Pathogenicity characterization of PRRSV-1 181187-2 isolated in China.

PRRSV-1 has caused more clinical infections in pigs in Chinese swine herds in recent years, however, the pathogenicity of PRRSV-1 in China is unclear. In order to study the pathogenicity of PRRSV-1, in this study, a PRRSV-1 strain, 181187-2, was isolated in primary alveolar macrophage (PAM) cells from a farm where abortions had been reported in China. The complete genome of 181187-2 was 14932 bp excluding Poly A, with 54-amino acid continuous deletion in the Nsp2 gene and 1 amino deletion in ORF3 gene compared with LV. Additionally, the piglets inoculated with strain 181187-2 by intranasal and intranasal plus intramuscular injection, animal experiments showed clinical symptoms including transient fever and depression, with no death. The obvious histopathological lesions including interstitial pneumonia and lymph node hemorrhage, and there were no significant differences in clinical symptoms and histopathological lesions with different challenge ways. Our results indicated that PRRSV -1 181187-2 was a moderately pathogenic strain in piglets.

[Simulator of Heart Rate and Respiratory Rate for Monitoring Accuracy of Intelligent Sleep Monitoring Devices].

OBJECTIVE: Invent a simulator which provides a simulation of heart rate and respiratory rate to the intelligent sleep monitoring devices based on precision pressure sensors. METHODS: The simulator was composed of control part and simulated silicone doll. The simulated silicone doll contains heartbeat simulator and breathing simulation airbag. Heartbeat and breathing combination pressure signal can be produced according to frequency set values. Frequencies of pressure signal of the simulator were compared with the monitoring results of intelligent sleep monitoring devices with known accuracy to verify the frequency accuracy of pressure signal of the simulator. Verified the repeatability and stability of the simulator with a stopwatch. RESULTS: The heart rate of the simulator was with in ±2 beats per minute of the monitoring results of intelligent sleep monitoring devices and the respiratory rate of the simulator was with in ±2 times per minute of the monitoring results. The repeatability and stability of the simulator was better than ±5% according to results with a stopwatch. CONCLUSIONS: It's practicable to use the simulator which provides a simulation of heart rate and respiratory rate to the accuracy test of the intelligent sleep monitoring devices based on precision pressure sensor.

Lethal Encephalitis in Seals with Japanese Encephalitis Virus Infection, China, 2017.

We isolated Japanese encephalitis virus (JEV) from brain samples of 2 seals with lethal encephalitis at Weihai Aquarium, Weihai, China, in 2017. We confirmed our findings by immunohistochemical staining and electron microscopy. Phylogenetic analysis showed this virus was genotype I. Our findings suggest that JEV might disseminate though infected zoo animals.

Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design.

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

Microfluidic techniques for tumor cell detection.

Cancer metastasis is the main cause of cancer-related death. Early detection of tumor cell in peripheral blood is of great significant to early diagnosis and effective treatment of cancer. Over the past two decades, microfluidic technologies have been demonstrated to have great potential for isolating and detecting tumor cell from blood. The present paper reviews microfluidic techniques for tumor cell detection based on various physical principles. The specific methods are categorized into active and passive methods depending on whether extra force field is applied. Working principles of the two methods are explained in detail, including microfluidics combined with optical tweezer, electric field, magnetic field, acoustophoresis, and without extra fields for tumor cell detection. Typical experiments and the results are reviewed. Based on these, research characteristics of the two methods are analyzed.

Isolation and genomic characterization of a canine parainfluenza virus type 5 strain in China.

A canine parainfluenza virus type 5 strain was isolated from a lung sample from a diseased dog. The genome sequene of this isolate, named HeN0718, was determined and compared tho those of other previously reported canine parainfluenza viruses. Unlike previously reported viruses, the HeN0718 strain contained several nucleotide mutations in the SH gene that led to a frame shift in the open reading frame. Phylogenetic analysis based on the complete virus genome and the P, F, and HN genes showed that HeN0718 was genetically closest to D277, a Korean strain that was isolated in 2008.

Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus.

