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Canine coronavirus (CCoV) generally causes an infection with high morbidity and low mortality in dogs. In recent years, studies on coronaviruses have gained a momentum due to coronavirus outbreaks. Mutations in coronaviruses can result in deadly diseases in new hosts (such as SARS-CoV-2) or cause changes in organ-tissue affinity, as occurred with feline infectious peritonitis virus, exacerbating their pathogenesis. In recent studies on different types of CCoV, the pantropic strains characterized by hypervirulent and multi-systemic infections are believed to be emerging, in contrast to classical enteric coronavirus infections. In this study, we investigated emerging hypervirulent and multi-systemic CCoV strains using molecular and bioinformatic analysis, and examined differences between enteric and pantropic CCoV strains at the phylogenetic level. RT-PCR was performed with specific primers to identify the coronavirus M (membrane) and S (spike) genes, and samples were then subjected to DNA sequencing. In phylogenetic analysis, four out of 26 samples were classified as CCoV-1. The remaining 22 samples were all classified as CCoV-2a. In the CCoV-2a group, six samples were in branches close to enteric strains, and 16 samples were in the branches close to pantropic strains. Enteric and pantropic strains were compared by molecular genotyping of CCoV in dogs. Phylogenetic analysis of hypervirulent pantropic strains was carried out at the amino acid and nucleotide sequence levels. CCoV was found to be divergent from the original strain. This implies that some CCoV strains have become pantropic strains that cause multisystemic infections, and they should not be ruled out as the cause of severe diarrhea and multisystemic infections.

As the outbreaks of COVID-19 in worldwide, coronavirus has once again caught the attention of people. Canine coronavirus is widespread among dog population, and sometimes causes even fatal cases. Here, to characterize the prevalence and evolution of current circulating canine coronavirus (CCoV) strains in China, we collected 213 fecal samples from diarrheic pet dogs between 2018 and 2019. Of the 213 samples, we found 51 (23.94%) were positive for CCoV. Co-infection with canine parvovirus (CPV), canine astrovirus (CaAstV), canine kobuvirus (CaKV), Torque teno canis virus (TTCaV) were ubiquitous existed. Mixed infection of different CCoV subtypes exists extensively. Considering the limited sequences data in recent years, we sequenced 7 nearly complete genomes and 10 complete spike gene. Phylogenetic analysis of spike gene revealed a new subtype CCoV-II Variant and CCoV-IIa was the most prevalent subtype currently circulating. Moreover, we identified strain B906_ZJ_2019 shared 93.24% nucleotide identifies with previous strain A76, and both of them clustered with CCoV-II Variant, which were not well clustered with the known subtypes. Recombination analysis of B906_ZJ_2019 indicated that strain B906_ZJ_2019 may a recombinant variant between CCoV-I and CCoV-II, which is consistent with strain A76. Furthermore, amino acid variations widely existed among current CCoV-IIa strains circulating in China and the classic CCoV-IIa strains, in spite of the unknown functions. In a word, we report a useful information as to the etiology and evolution of canine coronavirus in China based on the available sequences, which is urgent for the devise of future effective disease prevention and control strategies.

The current pandemic of COVID-19 has set off an urgent search for an effective vaccine. This search may well benefit from the experiences of the animal health profession in the development and use of coronavirus vaccines in domestic animal species. These animal vaccines will in no way protect humans against COVID-19 but knowledge of the difficulties encountered in vaccinating animals may help avoid or minimize similar problems arising in humans. Diverse coronaviruses can infect the domestic species from dogs and cats, to cattle and pigs to poultry. Many of these infections are controlled by the routine vaccination. Thus, canine coronavirus vaccines are protective in puppies but the disease itself is mild and self-limiting. Feline coronavirus infections may be mild or may result in a lethal immune-mediated disease – feline infectious peritonitis. As a result, vaccination of domestic cats must seek to generated protective immunity without causing immune-mediated disease. Vaccines against bovine coronavirus are widely employed in cattle where they protect against enteric and respiratory disease in young calves. Two major livestock species suffer from economically significant and severe coronavirus diseases. Thus, pigs may be infected with six different coronaviruses, one of which, porcine epidemic diarrhea, has proven difficult to control despite the development of several innovative vaccines. Porcine epidemic diarrhea virus undergoes frequent genetic changes. Likewise, infectious bronchitis coronavirus causes an economically devastating disease of chickens. It too undergoes frequent genetic shifts and as a result, can only be controlled by extensive and repeated vaccination. Other issues that have been encountered in developing these animal vaccines include a relatively short duration of protective immunity, and a lack of effectiveness of inactivated vaccines. On the other hand, they have been relatively cheap to make and lend themselves to mass vaccination procedures.

