Diverse amdoparvoviruses infection of farmed Asian badgers (Meles meles).

Amdoparvoviruses infect various carnivores, including mustelids, canids, skunks, and felids. Aleutian mink disease virus (AMDV) belongs to the prototypical species Amdoparvovirus carnivoran1. Here, we identified a novel amdoparvovirus in farmed Asian badgers (Meles meles), and we named this virus "Meles meles amdoparvovirus" (MMADV). A total of 146 clinical samples were collected from 134 individual badgers, and 30.6% (41/134) of the sampled badgers tested positive for amdoparvovirus by PCR. Viral DNA was detected in feces, blood, spleen, liver, lung, and adipose tissue from these animals. Viral sequences from eight samples were determined, five of which represented nearly full-length genome sequences (4,237-4,265 nt). Six serum samples tested positive by PCR, CIEP, and IAT, four of which had high antibody titers (> 512) against AMDV-G. Twenty-six of the 41 amdoparvovirus-positive badgers showed signs of illness, and necropsy revealed lesions in their organs. Sequence comparisons and phylogenetic analysis of the viral NS1 and VP2 genes of these badger amdoparvoviruses showed that their NS1 proteins shared 62.6%-88.8% sequence identity with known amdoparvoviruses, and they clustered phylogenetically into two related clades. The VP2 proteins shared 76.6%-97.2% identity and clustered into two clades, one of which included raccoon dog and arctic fox amdoparvovirus (RFAV), and the other of which did not include other known amdoparvoviruses. According to the NS1-protein-based criterion for parvovirus species demarcation, the MMADV isolate from farm YS should be classified as a member of a new species of the genus Amdoparvovirus. In summary, we have discovered a novel MMADV and other badger amdoparvoviruses that naturally infect Asian badgers and are possibly pathogenic in badgers.

First identification of canine adenovirus 1 in mink and bioinformatics analysis of its 100 K protein.

Introduction: Animal trade favors the spreading of emerging canine adenovirus 1 (CAdV-1) in mink. Because the 100K protein is not exposed to the viral surface at any stage, it can be used to differentiate the vaccine from wild virus infection. However, no related research has been conducted. This study aimed to find evidence of CAdV-1 in mink and predict the character of the 100K protein in the current circulating CAdV-1 strain of mink. Method: In this experiment, the identification of CAdV-1, the phylogenetic tree, homology, and bioinformatics analysis of 100K were conducted. Results: The results showed that the CAdV-1 was identified in the mink and that its Fiber was located in a separate branch. It was closely related to strains isolated from Norwegian Arctic fox and Red fox. 100K was located in a separate branch, which had the closest genetic relationship with skunks, porcupines, raccoons, and hedgehogs and a far genetic relationship with the strains in dogs. 100K protein is an unstable and hydrophobic protein. It had evidence of selective pressure and recombination, 1 glycosylation site, 48 phosphorylation sites, 60 dominant B cell epitopes, and 9 peptides of MHC-I and MHC-II. Its subcellular localization was mainly in the endoplasmic reticulum and mitochondria. The binding sites of 100K proteins were DBP proteins and 33K proteins. Discussion: The stains in the mink were different from fox. The exploration of its genomic characteristics will provide us with a deeper understanding of the prevention of canine adenovirus.

Cell adhesion function was altered during the seasonal regression of the seminiferous epithelium in the mink species Neovison vison.

Minks are seasonal breeders whose seminiferous epithelium undergoes regression through massive germ cell death, leaving only Sertoli cells and spermatogonial cells in the tubules. However, the molecular mechanisms that control this biological process remain largely unknown. This study describes a transcriptomic analysis of mink testes at various reproductive stages (active, regressing, and inactive). A comparison of seminiferous epithelium at different stages of reproduction shows that cell adhesion is altered during regression. In addition, genes and proteins involved in forming the blood-testis barrier (BTB) were examined in sexually active and inactive minks. The seminiferous epithelium in the testes of sexually inactive minks expressed occludin, but this expression was not discernibly observed in the testes of sexually active minks. There was no discernible expression of CX43 in the seminiferous epithelium in the testes of sexually inactive minks, but CX43 was expressed in the testes of sexually active minks. During the regression process, we observed a remarkable increase in the expression levels of Claudin-11, which is associated with Sertoli-germ cell junctions. In conclusion, these findings suggest a loss of Sertoli-germ cell adhesion, which may regulate postmeiotic cell shedding during testicular regression in mink.

