An update on canine coronaviruses: viral evolution and pathobiology

The emergence of human severe acute respiratory syndrome incited renewed interest in animal coronaviruses (CoVs) as potential agents of direct and indirect zoonoses. The reinforced epidemiological surveillance on CoVs has led to the identification of new viruses, genotypes, pathotypes and host variants in animals and humans. In dogs, a CoV associated with mild enteritis, canine coronavirus (CCoV), has been known since 1970s. CoV strains with different biological and genetic properties with respect to classical CCoV strains have been identified in dogs in the last few years, leading to a full reconsideration of the CoV-induced canine diseases. The genetic evolution of dog CoVs is paradigmatic of how CoVs evolve through accumulation of point mutations, insertions or deletions in the viral genome, that led to the emergence of new genotypes (CCoV type I), biotypes (pantropic CCoV) and host variants (canine respiratory coronavirus). This paper is a review of the current literature on the recent genetic evolution of CCoV and emergence of new CoVs in the dog. The significances of the newly acquired information for the canine health status and prophylaxis programmes are also discussed.     

M gene evolution of canine coronavirus in naturally infected dogs

Two stray pups (A and B), three and five months old, respectively, both naturally infected with canine coronavirus (CCoV), were studied for 180 days. The virus was detected intermittently in the pups' faeces by PCR for periods of 156 and 146 days, respectively. Sequence analysis of a fragment of the gene encoding the M protein revealed that the viruses detected at the onset of the infection were very similar to typical strains of CCoV, whereas from 42 days after infection in pup A and 40 days after infection in pup B the viruses had nucleotide and amino acid mutations resembling sequences in feline coronavirus.     

犬猫冠状病毒研究进展

犬冠状病毒（canine coronavirus,CCoV）与猫冠状病毒（feline coronavirus,FCoV）同属于冠状病毒科冠状病毒属α属,与其同属的病毒还有猪传染性胃肠炎病毒（porcine transmissible gastroenteritis virus,TGEV）、猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）等。遗传进化分析表明,该种属不同基因型病毒通过基因重组能产生新型变异毒株,为疾病的诊断与防控造成了很大的阻碍。β属冠状病毒包括牛冠状病毒（bovine coronavirus,BCoV）、犬呼吸道冠状病毒（canine respiratory coronavirus,CRCoV）、严重急性呼吸综合征冠状病毒（severe acute respiratory syndrome coronavirus,SARS-CoV）等,其中CRCoV与BCoV同源性较高,该类病毒与α属冠状病毒在基因组结构、致病机制、感染症状等方面差异较大。CCoV与FCoV在全球范围内广泛传播,具有发病率高、死亡率低的特点。由于RNA病毒本身的特点和环境选择压力的影响,这两种病毒不断变异进化,新的强致病力毒株相继出现。经FCoV基因突变演化而来的猫传染性腹膜炎病毒（feline infectious peritonitis virus,FIPV）毒力大大增强,病毒基因组中某些特异性的点突变使得针对宿主的细胞嗜性发生改变,该病毒的致病机制主要依赖于病毒感染后诱导机体产生的抗体依赖性增强作用（ADE）。针对犬猫冠状病毒的流行病学调查及防控不能仅依赖于疫苗免疫单一因素,还应综合考虑病毒毒力、环境条件、宠物自身免疫抵抗力状态等。针对犬猫冠状病毒的诊断应根据临床症状,结合常规血液学检查、血清生化检查和实验室诊断技术来进行全面的鉴定,防止出现假阳性及假阴性结果。     

Research Progress of Canine Coronavirus and Feline Coronavirus

Canine coronavirus (CCoV) and feline coronavirus (FCoV) belong to α-genus coronavirus of coronavirus family,porcine transmissible gastroenteritis virus (TGEV),porcine epidemic diarrhea virus (PEDV) also belong to the same genus.Genetic evolution analysis showed that different genotype of the virus could produce new variant strains through gene recombination,which caused great obstacles to the diagnosis and control of the disease.β-genus coronaviruses include bovine coronavirus (BCoV),canine respiratory coronavirus (CRCoV) and severe acute respiratory syndrome coronavirus (SARS-CoV).Among them,CRCoV has the highest homology with BCoV,but there are great differences in genomic structure,pathogenic mechanism and infection symptoms between this kind of coronavirus and α-coronavirus.CCoV and FCoV are widely spreading around the world,characterized by high morbidity and low mortality.Due to the characteristics of RNA virus and the influence of environmental selection pressure,the viruses continue to mutate and evolve,and new virulent strains appear one after another.The virulence of feline infectious peritonitis virus (FIPV) is greatly enhanced,some specific point mutations in the virus genome change the cellular tropism against the host.The pathogenesis of the virus mainly depends on the antibody-dependent enhancement (ADE) induced by virus infection.The epidemiological investigation and prevention and control of CCoV and FCoV should not only rely on the single factor of vaccine immunity,but also comprehensively consider the virulence of the virus,environmental conditions,pet self-immune resistance, and so on.The identification of CCoV and FCoV should be based on clinical symptoms,combined with routine hematological examination,serum biochemical examination and laboratory diagnosis techniques to prevent false positive and false negative results.     

