AbstractPorcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172–554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

Porcine enterovirus G (EV-G) is endogenous to most pig farming countries worldwide. Reports that a papain-like protease (PLP) gene has been naturally inserted into the 2C/3A junction region of the EV-G genome, has increased the potential public health threats from this virus. We constructed a full-length infectious cDNA clone of EV-G, CH/17GXQZ/2017, in order to determine the packaging capacity at the 2C/3A insertion site. Subsequently, recombinants viruses containing the coding tags, GFP, iLOV and His at the 2C/3A junction region, were synthesized. The infectious virus was successfully rescued only with the insertion of the His-tag, which displayed similar virological and molecular properties to its parental strain. This study determined the packaging capacity of the 2C/3A insertion site, and it provides a practical tool for studying the functions and pathogenic mechanisms of EV-G in pigs.

The current epidemic of porcine epidemic diarrhea virus (PEDV) G2 has caused huge economic losses to the pig industry. Evaluation for low virulence of the PEDV strains of subgroup G2a would be useful for the future development of effective vaccines.

纤突(Spike,S)蛋白是猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)诱导机体产生抗体的主要抗原,为制备其重组蛋白和多克隆抗体,本实验对分离株CHN/20GXNN-1/2020 的S基因进行序列分析,选择抗原指数较高的b区S1b,克隆至表达载体pET-32a进行原核表达,重组蛋白S1b主要以包涵体形式表达,大小约为 34.1 kDa.利用Ni-NTA琼脂糖树脂纯化的重组蛋白免疫新西兰大白兔,制备多克隆抗体.通过间接ELISA测定多克隆抗体的效价高于 1∶128000,Western blot和IFA均证明其可以与病毒S蛋白特异性结合.本研究制备的多克隆抗体特异性良好,为进一步研发PDCoV的检测方法和研究S蛋白的功能奠定了基础.

Since 2010, porcine epidemic diarrhea virus (PEDV) has swept across China and spread throughout the country, causing huge economic losses. In this study, 673 diarrhea samples from 143 pig farms in Guangxi during 2017–2022 were collected and detected for PEDV. Ninety-eight strains were selected for S1 gene analyses and these strains were classified into four subgroups (G1b, G2a, G2b and G2c), accounting for 1.02 (1/98), 75.51 (74/98), 16.33 (16/98) and 7.14% (7/98) of the total, respectively. Importantly, an increased number of strains in the G2c subgroup was found from 2019 onwards. Bayesian analysis revealed that Guigang may have been the epicenter of PEDVs in Guangxi. In addition, Guigang was identified as the primary hub from which PEDVs spread via two routes, namely Guigang–Wuzhou and Guigang–Laibin. Moreover, several coinfections of novel PEDV variants bearing large deletions in the partial S1 protein and PEDVs possessing an intact partial S1 protein were found in pigs. Further recombination analyses indicated that two of the strains, 18-GXNN-6 and 19-GXBH-2, originated from intra-genogroup recombination. Together, our data revealed a new profile of PEDV in Guangxi, China, which enhances our understanding of the distribution, genetic characteristics and evolutionary profile of the circulating PEDV strains in China.

针对猪德尔塔冠状病毒(PDCoV)的N基因建立检测PDCoV的SYBR Green Ⅰ荧光定量PCR方法,并验证了其特异性、敏感性和重复性等.结果显示,本方法特异性高,与猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪轮状病毒、猪肠病毒、伪狂犬病病毒、猪嵴病毒、猪繁殖与呼吸综合征病毒、猪星状病毒等病原无交叉反应.本方法Ct值与标准模板浓度在108.5～103.5PFU/mL范围内呈良好的线性关系,相关系数(R2)为0.994 9,直线方程为y=-3.594 9x+41.006,最低检测限为102.50 PFU/mL.运用该方法检测PDCoV感染不同来源细胞系的病毒载量,发现的PDCoV感染宿主谱广,能感染不同来源的细胞,且在LLC-PK细胞上的复制能力最强.本研究建立的方法为PDCoV的防控提供了技术支持.

为调查广西地区猪肠病毒G型(EV-G)基因组中是否存在PLpro基因的自然插入,并获得序列信息,本研究对2019-2021年广西区内347份临床腹泻样品进行EV-G PLpro基因的监测,发现32份EV-G阳性,其中4份EV-G阳性样品含有PLpro基因.扩增PLpro基因及VP1基因序列分析结果显示,4株PLpro序列与EV-G PLpro参考序列中均存在具有去泛素化酶活性的催化三联体残基,且蛋白两端都有3C蛋白酶水解位点,负责将外源蛋白水解至基因组外完成翻译.VP1基因序列分析结果显示,4株分别为EV-G1、EV-G10和EV-G14基因型.本研究首次在我国发现EV-G10及EV-G14基因型毒株的基因组中存在PLpro基因的插入,该结果为初步明确广西区内EV-G-PLpro毒株的流行概况及其遗传演化特点提供了数据支持.

