Duck Tembusu Virus Exhibits Pathogenicity to Kunming Mice by Intracerebral Inoculation

In this study, Kunming mice were used as the animal models to study the pathogenicity of TMUV. Three groups of 3-week-old female Kunming mice (n=15 mice per group) were infected with the SDSG strain of TMUV in 50μL allantoic fluid (104.8 ELD50/ 0.2ml) respectively by the intracerebral (i.c.), subcutaneous (s.c.) and intranasal (i.n.) routes. The control group (n=15 mice) was inoculated with 50μL sterile phosphate-buffered saline (PBS). Clinical signs, gross and microscopic lesions, viral loads in different tissues, and serum antibody titers were examined and recorded. Kunming mice infected intracerebrally showed typical clinical symptoms, including severe hindlimb paralysis, weight loss and death. Only dead mice presented severe intestinal mucosal edema. No gross lesions were observed in mice sequentially euthanized. However, microscopic lesions in the brain, spleen, liver, kidney and lung were very typical including varying degrees of viral encephalitis, lymphocytes depletion, liver cell necrosis and nephritis, etc. Viral loads in different tissues were detected by the SYBR Green I real-time PCR assay. Viral loads in the brain, liver and spleen were first detected and maintained a longer time, which indicated that these organs may be the target organs of TMUV. The level of viral loads was consistent with the severity of clinical signs and microscopic lesions in different tissues. The neutralizing antibody began to seroconvert at 8dpi. Clinical signs, microscopic lesions, viral loads and serum neutralizing antibodies weren’t observed in other groups. In summary, TMUV can cause systemic infections and death in Kunming mice by i.c., which provides some experimental basis for further study of the significance of TMUV in public health

Effect of Low Temperature Vacuum Slow Cooking on Muscle Quality Characteristics of Half Shell Scallop

This study utilized half-shell Chlamys farreri as the primary material to investigate the effects of different sous vide (SV) cooking temperatures (60, 65, 70, 75, 80 ℃) and cooking times (30, 45, 60, 75 min) on the quality attributes of the Chlamys farreri muscle. Sensory quality, drip loss, moisture content, water activity, texture, myofibrillar protein extraction, and microstructure were evaluated as key indicators. The results showed that as the cooking temperature and time increased, the sensory scores of SV Chlamys farreri initially rose and then declined, with the highest sensory score observed at 70 and 75 ℃ after 30 minutes of cooking. The drip loss of SV Chlamys farreri increased between 60 and 75 ℃ without significant differences (P0.05). Scanning electron microscopy and HE staining illustrated that as the cooking temperature increased, myofibrils underwent breakage, boundaries became indistinct, fractured myofibrils were cross-linked, and the disorder level escalated, ultimately leading to the aggregation of broken myofibrils into large masses. In conclusion, the optimal sensory quality of the scallops was achieved at 70 and 75 ℃ with 30 minutes of SV cooking, where drip loss and elasticity were lower than those in the control group (100 ℃, 15 min), while moisture content, water activity, hardness, and myofibrillar protein extraction rate were higher than those in the control group

Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb) 3G2 by indirect enzyme-linked immunosorbent assay (ELISA). In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays

A Study on the CO2-Enhanced Water Recovery Efficiency and Reservoir Pressure Control Strategies

CO2 geological storage (CGS) proved to be an effective way to mitigate greenhouse gas emissions, and CO2-enhanced water recovery (CO2-EWR) technology may improve the efficiency of CO2 injection and saline water production with potential economic value as a means of storing CO2 and supplying cooling water to power plants. Moreover, the continuous injection of CO2 may cause a sharp increase for pressure in the reservoir system, so it is important to determine reasonable reservoir pressure control strategies to ensure the safety of the CGS project. Based upon the typical formation parameters of the China Geological Survey CO2-EWR test site in the eastern Junggar Basin, a series of three-dimensional (3D) injection-extraction models with fully coupled wellbores and reservoirs were established to evaluate the effect of the number of production wells and the well spacing on the enhanced efficiency of CO2 storage and saline production. The optimal key parameters that control reservoir pressure evolution over time are determined. The numerical results show that a smaller spacing between injection and production wells and a larger number of production wells can enhance not only the CO2 injection capacity but also the saline water production capacity. The effect of the number of production wells on the injection capacity and production capacity is more significant than that of well spacing, and the simulation scenario with 2 production wells, one injection well, and a well spacing of 2 km is more reasonable in the demonstration project of Junggar Basin. CO2-EWR technology can effectively control the evolution of the reservoir pressure and offset the sharp increase in reservoir pressure caused by CO2 injection and the sharp decrease of reservoir pressure caused by saline production. The main controlling factors of pressure evolution at a certain spatial point in a reservoir change with time. The monitoring pressure drops at the beginning and is controlled by the extraction of water. Subsequently, the injection of CO2 plays a dominant role in the increase of reservoir pressure. Overall, the results of analysis provide a guide and reference for the CO2-EWR site selection, as well as the practical placement of wells

Western-blot identification of epitope.

<p>The 16-AA polypeptide of NS1-27 reacted with mAb 3G2 in Western-blot assay. Lane M, PageRuler Prestained Protein Ladder (Fermentas, Canada); Lane 1, GST-tag didn’t react with mAb 3G2; Lane 2, The band of NS1-27-GST fusion protein was visualized with mAb 3G2.</p

Sequences of the overlapping polypeptides from TMUV NS1 (SDSG strain, Accession number: KJ740747.1).

<p>Sequences of the overlapping polypeptides from TMUV NS1 (SDSG strain, Accession number: KJ740747.1).</p

The accurate mapping of one B cell epitope with mAb.

<p>NS1-27 polypeptide was truncated from the carboxy and amino terminals. After the truncated peptides were expressed as a GST fusion protein, they were probed with mAb 3G2 by indirect ELISA respectively. The minimal unit of the peptide was the sequence of 8 AA and 3 AA truncated from the carboxy and amino terminals of NS1-27.</p

Primary screening of epitope with mAb 3G2.

<p>One 16-AA polypeptide of TMUV NS1 protein (NS1-27) was screened with mAb 3G2 by indirect ELISA. Mouse serum against TMUV NS1 protein and normal mouse serum were used as positive and negative controls, respectively. Each sample was detected in triplicate. Error bars were expressed as standard deviation of the means (n = 3). The mean value was statistically significant, calculated by the two-tailed Student’s unpaired t-test (*P < 0.05).</p

Sequences of the truncated NS1-27 polypepide.

<p>Sequences of the truncated NS1-27 polypepide.</p

IFA and western-blot identification of mAb 3G2.

<p>A: Western-blot identification Lane 1, Control, GST-tag didn’t react with mAb 3G2; Lane 2, The band of NS1-GST fusion protein was reacted with mAb 3G2; Lane M, Blue plusIIprotein Marker (14-120kda, Transgen Biotech). B: IFA identification Monoclonal antibody against TMUV NS1 protein was used to perform IFA on TMUV-infected BHK-21 cells. BHK-21 cells infected with TMUV yielded significant fluorescence with six MAbs in the cytoplasm; Control BHK-21 cells didn’t yield any fluorescence.</p