An update on aspects of viral gastrointestinal diseases of dogs and cats

Viruses commonly cause gastrointestinal illnesses in dogs and cats that range in severity from mild diarrhoea to malignant neoplasia. Perpetual evolution of viruses is reflected in changing disease patterns, so that familiar viruses are sometimes discovered to cause new or unexpected diseases. For example, canine parvovirus (CPV) has regained the ability to infect felids and cause a panleucopenia-like illness. Feline panleucopenia virus (FPV) has been shown to cause fading in young kittens and has recently been implicated as a possible cause of feline idiopathic cardiomyopathy. Molecular scrutiny of viral diseases sometimes permits deeper understanding of pathogenesis and epizootiology. Feline gastrointestinal lymphomas have not, in the past, been strongly associated with retroviral infections, yet some of these tumours harbour retroviral proviruses. Feline leukaemia virus (FeLV) may play a role in lymphomagenesis, even in cats diagnosed as uninfected using conventional criteria. There is strong evidence that feline immunodeficiency virus (FIV) can also be oncogenic. The variant feline coronaviruses that cause invariably-fatal feline infectious peritonitis (FIP) arise by sporadic mutation of an ubiquitous and only mildly pathogenic feline enteric coronavirus (FECV); a finding that has substantial management implications for cat breeders and veterinarians. Conversely, canine enteric coronavirus (CECV) shows considerable genetic and antigenic diversity but causes only mild, self-limiting diarrhoea in puppies. Routine vaccination against this virus is not recommended. Although parvoviruses, coronaviruses and retroviruses are the most important known viral causes of canine and feline gastrointestinal disease, other viruses play a role. Feline and canine rotaviruses have combined with human rotaviruses to produce new, reassortant, zoonotic viruses. Some companion animal rotaviruses can infect humans directly. Undoubtedly, further viral causes of canine and feline gastrointestinal disease await discovery.     

Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 10¹ copies/μL for CCoV I and 6.91 × 10² copies/μL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.     

Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan

To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p < 0.0001 and p = 0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date.     

Western European epidemiological survey for parvovirus and coronavirus infections in dogs

An epidemiological survey for canine parvovirus (CPV) and canine coronavirus (CCoV) infections was conducted in Western Europe. A total of 156 faecal samples were collected from dogs with diarrhoea in Spain (n = 47), Italy (n = 39), France (n = 26), Germany (n = 21), the United Kingdom (n = 8), Belgium (n = 10), and the Netherlands (n = 5). Using molecular assays for virus detection and characterisation, CPV and CCoV were found to be widespread in European dog populations, either alone or in mixed infections. In agreement with previous reports, the original type CPV-2 was shown not to circulate in European dogs. The recently identified virus variant CPV-2c was predominant in Italy and Germany and present at high rates in Spain and France but was not detected in the UK or Belgium. Except for the UK, CCoV genotype I was identified in all European countries involved in the survey, albeit at a lower prevalence rates than CCoV genotype II.     

Safety and efficacy of a modified-live canine coronavirus vaccine in dogs

The safety and the efficacy of a modified-live (ML) canine coronavirus (CCoV) vaccine strain 257/98-3c was evaluated in 14 dogs seronegative and virus negative for CCoV. For the safety test, four dogs were inoculated, two by intramuscular and two by oronasal route, with 10 times the vaccinal dose. During the observation period (28 days) all dogs did not display any local or systemic reaction. For the efficacy test, eight dogs were vaccinated by intramuscular (four dogs-group A) or by oronasal route (four dogs-group B). Two dogs were maintained as non-vaccinated controls. In the dogs of group A, vaccinal virus was not detected in faecal samples by virus isolation (VI) and by PCR assay, while in the dogs of group B, the virus was revealed for six median days only by PCR. Twenty-eight days later, the vaccinated and control dogs were challenged with a field CCoV strain. After the challenge, the vaccinated dogs did not display clinical signs and the dogs of group A shed virus for 5.5 median days, evaluated by VI, and for 10 median days evaluated by PCR. Virus shedding was not observed, both by VI and PCR assay, in the dogs of group B. The two control dogs displayed moderate clinical signs and the virus was detected by VI for 14.5 median days starting from day 3 post-challenge (dpc 3) and by PCR assay for 23 median days starting from dpc 1.     