猪轮状病毒(porcine rotavirus,PoRV)感染仔猪可引起腹泻、脱水甚至死亡,是引起仔猪腹泻的常见病原,给养猪业造成巨大的经济损失.本研究为确定导致广西贵港某猪场仔猪腹泻的病原,采集腹泻仔猪的肠内容物,采用PCR方法检测猪流行性腹泻病毒(porcine epidemic virus,PEDV)、猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)以及PoRV病原,结果显示,仅PoRV为阳性,将其接种至Marc-145细胞,盲传4代后,可产生细胞皱缩、脱落等明显细胞病变.稳定盲传20代,每隔5代进行PoRV的检测,选取第10代的病毒上清液进行VP4、VP6和VP7基因测序及遗传进化分析,确定该分离株为G9 P[7]型.电镜观察,可见到大小约为70 nm的轮状病毒粒子,符合轮状病毒特征,进一步鉴定分离株为PoRV.结果 表明,PoRV的分离鉴定为其分子生物学特性研究以及该病的有效防控奠定了基础,也为疫苗的研发提供参考.

Enterovirus G (EV-G) infects porcine populations worldwide and the infections are generally asymptomatic, with the insertion of the papain-like cysteine protease gene (PLCP) increasing the potential public health threats. However, the genetic and pathogenic characteristics of EV-G itself are not fully understood as yet. In the present study, one EV-G strain, named CH/17GXQZ/2017, was isolated and purified from piglets with diarrheic symptoms from the Guangxi Province, China. This strain produced stable cytopathic effects on Marc-145 cells with a titer of 5 × 106 PFU/mL. The spherical enterovirus particles with diameters of 25–30 nm were observed by using transmission electron microscopy. The whole genome sequence of the CH/17GXQZ/2017 strain consists of 7,364 nucleotides, and the phylogenetic tree based on the amino acid sequences of VP1 indicated this strain was clustered to the G1 genotype. Seven-day-old piglets were inoculated orally with the CH/17GXQZ/2017 strain in order to evaluate its pathogenicity. Although none of the infected piglets died during the experiment, clinical neurological symptoms were observed manifesting as mild hyperemia and Nissl bodies vacuolization in the cerebrum. In addition, the infection with the CH/17GXQZ/2017 strain decelerated the weight gain of suckling piglets significantly. This study demonstrates that CH/17GXQZ/2017 is pathogenic to neonatal piglets and advance knowledge on the biological characteristics, evolution and pathogenicity of EV-G.

Aim To investigate the relationship between ClC-3 chloride channels and Ca2+ movement induced by thapsigargin(TG)in PC12 cells.Methods The concentration of intracellular free calcium([Ca2+]i)expressed as the ratio value(Intensity340/Intensity380)was determined with Fura-2/AM probe.Results The overexpression of ClC-3 protein caused a significant decrease in TG-induced Ca2+ influx compared with control cells,and cells transfected with pcDNA3.1,whereas the [Ca2+]i at resting level and at peak level was not different among all groups(P0.05).SK F96365 at the concentration of 5～20 μmol·L-1 inhibited the Ca2+ influx induced by 1.0 μmol·L-1 Thapsigargin in a concentration-dependent manner.The inhibitory effect of SK F96365 on Ca2+ influx was decreased by overexpression of ClC-3 protein.Conclusion ClC-3 chloride channel was involved in the regulation of store-operated Ca2+ entry(SOCE).

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">目的探讨ClC-3氯通道与Thapsigargin（TG）触发的Ca2+运动的关系。方法在PC12细胞中转染全长ClC-3cDNA,利用生物荧光影像分析系统测定胞质Ca2+技术探讨ClC-3氯通道对TG触发的Ca2+运动的影响。结果与对照组相比,ClC-3蛋白过表达对TG触发的PC12细胞静息[Ca2+]i的Ratio值和Ca2+释放的Ratio值无影响（P&gt;0.05）。但使Ca2+内流量明显降低（P&lt;0.05）。SK&amp;F96365可以浓度依赖的抑制TG触发的Ca2+内流,但与对照细胞及空载体转染细胞相比,SK&amp;F96365对ClC-3蛋白过表达细胞Ca2+内流的抑制作用明显减弱（P&lt;0.05）。结论ClC-3蛋白参与TG触发的经Ca2+池操纵的Ca2+内流（store-operated Ca2+entry,SOCE）调节。</span>