Here, we report for the first time the molecular mechanisms of testicular regression in mink. Our results, together with studies on other animals' characteristic reproductive features, identify a cluster of events crucial to the seminiferous epithelium regression process in mammalian seasonal breeders and highlight perspectives unique to the mink.

Immunological evaluation of recombination PRRSV GP3 and GP5 DNA vaccines in vivo.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a threat to the health of pigs worldwide, but commercially available vaccines offer limited protection against PRRSV infection. It is necessary to develop a more effective DNA vaccine. The immunological effects of DNA vaccines with three adjuvants were examined in pigs (Susscrofa domestica) challenged with PRRSV. These DNA vaccines, which encoded PRRSV GP3 and GP5, were formulated with A1, A2, and A3. Serum specific and neutralizing antibodies, IL-4, IFN-γ, IL-2, IL-10, CD4+ and CD8+T-lymphocytes, health status, histopathology, and viral loads were determined. The results showed that the use of adjuvant A3 led to higher levels of neutralizing antibodies and a lower viral load in pigs compared to the other adjuvants. The neutralizing antibody titers of the pVAX-GP35+A1 and pVAX-GP35+A3 groups reached a peak of 1:19 at 35 dpi. The maximum concentration of IL-4 was 136.77 pg/mL in the pVAX-GP35+A3 group. At 35 dpi, the IFN-γ concentration in the pVAX-GP35+A1 group was 227.4 pg/mL. pVAX-GP35+A3 group shows the highest IL-2 and IL-10 expression to the peak of 597.6 pg/mL and 189.1 pg/mL, respectively. We found a formulation demonstrated beneficial immune outcomes. This study provides an alternative vaccine to protect pigs from PRRSV.

Immunomodulatory Effect of Ginsenoside Rb2 Against Cyclophosphamide-Induced Immunosuppression in Mice.

Ginsenoside Rb2 (Rb2), a fundamental saponin produced and isolated from ginseng (Panax ginseng C.A. Meyer), has a wide range of biological actions. The objective of this investigation was to see if ginsenoside Rb2 has any immunomodulatory properties against cyclophosphamide (CTX)-induced immunosuppression. For the positive control group, levamisole hydrochloride (LD) was used. We discovered that intraperitoneal injection of Rb2 (5, 10, 20 mg/kg) could relieve CTX-induced immunosuppression by enhanced immune organ index, reduced the pathological characteristics of immunosuppression, promoted natural killer (NK) cells viability, improved cell-mediated immune response, boosted the IFN-γ (Interferon-gamma), TNF-α (Tumor necrosis factor-alpha), IL-2 (Interleukin-2), and IgG (Immunoglobulin G), as well as macrophage activity like carbon clearance and phagocytic index. Rb2 significantly elevated the mRNA expression of IL-4 (Interleukin-4), SYK (Tyrosine-protein kinase-SYK), IL-2, TNF-α, and IL-6 (Interleukin-6) in the spleen of CTX-injected animals. Molecular docking results showed that Rb2 had excellent binding properties with IL-4, SYK, IL-2, TNF, and IL-6, indicating the target protein might be strongly correlated with the immunomodulatory effect of Rb2. Taken together, ginsenoside Rb2 can improve the immune function that is declined in CTX-induced immunosuppressed mice, the efficacy maybe due to the regulation of related cytokine and mRNA expression.

Canine Adenovirus 1 Isolation Bioinformatics Analysis of the Fiber.

