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2016年31卷3期

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PERSPECTIVE

CRISPR-Cas-like system in giant viruses: why MIMIVIRE is not likely to be an adaptive immune system

2016, 31(3): 193 doi: 10.1007/s12250-016-3801-x

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Giant viruses from the Mimiviridae family (Mimivirus, Megavirus, etc.) replicate inside their Acanthamoeba host by the mean of a large intracytoplasmic virion factory within which DNA transcription and replication take place using the virus encoded machineries. Members of the Mimiviridae have been isolated in association with much smaller dsDNA viruses called "virophages" that replicate within the virion factory, behaving as parasites of the giant virus. In a recent work Levasseur et al. (2016) used a silencing approach to identify candidate genes possibly involved in the inhibition of the Zamilon virophage in a specific clade of Mimiviridae. Based on the presence of four copies of a short Zamilon sequence in one of the Mimivirus candidate gene, the authors proposed that resistance to virophages was conferred by a CRISPR-Cas-like system (called MIMIVIRE). Here we dispute this interpretation on the ground that 1) the simultaneous and co-localized replication of the virophage and Mimivirus genomes makes the acquisition of a nucleic acid-based immunity unlikely, 2) the Zamilonlike sequence allegedly acquired by Mimivirus are neither regularly spaced nor flanked by recognizable repeats, 3) the corresponding Zamilon sequence is devoid of distinct flanking sequences that may serve as Protospacer Adjacent Motifs (PAM) discriminating between the virophage and its host. We propose a simpler protein-based interaction model that explains the observed phenomena without having to extend the realm of adaptive immunity to the world of eukaryotic viruses, a revolutionary step that would require stronger experimental evidences.

Towards a coherent nomenclature of plant viruses

2016, 31(3): 197 doi: 10.1007/s12250-016-3741-5

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The International Committee on Taxonomy of Viruses (ICTV) has the decree to set the rules for the classification and naming of viruses. The species is the lowest level considered in the taxonomic hierarchy. In general, virus species are named according to the structure “ and the word ‘virus’”. A typical example is Tobacco mosaic virus [Genus: Tobamovirus]. With a few exceptions, this convention has been generally well accepted. As there is always room for improvement, virus nomenclature (King et al., 2011) can be managed more efficiently through several ways.
Research Article

HIV-1 B 和C亚型Tat的稳定性与C-末端有关

赵学潮, 钱玲玉, 周得玉, 齐迪, 刘畅, 孔晓红

2016, 31(3): 199 doi: 10.1007/s12250-016-3681-0

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多功能反式激活因子Tat是HIV-1复制所需的调控蛋白,具有高度序列多样性。尽管目前C亚型病毒引起的感染在全世界更普遍,但是大量的实验研究关注于HIV-1 B亚型Tat,而关于C亚型Tat的研究很少。我们认为不同亚型Tat之间序列的差异可能导致其功能存在差异。在本研究中,我们通过过表达融合蛋白Tat-Flag发现B亚型NL4-3 Tat和C亚型1084i Tat具有不同的蛋白稳定性。另外,与NL4-3 Tat相比,1084i Tat能够更有效地激活LTR和NF-κB。通过分析截短的Tat的活性,我们发现Tat的C-末端调控着它的稳定性和转录活性。根据我们的结果,我们推测B亚型Tat和C亚型Tat的稳定性差异导致了其转录活性的不同。

膜蛋白gp120的V1区在SIV和HIV的传播中被特异选择,并对膜蛋白的功能和病毒感染至关重要

李龑鹏 宋继萍 王燕 孙宾莲 杨荣阁, 宋继萍, 王燕, 孙宾莲, 杨荣阁,

2016, 31(3): 207 doi: 10.1007/s12250-016-3725-5

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HIV传播过程中会经历明显的传播瓶颈效应,传播瓶颈只选择某些特定的病毒并建立新的感染,而对于这些被选择传播的病毒的遗传特点还没有完全研究清楚。本研究中,我们对SIV和HIV传播历史,以及15-20年以来流行的HIV各个亚型的膜蛋白的氨基酸变化特征进行了分析。结果发现gp120蛋白的V1V2区,特别是V1区在传播及进化过程中被特异的选择,其中较短的V1区在传播中优先的选择,而在群体水平进化中,V1区则不断的增长从而逃逸宿主的免疫识别。我们接下来构建了HIV不同亚型膜蛋白的V1区突变体,并研究了V1区在膜蛋白中的功能。结果发现V1区的序列尽管高度的可变,它对于病毒的进入和感染时必不可少的。而这可能是因为V1区的缺失突变影响了膜蛋白前体gp160成熟切割为gp120和gp41,同时V1区也影响了病毒膜蛋白的包装水平。这些结果表明V1区在HIV的传播进化及感染中起到重要的作用。