Infectious diseases are a common cause of death in young dogs. Several factors are thought to predispose young dogs to microbiological infections. Identifying the cause of death is often a challenge, and broad diagnostic analysis is often needed. Here, we aimed to determine the infectious causes of death in young dogs aged up to 1 year, examining how it relates to age (under and over 6 months), lifestyle (owned versus ownerless), breed (purebred and crossbreed), and gender. A retrospective study was conducted in a 3-year period (2015–2017) on 138 dead dogs that had undergone necropsy and microbiological diagnostics. Enteritis and pneumonia were the most commonly observed lesions. Polymicrobism was more prevalent (62.3%) than single-agent infections and associated with a higher rate of generalised lesions. Ownerless dogs showed over a three-fold higher predisposition to viral coinfections than owned dogs. Above all, canine parvovirus was the most prevalent agent (77.5%), followed by canine coronavirus (31.1%) and canine adenovirus (23.9%); ownerless pups had a higher predisposition to these viruses. Escherichia coli (23.9%), Clostridium perfringens type A (18.1%), and Enterococcus spp. (8.7%) were the most commonly identified bacteria, which mostly involved in coinfections. A lower prevalence of CDV and Clostridium perfringens type A was observed in puppies under 6 months of age. In conclusion, this study is the first comprehensive survey on a wide panel of microbiological agents related to necropsy lesions. It lays the groundwork for future studies attempting to understand the circulation of infectious agents in a determined area.

The ongoing pandemic due to severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2, also known as COVID‐19) has led to unprecedented challenges for the global healthcare system. This novel coronavirus disease phenotype ranges from asymptomatic carriage to fulminant cytokine storm with respiratory failure, polyorgan dysfunction, and death. Severe disease is characterized by exuberant inflammation resulting from high circulating cytokines such as interleukin‐6 (IL‐6) and tumor necrosis factor (TNF). These inflammatory mediators are responsible for the detrimental effects on the immune, hematologic, respiratory, renal, gastrointestinal and other body systems. In addition to inhibition of viral replication, blunting this inflammatory response before overt cytokine storm is important to improve outcomes. Although there are upcoming promising agents such as remdesivir and convalescent plasma, inexpensive, safe, and widely available adjunct treatments to ameliorate disease burden would be welcome. Two potential antiinflammatory agents include indomethacin, which has been shown in experimental models to decrease canine coronavirus (CCoV) levels in dogs and exhibit antiviral activity against several other viruses, and the polyphenol, resveratrol, a potent antioxidant that has shown antiviral activity against several viruses.

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus 2), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102copies/μL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR(Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression., Highlights • In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea. • The limit of detection was up to 102copies/μL and performed well with high sensitivity and specificity. • Our results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.

Wild mammalian species, including bats, constitute the natural reservoir of betacoronavirus (including SARS, MERS, and the deadly SARS-CoV-2). Different hosts or host tissues provide different cellular environments, especially different antiviral and RNA modification activities that can alter RNA modification signatures observed in the viral RNA genome. The zinc finger antiviral protein (ZAP) binds specifically to CpG dinucleotides and recruits other proteins to degrade a variety of viral RNA genomes. Many mammalian RNA viruses have evolved CpG deficiency. Increasing CpG dinucleotides in these low-CpG viral genomes in the presence of ZAP consistently leads to decreased viral replication and virulence. Because ZAP exhibits tissue-specific expression, viruses infecting different tissues are expected to have different CpG signatures, suggesting a means to identify viral tissue-switching events. The author shows that SARS-CoV-2 has the most extreme CpG deficiency in all known betacoronavirus genomes. This suggests that SARS-CoV-2 may have evolved in a new host (or new host tissue) with high ZAP expression. A survey of CpG deficiency in viral genomes identified a virulent canine coronavirus (alphacoronavirus) as possessing the most extreme CpG deficiency, comparable with that observed in SARS-CoV-2. This suggests that the canine tissue infected by the canine coronavirus may provide a cellular environment strongly selecting against CpG. Thus, viral surveys focused on decreasing CpG in viral RNA genomes may provide important clues about the selective environments and viral defenses in the original hosts.

