Construction of recombinant fluorescent LSDV for high-throughput screening of antiviral drugs.

Lumpy skin disease virus (LSDV) infection is a major socio-economic issue that seriously threatens the global cattle-farming industry. Here, a recombinant virus LSDV-ΔTK/EGFP, expressing enhanced green fluorescent protein (EGFP), was constructed with a homologous recombination system and applied to the high-throughput screening of antiviral drugs. LSDV-ΔTK/EGFP replicates in various kidney cell lines, consistent with wild-type LSDV. The cytopathic effect, viral particle morphology, and growth performance of LSDV-ΔTK/EGFP are consistent with those of wild-type LSDV. High-throughput screening allowed to identify several molecules that inhibit LSDV-ΔTK/EGFP replication. The strong inhibitory effect of theaflavin on LSDV was identified when 100 antiviral drugs were screened in vitro. An infection time analysis showed that theaflavin plays a role in the entry of LSDV into cells and in subsequent viral replication stages. The development of this recombinant virus will contribute to the development of LSDV-directed antiviral drugs and the study of viral replication and mechanisms of action.

In situ observation of heterogeneous catalytic organic reactions via aggregation-induced emission luminogens.

Herein, we demonstrated for the first time that customized aggregation-induced emission luminogens can be utilized to directly visualize heterogeneous catalytic organic reactions, which further enables catalyst screening. We used the retro-Diels-Alder reaction as a model reaction for illustration, and identified montmorillonite K10 as the best catalyst in our catalyst screening.

Hepatitis B virus detected in a golden monkey fatal case, China.

Hepatitis B virus (HBV) is distributed worldwide and poses a significant threat to human health. Cross-species transmission of HBV from human to non-human primates could occur, which has been confirmed in three individual events. In this study, HBV DNA was detected in one golden monkey fatal case in China. The following genetic sequencing and analysis demonstrated the virus had a close genetic relationship with HBV genotype C in humans. To our knowledge, this is the first report suggested that HBV is related with a non-human primate fatal case in China.

Novel parvovirus in cats, China.

Parvovirus is a common element of the feline virus group and usually causes gastroenteritis and leukopenia in cats. In this study, we identified a novel protoparvovirus from the Chinese domestic cats, which is genetically similar to canine bufavirus (98.0%-99.8%), but sharing low amino acid identities in the viral structural proteins 2 (VP2) (36.1-37.2%) to the well-known canine parvovirus type 2 and feline panleukopenia virus. This virus was provisionally designated as feline bufavirus (FBuV). Screening of fecal samples revealed a prevalence of 7.4% (19/257) in domestic cats. Diarrhea was present in 52.6% (10/19) of cats positive for FBuV. However, statistical analysis showed no association between FBuV and clinical signs. VP2 gene of the 19 field FBuV was sequenced and phylogenetic analysis demonstrated that FBuV determined from China had a genetic diversity. This study will strengthen the understanding of the epidemiology and genetic diversity of bufavirus and provide a foundation for further studies.

An Adaptable Cryptosystem Enabled by Synergies of Luminogens with Aggregation-Induced-Emission Character.

The strong emission in the solid state and the feasibility of introducing stimuli responsiveness make aggregation-induced-emission luminogens promising for optical information encryption. Yet, the vast majority of previous reports rely on subtle changes in the molecular conformation or intermolecular interactions, limiting the robustness, multiplicity, capacity, and security of the resulting cryptosystems. Herein, a versatile cryptographic system is presented based on three interconnected and orthogonal covalent transformations concerning a tetraphenylethylene-maleimide conjugate. The cryptosystem is adapted into four configurations with different functionalities by organizing the reactions and molecules in different ways. These variants either balance the accessibility and security of the encrypted information or improve the security and density in data encryption. Significantly, they allow variable decryption from a single encryption and reconstruction of the chemical nature hidden in the fluorescent pattern can only be accessed through given algorithms. These results highlight the importance of multi-component synergies in advancing information encryption systems, which is enabled by the robustness and diversity stemming from the covalent nature of these transformations.

First identification and genomic characterization of equine hepacivirus sub-type 3 strain in China.

