Study on Anti-viral Effect of Rhubarb on Porcine Epidemic Diarrhea Virus in vitro

The inhibitory effects of rhubarb on cell lesion, which was caused by porcine epidemic diarrhea virus (PEDV), and its mechanism had been studied in this research.The maximum safe concentration of rhubarb solution applied to Vero cells was determined through morphological observation, and then the PEDV infected Vero cell model in vitro was established, by which the inhibitory effect of rhubarb on virus was studied.The experimental method had also been established based on this cell model.The result showed that the maximum safe concentration was 3.28 mg/mL.Both morphological observation and MTT assay were indicated that rhubarb could not only block the attachment of the virus to Vero cell and inhibit the synthesis and reproduction of the virus, but also could directly inactivate the virus.This research showed that rhubarb had an inhibitory effect on PEDV, and provided a theoretical basis for the prevention and treatment of clinical porcine epidemic diarrhea.     

甘肃黄河三峡景区旅游资源空间结构研究

旅游空间结构是旅游系统功能组织方式在空间上的投影或表象,其空间结构是否合理直接影响到旅游目的地旅游业的发展态势。黄河三峡景区凭借其丰富、独特的资源优势,已成为黄河流域最著名的景区之一。受河谷地带的基础性影响,其资源类型、空间结构、游览线路等表现出典型的空间特征,深刻影响着景区旅游空间格局。借助Arc GIS空间网络分析模块和Excel平台,对黄河三峡景区旅游资源空间结构进行定量分析,发现旅游资源空间分布呈集聚态势,基尼系数高达0.755,分布均匀度仅为0.245;采用最邻近指数和Voronoi多边形的变异系数分析空间分布类型;用地理集中指数和基尼系数分析空间分布均衡性,运用β指数、γ指数和α指数刻画空间连接度。结果表明:景区内旅游资源的空间分布均匀度低,呈多中心集聚分布。这既有利于景区内各景点之间的整合和旅游规模效应的产生,又可以减弱区间屏蔽作用等外部不经济性。景区内各景点之间的交通网络密度低,空间联系较弱,通达性较低。各景点之间的直接连接较少,旅游路线多为单线往返,缺少串联各个景点的环形线路。     

小麦TaeEF1β基因的克隆、同源性及表达分析

真核翻译延伸因子(eukaryotic translation elongation factor,eEFs)是一种重要的多功能调控蛋白,eEF1β是eEF1的组成部分,在蛋白质生物合成过程中发挥着重要的作用。本文通过RT-PCR扩增克隆小麦(Triticum aestivum L.)的eEF1β基因,并命名为TaeEF1β。氨基酸同源性分析发现,TaeEF1β具有高度保守性,且其保守结构域位于137~226 aa处。qRT-PCR结果表明,中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)侵染小麦植株后,可以诱导TaeEF1β基因转录水平的上调表达。另外,本文也进一步分析了TaeEF1β基因在小麦根、茎、叶的表达水平和CWMV侵染不同时间点的表达情况。     

草莓白化相关病毒中国分离物全基因组分析

草莓白化相关病毒(strawberrypallidosis-associatedvirus,SPa V)属于长线形病毒科(Closteroviridae)毛形病毒属(Crinivirus),可引起草莓病害,2017年在中国首次报道。采用高通量测序、RACE和RT-PCR技术获得了SPa V中国分离物(FJ)的基因组全长。该病毒含有两条正单链基因组RNA1和RNA2。RNA1全长8 048 nt,5′和3′非编码区序列分别为264和197 nt,含有3个开放阅读框(ORF),分别编码ORF 1a/1b融合蛋白和p9蛋白。RNA2全长7 977 nt,5′和3′非编码区序列分别为248和186 nt,含有8个开放阅读框(ORF),分别编码HSP70h、CPh、CP、CPm、p7、p6、p9和p28等8个蛋白。RNA1和RNA2与美国M1分离物分别具有98.5%和99.0%的核苷酸一致性;系统发育分析结果表明,SPa V中国分离物(FJ)单独处在一个分支。对SPa V来源的小RNA的分析表明,来源于SPa V的小RAN长度以21和22 nt为主。     

