Development and application of a cycleave dual-probe fluorescence quantitative PCR method for simultaneous detection of Mycoplasma gallisepticum ts-11 vaccine strain and non-ts-11 strains.

An attenuated vaccine against the Mycoplasma gallisepticum ts-11 strain has become an effective prevention and control method against MG infection. However, the ts-11 strain is usually difficult to distinguish from the non-ts-11 strain (including field isolates and other vaccine strains (F and 6/85)). Therefore, it is critical to establish a rapid and effective method to distinguish ts-11 strains from non-ts-11 strains. The gene sequences of the ts-11 strain (CP044225.1) and the non-ts-11 strain (including the wild-type (CP006916.3), 6/85 (CP044224.1), and F strains (NC_017503.1) were used to construct a conserved region containing a single point mutation in the potC gene in the ts-11 strain, after which a primer-probe combination method was designed. The primer-probe method was able to accurately and efficiently identify the ts-11 and non-ts-11 strains with minimum detection limits of 2.43 copies/µL and 1.65 copies/µL, respectively. Moreover, it could simultaneously distinguish the ts-11 strain from a non-ts-11 strain, and amplifications of avian influenza virus, infectious bronchitis virus, Newcastle disease virus, fowl adenovirus, infectious laryngotracheitis virus, infectious bursal disease virus, chicken anemia virus, Marek's disease virus, Mycoplasma synoviae, and Ornithobacter rhinotracheale were negative. The detection of clinical samples revealed that the established dual-probe fluorescence quantitative PCR method could be used to screen for mixed and single infections of the ts-11 strain and non-ts-11 strains effectively, with lower variation coefficients for intra- and interbatch repetition. The established cycleave dual-probe fluorescence quantitative PCR method showed good specificity, sensitivity, and repeatability and provides powerful technical support for the rapid and efficient differential diagnosis of the MG ts-11 strain from non-ts-11 strains.

Comprehensive quantitative determination of aquifer confinement based on tidal response of well water level and its application in North China.

Aquifer confinement represents a pivotal property that significantly influences the vulnerability and contamination risk of groundwater resources. Several methods have been proposed for determining aquifer confinement by analyzing the response of well water level to Earth tides and atmospheric tides. In this study, we evaluated the performance of the existing single methods and put forward an optimized comprehensive approach. We compared the determination results of the three single methods with those of a comprehensive method using water-level data from 39 earthquake precursor monitoring wells in North China. The results demonstrate that the comprehensive method effectively determined aquifer confinement, significantly reducing the uncertainty associated with the three single methods. The application of the comprehensive method in North China reveals that aquifer confinement may undergo temporal variations during long-term continuous observation, especially in areas where the confining properties of aquifers may vary due to human activities and earthquakes. In such areas, the comprehensive method facilitates accurate assessment of groundwater vulnerability, as well as the potential dispersion of underground pollutants.

Cellulose-Acetate Coating by Integrating Ester Group with Zinc Salt for Dendrite-Free Zn Metal Anodes.

Zinc (Zn) metal is considered a potential anode owing to its high theoretical capacity, safety, and low cost. However, the dendrites and corresponding side reactions in aqueous electrolytes hinder their further development in environmentally-friendly energy storage. Herein, ion-affiliative cellulose acetate (CA) coating with Zn(CF3 SO3 )2 is constructed on Zn anode (CAZ@Zn). Owing to the complexation effect between the polar ester group (CO) and Zn salt (Zn2+ ), the CAZ polymer coating enhances the hydrophilicity of the Zn anode and reduces the interfacial resistance, allowing the rapid Zn2+ diffusion and homogenizing the Zn deposition in an aqueous electrolyte to suppress zinc dendrite formation and growth. Therefore, the symmetric CAZ@Zn//CAZ@Zn battery achieves reversible plating/stripping over 2800 h at 1 mA cm-2 with 1 mAh cm-2 , about sevenfold higher than bare Zn. The full cell fabricated with an optimized Zn anode and the NH4 V4 O10 cathode achieves substantially stable performance, superior to that of bare Zn. This work provides a straightforward, effective, and scalable method to suppress the zinc dendrites and corresponding side reactions for aqueous Zn-ions batteries.