Aim: To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. Methods: Echocardiography was used to confirm the establishment of the cardiac hypertrophy model. The confocal microscopy and fluorescent indicator Fluo-3 was applied to examine the intracellular Ca2+ concentration ([Ca2+](i)), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results: L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+](i) increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. Conclusion: The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

Aim: To examine how the sarcoplasmic reticulum (SR) Ca 2+ content changes and the underlying mechanism in L -thyroxin-induced cardiac hypertrophy. Methods: Echocardiography was used to confirm the establishment of the cardiac hypertrophy model. The confocal microscopy and fluorescent indicator Fluo-3 was applied to examine the intracellular Ca 2+ concentration ([Ca 2+ ] i ), the Ca 2+ sparks, and the caffeine-induced Ca 2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca 2+ -ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results: L -thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca 2+ transient in the Ca 2+ -free solution was detected. The spontaneous Ca 2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca 2+ ] i increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca 2+ -ATPase was decreased in the hypertrophied ventricle tissue. Conclusion: The results suggested that the reduced SR Ca 2+ content may be associated with an increased Ca 2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca 2+ handling during hypertrophy in hyperthyroidism rats.

【Objective】 To investigate the effects of ClC-3 chloride channels on the Ca2+ movement induced by thapsigargin (TG) in PC12 cells transfected with ClC-3 oligonucleotides. 【Methods】 The concentration of intracellular free calcium ([Ca2+]i ) expressed as the ratio value (Intensity340 / Intensity380) was determined with Fura-2 / AM probe. 【Results】 The results showed that transient transfection of PC12 cells with antisense oligonucleotide specific to ClC-3 caused a significant increase in TG-induced Ca2+ influx compared with that in control cells, and cells transfected with lipofectamine, missense or sense oligonucleotide, whereas the [Ca2+]i at resting level and at peak level were not different among all groups (P>0.05). SK&F96365, a blocker for store- operated calcium channels (SOCC), inhibited the Ca2+ influx induced by 1.0 μmol / L thapsigargin in PC12 cells in a concentration-dependent manner. The inhibitory effect of SK&F96365 on Ca2+ influx was enhanced by antisense oligonucleotide as compared with Lipofectamine, sense or missense oligonuclotide. 【Conclusion】 ClC-3 chloride channels was involved in TG-induced store-operated Ca2+ entry(SOCE), but had no effects on the [Ca2+]i at resting level and Ca2+ release from Ca2+ stores.

Objectives: Previously, we have found that the ClC-3 chloride channel is involved in endothelin-1 (ET-1)-induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC-3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC-3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET-1-induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC-3 protein. Silence of ClC-3 by siRNA inhibited expression of ClC-3 protein, prevented an increase in BrdU incorporation and cell number induced by ET-1. Silence of ClC-3 also caused cell cycle arrest in G(0)/G(1) phase and prevented the cells' progression from G(1) to S phase. Knockdown of ClC-3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin-dependent kinase inhibitors (CDKIs) p27(KIP) and p21(CIP) expression. Furthermore, ClC-3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase-3 beta (GSK-3 beta) induced by ET-1. Conclusion: Silence of ClC-3 protein effectively suppressed phosphorylation of the Akt/GSK-3 beta signal pathway, resulting in down-regulation of cyclin D1 and cyclin E, and up-regulation of p27(KIP) and p21(CIP). In these BASMCs, integrated effects lead to cell cycle G(1)/S arrest and inhibition of cell proliferation.

Aim:To investigate the effect of ClC-3 antisense oligonucleotide on the proliferation of PC12 cells.Methods:PC12 cells were grouped to transfect 6.25,12.5,25.0,50.0,100.0 and 200.0 mg/L ClC-3 antisense oligonucleotide,50 mg/L ClC-3 sense oligonucleotide,50 mg/L ClC-3 missense oligonucleotide,respectively,cells without any transfection and transfected 10 mg/L Lipofectamine2000were the control,MTT and [3H]-TdR incorporation assay were used to measure the viability and DNA synthesize,and cell cycle was studied with flow cytometry analysis.Results:Compared with the control,ClC-3 antisense oligonucleotide could decreased the viability of PC12 cells,and the [3H]-TdR incorporation into DNA,and increased the numbers of the cells in G1 stage but decreased that in S stage(P0.05).Whereas lipofectamine,missense or sense oligonucleotide transfection had no effects on the proliferation of PC12 cells(P0.05).Conclusion:ClC-3 antisense oligonucleotide could arrest PC12 cells in G0/G1 stage which results in the inhibition of PC12 cells proliferation.