The pathogenicity of HNjz15, an NADC30-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), was investigated and compared to that of a highly pathogenic PRRSV JAX1 strain. Six-week-old pigs infected with each virus showed typical clinical symptoms, including high fever and respiratory disorders. Pigs infected with JXA1 had more-severe clinical manifestations than pigs infected with HNjz15. HNjz15 replicated in vivo with kinetics similar to those of JXA1 but induced a lower level of PRRSV-specific antibody at the beginning of virus infection. Histopathologically, JXA1 infection led to more-severe lung lesions and broader organ tropism than HNjz15 did. Different from what was observed with the previously reported NADC30-like PRRSV JL580 strain, all HNjz15-infected pigs survived until the end of the study. All of these results indicated that NADC30-like PRRSV HNjz15 is virulent to pigs but is less pathogenic than the JXA1 and JL580 PRRSV strains.

Screening and Stability Evaluation of Freeze-Dried Protective Agents for a Live Recombinant Pseudorabies Virus Vaccine.

Infection of pigs with the pseudorabies virus (PRV) causes significant economic losses in the pig industry. Immunization with live vaccines is a crucial aspect in the prevention of pseudorabies in swine. The TK/gE/gI/11k/28k deleted pseudorabies vaccine is a promising alternative for the eradication of epidemic pseudorabies mutant strains. This study optimized the lyophilization of a heat-resistant PRV vaccine to enhance the quality of a live vaccine against the recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k-. The A4 freeze-dried protective formulation against PRV was developed by comparing the reduction in virus titer after lyophilization and after seven days of storage at 37 °C. The formulation contains 1% gelatin, 5% trehalose, 0.5% poly-vinylpyrimidine (PVP), 0.5% thiourea, and 1% sorbitol. The A4 freeze-dried vaccine demonstrated superior protection and thermal stability. It experienced a freeze-dried loss of 0.31 Lg post-freeze-drying and a heat loss of 0.42 Lg after being stored at a temperature of 37 °C for 7 consecutive days. The A4 freeze-dried vaccine was characterized through XRD, FTIR, and SEM analyses, which showed that it possessed an amorphous structure with a consistent porous interior. The trehalose component of the vaccine formed stable hydrogen bonds with the virus. Long-term and accelerated stability studies were also conducted. The A4 vaccine maintained viral titer losses of less than 1.0 Lg when exposed to 25 °C for 90 days, 37 °C for 28 days, and 45 °C for 7 days. The A4 vaccine had a titer loss of 0.3 Lg after storage at 2-8 °C for 24 months, and a predicted shelf life of 6.61 years at 2-8 °C using the Arrhenius equation. The A4 freeze-dried vaccine elicited no side effects when used to immunize piglets and produced specific antibodies. This study provides theoretical references and technical support to improve the thermal stability of recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k- vaccines.

Disulfide linkages mediating nucleocapsid protein dimerization are not required for porcine arterivirus infectivity.

The nucleocapsid (N) proteins of the North American (type II) and European (type I) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) share only approximately 60% genetic identity, and the functionality of N in both genotypes, especially its role in virion assembly, is still poorly understood. In this study, we demonstrated that the ORF7 3' untranslated region or ORF7 of type I is functional in the type II PRRSV background. Based on these results, we postulated that the cysteine at position 90 (Cys90) of the type II N protein, which corresponds to an alanine in the type I protein, is nonessential for virus infectivity. The replacement of Cys90 with alanine confirmed this hypothesis. We then hypothesized that all of the cysteines in the N protein could be replaced by alanines. Mutational analysis revealed that, in contradiction to previously reported findings, the replacement of all of the cysteines, either singly or in combination, did not impair the growth of either type II or type I PRRSV. Treatment with the alkylating agent N-ethylmaleimide inhibited cysteine-mediated N dimerization in living cells but not in released virions. Additionally, bimolecular fluorescence complementation assays revealed noncovalent interactions in living cells among the N and C termini and between the N-terminal and C-terminal regions of the N proteins of both genotypes of PRRSV. These results demonstrate that the disulfide linkages mediating the N dimerization are not required for PRRSV viability and help to promote our understanding of the mechanism underlying arterivirus particle assembly.

Microstructural study on Kirkendall void formation in Sn-containing/Cu solder joints during solid-state aging.