间充质干细胞对人巨细胞病毒感染完全容许

乔冠华, 赵非, 程爽*, 罗敏华*

2016, 31(3): 219 doi: 10.1007/s12250-016-3754-0

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先天性HCMV感染是导致新生儿多器官发育异常等出生缺陷的重要病因。间充质干细胞(MSCs)是一类干细胞/前体细胞,具有多分化和自我更新的潜能,在多器官的形成过程中发挥重要作用。而HCMV感染所引起的非神经系统异常是否与MSCs相关尚未见报道。因此,本研究拟从人脐带的华顿氏胶中分离MSCs,分析MSCs对HCMV的易感性。首先,我们分离MSCs,并鉴定其吸附性、表面分子标记的表达和分化特性。然后,通过western blot、间接免疫荧光和噬斑形成试验,分析HCMV在MSCs中的感染特性,如病毒进入、复制、蛋白质表达、释放等,发现MSCs对HCMV感染完全容许。该研究的阐明为揭示先天性HCMV感染所引起的多系统发育异常的机制奠定了基础。

在非受纳细胞中与AcMNPV基因组同时存在的PlxyGV晚期基因启动子的表达活性

任禾林, 胡原, 郭亚君, 黎路林

2016, 31(3): 229 doi: 10.1007/s12250-015-3675-3

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在杆状病毒中,虽然对?-杆状病毒已有广泛的研究,但目前对?-杆状病毒复制的分子机制的了解很少。在本文的研究工作中,我们将属于β-杆状病毒属的小菜蛾颗粒体病毒 (Plutella xylostella granulovirus, PlxyGV) 的9个晚期基因启动子分别克隆到一个瞬时表达载体质粒和属于α-杆状病毒属的苜蓿银纹夜蛾核型多角体病毒 (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) 基因组分别构建相应的晚期基因报告质粒和报告病毒,对其与AcMNPV的各对应同源基因启动子在Sf9细胞中的表达活性做了对比分析。在瞬时表达实验中,这9个PlxyGV晚期启动子在由单个报告质粒和AcMNPV bacmid共转染的Sf9细胞中都能被激活。PlxyGV e18启动子、AcMNPV vp39和gp41启动子包含的晚期启动子基序的数目多于各自对应的AcMNPV和PlxyGV同源物。在感染实验中,由PlxyGV e18启动子、AcMNPV vp39启动子和gp41启动子驱动的报告基因的表达水平显著高于对应的AcMNPV和PlxyGV晚期基因启动子驱动的报告基因的表达。PlxyGV p6.9、pk1、gran、p10b和p10c启动子包含的晚期启动子基序的数目与各自对应的AcMNPV 同源启动子一致,但在被感染细胞中这些PlxyGV 启动子驱动的报告基因的表达水平明显低于对应的AcMNPV启动子;说明有些启动子包含了能被所属病毒编码的RNA聚合酶识别的特异性顺式元件,从而获得最佳表达效果。序列比对结果显示,AcMNPV 三个极晚期表达基因启动子都含有一个8-nt保守序列TAAATAAG, 其中包含晚期启动子基序ATAAG;在PlxyGV gran、p10c和pk1启动子的T/ATAAG上游4或5nt处存在一个5-nt 保守序列CAATT。本研究的实验结果显示PlxyGV晚期基因启动子可以被AcMNPV RNA聚合酶有效激活,提示β-杆状病毒晚期基因表达系统的调控机制可能与α-杆状病毒类似。

