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Goose nephritic astrovirus (GNAstV) has recently been identified, which causes kidney swelling and visceral gout in goslings. However, the pathological changes in kidney tissue due to GNAstV infection have not yet been described. In the study, fifty goslings were orally infected with GNAstV, and fifty goslings received PBS as a control. Kidney tissue was collected at different days following infection (dpi) to assess the injury. GNAstV infection reduced body weight, increased the relative weight of the kidney, and increased serum uric acid and creatinine levels. GNAstV was found within renal epithelial cells, and the viral load in the kidney peaked at 7 dpi. Pale and swollen kidney tissue was observed in infected goslings, especially at 5 and 7 dpi. GNAstV infection caused degeneration and necrosis of renal epithelial cells, structural destruction of the brush border, glycogen deposition in the glomerular mesangium, increased fibrosis, and infiltration of inflammatory cells into the renal interstitium. Moreover, swollen mitochondria, broken mitochondrial ridges, autophagosomes, and autophagolysosomes were observed under ultrahistopathological examination. GNAstV infection increased levels of LC3B, ATG5, and Beclin 1, and decreased p62, and downregulated WT1 mRNA and upregulated desmin mRNA. At early stages, GNAstV infection decreased expression of intercellular junction-related genes, including ZO-1, occludin, claudin-10, and catenin-α2. In conclusion, GNAstV infection causes renal epithelial cell autophagy, destruction of brush border and intercellular junctions, podocyte damage, and increased fibrosis, ultimately resulting in damage to the kidney.

A new canine picornavirus (CanPV) variant, designated as dog/SH1901/CHN/2019, was detected in a pool of various canine fecal samples in the mainland of China using a viral metagenomic analysis, and its nearly complete genome sequence was determined and analyzed. Sequence analyses revealed that it had a standard picornavirus genome organization, a type I internal ribosome entry site (IRES) in the 5'UTR. However, dog/SH1901/CHN/2019 has generated a serial of unique aa mutations and 7aa insertion when compared with the closely related CanPVs. Phylogenetic analysis and pairwise sequence comparisons based on the P1, 2C, 3C, and 3D protein sequences showed that dog/SH1901/ CHN/2019 was closely related to CanPV strains 244 F, 325 F and 6D, which clustered into an independent evolutionary clade and distantly related to CanPV strain A128thr of the genus Mischivirus, which indicated the unclassified CanPV strains may belong to a novel species or genus in the family Picornaviridae. This study extends our knowledge on the evolution and genetic diversity of CanPVs.

A novel duck picornavirus, designated as duck/AH15/CHN/2015, was isolated and identified from Cherry Valley ducks with short beak and dwarfism syndrome in 2015 in Anhui province of China. Duck/AH15/CHN/2015 has the highest degree of amino acid sequence identity (approximately 43 %) with duck hepatitis A viruses (DHAV) Complete genome analysis revealed that duck/AH15/CHN/2015 possesses a typical picornavirus-like genomic organization, 5' UTR-L-P1 (VP0-VP3-VP1)-P2 (2A1-2A2- 2B-2C)-P3 (3A-3B-3C-3D)-3'UTR-poly (A). The 5'UTR contains a potential type IV internal ribosome entry site, while a conserved "barbell"-like structure is found at the 3'UTR, which is similar to DHAV. Compared to the closest related DHAVs, two unrelated 2A proteins were predicted in duck/AH15/CHN/2015, while three unrelated 2A proteins were presented in DHAVs. Based on the amino acid identity comparison and phylogenetic analysis of P1, 2C, and 3CD (3C and 3D), duck/AH15/CHN/2015 was closely related to but distinct from DHAVs, and it was proposed to be a member of a novel species in the genus Avihepatovirus of the family Picornaviridae.