Canine adenovirus type 1 (CAdV-1) is a double-stranded DNA virus, which is the causative agent of fox encephalitis. The Fiber protein is one of the structural proteins in CAdV-1, which mediates virion binding to the coxsackievirus and adenovirus receptor on host cells. The suspected virus was cultured in the MDCK cells, and it was determined through the cytopathic effects, sequencing and electron microscopy. The informatics analysis of the Fiber was done using online bioinformatics servers. The CAdV-1-JL2021 strain was isolated successfully, and were most similar to the CAdV-1 strain circulating in Italy. The occurrence of negative selection and recombination were found in the CAdV-1-JL2021 and CAdV-2-AC_000020.1. Host cell membrane was its subcellular localization. The CAdV-1-JL2021 Fiber (ON164651) had 6 glycosylation sites and 107 phosphorylation sites, exerted adhesion receptor-mediated virion attachment to host cell, which was the same as CAdV-2-AC_000020.1 Fiber. The Fiber tertiary structure of the CAdV-1-JL2021 and CAdV-2-AC_000020.1 was different, but they had the same coxsackievirus and adenovirus receptor. "VATTSPTLTFAYPLIKNNNH" were predicted to be the potential CAdV-1 B cell linear epitope. The MHC-I binding peptide "KLGVKPTTY" were both presented in the CAdV-1-JL2021 and CAdV-2-AC_000020.1 Fiber and it is useful to design the canine adenovirus vaccine.

Difference Analysis Between Canine Adenovirus Types 1 And 2.

Canine adenoviruses (CAdVs) include type 1 (CAdV-1, virulent strain) and type 2 (CAdV-2, attenuated strain). In recent years, the incidences of CAdV infections are increasing. However, they are difficult to distinguish when the symptoms are untypical. It is pivotal to find the differences between the two virus types for scientific, epidemiological, and specific treatment. CAdV-1 (virulent strain) and CAdV-2 (attenuated strain) induced canine hepatitis (ICH) and tracheobronchitis (ITB), respectively, but the clinical symptom is not obvious. CAdV-1 and CAdV-2 have the same genome structure, diameter, morphological features, and cytopathic features, but the same character hinder the diagnose time of the serotypes. CAdV-1 and CAdV-2 have a difference in the genome sequence, coding proteins, viral activity, hemagglutination patterns. After infection, pathogenicity and transmission route are different between the two serotypes. Sequence alignment, PCR, Real time-PCR assay are useful methods to distinguish the two serotypes. The attenuated live CAdV-2 vaccine is currently used to protect against CAdV-1, but it also has a risk. The further research should focus on the pathogenicity mechanism and the useful vaccine for the two serotypes of canine adenovirus.

Immunogenicity of an Inactivated Canine Adenovirus Type 1 Vaccine for Foxes.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis. As with most viral agents, the best method of prevention is vaccination. In this study, the CAdV-1 strain F1301 strain was used to construct a new type 1 canine adenovirus inactivated vaccine candidate, and its safety and immunogenicity were evaluated in silver foxes. Next, animals were challenged and survival rates of animals vaccinated with either the commercially available or the current candidate vaccine were examined. The results confirmed that the inactivated CAdV-1 vaccine prepared in this study can effectively protect against challenge with virulent CAdV-1 in silver foxes, and the safety profile was improved relative to that of the commercial vaccine. This study confirmed that the fox CAdV-1 F1301 strain can be used as a platform for an inactivated CAdV-1 vaccine.

Identification and primary application of hybridomas cell secreting monoclonal antibodies against mink (Neovison vison) interferon-gamma.