Kirkendall void formation at the solder/metallization interface is an important reliability concern for Cu conductors and under-bump metallization in microelectronic packaging industry, whose mechanism is still hard to be understood for different individual cases. In the present work, two typical solder/Cu-diffusing couples, eutectic SnIn/Cu and SnBi/Cu, were studied by scanning/transmission electron microscopy to investigate the microstructural evolution and voiding process after soldering and then solid-state aging. It was concluded that Kirkendall voids formed between two sublayers within Cu2(In,Sn) phase in eutectic SnIn/Cu solder joint, whereas they appeared at the Cu3Sn/Cu interface or within Cu3Sn for eutectic SnBi/Cu solder joint. Besides the effect of impurity elements, the morphological difference within one intermetallic compound layer could change the diffusing rates of reactive species, hence resulting in void formation in the reaction zone.

iTRAQ-based proteomic analysis of imiquimod in the treatment of ulcerative colitis.

OBJECTIVE: In this study, we explored the potential mechanisms and the signaling pathways involved in the treatment of Ulcerative Colitis (UC) with imiquimod (IMQ). METHODS: The UC mouse model was established by treating C57BL/6J mice with 3% Dextran Sulfate Sodium (DSS). Then, the UC-related symptoms were examined. Disease Activity Index (DAI) was estimated based on weight loss, stool consistency, and occult bleeding or hematochezia. Histological changes were evaluated by Hematoxylin and Eosin (H&E) staining. Furthermore, we used multiplexed Isobaric Tagging for Relative and Absolute Protein Quantification (iTRAQ) technique coupled with high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the differentially expressed proteins (DEPs). RESULTS: Administration of 3% DSS for 7 days induced acute colitis associated with diarrhea, hematochezia, weight loss, and colon shortening. However, after IMQ administration, almost all the above symptoms were improved by different degrees. Specifically, the DAI, histological disorder, and colon shortening were attenuated. In iTRAQ analysis, a total of 4170 proteins were identified with a high confidence (≥ 95% confidence). The numbers of DEPs between the normal and UC model mice, between the normal and the IMQ-treated therapy mice, as well as between the model and the therapy mice were 317, 253, and 209, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the DEPs involved in the complement and coagulation cascades were downregulated in IMQ-treated therapy group. CONCLUSIONS: IMQ might ameliorate colitis by suppressing the complement and coagulation cascades pathway, which might serve as new therapeutic strategies for the treatment of patients with UC.

Use of reverse genetics to develop a novel marker porcine reproductive and respiratory syndrome virus.

Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the most abundant viral structural protein with high immunogenicity. Previously, the nonessential sequences for virus infectivity were identified at both N and C terminal ends of N protein. Here, by means of reverse genetics, a marker virus (v7APMa) was generated with a mutant N protein that differs from the wild-type strains (vAPRRS, type 2 PRRSV). v7APMa shows stable inheritance in cell culture and the virologic characteristics of the marker virus in vitro showed that v7APMa replicates well as its parental strain. In the pig model, the v7APMa marker virus induced a similar level of anti-N protein antibodies and robust antibodies against the marker peptide, from 14 days post infection. In addition, a peptide-based ELISA was developed to detect the specific antibodies for the introduced 7APMa peptide. This approach, using a rationally designed marker virus, provides a new potential mutant basis for further development of PRRSV novel vaccines.

Impact of urban innovation on urban green development in China's Yangtze River Economic Belt: perspectives of scale and network.

Understanding whether and how urban innovation offers a sound solution to the dilemma of urban green development is a crucial response to mitigate the detrimental effect on natural resources and environment for transitioning to sustainable urban development. To address the critical issue, we propose urban green development evaluation index system, and then examine how the urban innovation affects urban green development from the perspectives of government-scale, enterprise-scale, and spatial correlation network, all of which are originally applied in the 108 cities of Yangtze River Economic Belt of China (YREB) during period 2006-2018. The evaluation results show that urban innovation promotes urban green development, and both government-scale and enterprise-scale contribute to the effects. The constructed spatial correlation network of urban innovation illustrates the network structural form and reveals the network property, and further results tell that increasing network density and centrality would promote green development obviously. More specifically, the network density of urban innovation has been tied to the enhancement of urban green development, which is more significant in middle reaches than in lower and upper reaches of YREB. Similarly, optimizing the network's degree centrality and closeness centrality can help facilitate urban green development in whole YREB. Thus, the research findings would provide new insights into the essence and driving forces from various scale and hidden network when exploring and seeking urban green development path.