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 μL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

Current studies have revealed the immune effects of graphene oxide (GO) and have utilized them as vaccine carriers and adjuvants. However, GO easily induces strong oxidative stress and inflammatory reaction at the site of injection. It is very necessary to develop an alternative adjuvant based on graphene oxide derivatives for improving immune responses and decreasing side effects. Carnosine (Car) is an outstanding and safe antioxidant. Herein, the feasibility and efficiency of ultrasmall graphene oxide decorated with carnosine as an alternative immune adjuvant were explored. OVA@GO-Car was prepared by simply mixing ovalbumin (OVA, a model antigen) with ultrasmall GO covalently modified with carnosine (GO-Car). We investigated the immunological properties of the GO-Car adjuvant in model mice. Results show that OVA@GO-Car can promote robust and durable OVA-specific antibody response, increase lymphocyte proliferation efficiency, and enhance CD4(+) T and CD8(+) T cell activation. The presence of Car in GO also probably contributes to enhancing the antigen-specific adaptive immune response through modulating the expression of some cytokines, including IL-6, CXCL1, CCL2, and CSF3. In addition, the safety of GO-Car as an adjuvant was evaluated comprehensively. No symptoms such as allergic response, inflammatory redness swelling, raised surface temperatures, physiological anomalies of blood, and remarkable weight changes were observed. Besides, after modification with carnosine, histological damages caused by GO-Car in lung, muscle, kidney, and spleen became weaken significantly. This study sufficiently suggest that GO-Car as a safe adjuvant can effectively enhance humoral and innate immune responses against antigens in vivo.

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48–72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro. Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

Cancer theranostics, the ability to simultaneously diagnose and treat cancer, has become one of the major driving forces in nanobiotechnology. In the present work, a multifunctional system, methylene blue-incorporated folate-functionalized Fe3O4/mesoporous silica core–shell magnetic nanoparticles (MNPs), for simultaneous near-infrared (NIR) fluorescence imaging and targeting photodynamic therapy (PDT) has been developed. The core Fe3O4 MNPs offers the function of magnetically guided drug delivery, the mesoporous silica shell acts as an efficient drug loaded carrier, the photosensitizer methylene blue (MB) exhibits excellent NIR fluorescence imaging and PDT efficiency, and the folic acid (FA) can effectively enhance the delivery of MB to the targeting cancer cells, which overexpress the folate receptor. The results indicated that the multifunctional system could effectively be used in NIR fluorescence imaging. Moreover, it exhibited a synergistic effect of magnetic targeted PDT of cancer under NIR laser irradiation. Thus, the multifunctional system is promising for simultaneous cancer diagnosis and therapy.

Peste des petits ruminants (PPR) is an acute and contagious disease of some small ruminants caused by peste des petits ruminants virus (PPRV). Fusion (F) protein and hemagglutinin (H) protein are two glycoproteins of PPRV that might induce a protective immune response. In this study, three replication-defective recombinant adenoviruses were constructed and the immunogenicity was evaluated in goats (the natural host). The recombinant adenoviruses (rAds) expressing F, H, and F-H fusion protein were named rAd-F, rAd-H, and rAd-F-H, respectively. In vitro, the proteins expressed in AAV-293 cells infected with different rAds were identified by Western blotting and immunofluorescence. The results showed that the proteins could be expressed in vitro. Three groups of goats (6 goats per group) were inoculated subcutaneously twice at 3-week intervals with the rAds. As negative controls, two additional groups were inoculated with wild-type adenovirus (wtAd) or PBS. In vivo, goats immunized with the rAds developed PPRV-specific virus neutralizing antibody (VNA) by 3 weeks after primary immunization. Moreover, the seroconversions were maintained for approximately 21 weeks after primary immunization. Stronger lymphocyte proliferation responses were induced in goats immunized with the three rAds than in the negative controls (P

VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni(2+) affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. coli and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity.

Autophagy is a devouring process during which cytoplasmic proteins, organelles, or other contents are phagocytized and delivered into an autophagosome, which then fuses with a lysosome to become an autolysosome. Autophagy is involved in various human diseases, including cancers, and is triggered and regulated by different signaling pathways under various stimuli such as oxidative stress. Oxidative stress is caused by excessive reactive oxygen species (ROS). It is well known that mitochondria are the primary cellular sources of ROS, which play important roles in the induction of cell death after radiation treatment. It is unknown whether ROS participate in autophagy regulation, and the underlying mechanism is still unclear. In this review, the authors focus on the association between mitochondrial stress and autophagic cell death. They also discuss the roles of ROS and the lysosomal-mitochondrial axis.