Equine Hepacivirus (EqHV) is a newly discovered equine virus that is classified under the Hepacivirus genus of the Flaviviridae family. There are three sub-types of EqHV worldwide namely; sub-types 1-3. The majority of EqHV sub-type 1 strains were found in China. While different sub-types have been found in Japan and USA, therefore, to investigate whether the other sub-types of EqHV strains were present in China, a total of 60 horse serum samples were collected and screened for EqHV RNA through RT-PCR. The results revealed that 19 serum samples were RNA-positive (19/60) and the EqHV detection rate was 31.67%. One EqHV strain named GD23 was obtained and its near-complete genome sequence was acquired. Analysis of nucleotide p-distance with reference to the entire polyprotein gene revealed that GD23 was classified into sub-type 3. In addition, the phylogenetic analysis demonstrated that GD23 was clustered together with EqHV strains of sub-type 3 in other countries. The present study is the first to identify an EqHV sub-type 3 strain in China.

Genomic sequencing and characterization of a novel group of canine bufaviruses from Henan province, China.

Canine bufavirus (CBuV) is a novel protoparvovirus of dogs that was first reported in 2018 in Italy. The prevalence and genetic diversity of CBuV in China are not clear. In this study, a total of 115 canine fecal samples were collected from northern China in 2019, and two of the samples tested positive for CBuV DNA by PCR. These two field CBuV strains were designated Henan38 and Henan44. The complete genomic sequences of Henan38 and Henan44 were obtained by gap-filling PCR, sequenced, and assembled. Phylogenetic analysis demonstrated that the two strains clustered together in a novel group that was distant from previously reported CBuV strains. This study will strengthen our understanding of the epidemiology and genetic diversity of CBuV in China.

Synthesis of AB n -type colloidal molecules by polymerization-induced particle-assembly (PIPA).

Conventional synthesis of colloidal molecules (CMs) mainly depends on particle-based self-assembly of patchy building blocks. However, direct access to CMs by the self-assembly of isotropic colloidal subunits remains challenging. Here, we report the mass production of AB n -type CMs by polymerization-induced particle-assembly (PIPA), using a linear ABC triblock terpolymer system. Starting from diblock copolymer spheres, the association of spheres takes place in situ during the polymerization of the third block. The third blocks aggregate into attractive domains, which connect spheres into CMs. The stability of CMs is ensured, as long as the conversions are limited to ca. 50%, and the pH is low. The valence of AB n -type CMs (n = 2-6) is determined by the volume ratio of the polymer blocks. By tuning the volume ratio, 78.5% linear AB2-type CMs are yielded. We demonstrate that polymerization-induced particle-assembly is successful for the scalable fabrication of AB n -type CMs (50 g L-1), and can be easily extended to vastly different triblock terpolymers, for a wide range of applications.

Corrigendum: Emergence of Getah Virus Infection in Horse With Fever in China, 2018.

[This corrects the article DOI: 10.3389/fmicb.2019.01416.].

Emergence of Getah Virus Infection in Horse With Fever in China, 2018.

Getah virus (GETV) is a mosquito-borne virus that was first determined in Malaysia in 1955, and can infect humans and multiple other mammals. GETV infection in horses has been reported in Japan and India, and causes great economic losses. In China, GETV has been identified in mosquitoes, pigs, foxes, and cattle with a wide geographical distribution, but has not been detected in horses. In August 2018, a sudden onset of fever was observed in racehorse in an equestrian training center in Guangdong Province in southern China. Blood samples were collected from the sick horse, and PCR/RT-PCR analysis was performed to screen for equine viral pathogens associated with fever. The results indicated that the samples were GETV RNA positive. After RT-PCR, sequencing, and assembly, the genome of the first Chinese horse-derived GETV strain, GZ201808, was obtained. Compared with the genome sequences of other GETV strains, twelve unique nucleotide substitutions were observed in GZ201808. The genome of GZ201808 had the highest genetic identity (99.6%) with AH9192, which was detected in pigs in China in 2017. Phylogenetic analysis indicated that GZ201808 clustered in Group III, and was located in an independent branch distant from other horse-derived GETV strains, indicating a unique evolutionary pattern of GZ201808. This study first determined and described the disease course of horse infected with GETV in China, sequenced and characterized the genome of the field horse-derived GETV strain, and therefore presented an unequivocal report of GETV infection in horses in China.