Due to their susceptibility to several human viruses, the mink has been proposed as potential animal models for the study of human viral infections. However, there are no specific monoclonal antibody (mAbs) currently available for the detection of mink-specific interferon-gamma (miIFN-γ). The BALB/c mice were immunized intraperitoneally with purified recombinant miIFN-γ protein. The splenocytes were obtained and fused with murine myeloma cells. Five of 24 hybridoma clones were obtained to produce mAbs steadily with the strongest affinity to recombinant miIFN-γ protein. The isotype of the 31A, 31B and 31G were lgG 2b. The isotype of 44 and 46 were lgG 2a and 1. All five mAbs were κ light chains. Western blotting and indirect ELISA method showed that 5 mAbs were positive to miIFN-γ. Immunofluorescence showed that 2 mAbs (44 and 46) had a positive reaction to miIFN-γ. The hybridoma clone 46 had the highest sensitivity for the detection of miIFN-γ. Most importantly, our primary sandwich ELISA system (mAbs 46 and polyclonal antiserum) detected endogenous IFN-γ in mink lymphocytes infected with canine distemper virus (CDV). We have thus developed a novel mAbs could recognize miIFN-γ, and have demonstrated the first ELISA-based measurement of IFN-γ in lymphocyte of the mink.

Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies.

Canine adenovirus (CAdV) has a high prevalence in canine populations. High affinity neutralizing antibodies against conserved epitopes can provide protective immunity against CAdV and protect against future outbreaks. In this study, we identified two CAdV-2-specific neutralizing monoclonal antibodies (mAbs), 2C1 and 7D7, which recognized two linear-dependent epitopes. MAb 2C1 potently neutralized CAdV-2 with a 50% neutralization titer (NT50) of 4096, and mAb 7D7 partially neutralized CAdV-2 with a 50% NT50 of 64. Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing mAbs recognized the hexon protein (Hex) of CAdV-2. Through a 12-mer random peptide phage display and synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: 634RIKQRETPAL643 located on the surface region; and 736PESYKDRMYS745 located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid detection of all CAdVs.

The role of TGF-ß1/Smad3 signaling pathway and oxidative stress in the inhibition of osteoblast mineralization by copper chloride.

To explore the relationship of oxidative stress and TGF-ß 1/Smad3 pathway in the inhibition of osteoblast mineralization by copper chloride (CuCl2), the osteoblasts were treated with CuCl2 (0, 50 µM, 100 µM, 150 µM CuCl2 5H2O) for 24 h. We found that Cu impaired the osteoblast structure, inhibited the glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, alkaline phosphatase (ALP) content, mRNA expression of collagen I (COL-I), osteocalcin (OCN), insulin-like growth factor I (IGF-I), bone morphogenetic protein-2 (BMP-2), transforming growth factor ß1 (TGF-ß1) and core-binding factor α1 (Cbfα1), promoted the reactive oxygen species (ROS) production, inactivated the TGF-ß1/Smad3 pathway. It indicates that the inactivated TGF-ß1/Smad3 pathway leads to osteoblast impairment by CuCl2. It will contribute to clarify the influence of CuCl2 on the osteoblast mineralization.

Pathogenicity comparison of the SMPV-11 and attenuated mink enteritis virus F61 in mink.

Mink enteritis virus (MEV) is a major pathogen inducing acute hemorrhagic enteritis in mink. This study aims to determine the pathogenicity of the isolated MEV strain (SMPV-11) compared with the attenuated MEV strain (MEV-F61) in the mink. The two MEV strains were inoculated in the two mink groups, respectively. Then the clinical symptom, hematological, serological, and histopathological change were evaluated. Our findings showed that there were differences in the clinical features and pathological changes of the SMPV-11 and MEV-F61 in the mink. It indicates that SMPV-11 is a virulent strain, and it can be the potential MEV vaccine strain in the mink.

The biological characteristics of the canine adenovirus type 1 from fox and the transcriptome analysis of the infected MDCK cell.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related research are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30,535 bp and had higher homology to the other reported CAdV-1 strains. After 0, 12, and 36 h of CAdV-1 infection, the difference gene of the 592 long noncoding RNA and 11,215 microRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, Wnt, Herpes simplex, hepatitis C, and Epstein-Barr virus infection pathway in Madin-Darby canine kidney cell line (MDCK). The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified.

Cytoprotective effect of deferiprone against aluminum chloride-induced oxidative stress and apoptosis in lymphocytes.