Effect of mesalazine combined with probiotics on inflammation and immune function of patients with inflammatory bowel disease.

OBJECTIVE: To determine the effects of mesalazine combined with probiotics on inflammation and immune function of patients with inflammatory bowel disease (IBD). METHODS: In this retrospective study, a total of 116 patients with IBD treated in Renmin Hospital of Wuhan University from September 2018 to September 2021 were enrolled and divided into a control group (n=55, treated with mesalazine alone) and a research group (n=61, treated with mesalazine combined with probiotics) according to the treatment regimen. The two groups were compared in the levels of inflammatory factors, immune factors, adverse reactions, clinical efficacy and improvement of patients' disease condition before and after treatment. Logistic regression was used to analyze the independent risk factors of infection in patients with IBD at 6 months after admission. RESULTS: The research group showed a significantly higher the total effective rate than the control group (P<0.05), and there was no notable difference between the two groups in the incidence of adverse reactions (P>0.05). In addition, compared with the control group, the research group showed significantly lower levels of immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and had significantly lower scores of clinical activity index (CAI) and endoscopic activity index (EAI) after treatment (all P<0.05). Higher IgG, IgM, IL-6, CRP and EAI levels at admission were independent risk factors for infection in patients with IBD. CONCLUSION: Mesalazine combined with probiotics can substantially improve the disease condition of patients with IBD, improve their immune ability and reduce their inflammation level, with a good safety profile.

A therapeutic chimeric IgG/IgA expressed by CHO cells for oral treatment of PED in piglets.

Immunoglobulin A (IgA) of sows is critically important for assessing piglets' protective capacity against porcine epidemic diarrhea virus (PEDV). Here, we report a therapeutic chimeric anti-PEDV IgG/IgA expressed by Chinese hamster ovary (CHO) cells for oral treatment of PED. The chimeric anti-PEDV IgG/IgA was produced by the CHO cell lines, in which the heavy chain was constructed by combining the VH, Cγ1 and hinge regions of PEDV IgG mAb 8A3, and the Cα2 and Cα3 domains of a Mus musculus immunoglobulin alpha chain. The chimeric anti-PEDV IgG/IgA could neutralize the strains of CV777 (G1), P014 (G2) and HN1303 (G2) in vitro effectively, showing broad-spectrum neutralization activity. The in vivo challenge experiments demonstrated that chimeric anti-PEDV IgG/IgA (9C4) produced in the CHO cell supernatant could alleviate clinical diarrhea symptoms of the PEDV infection in piglets. In general, our study showed that chimeric anti-PEDV IgG/IgA produced from CHO cell line supernatants effectively alleviates PEDV infection in piglets, which also gives the foundation for the construction of fully functional secretory IgA by the J chain introduction to maximize the antibody therapeutic effect.

A canine-derived chimeric antibody with high neutralizing activity against canine parvovirus-2.

Canine parvovirus-2 (CPV-2) infection causes serious multisystemic disease in dogs and many animal species worldwide. Previously, a monoclonal antibody (MAb) of CPV-2, 10H4, showed high neutralizing activity and therapeutic effect against CPV-2 in dogs. However, the application of mouse MAb is limited in other animals due to immune rejection. Here, the variable regions of the heavy and light chains of 10H4 were cloned and ligated with constant canine antibody regions to produce a canine-derived chimeric MAb 11D9, in a CHO-S cell expression system. The cell supernatant of the CHO cell line 11D9 exhibited a HI titer of 1:2560 against all the variants of CPV-2 (new CPV-2a, new CPV-2b, and CPV-2c), and had the same average neutralization titer as the new CPV-2a (1:11,046.5) and new CPV-2b (1:11,046.5) variants, which was slightly higher than that of CPV-2c variants (1:10,615.7). In animal experiment, the treatment of chimeric MAb 11D9 had a high therapeutic effect in beagles infected with the new CPV-2a. Overall, the canine-derived chimeric MAb 11D9 produced by CHO-S cells showed a high HI and neutralization titer against CPV-2 and the therapeutic effects against the new CPV-2a in beagles, providing potential for the prevention or treatment of CPV-2 infections in dogs.

Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion.

In China, the re-emerging pseudorabies virus (PRV) variant has caused large-scale outbreaks of pseudorabies in swine herds with classical PRV vaccine immunization since late 2011. Here, a recombinant PRV with TK/gI/gE/11k/28k deletion was constructed based on variant HN1201 strain isolated in 2012, by the bacterial artificial chromosome infectious clones. Compared with the parental virus, the recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k- showed a similar virus grown curve and exhibited smaller plaques. The vaccination of rHN1201TK-/gE-/gI-/11k-/28k- could elicit an earlier and higher level of gB antibody, and the neutralizing antibodies elicited by rHN1201TK-/gE-/gI-/11k-/28k- were effective against both PRV classical and variant strains. Clinically, the body temperature of the pigs immunized with rHN1201TK-/gE-/gI-/11k-/28k- was significantly lower than that of the classical PRV vaccine immunized pigs, and the recombinant PRV could provide effective protection against the challenge with the PRV variant. These results imply that the rHN1201TK-/gE-/gI-/11k-/28k- could be a promising vaccine candidate for the prevention of the current epidemic of pseudorabies in China.

Sucrose transportation control mediates the fresh-keeping effects of burdock fructooligosaccharide in 'Crimson Seedless' grapes.

In 'Crimson Seedless' grapes, the appearance of senescence caused by abnormal dark red color, the loss of crisp taste caused by the decrease in firmness, and the fading of sweetness caused by the decrease in total soluble sugar (TSS) are the main problems affecting its edible qualities after storage. In the mesocarp, burdock fructooligosaccharide (BFO) restricted sucrose export; therefore, more carbohydrates were retained directly leading to higher TSS and sweetness, and cell osmotic pressure and firmness were retained indirectly. In the exocarp, BFO restricted sucrose import; therefore, the signal molecule sucrose was reduced and the senescence-related processes were inhibited. The downregulation of SUC12 and SUC27 by BFO may play an important role in restricting sucrose transportation. The opposing effects exhibited by exogenous sucrose treatments compared to those of BFO further verified these mechanisms. Based on the above mechanisms, sucrose transportation mediates the fresh-keeping effects of BFO in 'Crimson Seedless' grapes.

Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a.

Porcine reproductive and respiratory syndrome virus (PRRSV) has varied constantly and circulated in the pig industry worldwide. The prevention and control of porcine reproductive and respiratory syndrome (PRRS) is complicated. A visual, sensitive and specific diagnostic method is advantageous to the control of PRRS. The collateral cleavage activity of LwCas13a is activated to degrade non-targeted RNA, when crRNA of LwCas13a bond to target RNA. The enhanced Cas13a detection is the combination of collateral cleavage activity of LwCas13a and recombinase polymerase amplification (RPA). In this study, the enhanced Cas13a detection for PRRSV was established. The novel method was an isothermal detection at 37°C, and the detection can be used for real-time analysis or visual readout. The detection limit of the enhanced Cas13a detection was 172 copies/µl, and there were no cross-reactions with porcine circovirus 2, porcine parvovirus, classical swine fever virus and pseudorabies virus. The enhanced Cas13a detection can work well in clinical samples. In summary, a visual, sensitive and specific nucleic acid detection method based on CRISPR-Cas13a was developed for PRRSV.

Generating Recombinant Pseudorabies Virus for Use as a Vaccine Platform.

Pseudorabies virus (PRV) is a promising vaccine vector due to its distinctive features including many nonessential replication regions and a broad host range. Foreign genes of other viruses have been successfully inserted into and expressed in PRV and these recombinant viruses are very likely to induce humoral and/or cellular responses in immunized animals. This chapter offers an overview of methods for generating recombinant pseudorabies virus for use as a vaccine vector.