CO2-breathing and piercing polymersomes as tunable and reversible nanocarriers.

Despite numerous studies on utilizing polymeric vesicles as nanocapsules, fabrication of tunable molecular pathways on transportable vesicle walls remains challenging. Traditional methods for building penetrated channels on vesicular membrane surface often involve regulating the solvent polarity or photo-cross-linking. Herein, we developed a neat, green approach of stimulation by using CO2 gas as "molecular drill" to pierce macroporous structures on the membrane of polymersomes. By simply introducing CO2/N2 gases into the aqueous solution of self-assemblies without accumulating any byproducts, we observed two processes of polymeric shape transformation: "gas breathing" and "gas piercing." Moreover, the pathways in terms of dimension and time were found to be adjustable simply by controlling the CO2 stimulation level for different functional encapsulated molecules in accumulation, transport, and releasing. CO2-breathing and piercing of polymersomes offers a promising functionality to tune nanocapsules for encapsulating and releasing fluorescent dyes and bioactive molecules in living systems and also a unique platform to mimic the structural formation of nucleus pore complex and the breathing process in human beings and animals.

Self-polymerization of dopamine and polyethyleneimine: novel fluorescent organic nanoprobes for biological imaging applications.

The development of novel fluorescent nanoprobes has attracted great current research interest over the past few decades due to their superior optical properties and multifunctional capability as compared with small organic dyes. Although great advance has been made in the utilization of fluorescent nanoprobes for biomedical applications, development of novel fluorescent nanoprobes that possess good fluorescent properties, biocompatibility, biodegradability and water dispersibility through a convenient and effective route is still highly desirable. In this work, we reported for the first time that novel fluorescent organic nanoparticles (FONs) can be conveniently fabricated via self-polymerization of dopamine and polyethyleneimine at room temperature and in an air atmosphere within 2 h. These FONs exhibited strong green fluorescence, high water stability and excellent biocompatibility, making them highly potential for biological imaging applications. More importantly, due to the high reactivity of polydopamine, these FONs might also be further functionalized with other functional components through Michael addition or Schiff base reaction. Therefore the method described in this work would open new avenues for the fabrication of fluorescent nanoprobes for various biomedical applications.

Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling.

One-second-long increases in prefrontal cholinergic activity ("transients") were demonstrated previously to be necessary for the incorporation of cues into ongoing cognitive processes ("cue detection"). Nicotine and, more robustly, selective agonists at alpha4beta2* nicotinic acetylcholine receptors (nAChRs) enhance cue detection and attentional performance by augmenting prefrontal cholinergic activity. The present experiments determined the role of beta2-containing and alpha7 nAChRs in the generation of prefrontal cholinergic and glutamatergic transients in vivo. Transients were evoked by nicotine, the alpha4beta2* nAChR agonist ABT-089 [2-methyl-3-(2-(S)-pyrrolindinylmethoxy) pyridine dihydrochloride], or the alpha7 nAChR agonist A-582941 [2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole]. Transients were recorded in mice lacking beta2 or alpha7 nAChRs and in rats after removal of thalamic glutamatergic or midbrain dopaminergic inputs to prefrontal cortex. The main results indicate that stimulation of alpha4beta2* nAChRs evokes glutamate release and that the presence of thalamic afferents is necessary for the generation of cholinergic transients. ABT-089-evoked transients were completely abolished in mice lacking beta2* nAChRs. The amplitude, but not the decay rate, of nicotine-evoked transients was reduced by beta2* knock-out. Conversely, in mice lacking the alpha7 nAChR, the decay rate, but not the amplitude, of nicotine-evoked cholinergic and glutamatergic transients was attenuated. Substantiating the role of alpha7 nAChR in controlling the duration of release events, stimulation of alpha7 nAChR produced cholinergic transients that lasted 10- to 15-fold longer than those evoked by nicotine. alpha7 nAChR-evoked cholinergic transients are mediated in part by dopaminergic activity. Prefrontal alpha4beta2* nAChRs play a key role in evoking and facilitating the transient glutamatergic-cholinergic interactions that are necessary for cue detection and attentional performance.