BackgroundThe outbreak of SARS CoV-2 has caused ever-increasing attention and public panic all over the world. Currently, there is no specific treatment against the SARS CoV-2. Therefore, identifying effective antiviral agents to combat the disease is urgently needed. Previous studies found that indomethacin has the ability to inhibit the replication of several unrelated DNA and RNA viruses, including SARS-CoV.MethodsSARS CoV-2 pseudovirus-infected African green monkey kidney VERO E6 cells treated with different concentrations of indomethacin or aspirin at 48 hours post infection (p.i). The level of cell infection was determined by luciferase activity. Anti-coronavirus efficacy in vivo was confirmed by evaluating the time of recovery in canine coronavirus (CCV) infected dogs treated orally with 1mg/kg body weight indomethacin.ResultsWe found that indomethacin has a directly and potently antiviral activity against the SARS CoV-2 pseudovirus (reduce relative light unit to zero). In CCV-infected dogs, recovery occurred significantly sooner with symptomatic treatment + oral indomethacin (1 mg/kg body weight) daily treatments than with symptomatic treatment + ribavirin (10-15 mg/kg body weight) daily treatments (P =0.0031), but was not significantly different from that with symptomatic treatment + anti-canine coronavirus serum + canine hemoglobin + canine blood immunoglobulin + interferon treatments (P =0.7784).ConclusionThe results identify indomethacin as a potent inhibitor of SARS CoV-2.

In order to survey the infectious situation of canine coronavirus (CCV) in giant panda population, a virus neutralization test detecting specific antibodies against CCV in giant panda’s sera was established by using two-fold dilutions of serum and 100 TCID50 of the virus. The 62 sera samples of giant pandas, which were gathered from zoos and reserve region of Sichuan Province, China were detected. The neutralization antibody titer of 1:4 was recognized as the positive criterion, 8 sera samples were detected to be positive, and the positive rate was 12.9%. The titers of neutralizing antibody ranged from 1:8 to 1:32. It was the first comprehensive investigation on neutralization antibodies against CCV in giant panda population in China. The results of study showed that the infection of CCV in giant panda population was universal, which has posed a threat to the health of giant panda. Therefore, it is incumbent on us to study safe and effective vaccines to protect giant panda against CCV infection.

Canine coronavirus (CCoV) strains with the ability to spread to internal organs, also known as pantropic CCoVs (pCCoVs), have been detected in domestic dogs and wild carnivores. Our study focused on the detection and molecular characterization of pCCoV strains circulating in Italy during the period 2014–2017 in autochthonous dogs, in dogs imported from eastern Europe or illegally imported from an unknown country. Samples from the gut and internal organs of 352 dogs were screened for CCoV; putative pCCoV strains, belonging to subtype CCoV‐IIa, were identified in the internal organs of 35 of the examined dogs. Fifteen pCCoV strains were subjected to sequence and phylogenetic analyses, showing that three strains (98960‐1/2016, 98960‐3/2016, 98960‐4/2016) did not cluster either with Italian or European CCoVs, being more closely related to alphacoronaviruses circulating in Asia with which they displayed a 94%–96% nucleotide identity in partial spike protein gene sequences. The pCCoV‐positive samples were also tested for other canine viruses, showing co‐infections mainly with canine parvovirus.

The one-step polymerase chain reaction (one-step PCR) detection assay is an innovative PCR detection method, eliminating nucleic acid extraction steps, in which samples can be directly added to PCR reagents for testing. For simultaneous detection of CDV and CCoV, a sensitive and specific one-step duplex PCR (one-step dPCR) assay was developed with two pairs of primers that were designed based on H and M gene sequences of CDV and CCoV, respectively. The one-step dPCR with optimized detection conditions has high specificity and sensitivity; independent sequencing assays further verified these results.