Metal-Organic Frameworks-Mediated Assembly of Gold Nanoclusters for Sensing Applications.

Gold nanoclusters (AuNCs) are an emerging type of ultrasmall nanomaterials possessing unique physicochemical characteristics. Metal-organic frameworks (MOFs), a singular kind of porous solid and crystalline material, have attracted tremendous attention in recent years. The combination of AuNCs and MOFs can integrate and improve the prominent properties of both components, such as high catalytic activities, tunable optical properties, good biocompatibility, surface functionality and stability, which make the composites of MOFs and AuNCs promising for sensing applications. This review systematically summarizes the recent progress on the sensing of various analytes via MOFs-mediated AuNCs assemblies based on strategies of luminescence sensing, colorimetric sensing, electrochemiluminescence sensing, and electrochemical and photoelectrochemical sensing. A brief outlook regarding the future development of MOFs-mediated AuNCs assemblies for sensing application is presented as well.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

A Biomimetic Polymer-Based Composite Coating Inhibits Zinc Dendrite Growth for High-Performance Zinc-Ion Batteries.

Because of their low cost, safety, and green nature, aqueous Zn-ion batteries are promising candidates for energy storage. However, the appearance of Zn dendrites, hydrogen evolution reaction (HER), and corrosion limit the development of the aqueous Zn-ion batteries. Here, inspired by fibrous cartilage, a biomimetic poly(vinylidene fluoride) (PVDF)-based composite polymer coating layer, including aramid nanofiber (ANF) and zinc trifluoromethanesulfonate [Zn(CF3SO3)2], called ANFZ, was designed and fabricated. The high ionic conductivity (3.84 mS cm-1) of the flexible PVDF matrix, optimized by Zn(CF3SO3)2, combined with the highly mechanical ANF network can effectively guide the rate of Zn stripping/plating, homogenize the Zn2+ distribution, and suppress the dendrites. In addition, the high Coulombic efficiency is obtained due to the suppression of HER and corrosion by the biomimetic coating layer. Symmetric ANFZ@Zn//ANFZ@Zn can steadily work over 1000 h at 1 mA cm-2 with a high degree of reversibility, which is greater than that of bare Zn//bare Zn. Furthermore, the ANFZ@Zn//MVO batteries show a high specific capacity (400.2 mAh g-1, 0.1 A g-1) and a long cycle life. This work presents a novel method combined with bionics for designing and assembling Zn anodes without dendrites for zinc-ion batteries.

Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus.

African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

Fur Seal Feces-Associated Circular DNA Virus Identified in Pigs in Anhui, China.

Fur seal feces-associated circular DNA virus (FSfaCV) is an unclassified circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus that has been detected in mammals (fur seals and pigs). The biology and epidemiology of the virus remain largely unknown. To investigate the virus diversity among pigs in Anhui Province, China, we pooled 600 nasal samples in 2017 and detected viruses using viral metagenomic methods. From the assembled contigs, 12 showed notably high nucleotide acid sequence similarities to the genome sequences of FSfaCVs. Based on these sequences, a full-length genome sequence of the virus was then obtained using overlapping PCR and sequencing, and the virus was designated as FSfaCV-CHN (GenBank No. MK462122). This virus shared 91.3% and 90.9% genome-wide nucleotide sequence similarities with the New Zealand fur seal strain FSfaCV-as50 and the Japanese pig strain FSfaCV-JPN1, respectively. It also clustered with the two previously identified FSfaCVs in a unique branch in the phylogenetic tree based on the open reading frame 2 (ORF2), Rep-coding gene, and the genome of the reference CRESS DNA viruses. Further epidemiological investigation using samples collected in 2018 showed that the overall positive rate for the virus was 56.4% (111/197) in Anhui Province. This is the first report of FSfaCVs identified in pigs in China, and further epidemiological studies are warranted to evaluate the influence of the virus on pigs.

Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschussifanicus).

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD), and its infection results in mortality of 70-90% in farmed and wild rabbits. RHDV is thought to replicate strictly in rabbits. However, there are also reports showing that gene segments from the RHDV genome or antibodies against RHDV have been detected in other animals. Here, we report the detection and isolation of a RHDV from diseased Alpine musk deer (Moschussifanicus). The clinical manifestations in those deer were sudden death without clinical signs and hemorrhage in the internal organs. To identify the potential causative agents of the disease, we used sequence independent single primer amplification (SISPA) to detect gene segments from viruses in the tissue samples collected from the dead deer. From the obtained sequences, we identified some gene fragments showing very high nucleotide sequence similarity with RHDV genome. Furthermore, we identified caliciviral particles using an electron microscope in the samples. The new virus was designated as RHDV GS/YZ. We then designed primers based on the genome sequence of an RHDV strain CD/China to amplify and sequence the whole genome of the virus. The genome of the virus was determined to be 7437 nucleotides in length, sharing the highest genome sequence identity of 98.7% with a Chinese rabbit strain HB. The virus was assigned to the G2 genotype of RHDVs according to the phylogenetic analyses based on both the full-length genome and VP60 gene sequences. Animal experiments showed that GS/YZ infection in rabbits resulted in the macroscopic and microscopic lesions similar to that caused by the other RHDVs. This is the first report of RHDV isolated from Alpine musk deer, and our findings extended the epidemiology and host range of RHDV.

A Highly Sensitive Piezoresistive Pressure Sensor Based on Graphene Oxide/Polypyrrole@Polyurethane Sponge.

In this work, polyurethane sponge is employed as the structural substrate of the sensor. Graphene oxide (GO) and polypyrrole (PPy) are alternately coated on the sponge fiber skeleton by charge layer-by-layer assembly (LBL) to form a multilayer composite conductive layer to prepare the piezoresistive sensors. The 2D GO sheet is helpful for the formation of the GO layers, and separating the PPy layer. The prepared GO/PPy@PU (polyurethane) conductive sponges still had high compressibility. The unique fragmental microstructure and synergistic effect made the sensor reach a high sensitivity of 0.79 kPa-1. The sensor could detect as low as 75 Pa, exhibited response time less than 70 ms and reproducibility over 10,000 cycles, and could be used for different types of motion detection. This work opens up new opportunities for high-performance piezoresistive sensors and other electronic devices for GO/PPy composites.

Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

Implication of the Identification of an Earlier Pseudorabies Virus (PRV) Strain HLJ-2013 to the Evolution of Chinese PRVs.

Pseudorabies viruses (PRVs) pose a great threat to the pig industry of many countries around the world. Human infections with PRV have also been reported occasionally in China. Therefore, understanding the epidemiology and evolution of PRVs is of great importance for disease control in the pig populations and humans as well. In this study, we isolated a PRV designated HLJ-2013 from PRV-positive samples that had been collected in Heilongjiang, China, in 2013. The full genome sequence of the virus was determined to be ∼143 kbp in length using high-throughput sequencing. The genomic sequence identities between this isolate and 21 other previous PRV isolates ranged from 92.4% (with Bartha) to 97.3% (with SC). Phylogenetic analysis based on the full-length genome sequences revealed that PRV HLJ-2013 clustered together with all the Chinese strains in one group belonging to Genotype II, but this virus occurred phylogenetically earlier than all the other Chinese PRV strains. Phylogenetic trees based on both protein-coding genes and non-coding regions revealed that HLJ-2013 probably obtained its genome sequences from three origins: a yet unknown parent virus, the European viruses, and the same ancestor of all Chinese PRVs. Recombination analysis showed that HLJ-2013-like virus possibly donated the main framework of the genome of the Chinese PRVs. HLJ-2013 exhibited cytopathic and growth characteristics similar to that of the Chinese PRV strains SC and HeN1, but its pathogenicity in mice was higher than that of SC and lower than that of HeN1. The identification of HLJ-2013 takes us one step closer to understanding the origin of PRVs in China and provides new knowledge about the evolution of PRVs worldwide.