Enhancement of attentional performance by selective stimulation of alpha4beta2(*) nAChRs: underlying cholinergic mechanisms.

Impairments in attention are a major component of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Using an operant sustained attention task (SAT), including a distractor condition (dSAT), we assessed the putative pro-attentional effects of the selective alpha4beta2(*) nicotinic acetylcholine receptor (nAChR) agonist S 38232 in comparison with the non-selective agonist nicotine. Neither drug benefited SAT performance. However, in interaction with the increased task demands implemented by distractor presentation, the selective agonist, but not nicotine, enhanced the detection of signals during the post-distractor recovery period. This effect is consistent with the hypothesis that second-long increases in cholinergic activity ('transients') mediate the detection of cues and that nAChR agonists augment such transients. Electrochemical recordings of prefrontal cholinergic transients evoked by S 38232 and nicotine indicated that the alpha4beta2(*) nAChR agonist evoked cholinergic transients that were characterized by a faster rise time and more rapid decay than those evoked by nicotine. Blockade of the alpha7 nAChR 'sharpens' nicotine-evoked transients; therefore, we determined the effects of co-administration of nicotine and the alpha7 nAChR antagonist methyllycaconitine on dSAT performance. Compared with vehicle and nicotine alone, this combined treatment significantly enhanced the detection of signals. These results indicate that compared with nicotine, alpha4beta2(*) nAChR agonists significantly enhance attentional performance and that the dSAT represents a useful behavioral screening tool. The combined behavioral and electrochemical evidence supports the hypothesis that nAChR agonist-evoked cholinergic transients, which are characterized by rapid rise time and fast decay, predict robust drug-induced enhancement of attentional performance.

Lesions of the entorhinal cortex or fornix disrupt the context-dependence of fear extinction in rats.

Recent studies have shown that the hippocampus is critical for the context-dependent expression of extinguished fear memories. Here we used Pavlovian fear conditioning in rats to explore whether the entorhinal cortex and fornix, which are the major cortical and subcortical interfaces of the hippocampus, are also involved in the context-dependence of extinction. After pairing an auditory conditional stimulus (CS) with an aversive footshock (unconditional stimulus or US) in one context, rats received an extinction session in which the CS was presented without the US in another context. Conditional fear to the CS was then tested in either the extinction context or a third familiar context; freezing behavior served as the index of fear. Sham-operated rats exhibited little conditional freezing to the CS in the extinction context, but showed a robust renewal of fear when tested outside of the extinction context. In contrast, rats with neurotoxic lesions in the entorhinal cortex or electrolytic lesions in the fornix did not exhibit a renewal of fear when tested outside the extinction context. Impairments in freezing behavior to the auditory CS were not able to account for the observed results, insofar as rats with either entorhinal cortex or fornix lesions exhibited normal freezing behavior during the conditioning session. Thus, contextual memory retrieval requires not only the hippocampus proper, but also its cortical and subcortical interfaces.

Differential roles for hippocampal areas CA1 and CA3 in the contextual encoding and retrieval of extinguished fear.

Recent studies demonstrate that context-specific memory retrieval after extinction requires the hippocampus. However, the contribution of hippocampal subfields to the context-dependent expression of extinction is not known. In the present experiments, we examined the roles of areas CA1 and CA3 of the dorsal hippocampus in the context specificity of extinction. After pairing an auditory conditional stimulus (CS) with an aversive footshock (unconditional stimulus or US), rats received extinction sessions in which the CS was presented without the US. In Experiment 1, pretraining neurotoxic lesions in either CA1 or CA3 eliminated the context dependence of extinguished fear. In Experiment 2, lesions of CA1 or CA3 were made after extinction training. In this case, only CA1 lesions impaired the context dependence of extinction. Collectively, these results reveal that both hippocampal areas CA1 and CA3 contribute to the acquisition of context-dependent extinction, but that only area CA1 is required for contextual memory retrieval.

Stimulation of alpha2-adrenoceptors suppresses excitatory synaptic transmission in the medial prefrontal cortex of rat.