棉铃虫核多角体病毒成纤维细胞生长因子的功能研究

尹飞飞#, 杜瑞坤#, 匡文华, 阳光, 王华林, 邓菲, 胡志红, 王曼丽*

2016, 31(3): 240 doi: 10.1007/s12250-016-3710-z

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杆状病毒是唯一编码成纤维细胞生长因子(FGF)的病毒。组I alpha属杆状病毒vFGF在病毒感染中肠上皮细胞后,穿越基底膜感染气管进而建立系统感染过程中发挥重要作用。组II alpha杆状病毒编码的vFGF目前尚无功能研究, 它们在结构上与组I vFGF有较大不同,表现为具有较长C端和较多N-糖基化位点,推测其功能与组I vFGF有一定不同。本研究中,我们研究了组II alpha属杆状病毒棉铃虫核多角体病毒(HearNPV)vFGF的功能。vFGF的转录及表达时相结果表明,vFGF分别在感染后3小时和16小时检测到转录和表达。为进一步研究vFGF的功能,我们构建了vFGF缺失和回复的重组HearNPV病毒,并在细胞培养体系和虫体水平分别研究vFGF的功能。在细胞培养体系中,vFGF的缺失对出芽病毒粒子的产生和病毒DNA的复制没有显著影响。然而,生物测定实验表明,通过口服感染途径,vFGF显著提高了病毒的半数感染剂量,并且延长了感染虫子的死亡时间(推迟约12小时)。这些结果表明,vFGF是HearNPV虫体水平感染的重要毒力因子。

SAP功能域野生型和突变型口蹄疫病毒感染SK6细胞的转录组分析

倪子欣, 杨帆, 曹伟军, 张向乐, 靳野, 毛箬青, 杜晓莉, 李伟伟, 郭建宏, 刘湘涛, 朱紫祥, 郑海学

2016, 31(3): 249 doi: 10.1007/s12250-015-3709-x

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口蹄疫病毒可以引发家畜的快速发病,具有高度传染性。病毒蛋白酶L蛋白与口蹄疫病毒的致病性具有直接关联,突变L蛋白的SAP功能域可以降低病毒对猪的致病性。为了研究清楚哪些宿主基因的表达变化导致了病毒对猪源细胞的致病性下降,本研究利用二代测序技术对具有野生型SAP功能域和突变型SAP功能域的两株病毒感染的SK6细胞进行了转录谱测定,并对差异基因进行了分析。结果获得了1853个差异基因(表达倍数差异大于等于2倍)。进一步通过实时定量PCR对一些差异基因的表达进行了验证,证实了转录组测定的准确性和可重复性。通过对差异基因的系统分析发现许多差异基因与转录过程、免疫调控、细胞因子表达、炎症反应和凋亡存在直接联系。这些基因转录表达谱的变化或许是导致两株毒株致病性差异的原因。本研究为深入研究与L蛋白相关的抗病毒路径和致病机制提供了大量的潜在靶标基因。
Letter

一起由星状病毒I型1d亚型引起的幼儿园爆发性腹泻

侯美玲, 梁雪雪, 谭李倩, 刘鹏飞, 赵微

2016, 31(3): 258 doi: 10.1007/s12250-015-3701-5

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2010年11月,辽宁省某市一社区幼儿园35名3-4岁儿童爆发急性腹泻,全部患儿出现水样腹泻,平均体温37.5-38.3℃,持续腹泻2.5天以上,粪便镜检均无红细胞及脓细胞。患儿入院后经过补液均康复出院。所有患儿粪便上清液提取病毒RNA,经过RT-PCR与ELISA检测,结果表明:星状病毒感染率100%,其它肠道病毒如,轮状病毒、腺病毒、诺如病毒、沙波病毒等均为阴性。毒株进行分离及测序分析后,采用星状病毒衣壳蛋白ORF2序列348bp构建进化树,均属于HAstV-1d亚型,35株毒株之间相似性89.08–98.56%。采用RT-PCR对其中一阳性粪便样本进行全长序列分析,扩增毒株全长6771bp,分子生物学分析表明,其病毒RNA结构与其他已报道星状病毒I型毒株相似,同源性在89%-91.4%之间。星状病毒在我国婴幼儿腹泻群体中检出率在2-9%之间,均以住院胃肠炎患儿或合并其他肠道病毒感染患儿为主,单纯星状病毒感染的报道较少,且国内已报道的星状病毒I型感染均为HAstV-1b亚型。本研究首次报道了一起幼儿园爆发性HAstV-1d感染性腹泻,此次腹泻的爆发与幼儿园的卫生条件密切相关。

