Development and Evaluation of NanoPCR for the Detection of Goose Parvovirus.

Gosling plague (GP) is an acute and hemorrhagic infectious disease caused by goose parvovirus (GPV). The goose industry suffers significant economic losses as a result of GP, which is found to be widespread worldwide, with high rates of morbidity and mortality. Our group developed a novel technique for detecting GPV nanoparticle-assisted polymerase chain reaction (nanoPCR) and the characterization of its specificity and sensitivity. It was developed by using the traditional polymerase chain reaction (PCR) and nanoparticles. The findings of this study revealed that GPV nanoPCR products were 389 bp in length, and the lower limit of the nanoPCR assay was 4.68 × 102 copies/µL, whereas that of the conventional PCR assay was 4.68 × 104 copies/µL. A total of 230 geese suspected of GPV were detected using nanoPCR, with a positive rate of 83.0% and a specificity of 73%, respectively. Overall, we present a hitherto undocumented method for identifying GPV by using nanoPCR to aid in the evaluation of subclinical illness.

Identification of an IRES within the coding region of the structural protein of human rhinovirus 16.

As an alternative mechanism for cap-dependent (m7GpppN) translation, internal ribosome entry site (IRES)-dependent translation has been observed in the 5' untranslated regions (5' UTR) and coding regions of a number of viral and eukaryotic mRNAs. In this study, a series of 5' terminal truncated structural protein genes that were fused with GFP was used to screen for potential IRESs, and IRESs were identified using a bicistronic luciferase vector or GFP expression vector possessing a hairpin structure. Our results revealed that a putative IRES was located between nt 1982 and 2281 in the VP3 coding region of the human rhinovirus 16 (HRV16) genomes. We also demonstrated that effective IRES-initiated protein expression in vitro did not occur through splicing sites or cryptic promoters. We confirmed that thapsigargin (TG), an inducer of endoplasmic reticulum stress (ERS), facilitated increased IRES activity in a dose-dependent manner. Additionally, the secondary structure of the IRES was predicted online using the RNAfold web server.

The first detection and characterization of goose parvovirus (GPV) in Turkey.

Derzsy's disease, which is seen in goslings (Anser anser domestica) and Muscovy ducks (Cairina moschata), progresses to high mortality and causes significant yield losses. The disease agent is goose parvovirus (GPV), which is common in countries with waterfowl production. It has not previously been reported in Turkey. Using qPCR and sequencing of the VP3 protein-encoding gene, GPV is identified as the causative agent of high mortality among geese between 2018 and 2019. The VP3 sequences were also compared with the similar GenBank sequences phylogenetically. All the sequences were found to be most similar (98.90%) with Polish and Taiwan GPV strains. Phylogenetic analysis of the VP3 gene in strains in Turkey and comparison with strains from other countries demonstrated that the Turkish strains are native to the geography and circulated locally. This study detected the presence of the GPV gene for the first time in Turkey and demonstrated the importance of comparing the vaccine strain and wild type.

Application of high-resolution melting curve analysis for identification of Muscovy duck parvovirus and goose parvovirus.

This study reports the findings of a high-resolution melting (HRM) curve analysis combined with PCR technique (PCR-HRM) to differentiate between Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV). A degenerate primer set was designed based on the VP3 gene of MDPV and GPV. The PCR HRM assay was able to discriminate between MDPVs and GPVs by differences in melting curve shapes and melting temperatures. A total of forty-five clinical samples, passaged in the allantoic cavity of Muscovy duck eggs, were detected by the PCR-HRM assay. Among the 12 positive samples, two were identified as MDPV and two as GPV with high genotype confidence percentage (GCP) values. Seven positive samples had low GCP values for the melting curve analysis and were identified as co-infection samples. One of the 12 positive samples, designed GDNX strain, was identified as a variant strain with a divergent melting curve profile. To assess the capability of PCR-HRM assay to distinguish MDPV and GPV, fifty-two field samples were collected and examined. Seven samples were positive for MDPV and/or GPV. Thus, this developed assay was useful for discrimination of MDPVs and GPVs and can also be suitable for detecting co-infection samples.

Molecular Characterization and Comparative Pathogenicity of Goose Parvovirus Isolated from Jilin Province, Northeast China.

Goose parvovirus (GPV) is a highly contagious disease caused by GPV in goslings and young Muscovy ducklings. In recent years, frequent GPV outbreaks have occurred in many regions of Jilin Province, China. In this study, to understand the immune-related characteristics of the currently prevailing GPV strains in some regions of Jilin Province, six GPV strains were isolated from six different regions of Jilin Province in 2016-2018. The results of phylogenetic analysis, clinical signs, and histopathologic analysis showed that four strains were virulent and two strains were attenuated. Specifically, we found that the two attenuated strains have the same amino acid mutation at the same position in the virus protein 3 (VP3) gene, and the virulent strains have more mutation sites than the attenuated strains. This finding suggests that changes in these sites may result in GPV replication or reduction in the immune response in goslings, thereby producing strong pathogenicity, and that attenuated strains are more conservative than virulent strains.