One-step triplex PCR/RT-PCR to detect canine distemper virus, canine parvovirus and canine kobuvirus.

To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 102.1 TCID50 for CDV, 101.9 TCID50 for CPV and 103 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs.

A High Compressibility Pressure-Sensitive Structure Based on CB@PU Yarn Network.

In this work, a piezoresistive sensor structure based on carbon black (CB)@polyurethane (PU) yarn material was developed. Specifically, CB@PU yarn was constructed by the polymer-mediated water-based electrostatic deposition method. The distribution of the yarn was artificially controlled to fabricate conductive networks. The CB conductive layer was efficiently supported by the net-like structure of PU yarn, thus generating collaborative advantage. The as-fabricated pressure sensor not only displayed compressibility of over 97%, but also detected a wide pressure change from 25 Pa to 20 kPa. Furthermore, this sensor exhibited response time of less than 70 ms and reproducibility of over 10,000 cycles. The advantages of the CB@PU network ensured this pressure-sensitive structure enormous potential application in pressure sensitive equipment.

First report of feline bocavirus associated with severe enteritis of cat in Northeast China, 2015.

Feline bocavirus (FBoV) is a newly identified bocavirus of cats in the family Parvoviridae. A novel FBoV HRB2015-LDF was first identified from the cat with severe enteritis in Northeast China, with an overall positive rate of 2.78% (1/36). Phylogenetic and homologous analysis of the complete genome showed that FBoV HRB2015-LDF was clustered into the FBoV branch and closely related to other FBoVs, with 68.7-97.5% identities. And the genes of VP1, NPA and NS1 shared 70.7-97.6, 72.4-98.6 and 67.2-98.0% nucleotide identities with other FBoVs, respectively. The results suggested that the FBoVs could be divided into two distinct lineages, and the difference of nucleotide identities was >20-30% between the lineages.

Deletion of the B-B' and C-C' regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression.

Inverted terminal repeats (ITRs) of the adeno-associated virus (AAV) are essential for rescue, replication, packaging, and integration of the viral genome. While ITR mutations have been identified in previous reports, we designed a new truncated ITR lacking the B-B' and C-C' regions named as ITRΔBC and investigated its effects on viral genome replication, packaging, and expression of recombinant AAV (rAAV). The packaging ability was compared between ITRΔBC rAAV and wild-type (wt) ITR rAAV. Our results showed the productivity of ITRΔBC rAAV was reduced 4-fold, which is consistent with the 8-fold decrease in the replication of viral genomic DNA of ITRΔBC rAAV compared with wt ITR rAAV. Surprisingly, transgene expression was significantly higher for ITRΔBC rAAV. A preliminary exploration of the underlying mechanisms was carried out by inhibiting and degrading the ataxia telangiectasia mutated (ATM) protein and the Mre11 complex (MRN), respectively, since the rAAV expression was inhibited by the ATM and/or MRN through cis interaction or binding with wt ITRs. We demonstrated that the inhibitory effects were weakened on ITRΔBC rAAV expression. This study suggests deletion in ITR can affect the transgene expression of AAV, which provides a new way to improve the AAV expression through ITRs modification.

Feline Panleucopenia Virus NS2 Suppresses the Host IFN-ß Induction by Disrupting the Interaction between TBK1 and STING.

