Nucleotide composition and synonymous codon usage of open reading frames in Norovirus GII.4 variants

Abstract

Norovirus GII.4 variants, a genotype in genogroup II belonging to the genus Norovirus, is a single-strand positive sense RNA containing three open reading frames (ORF1, ORF2 and ORF3) and is the most important pathogen causing nonbacterial gastroenteritis outbreaks. By using bioinformatic softwares such as Codon W, SPSS and so on, a total of 292 strains of the viruses isolated from 1974 to 2016 were analyzed for nucleotide composition and synonymous codon usage in each ORF. The result shows that it is enriched for A over the other bases in nucleotide composition, G behind the other bases in the 3^rd site of all synonymous codons in the three ORFs. The patterns of nucleotide composition and codon bias of ORF2 are similar to those of ORF3 and different from those of ORF1. There are generally UpA motif and CpG motif in the codons with the lowest proportion. Correspondence analysis indicates that the codon usage may be changing over a certain time period for ORF1 in 2006 and 2012, ORF2 in 2012, and ORF3 in 2013. ENC (effective number of codons) plot and other analyses indicate that both natural selection and mutational pressure play partly roles in the ORFs, but natural selection is more important for ORF2 and ORF3. Besides, we also found all optimal codons in the ORFs. The study provides a basic understanding of the mechanism for norovirus GII.4 codon usage bias. Abbreviations ORF Open Reading Frame

ENC Effective Number of Codons

COA correspondence analysis

RSCU Relative Synonymous Codon Usage

CAI Codon Adaptation Index

CBI Codon Bias Index

Fop frequency of optimal codons

L_sym number of synonymous codons

L_aa length amino acids

GRAVY grand average of hydropathicity

Aroma aromaticity

Communicated by Ramaswamy H. Sarma     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

诺如病毒的致病机制及诊断

诺如病毒(Norovirus, NoV)是引起全人群急性胃肠炎暴发和流行的主要病原体,也是引起食源性疾病的最常见非细菌性病原体。由于缺少有效的小型动物及培养细胞研究模型,目前对NoV感染的致病机制尚不清楚。NoV实验室诊断技术发展成熟,尤其是近年来新技术的应用将进一步推动其诊断水平的提高,满足公共卫生和临床诊疗的新需求。     

诺如病毒GII.6 P粒子原核表达与纯化

原核表达的诺如病毒(Noroviruses,NoV)衣壳蛋白亚基P粒子与No V有相同的抗原类型,可以代替NoV在体外进行结合,这对于研究该病毒与宿主和环境载体结合的相关机理有重大意义。本研究成功构建GII.6 P粒子基因表达载体,并对原核表达体系中诱导剂浓度、诱导温度及诱导时间进行优化。结果表明表达载体在22℃、2×10~(-4)mol/L IPTG诱导22 h后表达量最高。随后,在亲和层析的基础上结合阴离子交换以及凝胶过滤层析对表达产物进行纯化,最终获得高纯度GII.6 P粒子。     

诺如病毒：“完美的人类病原”

诺如病毒是近来在我国北京、上海、浙江、广东等地导致病毒性胃肠炎暴发的病原体。本文对该病毒的生物学特征与流行病学进行简短综述,并对正在研发的疫苗作介绍。     

2010年深圳地区诺如病毒的基因分型

了解2010年深圳地区诺如病毒的基因型别及分子流行病学特点。 用诺如病毒特异性引物（GI-SKF/GI-SKR、COG2F/G2-SKR）,通过逆转录-聚合酶链反应（RT-PCR）方法进行诺如病毒核酸扩增,阳性产物回收纯化并测序,用Clustal W和MEGA4.0生物软件对诺如病毒序列进行序列比对和系统进化分析。 85份阳性标本中有79株诺如GⅡ型和6株诺如GⅠ型,其中55株为GⅡ/4(2006b)型,16株为GⅡ/4(2008variant)型,2株为GⅡ/1型,4株为GⅡ/5型,2株为GⅡ/11型,1株为GI/4型,2株为GI/5型,3株为GI/6型。 2010年深圳地区诺如病毒的主要型别是GI和GⅡ,并且以GⅡ/4型为主,流行优势株为GⅡ/4（2006b）。     

A nucleic acid sequence-based amplification assay for real-time detection of norovirus genogroup II