犬冠状病毒诊断技术研究进展

犬冠状病毒（canine coronavirus,CCoV）是导致犬胃肠道疾病的常见病原,与其他肠道病原共同感染时犬死亡率显著升高,严重影响养犬业的健康发展。快速、灵敏和特异的诊断技术对该病的防控起到至关重要的作用。除临床诊断外,常见的CCoV实验室诊断技术主要有病毒分离培养、电镜观察及血清学诊断技术,然而这些技术往往耗时长且工作量较大,分子诊断技术主要包括普通RT-PCR、实时荧光定量RT-PCR、纳米PCR等检测方法,这些诊断技术在准确性、敏感性及特异性上有极大提高,诊断效率也显著提高。近年来,随着分子免疫诊断技术与新型材料研发技术的迅速发展,新的检测方法应运而生,纳米金、核酸探针、芯片技术及纳米生物传感器等技术更加敏感、便捷、高效且都具有制备试剂盒的可能性,从而在诊断上具有简洁性和普适性。文章将针对CCoV的诊断技术进行全面综述,以期为CCoV诊断试剂的研发提供参考。     

Experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia

A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8-9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions.     

Molecular characterization of canine coronavirus strains circulating in Brazil

To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil.     

Viral infections and bovine mastitis: a review

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.     

溶瘤病毒在犬肿瘤治疗中的研究进展

溶瘤病毒疗法是利用病毒治疗恶性肿瘤的一种较有前景的新型生物疗法。溶瘤病毒不仅能直接特异性感染和溶解肿瘤细胞，也能刺激肿瘤部位的免疫反应来间接地溶解肿瘤组织细胞，且对正常组织细胞无杀伤或仅有较弱的杀伤作用，从而能达到理想的抗肿瘤效果。文章综述了溶瘤病毒的发展史及溶瘤病毒介导肿瘤消融术的机制，分析麻疹病毒、犬瘟热病毒、新城疫病毒、仙台病毒等8种病毒作为抗肿瘤药物在体外试验、异种移植肿瘤动物模型试验、肿瘤患犬治疗试验等不同阶段对各种犬肿瘤细胞的杀伤效果，提示溶瘤病毒疗法可能是一种安全可靠的恶性肿瘤治疗新方法，但真正将溶瘤病毒疗法应用在犬肿瘤临床治疗中时病毒肿瘤特异性、病毒使用剂量等问题仍需解决，本综述可为溶瘤病毒在犬肿瘤临床治疗中的安全使用提供新的思路和技术途径。     

Canine coronavirus induces apoptosis in cultured cells

Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection.     

The prevalence of a group 2 coronavirus in dogs in Japan

Canine coronavirus (CCoV) has been reported to cause acute diarrhea mainly in young pups. CCoV and feline coronavirus are classified as group 1 coronaviruses. However, it has recently been reported in the United Kingdom that the group 2 coronavirus gene, which is more closely related to the bovine coronavirus (BCoV) and human coronavirus strain OC43, has been detected in respiratory tract tissue samples from dogs with respiratory disease. In this study, we examined the prevalence of antibodies to group 2 coronaviruses in domestic dogs and cats in Japan by a neutralization test using BCoV. All 104 feline serum samples were negative (<1:5) for anti-BCoV antibodies. In contrast, of the 898 canine serum samples, 160 (17.8%) were positive for anti-BCoV antibodies, and the antibody titers ranged from 1:5 to more than 1:640, with 1:160 being the most frequent. No correlation was found between the titers of the anti-BCoV and anti-CCoV antibodies in the 198 serum samples of dogs with a known history of CCoV vaccination. We amplified, by RT-PCR, group 2 coronavirus-specific hemagglutination/esterase genes in the oral swabs of a total of 10 young pups presenting with or having recovered from respiratory signs, or having anti-BCoV antibodies, with the result that 2 pups were positive for the hemagglutination/esterase genes. These results strongly suggest that an unknown group 2 coronavirus as well as the known enteritis-causing CCoV (group 1 coronavirus) is prevalent among domestic dogs in Japan.     