鸭坦布苏病毒cDNA感染性克隆的构建和病毒拯救

陈浩, 张英, 张欣, 提金凤, 刁有祥

2016, 31(3): 262 doi: 10.1007/s12250-015-3678-0

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近年来,坦布苏病毒感染是在我国水禽养殖业中新发的一种传染性疾病。我们成功构建了坦布苏病毒的cDNA感染性克隆。将病毒的cDNA克隆至低拷贝载体pwsk29载体中,测序无误后,将该重组质粒转染至稳定表达T7 RNA聚合酶的BHK21细胞中。经RT-PCR、间接免疫荧光技术证实成功拯救该病毒,并对拯救的子代病毒进行生长曲线和致病性试验。结果显示,拯救毒株与亲本株具有相似的生物学特性。

Recombinant canarypox virus expressing the VP2 protein of infectious bursal disease virus induces protection in vaccinated SPF chickens

2016, 31(3): 266 doi: 10.1007/s12250-015-3680-6

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Infectious bursal disease virus (IBDV) causes infectious bursal disease, a highly contagious immunosuppressive disease that affects young chickens and causes economic losses in the poultry industry worldwide. IBDV replicates mainly in actively dividing B lymphocytes within the bursa of Fabricius (BF), leading to immunosuppression in affected flocks (Mahgoub et al., 2012). Viral protein 2 (VP2), the only structural component of the IBDV icosahedral capsid, is the major antigen responsible for inducing protective immunity in the host (Mahgoub et al., 2012).

Diagnosis and phylogenetic analysis of orf virus in Aleppo and Saanen goats from an outbreak in Turkey

2016, 31(3): 270 doi: 10.1007/s12250-015-3684-2

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In this study, multiplex PCR was used to amplify both the B2L gene partial coding region and a housekeeping gene. A substantial amount of research has been conducted on the B2L gene, and this presents opportunities for comparisons between different strains around the world. Field strains have been compared with human, sheep, and goat orf viruses and with other parapoxviruses such as PCPV and BPSV. Although the presence of the virus in Turkey was previously reported, its relationship with other orf viruses around the world has not yet been demonstrated, in contrast to parapoxvirus infections of cattle and humans in Turkey (Karakas et al., 2013; Oguzoglu et al., 2014). This study provides diagnostic and molecular characterization data for the economically damaging parapoxvirus infection that circulated among goat flocks in Turkey in the 2013–2014 lambing season.

Characterization of a recombinant Akabane mutant virus with knockout of a nonstructural protein NSs in a pregnant goat model

2016, 31(3): 274 doi: 10.1007/s12250-015-3704-2

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Akabane virus (AKAV), an orthobunyavirus, is transmitted primarily by biting midges and is widely distributed throughout the world except the Europe. AKAV was first isolated from mosquitoes in Japan (Oya et al., 1961). Although pregnant cows, ewes, and goats infected with AKAV exhibit no clinical signs of disease, in utero infections result in abortion, premature birth, stillbirth, and congenital deformities such as arthrogryposis-hydranencephaly syndrome (Kurogi et al., 1976), causing economic losses in the livestock industry. The live, attenuated vaccine strain, TS-C2, was derived from the OBE-1 strain as a temperature sensitive mutant (Kurogi et al., 1979). Although vaccination has reduced the prevalence of the disease, antigenic and pathogenic variants of AKAV have been isolated (Lee et al., 2002; Ogawa et al., 2007a); for example, a variant Iriki strain was isolated from a calf with nonsuppurative encephalitis and neurological symptoms in Japan (Miyazato et al., 1989) and it shows low antigenic cross-reactivity with the reference strain in neutralization tests (Akashi and Inaba, 1997). Therefore, it is necessary to reconsider the vaccination strategy to effectively control the disease. Here we evaluated characters of a mutant virus with knockout of a nonstructural protein NSs, which acts as type I interferon antagonist and is involved in the regulation of host protein synthesis (Weber et al., 2002), by experimentally infecting pregnant goats. The pregnant goat model might be useful for AKAV studies, as suggested by previous reports of experimental transplacental infection of caprine fetuses (Kurogi et al., 1977).