Specific pathogen free kittens were vaccinated with an unattenuated field isolate of canine coronavirus (CCV) either by aerosol or subcutaneously, and received boosting vaccinations four weeks later. Aerosolisation elicited a homologous virus-neutralising (VN) antibody response that increased steadily over a four-week period and levelled off one to two weeks after revaccination. The initial aerosolised dose produced an asymptomatic infection with excretion of CCV from the oropharynx up to eight days after vaccination; virus shedding was not detected, however, after the second inoculation. Cats vaccinated subcutaneously developed low VN antibody titres after the first CCV dose and experienced a strong anamnestic response after the second dose. Neutralising antibody titres then levelled off one to two weeks after revaccination at mean values somewhat lower than in cats vaccinated by aerosol. CCV was not isolated from the oropharynx after either subcutaneous dose. Four weeks after CCV boosting inoculations, vaccinated cats and sham-vaccinated control cats were divided into three subgroups and challenged by aerosol with the virulent UCD1 strain of feline infectious peritonitis virus (FIPV UCD1) at three different dosage levels. Five of six cats (including sham-vaccinated controls) given the lowest challenge dose showed no signs of disease, while all other cats developed lesions typical of feline infectious peritonitis (FIP). The five surviving cats developed FIP after subsequent challenge with a fivefold higher dose of FIPV. Thus heterotypic vaccination of cats with CCV did not provide effective protection against FIPV challenge.

AIMS To determine which of the common canine respiratory pathogens circulate among selected populations of healthy and diseased dogs in New Zealand. METHODS Coagulated blood samples for serology and oropharyngeal swabs for virology were collected from healthy dogs (n=47) and from dogs with acute respiratory disease (n=49). For diseased dogs a convalescent blood sample was also collected 3-4 weeks later. Oropharyngeal swabs were subjected to virus isolation and tested for canine parainfluenza virus (CPIV), canine adenovirus (CAdV) 2, canine herpesvirus (CHV), canine respiratory coronavirus (CRCoV), canine influenza virus (CIV), canine distemper virus (CDV), Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, and Mycoplasma cynos nucleic acids by quantitative PCR (qPCR). Sera were tested for CRCoV antibody using competitive ELISA and results expressed as percent of inhibition (POI). RESULTS The mean age of diseased dogs (2.7, min

The aim of this study was to investigate canine astrovirus (CaAstV) infection in southwest China. We collected 107 faecal samples from domestic dogs with obvious diarrhoea. Forty-two diarrhoeic samples (39.3 %) were positive for CaAstV by RT-PCR, and 41/42 samples showed co-infection with canine coronavirus (CCoV), canine parvovirus-2 (CPV-2) and canine distemper virus (CDV). Phylogenetic analysis based on 26 CaAstV partial ORF1a and ORF1b sequences revealed that most CaAstV strains showed unique evolutionary features. Interestingly, putative recombination events were observed among four of the five complete ORF2 sequences cloned in this study, and three of the five complete ORF2 sequences formed a single unique group, suggesting that these strains could be a novel genotype. We successfully sequenced the complete genome of one CaAstV strain (designated 2017/44/CHN), which was 6628 nt in length. The features of this genome include putative recombination events in the ORF1a, ORF1b and ORF2 genes, while the ORF2 gene had a continuous insertion of 7 aa in region II compared with the other complete ORF2 sequences available in GenBank. Phylogenetic analysis showed that 2017/44/CHN formed a single group based on genome sequences, suggesting that this strain might be a novel genotype. The results of this study revealed that CaAstV circulates widely in diarrhoeic dogs in southwest China and exhibits unique evolutionary events. To the best of our knowledge, this is the first report of recombination events in CaAstV, and it contributes to further understanding of the genetic evolution of CaAstV.