Recent advances in canine virus research

The widespread application of techniques such as electron microscopy and immunofluorescence in the study of canine viruses has significantly advanced our knowledge particularly with regard to CAV–ICH and CDV. The isolation of new viruses particularly from the respiratory tract has stimulated renewed interest in upper respiratory disease in the dog. The fact that a single virus may cause a number of diseases with, for example, CAV–ICH being associated with respiratory, liver and renal disease while some infections, e.g. upper respiratory disease, can be associated with a number of viruses emphasizes the complexity of canine virus infections. Although many aspects of the pathogenesis of canine virus infections have been carefully studied, it is hoped that more attention will be directed to many of the unanswered questions concerning the role of immunological factors. Résumé. Résumé. L'application universelle de techniques telles que la microscopie électronique et l'immunonuorescence dans l'tude des virus canins a accru nos connaissances de façon significative, particulièrement en ce qui concerne GAV-ICH et CDV. L'isolation de nouveaux virus en provenance des voies respiratoires a stimulé un intérět renouvelé dans les maladies des voies respiratoires chez les cheins. Le fait qu'un seul virus puisse causer un certain nombre de maladies avec, par exemple GAV-ICH étant associé avec des maladies de la respiration, du foie et des reins tandis que des infections des maladies de voies respiratoires supérieurs peuvent ětre associées à un certain nombre de virus, souligne la complexité des infeptions de virus canins. Bien que de nombreux aspects de la pathogenèse d'infections de virus canins aient été soigneusement étudiés, on espère apporter plus d'attention à beacoup de questions restées sans réponses concernant le rǒle des facteurs immunologiques. Zusammenfassung. Die weitverbreitete Anwendung von verschiedener Technik iwe Elek-tonenmikroskopie und Immunfluoreszenz in der Studie von Hundeviren hat unser Wissen erheblich gesteigert, besonders in Hinsicht auf CAV-ICH und CDV. Die Isolierung von neuen Viren, besonders vom Atefnweg hat émeutes Interesse an der Erkrankung des oberen Luftweges von Hunden, angeregt. Die Tatsache, dass ein einzelner Virus eine Anzahl von Krankheiten verursachen kann, z.B. CAV-ICH ist assoziiert mit Atemweg- Leber- und Nierenerkrankung, wàhrend einige infektionen z.B. obère Atemwegerkrankung mit einer Anzahl von Viren in Verbindung gebracht werden kann, bekràftigt die Verwicklung der Hundevirusinfektionen. Obwohl viele Ansichten der Pathogenese von Hundevirusinfektion sorgfǎltig studiert wurden, hofft man dass mehr Aufmerksamkeit auf die vielen unbeantworteten Fragen gerichtet wird, die die Rolle der immunologischen Faktoren betrifft.     

Genetic characterization of a novel influenza A virus H5N2 isolated from a dog in China

An influenza virus, A/canine/Shandong/JT01/2009, has been isolated from a dog exhibiting classical flu signs in China. HAI and NAI assays subtyped A/canine/Shandong/JT01/2009 as a H5N2 like virus. Phylogenetic reconstructions indicated strong relationships with viruses from various hosts and dispersed geographic locations. These analyses indicate A/canine/Shandong/JT01/2009 is a novel virus generated by complex reassortment of the viral segments.     

Action of disinfectants on canine coronavirus replication in vitro

Canine coronavirus (CCoV) is responsible for enteric disease in pups. Infected dogs generally have a rapid recovery, so the virus is highly contagious and the spread of infection is difficult to control. Chemical disinfectants have been widely used in human disease-control programmes to prevent viral infectious diseases from spreading, but to date, there are no studies in the literature on the sensitivity of CCoV to chemical biocides. The present study investigated the sensitivity of CCoV to disinfectants currently used for prophylaxis in kennel and dog breeding locations. The effects of three agents: alkyl-dimethyl-benzyl-ammonium chloride, benzalkonium chloride and didecyl-dimethyl-ammonium chloride, on the infectivity titre of CCoV in A72 cell lines, were studied at different concentrations. Although they may regard a small number of agents, the findings showed that the sensitivity of CCoV to disinfectants varies and the differences are dose correlated. In general, virus inactivation implies a permanent loss of infectivity which can be evaluated in suspensions and hand disinfection tests.