Nasal virome of dogs with respiratory infection signs include novel taupapillomaviruses.

Using viral metagenomics, we characterized the mammalian virome of nasal swabs from 57 dogs with unexplained signs of respiratory infection showing mostly negative results using the IDEXX Canine Respiratory Disease RealPCR™ Panel. We identified canine parainfluenza virus 5, canine respiratory coronavirus, carnivore bocaparvovirus 3, canine circovirus and canine papillomavirus 9. Novel canine taupapillomaviruses (CPV21-23) were also identified in 3 dogs and their complete genome sequenced showing L1 nucleotide identity ranging from 68.4 to 70.3% to their closest taupapillomavirus relative. Taupapillomavirus were the only mammalian viral nucleic acids detected in two affected dogs, while a third dog was coinfected with low levels of canine parainfluenza 5. A role for these taupapillomavirues in canine respiratory disease remains to be determined.

Phylogeny of canine coronavirus (CCoV) from Brazilian dogs based on membrane protein partial sequences / Filogenia de Coronavírus canino (CCoV) de cães brasileiros com base em sequências parciais da proteína de membrana

Este artigo descreve a anteriormente desconhecida diversidade molecular de amostras brasileiras de Coronavírus canino (CCoV). Vinte e duas amostras foram submetidas à análise da sequência parcial do gene codificador da proteína de membrana, sendo 12 classificadas como CCoV Tipo II e 10 como CCoV Tipo I e uma possível sublinhagem tipicamente brasileira foi encontrada para o CCoV Tipo II.

Involvement of FOXO transcription factors, TRAIL-FasL/Fas, and sirtuin proteins family in canine coronavirus type II-induced apoptosis.

n our previous study, we have shown that canine coronavirus type II (CCoV-II) activates both extrinsic and intrinsic apoptotic pathway in a canine fibrosarcoma cell line (A-72 cells). Herein we investigated the role of Sirtuin and Forkhead box O (FOXO) families in this experimental model using Nortern Blot and Western Blot analysis. Our results demonstrated that mitochondrial SIRT3 and SIRT4 protein expression increased from 12 and 24 h post infection (p.i.) onwards, respectively, whereas the nuclear SIRT1 expression increased during the first 12 h p.i. followed by a decrease after 36 h p.i., reaching the same level of control at 48 h p.i. Sirtuins interact with/and regulate the activity of FOXO family proteins, and we herein observed that FOXO3A and FOXO1 expression increased significantly and stably from 12 h p.i. onwards. In addition, CCoV-II induces a remarkable increase in the expression of TNF-related apoptosis-inducing ligand (TRAIL), while we observed a slight up-regulation of FasL/Fas at 36 p.i. with a decrease of both proteins at the end of infection. Furthermore, we found that virus infection increased both bax translocation into mitochondria and decreased bcl-2 expression in cytosol in a time-dependent manner.These data suggest that FOXO transcription factors mediate pro-apoptotic effects of CCoV-II, in part due to activation of extrinsic apoptosis pathway, while some Sirtuin family members (such as SIRT3 and SIRT4) may be involved in intrinsic apoptotic pathway. Moreover, these results propose that TRAIL is an important mediator of cell death induced by CCoV-II during in vitro infection.

Basic science track. Entry and release of canine coronavirus from polarized epithelial cells.

Virus entry into and release from epithelial cells are polarized as a result of the distribution of the viral receptors. In order to establish the polarity of entry and release of CCoV from epithelial cells, the interactions of the virus with A72 and CrFK cells grown on permeable supports was evaluated, and the amount of infective virus in the apical and in the basolateral media was determined and compared. Infection of A72 cells after different times post seeding demonstrated that CCoV grow after infection from both apical and basolateral sides. In CrFK cells, CCoV was observed in both compartments only in the later phase of the infection. To establish the reciprocal binding of CCoV on plasma membrane, A72 cells on a permeable support were preincubated with a mAb specific for CCoV. Infection from the apical side was blocked by mAb applied to that side; in contrast, such treatment on the basolateral side had no effect on the infectious process. Similarly, the low levels of CCoV observed after basolateral exposure to virus was abolished following mAb treatment of that side. The identification of CCoV into the basolateral medium could play an important role in the viral pathogenesis.

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.

Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA.

In this study feline (FECV and FIPV) and canine (CCoV) coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virus entry appeared to occur preferentially through the apical membrane, similar to the entry of the related porcine coronavirus transmissible gastroenteritis virus (TGEV) into these cells. However, whereas TGEV is released apically, feline and canine coronaviruses were found to be released from the basolateral side of the epithelial cells. These observations indicate that local infections as caused by TGEV, FECV and CCoV do not strictly correlate with apical release, as suggested by earlier work.

Differences in virus receptor for type I and type II feline infectious peritonitis virus.

Feline infectious peritonitis viruses (FIPVs) are classified into type I and type II serogroups. Here, we report that feline aminopeptidase N (APN), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, is a receptor for type II FIPV but not for type I FIPV. A monoclonal antibody (MAb) R-G-4, which blocks infection of Felis catus whole fetus (fcwf-4) cells by type II FIPV, was obtained by immunizing mice with fcwf-4 cells which are highly susceptible to FIPV. This MAb also blocked infection of fcwf-4 cells by type II feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV). On the other hand, it did not block infection by type I FIPVs. MAb R-G-4 recognized a polypeptide of relative molecular mass 120-130 kDa in feline intestinal brush-border membrane (BBM) proteins. The polypeptide possessed aminopeptidase activity, and the first 15 N-terminal amino acid sequence was identical to that of the feline APN. Feline intestinal BBM proteins and the polypeptide reacted with MAb R-G-4 (feline APN) inhibited the infectivity of type II FIPV, type II FECV, CCV and TGEV to fcwf-4 cells, but did not inhibit the infectivity of type I FIPVs.