AIMS: To use molecular beacon based nucleic acid sequence-based amplification (NASBA) to develop a rapid, sensitive, specific detection method for norovirus (NV) genogroupII (GII). METHODS AND RESULTS: A method to detect NV GII from environmental samples using real-time NASBA was developed. This method was routinely sensitive to 100 copies of target RNA and intermittent amplification occurred with as few as 10 copies. Quantitative estimates of viral load were possible over at least four orders of magnitude. CONCLUSIONS: The NASBA method described here is a reliable and sensitive assay for the detection of NV. This method has the potential to be linked to a handheld NASBA device that would make this real-time assay a portable and inexpensive alternative to bench-top, lab-based assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of the real-time NASBA assay described here has resulted in a simple, rapid (<1 h), convenient testing format for NV. To our knowledge, this is the first example of a molecular beacon based NASBA assay for NV.     

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT‐PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9–92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1–65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7–92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5–86.5%) were higher than those with other mammal SaVs (20.7–58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV‐ and SaV‐infected dogs and cats in Japan. The findings suggest there are species‐specific circulations of NoV and SaV among dogs and cats, in Japan.     

EV71病毒中和表位和诺如病毒P结构域嵌合蛋白的原核表达

目的:构建肠道病毒71型(Enterovirus71,EV71)的线性中和抗原表位与诺如病毒P结构域融合基因的重组质粒,在大肠杆菌中表达诺如病毒P结构域与EV71中和抗原表位的嵌合蛋白。方法:根据已报道的3个EV71线性中和抗原表位的氨基酸序列,按大肠杆菌密码子表达使用的偏好性优化和设计各线性中和抗原表位的核苷酸序列,将这些表位以单个或不同的组合克隆至含诺如病毒P结构域和GST标签的质粒中,经测序确认后,分别转化到E.coli BL21(DE3)感受态细胞中,通过IPTG诱导融合蛋白表达。用GST融合蛋白纯化磁珠对融合蛋白进行纯化,最后通过免疫印迹法确认融合蛋白的表达及嵌合蛋白的抗原性。结果:测序结果表明,成功地构建了含EV71病毒3个单表位和4个串联中和抗原表位的诺如病毒P结构域重组质粒,而且这7个含线性中和抗原表位的嵌合蛋白在大肠杆菌中都以可溶形式得到了表达。免疫印迹分析表达蛋白的抗原性结果表明,表达的嵌合蛋白都能与抗诺如病毒P结构域抗血清反应。除了含单表位的SP55和SP28嵌合蛋白外,其它的嵌合蛋白均能与抗EV71病毒的抗血清反应。结论:成功地在大肠杆菌中表达了诺如病毒P结构域和EV71病毒中和抗原表位的嵌合蛋白,且具有抗原性,这为诺如病毒和EV71病毒的二价疫苗及检测方法的研发奠定了基础。     

深圳地区环境水体和人群中诺如病毒监测与分析

【目的】检测深圳地区环境水体和人群的诺如病毒(No Vs),探讨其是否在两者之间循环传播。【方法】2014年3月至2015年2月检测24份深圳茅洲河河水标本和287份腹泻患者粪便标本。河水标本经过混合纤维素酯膜和PEG法二次浓缩后提取核酸,粪便标本无需浓缩,稀释离心后直接提取核酸。应用实时荧光逆转录PCR初步分型,RT-PCR扩增ORF2保守区域衣壳蛋白(VP1)基因后测序确定亚型、分析不同来源病毒的同源性;同时建立基因进化树,进一步分析不同来源No Vs的亲缘关系。【结果】在河水标本和腹泻患者粪便标本中,No Vs阳性率分别为23.1%和17.4%,其中上下游河水标本阳性率分别为8.3%和41.7%,河水中No VGI型和No VGII型的阳性率为16.7%和8.3%。扩增阳性样本发现茅洲河水中检出No V主要是GI.6型,其次是GII.4 Sydney_2012型,而从腹泻胃肠炎患者粪便标本中检出No Vs主要是No VGII.4Sydney_2012型和少量的GII.3型。同时期水体和人群粪便标本来源的No VGII.4 Sydney_2012型VP1基因相似性98.2%–100.0%。【结论】No VGII.4 Sydney_2012型是深圳地区主要的流行株。No Vs在环境水体和人群中于某种程度上存在循环传播。