Feline panleucopenia virus (FPV) is a highly infectious pathogen that causes severe diseases in pets, economically important animals and wildlife in China. Although FPV was identified several years ago, little is known about how it overcomes the host innate immunity. In the present study, we demonstrated that infection with the FPV strain Philips-Roxane failed to activate the interferon ß (IFN-ß) pathway but could antagonize the induction of IFN stimulated by Sendai virus (SeV) in F81 cells. Subsequently, by screening FPV nonstructural and structural proteins, we found that only nonstructural protein 2 (NS2) significantly suppressed IFN expression. We demonstrated that the inhibition of SeV-induced IFN-ß production by FPV NS2 depended on the obstruction of the IFN regulatory factor 3 (IRF3) signaling pathway. Further, we verified that NS2 was able to target the serine/threonine-protein kinase TBK1 and prevent it from being recruited by stimulator of interferon genes (STING) protein, which disrupted the phosphorylation of the downstream protein IRF3. Finally, we identified that the C-terminus plus the coiled coil domain are the key domains of NS2 that are required for inhibiting the IFN pathway. Our study has yielded strong evidence for the FPV mechanisms that counteract the host innate immunity.

Molecular characterization of a feline calicivirus isolated from tiger and its pathogenesis in cats.

Feline calicivirus (FCV) is a virus that causes respiratory disease in cats. In this study, the FCV TIG-1 was isolated from Siberian tiger feces collected in 2014 in Heilongjiang Province, China. Phylogenetic analysis among TIG-1 and other FCVs showed that TIG-1 does not share the same lineage with other FCV isolates from Heilongjiang or other regions in China but is located in the same cluster with the FCV strain Urbana, which was isolated from the United States. The growth kinetics in vitro and the pathogenicity in cats between TIG-1 and the domestic cat-origin FCV strain F9 (vaccine strain) and strain 2280 were compared. We found that the growth kinetics of strains TIG-1 and 2280 were faster than that of strain F9 from 12h to 36h post-infection, indicating that strains TIG-1 and 2280 produce infectious virions and reach peak yields earlier. Challenge experiments in cats showed that TIG-1 grew faster than the other two strains in the lungs of cats and that TIG-1 is a virulent FCV with 100% morbidity and lethality. In addition, the histopathological results showed that the virulent TIG-1 strain directly led to severe lung tissue damage and indirectly led to intestinal damage. The results presented here show that a tiger-origin FCV exhibits high virulence in cats.

Characterization and pathogenicity of a novel mammalian orthoreovirus from wild short-nosed fruit bats.

Mammalian orthoreoviruses (MRVs) have a wide range of geographic distribution and have been isolated from humans and various animals. This study describes the isolation, molecular characterization and analysis of pathogenicity of MRV variant B/03 from wild short-nosed fruit bats. Negative stain electron microscopy illustrated that the B/03 strain is a non-enveloped icosahedral virus with a diameter of 70nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration patterns showed that the B/03 viral genome contains 10 segments in a 3:3:4 arrangement. The isolate belongs to MRV serotype 1 based on S1 gene nucleotide sequence data. BALB/c mice experimentally infected with B/03 virus by intranasal inoculation developed severe respiratory distress with tissue damage and inflammation. Lastly, B/03 virus has an increased transmission risk between bats and humans or animals.

In vitro antiviral effect of germacrone on feline calicivirus.

Feline calicivirus (FCV) often causes respiratory tract and oral disease in cats and is a highly contagious virus. Widespread vaccination does not prevent the spread of FCV. Furthermore, the low fidelity of the RNA-dependent RNA polymerase of FCV leads to the emergence of new variants, some of which show increased virulence. Currently, few effective anti-FCV drugs are available. Here, we found that germacrone, one of the main constituents of volatile oil from rhizoma curcuma, was able to effectively reduce the growth of FCV strain F9 in vitro. This compound exhibited a strong anti-FCV effect mainly in the early phase of the viral life cycle. The antiviral effect depended on the concentration of the drug. In addition, germacrone treatment had a significant inhibitory effect against two other reference strains, 2280 and Bolin, and resulted in a significant reduction in the replication of strains WZ-1 and HRB-SS, which were recently isolated in China. This is the first report of antiviral effects of germacrone against a calicivirus, and extensive in vivo research is needed to evaluate this drug as an antiviral therapeutic agent for FCV.