Aluminum (Al) is a toxic metal, and excessive Al accumulation causes immunosuppression. Deferiprone (DFP) is a well-known chelator and used in dialysis patients for removing Al from tissues. The present study aimed to investigate whether DFP treatment can attenuate immunotoxicity induced by aluminum chloride (AlCl3) in cultured lymphocytes. Lymphocytes were treated with 0 and 0.6 mmol/L AlCl3∙6H2O (pH 7.2) and/or 1.8 mmol/L DFP, respectively. Immune function of lymphocytes was assessed by T and B lymphocytes proliferation rates, T lymphocyte subpopulations and IL-2, IL-6 and TNF-α contents. In addition, lymphocyte damage was assessed by LDH activity, NO and MDA contents, NOS, SOD and GSH-Px activities, lymphocyte apoptosis index. These results showed that AlCl3 exposure reduced T and B lymphocyte proliferation rates, CD3+ and CD4+ T lymphocyte subpopulations, CD4+/CD8+ ratio, IL-2, IL-6 and TNF-α contents, SOD and GSH-Px activities, early and later lymphocyte apoptosis indexes while enhanced CD8+ T lymphocyte subpopulation, NO and MDA contents, LDH activity. DFP treatment attenuated the immunotoxicity of lymphocytes and reduced oxidative stress and lymphocyte apoptosis induced by AlCl3, indicating that DFP could protect lymphocytes against immunosuppression induced by AlCl3 through attenuating oxidative stress and apoptosis.

Aluminum Chloride Causes the Dysfunction of Testes Through Inhibiting the ATPase Enzyme Activities and Gonadotropin Receptor Expression in Rats.

The aim of this experiment is to explore the effects of aluminum chloride (AlCl3) on the ATPase enzymes and gonadotropin receptors in the testes. Eighty male Wistar rats were orally exposed to 0 mg/kg body weight (BW) (control group, CG), 64 mg/kg BW (low-dose group, LG), 128 mg/kg BW (mid-dose group, MG), or 256 mg/kg BW (high-dose group, HG) for 120 days. The microstructure and ultrastructure of testes; the activities of Na+-K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase; and the mRNA and protein expressions of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptors (LHR) in the testes were examined. The results showed that the testes histological structure were damaged; the activities of Na+-K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase, the mRNA and protein expressions of FSHR and LHR in the testes were all decreased in the rats with AlCl3 exposure. It indicates that AlCl3 causes the dysfunction of testes in rats.

Aluminum chloride caused liver dysfunction and mitochondrial energy metabolism disorder in rat.

Aluminum (Al) is known to exert hepatotoxicity. However, the mechanisms mostly are unclear. Liver is a metabolism organ that maintains the energy level and structural stability of body, mitochondria are the main sites of energy metabolism, thus, we hypothesized that mitochondrial energy metabolism disorder contributes to liver dysfunction in aluminum chloride (AlCl3) treatment rat. To verify the hypothesis, forty male Wistar rats were randomly allocated and orally exposed to 0, 64mg/kg, 128mg/kg and 256mg/kg body weight AlCl3 in drinking water for 120days, respectively. We found that AlCl3 exposure reduced the electron transport chain complexes I-V activities and adenosine triphosphate (ATP) level, as well as disturbed mitochondrial DNA transcript, presenting as the inhibited mRNA expressions of NADH dehydrogenase 1, NADH dehydrogenase 2, cytochrome b, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 3 and ATP synthase 6, indicating that AlCl3 exposure disturbs the mitochondrial energy metabolism, and it caused an increase in liver enzymes (Aspartate aminotransferase and Alanine aminotransferase) and histopathological lesions. Additionally, we found that reactive oxygen species accumulation and decreased superoxide dismutase activity in mitochondria, and increased 8-Hydroxydeoxyguanosine levels in mitochondrial DNA, demonstrating AlCl3 exposure promotes mitochondrial oxidative stress, which may be a contributing factor to mitochondrial energy metabolism disorder and liver dysfunction. The study displayed that mitochondria are the potential target of liver damage induced by AlCl3, providing considerable direction for the prevention and clinical intervention of liver diseases.