Caracterización molecular y patogenicidad comparativa de parvovirus de ganso aislados en la provincia de Jilin, noreste de China. El parvovirus del ganso (GPV, por sus siglas en inglés) es una enfermedad altamente contagiosa causada por el parvovirus de ganso en gansitos y patitos reales jóvenes. En los últimos años, se han presentado brotes frecuentes por el parvovirus del ganso en muchas regiones de la provincia de Jilin, en China. En este estudio, para comprender las características relacionadas con el sistema inmunológico de las cepas del parvovirus del ganso prevalentes actualmente en algunas regiones de la provincia de Jilin, se aislaron seis cepas de parvovirus del ganso de seis regiones diferentes de la provincia de Jilin entre los años 2016 al 2018. Los resultados del análisis filogenético, los signos clínicos y el análisis histopatológico mostraron que las cuatro cepas fueron virulentas y dos fueron atenuadas. Específicamente, se encontró que las dos cepas atenuadas tienen la misma mutación de aminoácidos en la misma posición en el gene de la proteína viral 3 (VP3) y las cepas virulentas tienen más sitios de mutación que las cepas atenuadas. Este hallazgo sugiere que los cambios en estos sitios pueden resultar en la replicación o reducción de la respuesta inmune en los gansitos, lo que induce una fuerte patogenicidad y que las cepas atenuadas son más conservadas que las virulentas.

Evaluation of chemotherapy with nanosomal paclitaxel and gene therapy expressing apoptosis-inducing proteins in the management of spontaneous canine mammary neoplasm.

Mammary gland tumours are the second most common neoplasm representing about 40-50% of all neoplasm after skin tumour, but the majority of these tumours occur in intact/ non spaying female dogs. Surgical excision of the benign tumour is the standard treatment of canine mammary tumours. Chemotherapy is the choice of treatment if the tumour is malignant or shows evidence of invasion into lymph or blood vessels, however, they showed different side effects and their success rate is varied. Taxanes are now the most promising anti-cancer drugs with little side effects. Gene therapy expressing apoptosis-inducing proteins have ability to kill cancer cells while sparing normal cells. The present study was conducted for exploring the oncolytic effect of viral gene therapy expressing apoptosis-inducing proteins construct (ns1 +vp3), nanosomal paclitaxel as chemotherapeutic agent and surgical therapy in the management of spontaneous canine mammary tumours. Chemotherapy (nanosomal paclitaxel) (n=10), viral gene construct (ns1 +vp3) (n=10) and surgical therapy (n=10) were used in 30 female dogs of different breeds having different types of spontaneous mammary tumours. Chemotherapeutic drug and viral gene construct (ns1 +vp3) induced apoptosis in canine mammary neoplasms were studied using fluorescent activated cell sorting analysis. However, apoptotic percentage was significantly higher in chemotherapeutic group than viral gene construct therapy. No major side effects were observed in any groups. Matrix metalloproteinase-2 was found as an important prognostic tool in the management of canine mammary tumours. In conclusion, chemotherapy with nanosomal paclitaxel proved better than viral gene construct (ns1 +vp3) in the treatment of canine mammary neoplasm.

Development of a Quantitative Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Novel Goose Parvovirus.

An infectious disease characterized with short bills and protruding tongues has attacked to meat ducks in China since March 2015, which has caused ducks poor growth and enormous economic losses to duck industry of China. It was eventually proved to be caused by parvovirus after pathogen isolation and identification. As the genomic sequence analysis showed, this pathogen shared 90.8-94.6% of nucleotide identity with goose parvovirus (GPV), and it was called duck-origin novel goose parvovirus (N-GPV). In this study, a quantitative loop-mediated isothermal amplification (qLAMP) assay was developed for the rapid diagnosis of N-GPV. A set of four specific primers, two inner and two outer, were designed targeting at VP3 gene, which could be completed within 60 min at 65°C in water bath or on a real-time PCR instrument for quantitative analysis. Specificity test of LAMP assay showed that there was no cross-reactivity between N-GPV and other duck pathogens, and the detection limit of qLAMP assay was 1.0 × 102 copies/µL. The repeatability of this method was confirmed by inter-assay and intra-assay tests with variability ranging from 0.74 to 2.25%. The results have indicated that the qLAMP assay was a simple, rapid, accurate, sensitive, and specific method for detecting N-GPV, especially on field detection.

