Development of TaqMan® MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1

<p>Abstract</p> <p>Background</p> <p>Anatid herpesvirus 1 (AHV-1) is an alphaherpesvirus associated with latent infection and mortality in ducks and geese and is currently affecting the world-wide waterfowl production severely. Here we describe a fluorescent quantitative real-time PCR (FQ-PCR) method developed for fast measurement of AHV-1 DNA based on TaqMan MGB technology.</p> <p>Results</p> <p>The detection limit of the assay was 1 × 10¹standard DNA copies, with a sensitivity of 2 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation.</p> <p>Conclusion</p> <p>The high sensitivity, specificity, simplicity and reproducibility of the AHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method suitable for a broad spectrum of AHV-1 etiologically related application.</p

Characterization of subcellular localization of duck enteritis virus UL51 protein

<p>Abstract</p> <p>Background</p> <p>Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells.</p> <p>Results</p> <p>The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism.</p> <p>Conclusion</p> <p>In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.</p

Expression and characterization of recombinant VP19c protein and N-terminal from duck enteritis virus

<p>Abstract</p> <p>Background</p> <p>Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all <it>Alphaherpesvirus </it>subfamily. However, there is no report on the VP19c protein of duck enteritis virus (DEV). In this study, we expressed the DEV VP19c protein and presented its antigenic properties. Moreover, we developed polyclonal antibody against the VP19c protein and characterized it.</p> <p>Methods</p> <p>A recombinant VP19c (rVP19c) and N-terminal were expressed in <it>Escherichia coli </it>(E.coli) and purified by Ni2+-affinity chromatography. The antigenic properties of the recombinant protein were determined by Western blot and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the polyclonal antibodies against the purified recombinant proteins were produced and the titer of polyclonal antibody was determined by ELISA analysis. Finally, the antibody was used to recognize the VP19c in the cells infected with DEV in the immunofluorescence assay.</p> <p>Results</p> <p>The N-terminally His-tagged rVP19c and rVP19c(N) were produced as inclusion bodies in E. coli strain BL21 (DE3) with molecular weight of about 66 and 46 kDa. Then the proteins were purified to reach the level of homogeneity. Western blot and ELISA analysis that the rVP19c seems to be structurally and antigenically very similar to native VP19c and the N-terminus of VP19c may contain most antigenic linear-epitopes. Furthermore, ELISA analysis demonstrated that the titer of polyclonal antibody was approximately 1:12800, and in the immunofluorescence assay, the antibody was able to recognize the VP19c in the cells infected with DEV.</p> <p>Conclusions</p> <p>To our knowledge, this was the first report on basic properties of DEV VP19c protein. In the present study, we obtained a high-level expression of the recombinant VP19c protein as well as high titers of rabbit polyclonal antibody against to VP19c protein. The anti-rVP19c serum was able to detect the VP19c protein in DEV infected cells and the VP19c protein targeted to the nucleus as distinct punctate speckles. This specific polyclonal antibody provides a good tool for further studying structural and functional characterization of DEV VP19c.</p

Development and evaluation of an immunochromatographic strip test based on the recombinant UL51 protein for detecting antibody against duck enteritis virus

<p>Abstract</p> <p>Background</p> <p>Duck enteritis virus (DEV) infection causes substantial economic losses to the worldwide duck-producing areas. The monitoring of DEV-specific antibodies is a key to evaluate the effect of DEV vaccine and develop rational immunization programs. Thus, in this study, an immunochromatographic strip (ICS) test was developed for detecting DEV serum antibodies.</p> <p>Results</p> <p>The ICS test is based on membrane chromatography, and uses both the purified recombinant UL51 protein conjugated with colloidal gold and goat anti-rabbit IgG conjugated with colloidal gold as tracers, the purified recombinant UL51 protein as the capture reagent at the test line, and rabbit IgG as the capture reagent at the control line. The specificity of the ICS was evaluated by sera against DEV, Duck hepatitis virus (DHV), Riemerella anatipestifer (RA), Duck E. coli, Muscovy duck parvovirus (MPV), or Duck Influenza viruses (DIV). Only sera against DEV showed the strong positive results. In order to determine the sensitivity of the ICS, anti-DEV serum diluted serially was tested, and the minimum detection limit of 1:128 was obtained. The ICS components, which are provided in a sealed package, require no refrigeration and are stable for 12 months. To evaluate the effect of the ICS, 110 duck serum samples collected from several non-immune duck flocks were simultaneously tested by the ICS test, enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). The results showed that the sensitivity of the ICS test was almost consistent with ELISA and much higher than NT, has low cost, and is rapid (15 min) and easy to perform with no requirement of specialized equipment, reagent or technicians.</p> <p>Conclusions</p> <p>In this work, we successfully developed a simple and rapid ICS test for detecting DEV serum antibodies for the first time. The ICS test was high specific and sensitive for the rapid detection of anti-DEV antibodies, and has great potential to be used for the serological surveillance of DEV infection in the field.</p