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 104 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Canine parvovirus (CPV) is an important pathogen in domestic dogs, and the original antigenic types CPV-2 and its variants, CPV-2a, 2b and 2c, are prevalent worldwide. A multiplex TaqMan real-time PCR method was developed for the detection and differentiation of four antigenic types of CPV. A set of primers and probes, CPV-305F/CPV-305R and CPV-2-305P (for CPV-2)/CPV-2a-305P (for CPV-2a, 2b and 2c), was able to differentiate CPV-2 and its variants (CPV-2a, 2b and 2c). Another set of primers and probes, CPV-426F/CPV-426R and CPV-2-426P (for CPV-2 and 2a)/CPV-2b-426P (for CPV-2b)/CPV-2c-426P (for CPV-2c), was able to differentiate CPV-2a (2), CPV-2b, and CPV-2c. With these primers and probes, the multiplex TaqMan real-time PCR assay detected effectively and differentiated CPV-2, 2a, 2b and 2c by two separate real-time PCRs. No cross reactivity was observed with canine distemper virus, canine adenovirus, and canine coronavirus. The detection limit of the assay is 101 genome copies/μL for CPV-2, CPV-2a, CPV-2b, and 102 copies/μL for CPV-2c. The multiplex real-time PCR has 100% agreement with DNA sequencing. We provide a sensitive assay that simultaneously detects and differentiate four antigenic types of CPV and the method was also used for quantification of CPVs viral genome., Highlights • The Multiplex TaqMan real-time PCR can simultaneously detect and differentiate four antigenic types of CPV. • The method is suit for using in detection of CPVs in China. • The method showed a high specificity and sensitivity. • The method was also used for quantification of CPVs viral genome.

Canine coronavirus is a single-stranded RNA virus that causes enteritis in dogs of any age. Coronaviral enteritis is seldom definitively diagnosed, since it is usually much less severe than many other types of enteritis and is self-limiting. Conventional diagnostics for the canine coronaviral enteritis such as polymerase chain reaction (PCR), virus isolation, and electron microscopic examination are inappropriate for small animal clinics due to the complicated experimental processes involved. Therefore, a commercially available lateral flow test kit based on chromatographic immunoassay techniques was tested to evaluate its performance as a first-line diagnostic test kit that could be used in clinics. The coronavirus antigen test kit detected canine coronavirus-infected dogs with 93.1% sensitivity and 97.5% specificity. The detection limit of the test kit was between 1.97 × 104/mL and 9.85 × 103/mL for samples with a 2- fold serial dilution from 1.25 × 106 TCID50 (TCID50, 50% tissue culture infectious dose). Additionally, the test kit had no cross-reactivity with canine parvovirus, distemper virus, or Escherichia coli. Overall, the commercially available test kit showed good diagnostic performance in a clinical setting, with results similar to those from PCR, confirming their potential for convenient and accurate use in small animal clinics.

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 μL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

In dogs, canine parvovirus (CPV) enteritis is associated with high morbidity and fatality rates requiring early diagnosis to facilitate treatment and reduce its spread. In recent times, various commercial immunochromatographic (IC) test kits are available for its rapid diagnosis, which require an assessment of their accuracy. Therefore, precision of a point-of-care IC combination test kit for canine coronavirus (CCoV)/CPV faecal antigen detection was evaluated in this study. Multicentred random faecal samples from 115 dogs with gastroenteritis were checked for the presence of CPV antigens using the SensPERT IC combination test kit and the result was compared with polymerase chain reaction (PCR) as a reference test. Parvovirus was detected in 105 (91.3%) and 108 (93.9%) faecal samples by the point-of-care test kit and PCR, respectively. The point-of-care IC test kit showed 95.4% relative sensitivity, 71.4% specificity, 98.1% positive predictive value, 50.0% negative predictive value, and 93.9% accuracy comparable to conventional PCR in the samples tested. This point-of-care test kit also demonstrated a fair positive likelihood ratio (3.34), a very low negative likelihood ratio (0.07) and a moderate agreement (Kappa = 0.6) compared with conventional PCR. This test kit has shown to be very useful in the screening of dogs for CPV infection, and is a reliable alternative for diagnosing CPV both in conventional laboratories and remote areas without laboratories. Negative results in the IC testing with high suspicion of CPV infection should be further confirmed using superior test such as PCR.