An unknown infectious disease in Cherry Valley ducks (Anas platyrhynchos domesticus) characterized by short beak and strong growth retardation occurred in China during 2015. The causative agent of this disease, tentatively named duck short beak and dwarfism syndrome (DSBDS), as well as the evolutionary relationships between this causative agent and all currently known avian-origin parvoviruses were clarified by virus isolation, transmission electron microscope (TEM) observation, analysis of nuclear acid type, (RT-)PCR identification, whole genome sequencing, and NS1 protein sequences-based phylogenetic analyses. The results indicated that the causative agent of DSBDS is closely related with the goose parvovirus-like virus, which is divergent from all currently known avian-origin parvoviruses and should be a novel duck parvovirus (NDPV).

We investigated the ability of the rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) to interact specifically with the minor structural protein VP10, using an in vivo cell-based CheckMate™ Mammalian Two-Hybrid System. RHDV VP60 protein interacted specifically with VP10. Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes. However, when VP60 was divided into two fragments, the interaction between VP60 and VP10 was impaired dramatically. These results will be helpful for further investigating the mechanism of RHDV particle assembly.

In this study, virus-like particles (VLPs) derived from rabbit hemorrhagic disease virus (RHDV) were evaluated for the development of a vaccine against RHDV infection. The VP60 gene was cloned and inserted into a pSMK expression vector containing a small ubiquitin-like modifier (SUMO) tag that can promote the soluble expression of heterologous proteins in Escherichia coli cells. After expression and purification of His-SUMO-VP60 and cleavage of the SUMO tag, we found that the RHDV VP60 protein had self-assembled into VLPs with a similar shape and smaller size compared with authentic RHDV capsid. Next, the antigenicity and immunogenicity of the VLPs were examined. The results showed that RHDV-specific responses were clearly induced in rabbits and that all rabbits in the VLP group survived while those in the negative control group died within 72 h post-infection. These results suggest that VLP-based RHDV could be a promising RHDV vaccine candidate.

Lysosome is a highly membrane-bound organelle which possesses a sequence of biological functions including protein degradation, cell signal transduction, plasma membrane repairment, homoeostasis, and autophagy. The lysosome contains more than 50 soluble acid hydrolases, and the acidification of lysosome is the most important biological characteristic. The integrity of lysosome is of vital importance. During the past few years, it was reported that the destabilization of lysosomal membrane can result in the release of lysosomal contents into cytosol and trigger cell death in a caspase-dependent or caspase-independent pathway. Lysosome functions at the late stage of autophagy and degrades cellular components delivered by autophagosome, which is a complicated process. The present article will summarize the current knowledge on the role of lysosome in cell death regulation and the underlying mechanisms.

Mitochondria as the critical powerhouse of eukaryotic cells play important roles in regulating cell survival or cell death. Under numerous stimuli, impaired mitochondria will generate massive reactive oxygen species (ROS) which participate in the regulation of vital signals and could even determine the fate of cancer cells. While the roles of mitochondria in radiation-induced autophagic cell death still need to be elucidated. Human cervical cancer cell line, Hela, was used, and the SOD2 silencing model (SOD2-Ri) was established by gene engineering. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assays, MitoTracker Green staining was used to detect mitochondrial mass, Western blot was used to detect protein expression, and the level of ROS, autophagy, and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. Ionizing radiation (IR) could induce the increase of MAPLC3-II/MAPLC3-I ratio, Beclin1 expression, and ROS generation but decrease the MMP in a time-dependent manner. After SOD2 silencing, the IR-induced changes of ROS and the MMP were significantly enhanced. Moreover, both the radio sensitivity and autophagy increased in SOD2-Ri cells. Whereas, compared with SOD2-Ri, the opposite results were obtained by NAC, an antioxidant. After the treatment with the inhibitor of mitochondrial electron-transport chain complex II, thenoyltrifluoroacetone (TTFA), the rate of autophagy, ROS, and the total cell death induced by IR increased. In addition, the decrease of MMP was more obvious. However, these results were reversed by cyclosporine A (CsA). IR could induce ROS generation and mitochondrial damage which lead to autophagic cell death in Hela cells.