番木瓜环斑病毒弱毒株的构建及分析

弱毒株是利用交叉保护防治植物病毒病害的关键限制因子。通过对马铃薯Y病毒属病毒(强、弱毒株)的全长及部分氨基酸序列比对分析,筛选出在亲本强毒株PRSV-LM的P1和HC-Pro基因上的8个可能与致病性相关的氨基酸突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))。采用定点突变和Gibson拼接法,分别成功构建了4种含有2个突变位点(L_(754)和N_(787))、4个突变位点(I_(309)、K_(481)、F_(753)和D_(944))、6个突变位点(I_(309)、K_(481)、F_(753)、L_(754)、N_(787)和D_(944))和8个突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))的PRSV-LM全长c DNA侵染性克隆突变体:p Gprsvm~2、p Gprsvm4、p Gprsvm6和p Gprsvm8。经人工接种、病症观察和RT-PCR检测,4种突变体克隆均能系统性侵染非转基因番木瓜植株,除克隆p Gprsvm~2的病症表现与亲本强毒株PRSV-LM一致外,其他多位点突变的克隆p Gprsvm4、p Gprsvm6和p Gprsvm8在番木瓜植株上的病症严重程度出现依次减弱,而克隆p Gprsvm8的病症最弱,且延迟2周发病。本实验初步验证了6个氨基酸突变位点(I_(309)→S、K_(481)→Q、K_(598)→E、F_(753)→L、R_(728)→I和D_(944)→N)与PRSV-LM的病症表现及致病力相关,其中K_(598)→E和R_(728)→I可能是影响致病性的关键位点。     

Identification and Molecular Characteristics Analysis of Porcine Epidemic Diarrhea Virus Variants

To ascertain a diarrhea case in a pig farm in Shandong province,the pathological changes of dead piglets were observed and nested RT-PCR test was carried out on 7 diarrhea samples for porcine epidemic diarrhea virus (PEDV).Pathological examination revealed that intestine was detected as enlargement,hyperemia and edema.After histological examination,the typical microscopic lesions of intestine were disappearance of epithelial cells,villus shrinkage and shortening.The result of nested RT-PCR showed that all of the 7 samples could amplify a specific target band of PEDV.Molecular characteristics of two field strains showed that they had an amino acid homology of 92.3% to 92.4% with vaccine CV777 and 96.6% to 98.6% with other previous field strains which sequences were downloaded from GenBank.Phylogenetic tree analysis further revealed that all of PEDV strains could be mainly divided into two clusters of G1 and G2. G2 consisted of the field strains of our study and other field strains from USA,China and so on,which had the same sequence characteristics of two insertions and one deletion,while G1 consisted of all vaccines of CV777 and several older field strains from China and Korea.These results indicated that our field strains were the dominant strains in recent epidemic diarrhea occurrence.Moreover,its molecular characteristics might be a characterization of differentiating the field and vaccine strains.     

基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

最近，笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒（PEDV），为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行，对实验室2018-2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因（spike）分子特征研究。结果表明：腹泻样本的PEDV检出率为42.2%（49/116，95% CI=33.1%~51.8%），并获得了13条完整的S基因序列，全长为4 149~4 170 bp，序列相似性为94.2%~99.9%，其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%~98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群，其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群；SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支，且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程，以贝叶斯进化分析软件包（BEAST）进行分歧时间估算，结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年，早于藏猪源新G1亚群其余毒株的最早分歧时间（2015.7年）；SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年，早于G2亚群的藏猪源毒株2014.7年，所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV，并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川，为新型PEDV分子遗传进化的监测提供了依据。     

延边地区烟草病毒病与气象因子关系的研究

为探明延边地区烟草病毒病的发病规律,为生产优质高产烟叶提供科学有效的病毒病预防措施,简要分析了延边地区2010和2011年病毒病与气象因子的关系.结果表明,延边地区进入6月份,当日平均温度高于16.0℃,烟草病毒病就可以发生.病毒病发病前10d的日平均温度高,日照时数大于6h的天数多,降雨量少的情况下,病毒病发生严重.     

Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10^-3 to 3×10^-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.