The present study was carried out in the region of South Gujarat, India, to determine the prevalence and predisposing factors of canine parvovirus-2 (CPV-2) infection in acute gastroenteritis of pups. Further, haemagglutination (HA) test, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and rapid immunochromatography test were compared for diagnosis and prevalent CPV-2 types were ascertained. A total of 73 diarrhoea samples were collected and out of those 32, 33 and 35 were found positive via HA, ELISA and PCR, respectively. In rapid test, 26/52 samples were found positive. Among different age-groups, 11/24 and 13/21 animals were positive in pups aged 4–8 and 8–12 weeks, respectively. All but one (34/35) positive samples were from unvaccinated animals. Labrador was found to be the most susceptible breed (n = 13) to infection. Considering PCR as the best test, 47.94% (35/73) prevalence of CPV was recorded. Among PCR positive samples, 3 and 32 belonged to type CPV-2a and CPV-2b, respectively. Type CPV-2c was not detected among the examined samples. Sequencing analysis of 9/10 CPV-2b isolates revealed single nucleotide polymorphism (SNP) (A-G) at position 4106 (alanine to threonine) and suggested the occurrence of mutant, new CPV-2b in this area. As other major pathogen canine coronavirus was detected in 7/38 CPV negative samples. Conclusively, CPV-2 infection was detected in 47.97% cases of AGE of pups which warrants search for other pathogens in the diagnostic procedure. This work is among the few recent reports which depict the occurrence of a novel mutant (new CPV-2b) in India.

Background Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. Methods A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. Results The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min–12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R2 value of the positive results was 0.947. Conclusions The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

Highlights • A molecular survey for selected viral enteropathogens was conducted in dogs with and without acute gastroenteritis. • Canine parvovirus and coronavirus were strongly associated with occurrence of enteric disease. • Canine circovirus infections correlated with acute gastroenteritis only when associated with other enteric viruses., Canine circovirus (CanineCV) is a canine virus, whose pathogenetic role is still uncertain. Based on recent data suggesting its role as entheropathogen, a case-control study was conducted between 2013 and 2016 to investigate the association of CanineCV with gastroenteritis in dogs, alone or in combination with other viral pathogens, including canine parvovirus (CPV), canine coronavirus (CCoV) and canine distemper virus (CDV). A total of 219 dogs suffering from acute gastroenteritis disorders and 67 controls randomly recruited among healthy dogs or patients presenting without enteric signs were screened by a panel of real-time (RT-)PCR assays for CanineCV, CPV, CCoV and CDV. A high prevalence of viral infections was detected in dogs with gastroenteritis (77.16%), with CPV representing the most frequently detected enteropathogen, followed by CanineCV and CCoV. While CPV and CCoV infections displayed a strong association with occurrence of acute gastroenteritis (p

MSc (Animal Health), North-West University, Mafikeng Campus Diarrhoea is a complex condition mostly encountered in small animal practices incriminating some common enteric pathogens including canine parvovirus type 2, canine coronavirus, canine distemper virus, canine rotavirus, endoparasites, bacteria as well as life style and diet. The CPV, CCoV and CRV are the main viral enteric pathogens responsible for severe gastroenteritis including diarrhoea; whereas CPV-2 is the cause of high morbidity and high mortality in dog populations. Since its appearance in the late 1970, several evolutions and mutations of CPV have been observed, resulting in the replacement of the original type by its new variants CPV-2a, CPV-2b and later on CPV-2c, circulating worldwide. Nowadays, in severe cases of CPV-2 infection, it was found that there is mix infection between CPV-2 and CCoV, or CPV-2 and CRV infection. Therefore, the monitoring of these infectious diseases is critical for control and prevention. Our study aimed to determine the epidemiology of bloody diarrhoea, to identify and discriminate viral enteric pathogens circulating among puppies presented to the North West University, Animal Health hospital, Mafikeng campus by means of Immunochromatography and molecular techniques. Faecal samples of 84 diarrhoeic dogs presented with signs suspected to be due to CPV-2 infection were collected at consultation. In addition, other demographic informations were collected for each dog. Two diagnostic methods were used: Immunochromatography assay (IC) for antigen detection and conventional PCR using the universal CPV primers for characterisation. Of the 84 samples, IC tested 66/84, 2/84 and 2/84 positive CPV-2, CCoV and CRV antigens respectively. Conventional PCR revealed 80/84, 0/84 and 12/84 positive CPV-2 DNA, CCoV-RNA and CRV-RNA respectively. The analysis of the sequences confirmed the presence of 80 CPV-2 strains and 12 CRV strains. The predominant circulating variant was the CPV-2c followed by CPV-2b (minor); and G3 rotavirus serotype. The statistical results showed a significance (P