Aim To investigate the method of cell culture for smooth muscle cells from rat cerebral basilar artery and understand cells growth and biological characteristics.Methods The explant attached method was applied for cell culture of rat basilar artery smooth muscle cells(BASMCs).The cultured BASMCs were identified by immunocytochemical staining.The activities of cells were indicated by the dynamic changes of intracellular calcium concentration observed by RF-5 000 fluorospectro-photometer.Results BASMCs grew out of tissue blocks by 5 days,reached confluency could be subcultured after 2 weeks.Cultured cells were identified by intensely positive immunocytochemical staining to smooth muscle actin-specific.Introduction of calcium channel agonists induced significant increase in Fura-2 fluorescence ratio(F340/F380)and cells were in good condiction.Conclusion Explant attached method is simple,efficient and economic.It provides an ideal cell model for the study of pathogenesis of the cerebral vascular diseases.

The cerebrovascular remodeling is a prominent feature of hypertension and considered a major risk factor for stroke. Cerebrovascular smooth muscle cells meet volume challenge during this pathophysiological process. Our previous studies suggest that volume regulated chloride channels may be critical to the cell cycle of vascular smooth muscle cells. However, it is unknown whether the volume-regulated chloride movement is altered in hypertension. Therefore, we directly measured the concentration of intracellular chloride ([Cl(-)](i)) in rat basilar arterial smooth muscle cells isolated from control rats and rats that were made hypertensive for 1 to 12 weeks after partial renal artery constriction (2-kidney, 2-clip method) using a 6-methoxy-N-ethylquinolinium iodide fluorescence probe. The [Cl(-)](i) in isotonic solution showed no difference in all of the groups. After hypotonic perfusion, the reduction in [Cl(-)](i) was more prominent in hypertensive cerebrovascular smooth muscle cells than in sham control cells. Genistein, a protein tyrosine kinase inhibitor, inhibited hypotonic-induced reduction in [Cl(-)](i), whereas sodium orthovanadate, a protein-tyrosine phosphatase inhibitor, enhanced hypotonic-induced reduction in [Cl(-)](i) in both groups. The percentage inhibition of reduction in [Cl(-)](i) by genistein on volume-regulated chloride movement has a positive correlation with blood pressure levels in the 2-kidney, 2-clip hypertensive group, as is the case for the percentage increase of reduction in [Cl(-)](i) by sodium orthovanadate. Antihypertensive therapy with the angiotensin-converting enzyme inhibitor captopril completely reversed abnormal volume-regulated chloride movement in hypertensive rats. We conclude that volume-regulated chloride movement is augmented in rat cerebrovascular smooth muscle cells in proportion to the severity of hypertension.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">目的探讨Cd2+在Fura-2荧光测定胞质Ca2+过程中对荧光的干扰作用。方法培养的PC12细胞,在无钙溶液中利用Fura-2荧光探针测定胞质内Ca2+浓度。结果在thapsigargin诱导Fura-2荧光比值（F340/F380）上升后,CdC l2可以导致F340/F380进一步升高。将细胞膜破坏,Fura-2从胞质内溢出,CdC l2依然可以导致荧光比值明显上升。在无PC12细胞,无Ca2+的溶液中,加入Fura-2/AM,100μmol.L-1CdC l2可以导致F340/F380明显升高。结论Cd2+对Fura-2及Fura-2/AM都存在荧光干扰现象,使荧光比值增加。</span>

Aim: To test the hypothesis that Cl − channel blockers affect T cell proliferation through Ca 2+ -release-activated Ca 2+ (CRAC) signaling and examine the effects of the combination of a CRAC channel blocker and a Cl − channel blocker on concanavalin A (ConA; 5 mg/mL)-induced Ca 2+ signaling, gene expression and cellular proliferation in human peripheral T lymphocytes. Methods: [ 3 H]Thymidine incorporation, Fura-2 fluorescent probe, RNase protection assay, and reverse transcription-polymerase chain reaction were used. Results: The Cl − channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited ConA-induced Ca 2+ influx, interleukin-2 mRNA expression and T lymphocyte proliferation in a concentration-dependent manner, and also enhanced the inhibitory effects of 1-{beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1 H -imida-zole (SK&F96365) on the above key events during T cell activation. A combination of DIDS (1 μmol/L) and SK&F96365 (1 μmol/L) significantly diminished ConA-induced ClC-3 mRNA expression by 64%, whereas DIDS(1 μmol/L) or SK&F96365 (1 μmol/L) alone decreased ConA-induced ClC-3 mRNA expression by only 16% and 9%, respectively. Conclusion: These results suggest that there is an interaction between CRAC-mediated Ca2+ signaling and DIDS-sensitive Cl − channels during ConA-induced T cell activation and proliferation. Moreover, the DIDS-sensitive Cl − channels may be related to the ClC-3 Cl − channels.