Equine coronavirus induces apoptosis in cultured cells

Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cytopathic effect in ECoV infection. Morphologic and biochemical features of apoptosis, such as nuclear fragmentation and DNA ladder formation, were also detected in ECoV-infected cells. Moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases – caspase-3/7 – and initiator caspases – caspase-8 and caspase-9 – that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ECoV-infected MDBK cells. Therefore, it was suggested that ECoV can induce apoptosis in MDBK cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. Although the pathological characteristics of ECoV are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ECoV infection.     

Clinical,hematological, and biochemical findings in puppies with coronavirus and parvovirus enteritis

The clinical and laboratory findings in puppies naturally infected with canine coronavirus (CCoV) and/or canine parvovirus (CPV) were compared with findings in uninfected puppies. Lymphopenia was the only parameter related to CCoV infection that was statistically significant; vomiting, anorexia, lethargy, hemorrhagic fluid diarrhea, leukopenia, lymphopenia, thrombocytopenia, hypoglycemia, and hypoproteinemia were correlated with CPV infection.     

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.     

应用流式细胞术研究犬瘟热病毒感染细胞的凋亡

用犬瘟热病毒 ( CDV) 2种不同毒株感染非洲绿猴肾细胞 ( Vero) ,用流式细胞术分析了病毒诱导的感染细胞的凋亡。结果发现 ,2种毒株感染引起的细胞病变类型不同 ,分为圆缩型与合胞体型 ;2种毒株均可诱导感染细胞凋亡 ,但毒株间的凋亡细胞比例存在差异 ,即产生合胞体型细胞病变的毒株凋亡出现较晚 ,凋亡细胞比例显著低于产生圆缩型细胞病变的毒株 ( P<0 .0 1 )。结果提示 ,不同毒株导致细胞死亡的机制可能不同     

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.     

Genetic evolution of canine coronavirus and recent advances in prophylaxis

Since the first identification of the virus in 1971, the disease caused by canine coronavirus (CCoV) has not been adequately investigated and the role that the virus plays in canine enteric illness has still not been well established. In the last decade, as a consequence of the relatively high mutation frequency of RNA positive stranded viruses, CCoV has evolved and a new genotype has been identified in the faeces of infected dogs. The several studies carried out by different researchers have focused upon the epidemiological relevance of these viruses and, considering the wide diffusion of CCoV infections among dog populations, the author underlines the need for further investigation on the biology of CCoV and on the pathogenetic role of their infections.     

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.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

Apoptosis and anti-apoptotic heat shock proteins in canine cutaneous infundibular keratinizing acanthomas and squamous cell carcinomas

Cell stress and death are linked in the neoplastic process, and heat shock proteins appear to play an important role by inhibiting apoptotic pathways. The apoptotic rates in 9 canine infundibular keratinizing acanthomas (IKAs) and 17 canine squamous cell carcinomas (SCCs) were correlated with the immunohistochemical expression of caspase-3 and the antiapoptotic heat shock proteins Hsp27, 72 and 73. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) method. The absence of a correlation between the TUNEL index and active-caspase-3 expression, a paucity of active-caspase-3-positive cells and Hsp72 over-expression were considered to be indicative of inhibition of apoptosis, and suggestive that inhibition of cell death plays a key role in oncogenesis and tumour growth of some canine skin neoplasms.     

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.     

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.     

High level expression of biologically active canine interferon-alpha subtype 4 using a baculovirus

In this study, a high amount of bioactive recombinant canine interferon-alpha subtype 4 (CaIFN-alpha4) was expressed in a baculovirus system. For easy purification, it was expressed as a CaIFN-alpha4 bearing histidine hexamer at the C-terminal region, designated CaIFN-alpha4His. CaIFN-alpha4His was detected in culture supernatants of insect cells infected with the recombinant virus using sodium dodecyl sulfate-polyarcylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining. The level of expression was very high, and approximately 1 mg of purified protein, with 5.0 x 10(7) units/mg, was obtained from 300 ml of culture supernatant. The purified product showed antiviral activity against Vesicular stomatitis virus on canine tumor cell line A72 and chicken embryo fibroblast cells.     

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。     

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.     

Canine coronavirus inactivation with physical and chemical agents

Canine coronavirus (CCoV) is responsible for mild or moderate enteritis in puppies. The virus is highly contagious and avoiding contact with infected dogs and their excretions is the only way to ensure disease prevention. Since no studies have yet focused on the sensitivity of CCoV to chemical biocides the present investigation examined the efficiency of physical and chemical methods of viral inactivation. CCoV infectivity was stable at +56 degrees C for up to 30 min, but tended to decrease rapidly at +65 degrees C and +75 degrees C. Germicidal ultra-violet (UV-C) light exposure demonstrated no significant effects on virus inactivation for up to 3 days. CCoV was observed to be more stable at pH 6.0-6.5 while extreme acidic conditions inactivated the virus. Two tested aldehydes inactivated the virus but their action was temperature- and time-dependent. The methods for CCoV inactivation could be applied as animal models to study human coronavirus infection, reducing the risk of accidental exposure of researchers to pathogens during routine laboratory procedures.     

Anatid herpesvirus 1 CH virulent strain induces syncytium and apoptosis in duck embryo fibroblast cultures

Anatid herpesvirus 1 (AHV-1) CH virulent strain was first isolated from an infected duck and it was found that this virus strain could induce cytopathic effect (CPE) in duck embryo fibroblast (DEF). Following AHV-1 infection, DEF showed morphological changes such as cell rounding, improved refractivity and detachment from the culture surface. However, its pathological characteristics were not adequately known. Related studies were performed and the results showed that syncytium formation could be observed as the other type of CPE in AHV-1 infection. Hematoxylin-eosin staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining of infected DEF were each used to visualize the shape and distribution of chromatin within nuclei and nuclear fragmentation was observed. Chromatin condensation and margination, as well as formation of apoptotic bodies were observed by transmission electron microscopy (TEM). DNA ladder formation was detected in AHV-1 infected cells and apoptosis of the infected DEF was also detected by flow cytometry analysis of Annexin V-FITC/PI staining method. Therefore, it was suggested that AHV-1 virulent strain can induce syncytium and apoptosis in DEF. Syncytium formation and apoptosis observed in this study may contribute to the elucidation of AHV-1 pathogenesis.