Nowadays, heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, has become the target of antitumor drugs as a result of its close relationship with the occurrence and development, biological behavior, and prognosis of a tumor. Autophagy has attracted big attention recently for its paradoxical roles in cell survival and cell death, especially in the pathogenesis and treatment of cancer. Moreover, it has been verified that Hsp90 plays a role in autophagy via regulating the stability and activity of signaling proteins, and some Hsp90 inhibitors can induce autophagy. However, the underlying mechanisms for these important processes have not been clarified so far. In this study, we focus on the roles of Hsp90 in the regulation of autophagy, such as toll-like receptor (TLR)-mediated autophagy, Ulk1-mediated mitophagy, and chaperone-mediated autophagy (CMA). The roles of Hsp90 inhibitors in cancer therapy will also be elucidated.

The complete genome sequence of a novel duck orthoreovirus, designated DRV strain TH11(DRV-TH11), was determined and characterized. The DRV-TH11 genome is comprised of 23,417 bp and its genome organization is more similar to that of avian orthoreoviruses (ARVs) of chicken origin than other reoviruses. The results of comparative sequence analysis and dendrograms based on the µB- and σC-encoding genes indicated that TH11 may be derived from the reassortment of ARVs and classic Muscovy duck reovirus (MDRV). A possible recombinant event was identified using the SimPlot program, and it occurred in the M2 segment. The results indicated that reassortment and mutation play a role in the evolution of duck reovirus.

Nanotechnology has given scientists new tools for the development of advanced materials for the detection and diagnosis of various types of diseases. In particular, ultrasmall superparamagnetic iron oxides (USPIOs) have been investigated in many biological applications, both in vitro and in vivo. Due to their small size (diameter < 20 nm), these particles are not immediately removed from the circulation by the reticuloendothelial system (RES), have a longer blood half-life, a wider biodistribution and allow potential targeting with appropriate bioconjugates to specific tissues both normal and tumorous. This review will mainly discuss the synthesis of USPIOs and their applications as MRI contrast agent for disease detection.

The goose parvovirus (GPV) Rep1 protein is both essential for viral replication and a potential target for GPV diagnosis, but its protein characterization and intracellular localization is not clear. We constructed a recombinant plasmid, pET28a/GPV-Rep1, and expressed the Rep1 gene in BL21 (DE3) Escherichia coli. A protein approximately 75 kDa in size was obtained from lysates of E. coli cells expressing the recombinant plasmid. SDS-PAGE analysis showed that after induction with 0.6 mM isopropyl β-D-thiogalactosidase (IPTG) at 30°C for 5 h, the Rep1 protein was highly overexpressed. Two methods used to purify proteins, a salinity-gradient elution and Ni-NTA affinity chromatography, were performed. The amount of Rep1 protein obtained by Ni-NTA affinity chromatography was 41.23 mg, while 119.9 mg of Rep1 protein was obtained by a salinity-gradient elution from a 1 L E. coli BL21 (DE3) culture. An immunogenicity analysis showed that the protein could significantly elicit a specific antibody response in immunized goslings compared to control groups. Antibody titers peaked to 1:5120 (optical density (OD) 450 = 3.9) on day 28 after immunization but had mean titers of 1:10,240 (OD450 = 4.2) in gosling groups immunized with a commercially available GPV-attenuated vaccine strain. Experiments examining subcellular localization showed that the Rep1 protein appeared to associate predominantly with the nuclear membrane, especially during later times of infection. This work provides a basis for biochemical and structural studies on the GPV Rep1 protein.

Until recently, there was no cell line that could produce continuously high-titer duck hepatitis virus type 1 (DHV-1). In this study, a duck embryo fibroblast (DEF) cell line was established, and the susceptibility of this cell line to DHV-1 was determined. The primary culture of DEF cells was from a duck embryo that was partially digested with trypsin. Digested tissue pieces were cultured at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. The cultured DEF cells, which had the morphology of fibroblast, proliferated to 100% confluence four days later. An immortalized DEF cell line, named DEF-TA, was established and subcultured to passage 33, and the susceptibility of that cell line to DHV-1 was determined. In the DHV-1 susceptibility tests, cytopathic effects and the propagation of virus were observed in DEF-TA cells after DHV-1 infection. This continuous DHV-1-susceptible DEF cell line may serve as a valuable cell line for studies of cell-virus interactions and the pathogenesis of DHV-1 and may be useful for the development of an inactivated vaccine.