the patient's samples paired-transmission was highly unlikely. CONCLUSION: The results of this case support current knowledge about the low risk of secondary zoonotic dog-to-human transmissions of SARS-CoV-2 and emphasizes the strength of genomic sequencing in deciphering viral transmission chains.

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.