A retrospective analysis of factors associated with the length of hospital stay in COVID-19 patients treated with Nirmatrelvir / Ritonavir

Objectives: This study reviewed factors influencing the length of hospital stay in adult inpatients with confirmed Coronavirus disease (COVID-19) who were treated with Nirmatrelvir/Ritonavir.Methods: We did a retrospective analysis of data from a cohort of inpatients with confirmed diagnosis of Omicron variant of SARS-CoV-2 infection who were treated with Nirmatrelvir/Ritonavir. We included patients who were treated from 13th March 2022 to 6th May 2022 in various in-patient treatment units in Quanzhou, Fujian Province, China. The primary study outcome was the length of hospital stay. Secondary study outcome was viral elimination defined as negative for ORF1ab and N genes [cycle threshold (Ct) value ≥35 in real-time PCR], according to local guidelines. Hazard ratios (HR) of event outcomes were analyzed using Multivariate Cox regression models.Results: We studied 31 inpatients with high risk for severe COVID-19 who were treated with Nirmatrelvir/Ritonavir. We found that inpatients with shorter length of hospital stay (≤17 days) were mostly females with lower body mass index (BMI) and Charlson Comorbidity Index (CCI) index. Their treatment regimen with Nirmatrelvir/Ritonavir was started within 5 days of diagnosis (p &lt; 0.05). Multivariate Cox regression indicated that inpatients starting treatment of Nirmatrelvir/Ritonavir within 5 days had a shorter length of hospital stay (HR 3.573, p = 0.004) and had a faster clearance of viral load (HR 2.755, p = 0.043).Conclusion: This study assumes relevance during the Omicron BA.2 epidemic as our findings suggest that early treatment with Nirmatrelvir/Ritonavir within 5 days of diagnosis (≤5 days) was highly effective in shortening the length of hospital stay and faster viral load clearance

A Thymidine Kinase recombinant protein-based ELISA for detecting antibodies to Duck Plague Virus

<p>Abstract</p> <p>Background</p> <p>Duck plague virus (DPV) is the causative agent of Duck Plague (DP) that causes significant morbidity and mortality throughout duck-producing areas of the world. The diagnosis of DP currently relies on the use of live or inactivated whole DPV virion as antigens in ELISA, but it is too laborious and expensive for routine application, and it is still difficult to get purified DPV virion with current technology.</p> <p>Results</p> <p>In this study, we describe the expression and purification of a recombinant Thymidine Kinase (TK) protein which makes antigen in an in-house developed, optimized and standardized ELISA. The specificity of the optimized TK-ELISA was evaluated by antisera against Duck Plague Virus (DPV), Duck Hepatitis B Virus (DHBV), Duck Hepatitis Virus (DHV), <it>Riemerella Anatipestifer</it>(<it>R. A</it>), <it>Escherichia coli </it>(<it>E. coli</it>) and <it>Salmonella anatum </it>(<it>S. anatum</it>). Only antisera against DPV yielded a specific and strong signal. In order to determine the sensitivity of the TK-ELISA, a panel of diluted sera was tested, and the minimum detection limit of 1:2560 (OD450 nm = 0.401) was obtained according to the endpoint cut-off (0.2438). The repeatability and reproducibility under the experimental conditions demonstrates a low variability (P > 0.05). The suspected sera samples (n = 30) were determined by TK-ELISA and the positive rate is 90% (27/30), and the TK-ELISA showed 83.33% (22+3/30) coincidence rate with the Serum Neutralization Test (SNT) and 90% (24+3/30) coincidence rate with the whole DPV virion based-ELISA (DPV-ELISA). When defining the dynamics of antibody response to attenuated live DPV vaccine, the maximum antibodies is reached after 4 weeks.</p> <p>Conclusions</p> <p>The results suggest that the TK-ELISA provides high specificity, sensitivity, repeatability and reproducibility for detection of anti-DPV antibodies in duck sera, and has the potential to be much simpler than DPV-ELISA and SNT for the sera epidemiological investigation.</p