Stimulation of alpha2-, especially alpha2A-adrenoceptor (AR), in the prefrontal cortex (PFC) produces a beneficial effect on cognitive functions such as working memory. Alpha2-adrenergic agonists like clonidine and guanfacine have been used experimentally and clinically for treatment of psychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD) and schizophrenia. However, the neurophysiological actions of alpha2-ARs in the PFC are poorly understood. In the present study, we recorded field excitatory post-synaptic potential (fEPSP) and evoked excitatory post-synaptic current (eEPSC) in the medial prefrontal cortex (mPFC) of rats, using in vivo field-potential recording and in vitro whole-cell patch-clamp recording techniques, and examined the effects of the alpha2-AR agonist clonidine and the selective alpha2A-AR agonist guanfacine on fEPSP and eEPSC. Systemic or intra-mPFC application of clonidine or guanfacine significantly reduced fEPSP in the mPFC, either in anesthetized or freely moving rats. Consistently, bath-application of guanfacine suppressed eEPSC in layer V/VI pyramidal neurons, and this effect was blocked by the alpha2-AR antagonist yohimbine or the Gi inhibitor NF023. Moreover, treatment with guanfacine had no effect on paired-pulse facilitation (PPF) of fEPSP and eEPSC. The present study provides the first electrophysiological evidence that stimulation of alpha2A-AR inhibits excitatory synaptic transmission in the mPFC through a post-synaptic mechanism.

Hippocampal involvement in contextual modulation of fear extinction.

Extinction of fear conditioning in animals is an excellent model for the study of fear inhibition in humans. Substantial evidence has shown that extinction is a new learning process that is highly context-dependent. Several recovery effects (renewal, spontaneous recovery, and reinstatement) after extinction suggest that the contextual modulation of extinction is a critical behavioral mechanism underlying fear extinction. In addition, recent studies demonstrate a critical role for hippocampus in the context control of extinction. A growing body of evidence suggests that the hippocampus not only plays a role in contextual encoding and retrieval of fear extinction memories, but also interacts with other brain structures to regulate context-specificity of fear extinction. In this article, the authors will first discuss the fundamental behavioral features of the context effects of extinction and its underlying behavioral mechanisms. In the second part, the review will focus on the brain mechanisms for the contextual control of extinction.

Ventral hippocampal muscimol disrupts context-specific fear memory retrieval after extinction in rats.

Research aimed at understanding Pavlovian fear memory extinction has yielded considerable insight into the conditions under which fear memories may become inhibited. After extinction, Pavlovian fear memory retrieval is context-specific. Fear memories are not expressed in the extinction context, but they are expressed in every other context. Research indicates that the dorsal hippocampus mediates the context-specific expression of fear memory, but the role of the ventral hippocampus in mediating this process is unknown. Insofar as the ventral hippocampus is involved in the acquisition and expression of both context and tone fear, we asked whether GABA systems in the ventral hippocampus mediate context-specific fear memory retrieval after extinction. Experiment 1 showed that ventral hippocampal inactivation with muscimol disrupted context-specific fear memory retrieval. Experiment 2 showed that rats infused with muscimol can discriminate a context in which they were shocked from a neutral context. Nonetheless, they do appear to have a mild impairment in this task. Experiment 3 showed that ventral hippocampal muscimol did not disrupt locomotor activity, but did result in a slight increase in freezing and grooming, an effect that cannot explain the context-specific retrieval deficit demonstrated in experiment 1. These data are consistent with a role for the ventral hippocampus in mediating context-specific fear memory retrieval.

Electrolytic lesions of the dorsal hippocampus disrupt renewal of conditional fear after extinction.

There is a growing body of evidence that the hippocampus is critical for context-dependent memory retrieval. In the present study, we used Pavlovian fear conditioning in rats to examine the role of the dorsal hippocampus (DH) in the context-specific expression of fear memory after extinction (i.e., renewal). Pre-training electrolytic lesions of the DH blunted the expression of conditional freezing to an auditory conditional stimulus (CS), but did not affect the acquisition of extinction to that CS. In contrast, DH lesions impaired the context-specific expression of extinction, eliminating the renewal of fear normally observed to a CS presented outside of the extinction context. Post-extinction DH lesions also eliminated the context dependence of fear extinction. These results are consistent with those using pharmacological inactivation of the DH and suggest that the DH is required for using contextual stimuli to regulate the expression of fear to a Pavlovian CS after extinction.