Alphacoronavirus 1 (subgenus Tegacovirus, genus Alphacoronavirus, family Coronaviridae), which encompasses transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV) and canine coronavirus (CCoV), is an important pathogen that can cause severe gastroenteritis and is distributed worldwide. CCoV has two different genotypes: CCoV type I, which has a high identity with FCoV-I, and CCoV type II, which is divided into two subtypes, CCoV IIa (pantropic) and CCoV IIb, which is related to FCoV-II and has been involved in multiple recombination events. Between 2014 and 2018, 43 fecal samples from puppies and young dogs under 1 year of age with hemorrhagic enteritis and from 5 cats under 2 years of age with ascites or thoracic effusion were collected by a private veterinary practice in Bogotá, Colombia. A screening for Coronavirus via RT-PCR (nsp12) and PCR amplification of Canine protoparvovirus (VP1) revealed 27.1% (13/49) and 72.9% (35/49) positive samples, respectively. Positive samples for coronavirus were tested for M, N, S and the sequences grouped in the FCoV, CCoV-I and CCoV-IIb clusters that were distant from the pantropic type (IIa). The N gene formed two clusters, one exclusively with samples from this study in subtype II and another with strains in subtype I. For gene S (subtype I), the samples clustered with the Brazilian samples, while samples positive for S subtype IIb grouped into a cluster distinct from the other reference sequences. The prevalence of coronaviruses identified in this study is within the range reported by different countries worldwide., Genetics; Microbiology; Virology; Biocomputational method; Viral disease; Viral genetics; Phylogeny; Epidemiology; Coronavirus; Alphacoronavirus 1; Dogs; Cats; Colombia; Phylogenetic relationship.

Background Canine parvovirus (CPV) and feline parvovirus (FPV) are causative agents of diarrhea in dogs and cats, which manifests as depression, vomiting, fever, loss of appetite, leucopenia, and diarrhea in young animals. CPV and FPV can single or mixed infect cats and cause disease. To diagnose sick animals effectively, an effective virus diagnostic and genome typing method with high sensitivity and specificity is required. Results In this study, a conserved segment containing one SNP A4408C of parvovirus was used for real-time PCR amplification. Subsequently, data were auto-analyzed and plotted using Applied Biosystems® High Resolution Melt Software v3.1. Results showed that CPV and FPV can be detected simultaneously in a single PCR reaction. No cross-reactions were observed with canine adenovirus, canine coronavirus, and canine distemper virus. The assay had a detection limit of 4.2 genome copies of CPV and FPV. A total of 80 clinical samples were subjected to this assay, as well as to conventional PCR-sequence assay and virus isolation. Results showed that the percentage of agreement of the assay and other methods are high. Conclusions In short, we have developed a diagnostic test for the accurate detection and differentiation of CPV and FPV in fecal samples, which is also cost effective.