Value of deep learning models based on ultrasonic dynamic videos for distinguishing thyroid nodules

ObjectiveThis study was designed to distinguish benign and malignant thyroid nodules by using deep learning(DL) models based on ultrasound dynamic videos.MethodsUltrasound dynamic videos of 1018 thyroid nodules were retrospectively collected from 657 patients in Zhejiang Cancer Hospital from January 2020 to December 2020 for the tests with 5 DL models.ResultsIn the internal test set, the area under the receiver operating characteristic curve (AUROC) was 0.929(95% CI: 0.888,0.970) for the best-performing model LSTM Two radiologists interpreted the dynamic video with AUROC values of 0.760 (95% CI: 0.653, 0.867) and 0.815 (95% CI: 0.778, 0.853). In the external test set, the best-performing DL model had AUROC values of 0.896(95% CI: 0.847,0.945), and two ultrasound radiologist had AUROC values of 0.754 (95% CI: 0.649,0.850) and 0.833 (95% CI: 0.797,0.869).ConclusionThis study demonstrates that the DL model based on ultrasound dynamic videos performs better than the ultrasound radiologists in distinguishing thyroid nodules

Association of IGF-I gene polymorphisms with milk yield and body size in Chinese dairy goats

The association of IGF-I gene polymorphisms with certain traits in 708 individuals of two Chinese dairy-goat breeds (Guanzhong and Xinong Saanen) was investigated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods were employed in screening for genetic variation. Two novel mutations were detected in the 5'-flanking region and in intron 4 of IGF-I gene, viz., g.1617 G > A and g.5752 G > C (accession D26119.2), respectively. The associations of the g.1617 G > A mutation with milk yield and the body size were not significant (p > 0.05). However, in the case of g.5752 G > C, Xinong Saanen dairy goats with the CG genotype presented longer bodies (p < 0.05). Chest circumference (p < 0.05) was larger in Guanzhong goats with the GG genotype. In Xinong Saanen dairy goats with the CC genotype, milk yields were significantly higher during the first and second lactations (p < 0.05). Hence, the g.5752 G > C mutation could facilitate association analysis and serve as a genetic marker for Chinese dairy-goat breeding and genetics

Dominant role of GABAB2 and Gbetagamma for GABAB receptor-mediated-ERK1/2/CREB pathway in cerebellar neurons

gamma-aminobutyric acid type B (GABA(B)) receptor is an allosteric complex made of two subunits, GABA(B1) and GABA(B2). GABA(B2) plays a major role in the coupling to G protein whereas GABA(B1) binds GABA. It has been shown that GABA(B) receptor activates ERK(1/2) in neurons of the central nervous system, but the molecular mechanisms underlying this event are poorly characterized. Here, we demonstrate that activation of GABA(B) receptor by either GABA or the selective agonist baclofen induces ERK(1/2) phosphorylation in cultured cerebellar granule neurons. We also show that CGP7930, a positive allosteric regulator specific of GABA(B2), alone can induce the phosphorylation of ERK(1/2). PTX, a G(i/o) inhibitor, abolishes both baclofen and CGP7930-mediated-ERK(1/2) phosphorylation. Moreover, both baclofen and CGP7930 induce ERK-dependent CREB phosphorylation. Furthermore, by using LY294002, a PI-3 kinase inhibitor, and a C-term of GRK-2 that has been reported to sequester Gbetagamma subunits, we demonstrate the role of Gbetagamma in GABA(B) receptor-mediated-ERK(1/2) phosphorylation. In conclusion, the activation of GABA(B) receptor leads to ERK(1/2) phosphorylation via the coupling of GABA(B2) to G(i/o) and by releasing Gbetagamma subunits which in turn induce the activation of CREB. These findings suggest a role of GABA(B) receptor in long-term change in the central nervous system