Study on cellular localization of VP3 and its apoptosis-inducing effect on breast cancer cells / 中国免疫学杂志

Objective:To observe the localization of chicken infectious anemia virus VP3 gene in normal cells and breast cancer cells in different times and its apoptosis-inducing effect.Methods: The fundamental cloning method,inserting the VP3 gene of chicken anemia virus into the eukaryotic expression vector pEGFP-C1 was used.Then,the positive recombinant containing VP3 gene pEGFP-C1-VP3 was transfected into human breast cancer cell line MCF-7 and mouse fibroblasts L929 by FuGENE(R)6 transfection reagent in vitro respectively.After 24 hours,48 hours and 72 hours,fluorescence microscope was used to observe the distribution of VP3 in cells and the rate of apoptosis was studied on the treated MCF-7 cells by FCM(Flow Cytometry).Results: The recombinant plasmid pEGFP-C1-VP3 could be localized in the nuclei of breast cancer cells,which showed the typical nuclear changes in different stages of apoptosis.In L929 cells,pEGFP-C1-VP3 underwent a process of migration from the nucleus to the cytoplasm,which didn′t induce apoptosis of L929 cells.Conclusion: VP3 located in the nucleus of MCF-7 breast cancer cells,which can led to cancer cell death by inducing apoptosis,and the apoptosis rate was higher than the control group with time dependence.VP3 located in the cytoplasm of normal cells,and didn′t induce apoptosis.

Development of a TaqMan-based real-time PCR assay for the detection of Novel GPV.

The newly emerged disease, duck beak atrophy and dwarfism syndrome (BADS), is caused by novel goose parovirus (N-GPV). Although N-GPV infection has severe consequences, few methods for detecting this virus have been developed. Therefore, the availability of rapid and reliable molecular diagnostic methods would aid future studies of this novel virus. Clinical specimens from 138 suspected cases of N-GPV infection and 120 cloacal swabs from breeding ducks were used in this study. The targeted sequence of N-GPV cloned into the pMD18-T vector was used to generate the N-GPV DNA standard curve. The specificity of the assay was validated using duck plague virus, GPV, duck hepatitis virus, avian influenza virus, duck reovirus, tembusu virus, and fowl adenovirus. The lowest limit of detection was 8.8×101copies/µL with a good linear standard curve (Y=-3.3682X+37.220, R2=0.9953) over a wide range of input DNA, of which the concentration was between 8.8×101 to 8.8×108copies/µL. The results show that the real-time PCR assay is a highly sensitive, specific, reproducible, and versatile method for quantitatively detecting N-GPV DNA, and thus can be used to detect this virus, thereby facilitating epidemiological investigations of animals with BADS.

Inhibitory effects of attenuated Salmonella typhimurium-mediated TRAIL and VP3 gene against gastric cancer cells / 第二军医大学学报

Objective: To investigate the inhibitory effect of eukaryotic expression vector (attenuated salmonella typhimurium) carrying tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and Chicken anemia virus VP3 gene on gastric cancer cells in vitro and in vivo. Methods: The cloning vectors pBud-TRAIL, pBud-VP3, and pBud-TRAIL-VP3 were transformed into attenuated Salmonella typhimurium by electric transformation technique. The S. typhimurium-based carriers were then transfected into gastric cancer cells, line SGC-7901 after stability assay. The expression of fusion green fluorescent protein was examined using fluorescent microscopy after 24 h. MTT assay was used to examine the inhibition of cell growth. Flow cytometry was used to detect cycle distribution and apoptosis rates of cells. The expression of caspase-3 and caspase-9 was assayed by immunohistochemistry method. Salmonella typhimurium carrying recombinant plasmid was administrated orally in sarcoma-bearing mice; 8 weeks later RT-PCR was used to detect the expression of cloning vectors in tumor tissue. Meanwhile, the sizes of tumors were also determined. Results: The recombinant plasmids were stably transformed into attenuated Salmonella typhimurium, and the plasmids was satisfactorily expressed in gastric cancer cells via attenuated Salmonella typhimurium. TRAIL and VP3 inhibited the proliferation of gastric cancer cells after 48 h. Flow cytometry analysis showed that the pBud-TRAIL-VP3 obviously enhanced apoptosis rates of gastric cancer cells. TRAIL and VP3 jointly increased the expression of caspase-3 and caspase-9. In vivo study showed that TRAIL and VP3 genes were expressed in tumor tissue and could inhibit the tumor growth(P<0.05). Conclusion: Attenuated Salmonella typhimurium-mediated TRAIL and VP3 transfection of gastric cancer cells can inhibit cell growth in vitro and in vivo. The joint effect of TRAIL and VP3 is correlated with the increase of caspase-3 and caspase-9 expression.

Feasibility on Systemic Delivery of Asialoorosomucoid Complex to Hepatic Origin Cells Mediated by Asialoglycoprotein Receptor / 华中科技大学学报（医学）（英德文版）

Summary: Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After intravenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.