Canine coronavirus (CCoV) is an important pathogen that causes enteritis in dogs, but there is no information on CCoV infection in Vietnam. To examine the prevalence of CCoV infection among Vietnamese dogs, 201 serum samples were analyzed by virus-neutralization (VN) test. The results showed that antibody against CCoV-II was present in 87 dogs (43.3%). To detect genes of CCoV, fecal samples collected from 30 diarrheic and 50 healthy dogs were examinated by RT-PCR, confirming that 2 diarrheic dogs and 5 healthy dogs were positive for CCoV. Nucleotide sequences of N-terminal region of spike (S) gene indicated that CCoV strains were divided into two subgenotypes, CCoV-IIa and -IIb, respectively. Furthemore, we succeeded in isolating CCoV/dog/HCM47/2015, the isolate was plaque-purified three times, and 3'-terminal one-third of the genome was analyzed. Interestingly, the plaque-purified virus had a large deletion in ORF3abc and E genes (1,165 nt), and a short deletion in ORF7b gene (60 nt), suggesting that these regions are not necessary for in vitro replication of CCoV. Next, the antigenicity between the isolated CCoV-IIb and the other CCoV-IIa was compared by VN test, revealing that antigenicty of the isolated CCoV is equal or higher than that of the other CCoV. In summary, two subgenotypes of CCoV-II are spreading among Vietnamese dogs. The isolated virus with a large deletion after in vitro passage may be useful for the development of vaccine, owing to its antigenicity and efficient viral growth in vitro.

Highlights • CPV, CCoV and β-hemolytic E. coli are associated with (severe) acute diarrhoea in puppies. • Puppies from high-volume breeders are more likely to be infected with CPV, CCoV and Cystoisospora spp. • CPV and CCoV are more often detected in winter/spring. • Multiple enteric infections are common in puppies with and without diarrhoea. • Screening of CPV, CCoV and β-hemolytic E. coli is advised in diarrhoeic puppies., Laboratory confirmation of the causative agent(s) of diarrhoea in puppies may allow for appropriate treatment. The presence of potential pathogens however, does not prove a causal relationship with diarrhoea. The aim of this study was to identify specific enteropathogens in ≤12 month old puppies with and without acute diarrhoea and to assess their associations with clinical signs, putative risk factors and pathogen co-occurrence. Faecal samples from puppies with (n = 113) and without (n = 56) acute diarrhoea were collected and screened for Canine Parvovirus (CPV), Canine Coronavirus (CCoV), Salmonella spp., Campylobacter spp., Clostridium perfringens, Clostridium difficile, β-hemolytic Eschericha coli (hEC), Giardia spp., Toxocara spp., Cystoisospora spp., and Cyniclomyces guttulatus. One or more pathogens were detected in 86.5% of diarrhoeic puppies and in 77.8% of asymptomatic puppies. Significant positive associations were found between CPV and CCoV, CPV and Cystoisospora spp., Toxocara spp. and hEC, Giardia spp. and C. guttulatus. Only CPV and CCoV were significantly associated with diarrhoea, hEC with a subset of puppies that had diarrhoea and severe clinical signs. CPV was more prevalent in puppies under 3 months of age. Puppies from high-volume dog breeders were significantly at increased risk for CPV (OR 4.20), CCoV (OR 4.50) and Cystoisospora spp. (OR 3.60). CCoV occurred significantly more often in winter (OR 3.35), and CPV in winter (OR 3.78) and spring (OR 4.72) as compared to summer. We conclude that routine screening for CPV, CCoV and hEC is recommended in puppies with acute diarrhoea, especially if they are under 3 months of age and originate from high-volume dog breeders. Routine screening for other pathogens may lead to less conclusive results.

We collected blood samples from wolves ( Canis lupus ) on the Alaska Peninsula, southwest Alaska, US, 2006-11 and tested sera for antibodies to canine adenovirus (CAV), canine coronavirus (CCV), canine distemper virus (CDV), canine herpesvirus (CHV), canine parainfluenza (CPI), canine parvovirus (CPV), Neospora caninum , and Toxoplasma gondii . Detected antibody prevalence was 90% for CAV, 28% for CCV, 12% for CDV, 93% for CHV, 0% for CPI, 20% for CPV, 0% for N. caninum, and 86% for T. gondii . Prevalence of CCV antibodies suggested a seasonal pattern with higher prevalence during spring (43%) than in fall (11%). Prevalence of CCV antibodies also declined during the 6-yr study with high prevalence during spring 2006-08 (80%, n=24) and low prevalence during spring 2009-11 (4%, n=24). Prevalence of N. caninum and T. gondii antibodies were highly variable in the study area during 2006-11. Results suggested that some pathogens might be enzootic on the Alaska Peninsula (e.g., CAV and CHV) while others may be epizootic (e.g., CCV, N. caninum , T. gondii ).

To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.