Complete Genomic Sequence of Transmissible Gastroenteritis Virus TS and 3＇ End Sequence Characterization Following Cell Culture

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains.Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster.Continued culture in different cell types indicated that TGEV TS virulence could be attenuated alter fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.     

猪传染性胃肠炎病毒S蛋白抗原位点分子特征分析

为了进一步研究猪传染性胃肠炎病毒S蛋白抗原位点的分子特征,选取GenBank中22株TGEV分离株,采用生物信息学方法对抗原位点氨基酸序列进行同源性比对分析。结果表明,不同分离株的A和C位点高度保守,B和D位点则有一些变化。     

猪传染性胃肠炎病毒n基因的原核表达及重组蛋白的免疫活性分析

猪传染性胃肠炎(transmissible gastroenteritis,TGE)是由猪传染性胃肠炎病毒(transmissible gas-troenteritis virus,TGEV)引起的一种急性、高度接触性传染病,以呕吐、水样腹泻、脱水和对2周龄以内仔猪高度致死率为特征[1]。猪传染性胃肠炎病毒隶属于冠状病毒科冠状病毒属,是引起仔猪病毒性腹泻的重要病原,其基因组为单股正链的有感染性不分节段的RNA,TGEV结构蛋白主要由S、N、Ms、M蛋白组成[2]。其中n基因指导合成病毒的核衣壳蛋白(N),它是一种磷酸化的蛋白,存在于病毒粒子的内部,其分子质量为47kD[3],与病毒基因组组成核衣壳;N…     

An episulfide cation (thiiranium ring) trapped in the active site of HAV 3C proteinase inactivated by peptide-based ketone inhibitors

We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

The ORF4a protein of human coronavirus 229E functions as a viroporin that regulates viral production

In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li⁺ partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

猪传染性胃肠炎病毒S基因全抗原位点片段及猪呼吸道冠状病毒缺失片段表达产物的反应原性比较

猪呼吸道冠状病毒（PRCV）是猪传染性胃肠炎病毒（TGEV）的自然缺失株,二者主要区别在于PRCV的S基因缺失B、C两个主要抗原位点。因此,利用RT—PCR技术分别扩增TGEV Purdue毒株S基因近N端的S1和S1-2两个片段,将之克隆到pOEX—KG原核表达载体的GST基因下游,经表达,获得大小约52kD和108kD两种融合蛋白,表达量分别占菌体总蛋白的45％和35％,主要以包涵体的形式存在。利用TGEV和PRCV两种阳性血清进行Western blot检测,当第一抗体为TGEV阳性血清时,可检测到52kD和108kD两条带;而当第一抗体为PRCV阳性血清时,只检测到108kD一条带。结果表明这两种融合蛋白具有良好的反应原性,且可用于TGEV和PRCV的鉴别诊断,为进一步研制开发TGEV和PRCV的鉴别诊断试剂盒奠定了基础。     

猪传染性胃肠炎病毒S蛋白在乳酸菌中的表达

目的：构建猪传染性胃肠炎病毒S蛋白的细胞内表达重组乳酸乳球菌,确定其最佳表达条件,为重组乳酸菌作为口服疫苗防治猪传染性胃肠炎奠定基础。方法：根据猪传染性胃肠炎病毒纤突（S）蛋白的全基因序列及表达载体质粒的基因融合特点,设计一对引物,进行PCR,获得含有TGEV S基因4个主要抗原位点的约2000bp目的片段,将其与表达载体质粒pNZ8048进行连接,通过电转化进入宿主菌乳酸乳球菌NZ9000细胞内,在乳链菌肽（Nisin）的诱导下进行表达,确定最佳表达条件;并通过SDS-PAGE进行检测和Western-blot分析表达蛋白活性。结果：成功获得了TGEV S蛋白在乳酸乳球菌细胞内的表达并且表达的蛋白具有TGE全病毒的抗原性。确定了乳酸乳球菌表达TGEV S蛋白的最佳表达条件为在以1ng/ml的乳链杆菌肽nisin诱导下,诱导后3h,重组蛋白表达效率达最高,重组蛋白约占菌体总蛋白含量的8．7％。结论：在乳酸乳球菌细胞内表达的重组TGEV S蛋白获得了理想表达,为进一步研制开发防治TGE口服疫苗提供物质基础。     

反义RNA抑制猪传染性胃肠炎病毒复制的研究

根据反义RNA作用原理,设计一条互补猪传染性胃肠炎病毒基因(26888—27184)区的反义RNA序列。将该序列与逆转录病毒表达载体构建成质粒PLXSN—N5’,并与质脂体共转染PA317细胞,经G418(500μg／ml)筛选出稳定的产毒细胞克隆。取其上清液感染小鼠成纤维细胞NIH3T3,测定细胞克隆产生的假病毒滴度,用高滴度假病毒感染IBRS2细胞。提取被感染的IBRS2细胞总DNA和RNA,通过PCR和RT_PCR证明PL,XSN—N5’整合到IBRS2细胞基因组。病毒感染细胞病变表明,反义RNA有明显抑制TGEV复制的作用。     

Effects of humidity and other factors on the generation and sampling of a coronavirus aerosol

Suspensions of transmissible gastroenteritis virus (TGEV), a porcine coronavirus, were nebulized at rates of 0.1–0.2 ml/min into moving air using a Collison nebulizer or a plastic medical nebulizer operating at pressures ranging from 7 to 15 psi. The airborne viruses were collected on heating, ventilating, and air conditioning (HVAC) filters in an experimental apparatus and also sampled upstream of these test filters using AGI-30 and BioSampler impinger samplers. To study the effects of relative humidity (RH) on TGEV collection by the filters and samplers, the virus was nebulized into air at 30, 50, 70, and 90% RH. There were no significant changes in virus titer in the nebulizer suspension before and after nebulization for either nebulizer at any of the pressures utilized. Aerosolization efficiency – the ratio of viable virus sampled with impingers to the quantity of viable virus nebulized – decreased with increasing humidity. BioSamplers detected more airborne virus than AGI-30 samplers at all RH levels. This difference was statistically significant at 30 and 50% RH. Nebulizer type and pressure did not significantly affect the viability of the airborne virus. Virus recovery from test filters relative to the concentration of virus in the nebulizer suspension was less than 10%. The most and the least virus were recovered from filter media at 30% and 90% RH, respectively. The results suggest that TGEV, and perhaps other coronaviruses, remain viable longer in an airborne state and are sampled more effectively at low RH than at high humidity.