BackgroundBiomarkers for the early prediction of canine acute kidney injury (AKI) are clinically important. Recently, neutrophil gelatinase-associated lipocalin (NGAL) was found to be a sensitive biomarker for the prediction of human AKI at a very early stage and the development of AKI after surgery. However, NGAL has not yet been studied with respect to dog kidney diseases. The application of NGAL canine AKI was investigated in this study.ResultsThe canine NGAL gene was successfully cloned and expressed. Polyclonal antibodies against canine NGAL were generated and used to develop an ELISA for measuring NGAL protein in serum and urine samples that were collected from 39 dogs at different time points after surgery.AKI was defined by the standard method, namely a serum creatinine increase of greater than or equal to 26.5 μmol/L from baseline within 48 h. At 12 h after surgery, compared to the group without AKI (12 dogs), the NGAL level in the urine of seven dogs with AKI was significantly increased (median 178.4 pg/mL vs. 88.0 pg/mL), and this difference was sustained to 72 h.ConclusionAs the increase in NGAL occurred much earlier than the increase in serum creatinine, urine NGAL seems to be able to serve as a sensitive and specific biomarker for the prediction of AKI in dogs.

An association between Epstein-Barr virus (EBV) infection and systemic lupus erythematosus (SLE) has been suggested from previous serologic evidence. Since most adults in Taiwan are EBV-infected, seroepidemiologic studies based on standard assays for EBV are unlikely to dissociate SLE patients and control groups. We reexamine this question by using novel methodologies in which IgA anti-EBV-coded nuclear antigens-1 (EBNA-1) and IgG anti-EBV DNase antibodies were analysed by ELISA, and EBV viral loads were detected by real-time quantitative PCR for 93 adult SLE patients and 370 age-, sex- and living place-matched healthy controls in Taiwan. The specificities of antibodies for extractible nuclear antigens were determined by Western blot. Our results show that IgA anti-EBV EBNA1 antibodies were detectable in 31.2% SLE patients but only in 4.1% of controls (odds ratio [OR] = 10.72, 95% confidence interval [CI] = 5.19-22.35; P < 10(-7)), IgG anti-EBV DNase antibodies were detected in 53.8% SLE patients but only in 12.2% controls (OR = 8.40, 95% CI = 4.87-14.51; P < 10(-7)). EBV DNA was amplifiable from the sera of 41.9% SLE patients but from only 3.24% controls (P < 0.05). A significant association of IgG anti-EBV DNase antibodies with anti-Sm/RNP antibodies was observed (P < 0.005). The higher seroreactivity and higher copy numbers of EBV genome indicated association of EBV infection with SLE in Taiwan.

Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2␣ subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCEThe OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. Orf virus (ORFV), a member of the Parapoxvirus genus and the Poxviridae family, is the causative agent of contagious ecthyma in sheep, goats, and other ruminants. The disease is characterized by the development of pustular lesions around the nostrils and mouth with a high incidence rate and a low mortality rate in healthy adult animals. In contrast, infection in immunosuppressed animals or in lambs may be fatal (1). ORFV is also of concern as a source of zoonotic infection because it can cause cutaneous lesions in humans in contact with infected animals. Persistent infection with ORFV can be observed in goats and sheep, and while the severity of lesions is reduced compared with that seen in primary infection, this persistence suggests that the virus is able to evade host immunity (2-4). In line with this observation, ORFV has been shown to encode several proteins that modulate the host response to infection. These include viral homologues of ovine cytokines, such as vascular endothelial growth factor, interleukin-10 (IL-10), and a granulocyte-macrophage colony-stimulating factor (GM-CSF)-inhibiting protein, as well as an apoptosis inhibitor (5-7). ORFV also antagonizes interferon (IFN) signaling, and this is done by the product...

BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a useful biomarker for the early prediction of renal diseases. NGAL may exist as monomer, dimer and/or NGAL/MMP-9 complex forms in humans. In this study, the existence of various forms of NGAL in urine (uNGAL) was determined and whether these forms are related to the different urinary diseases found in dogs is further discussed.ResultsEighty-one urine samples from dogs with different forms of renal disease (41), pyuria (19) and a number of non-renal related diseases (10), as well as healthy dogs (11), were collected. uNGAL concentrations and their molecular forms in dogs were measured by ELISA and Western blot analysis, respectively. The uNGAL concentrations of dogs with pyuria (median: 15.35 ng/mL) were significantly higher than those of the healthy control animals (median: 3.92 ng/mL) (p < 0.01), but lower than those of dogs with renal diseases (median: 23.77 ng/mL). Each NGAL molecular form could be detected in dog urine. In particular, monomer was detected more frequently in patients with renal disease than those with non-renal diseases; while the dimer form appeared in a significantly higher percentage of cases with pyuria compared to those without pyuria. The NGAL/MMP-9 complex was found to exist not only in the patients with cystitis, but also in the cases with renal injury.ConclusionDifferent molecular forms of uNGAL can indicate different origins of the urinary abnormalities. Determining the molecular forms of uNGAL present in diseased dogs may provide clinical workers with a tool that will help the early and more precise detection of different urinary diseases.

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BackgroundCanine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions.ResultsSequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains.ConclusionsThe method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

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16Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral 17 protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA 18 (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a 19 direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR 20 activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, 21we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 22 3 interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts 34 with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. 35The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. 36The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-37to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 38 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to 39 the stability of the genetic code and the structure of RNA, these observations suggest that in addition 40 to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to 41 combat antiviral responses or achieve efficient viral infection. 42 43 44 4 7Previous reports revealed that OV20.0 acts as a PKR antagonist by targeting its activators (5, 6). In 102 this study, we discovered a novel interaction of OV20.0 with ADAR1 and investigated its 103 consequences for ORFV replication. Initially, the association of OV20.0 and ADAR1 was 104 investigated. Subsequently, a series of deletion constructs of OV20.0 and ADAR1 were made to 105 map the domains involved in the interaction between these two proteins. Moreover, whether 106 OV20.0 interferes with the RNA-editing activity of the ADAR1 enzyme, and the effects of ADAR1 107 on PKR activation were investigated. Since OV20.0 can associate with both PKR and ADAR1, the 108 hierarchy of these associations was also examined. Finally, the impact of ADAR1 on ORFV 109 infection was explored for the first time. 110 111 Materials and methods 112 Cell culture. Human embryonic kidney cell line 293T (HEK 293T), human lung carcinoma A549 113 cells, and primary goat fibroblasts were propagated in Dulbecco's modified Eagle's medium 114 (DMEM; Gibco BRL, Life Technologies Corporation Carlsbad, CA, USA) with 10% fetal bovine 115 serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Gibco BRL). All cells 116 were incubated at 37 o C with 5% CO2. 117 Virus and infection. The recombinant ORFV expressing enhanced green fluorescent protein 118 (eGFP) was described previously (5). For infection, cells at 80% confluency were washed with 119 containing target sequence of ADAR1, i.e. sense: 5'-GGACAGGA GACGGAATTCGC-3' was 131 introduced into the sites of BsmbI of lentiCRISPR v2-mCherry. HEK 293T cells were transfect...