Phylogenetic Characterisation of the Full Genome of a Bagaza Virus Isolate from Bird Fatalities in South Africa

Bagaza virus (BAGV), a member of the Ntaya serogroup in the Flavivirus genus of the Flaviviridae, was isolated from the brain tissue of a Himalayan monal pheasant that died following neurological signs in Pretoria, South Africa in 2016. Next-generation sequencing was carried out on this isolate resulting in a genome sequence of 10980nt. The full genome sequence of this isolate, designated ZRU96-16, shared 98% nucleotide identity with a BAGV isolate found in Culex univitattus mosquitoes from Namibia and 97% nucleotide identity with a Spanish BAGV sequence isolated from an infected partridge. In total, seven amino acid variations were unique to ZRU96-16 after alignment with other BAGV and Israel turkey meningoencephalomyelitis (ITV) genomes. The 3′UTR sequence of ZRU96-16 was resolved with sufficient detail to be able to annotate the variable and conserved sequence elements within this region. Multiple sequence alignment of the 3′UTR suggested that it could be useful in lineage designation as more similar viruses carried similar mutations across this region, while also retaining certain unique sites. Maximum likelihood phylogenetic analysis revealed two clusters containing both BAGV and ITVs from Europe, the Middle East and Africa. Broadly, temporal clustering separated isolates into two groups, with one cluster representing viruses from the 1960–2000’s and the other from 2010 onwards. This suggests that there is consistent exchange of BAGV and ITV between Europe and Africa. This investigation provides more information on the phylogenetics of an under-represented member of the Flaviviridae and provides an avenue for more extensive research on its pathogenesis and geographic expansion.     

Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9

The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.     

可视化仿真手术在脾脏手术中的应用

目的研究基于64排螺旋CT脾脏扫描数据的三维重建和脾切除的动态可视化仿真手术。方法采集脾脏64排螺旋CT的原始扫描数据,通过一种自适应的区域生长法对CT序列图像进行图像程序分割和自动提取,再采用自行研发的医学图像处理软件分割后的图像数据进行三维重建,然后利用Freeform Modeling System和PHANTOM软件系统进行脾切除的可视化仿真手术研究。结果利用自适应区域生长法进行脾脏图像程序分割速度快、效果好,可获得满意的脾脏分割数据。采用自行研发的图像处理软件对脾脏的分割数据进行三维重建图像结构清晰,可真实再现脾脏和周围的结构。利用Freeform Modeling System和PHANTOM软件系统可以有助于术前进行模拟的脾脏可视化仿真手术。结论利用脾脏CT扫描数据进行的可视化三维重建和脾切除仿真手术研究,对脾脏手术前的风险评估和临床教学等方面都有很大的应用价值。     

加强临床检验质量控制的思考

临床检验是疾病诊断和治疗的重要参考。提高临床检验质量一直都是临床检验人员、医疗机构和卫生行政部门关注的重要问题。临床检验水平的高低,不仅体现在检验设备、人员素质上,也同样体现在临床检验政策法规和操作规范、医疗机构临床检验操作管理上。但我国的临床检验质量控制尚不完善,临床检验质量不高。本文从卫生行政部门、医疗机构以及临床检验人员的角度提出加强临床检验质量控制,提高临床检验质量的建议和措施。     

术前护理访视在择期手术中的应用体会

随着现代护理学科的快速发展,手术室护理工作不再局限于配合手术等单纯的技术操作,而更应注意“以病人为中心”的手术全过程护理[1 ] 。自从我院开展手术室整体护理工作以来,我们加强围手术期每个环节的护理工作,选择2 81例择期手术患者针对术前访视,仔细总结工作经验及护理评价,取得了满意的效果,现报告如下。1　对象访视对象为2 0 0 0年4月至2 0 0 1年1月,我院择期手术患者中2 81例,男15 3例,女12 8例,年龄15岁～73岁,其中妇产科手术6 5例,普外科82例,骨外科78例,胸外科39例,其他17例,文化程度有大学、初中、文盲。2　方法2 .1　访视准备…     

后颅窝肿瘤术后早期脑积水

目的 分析后颅窝肿瘤术后早期脑积水的原因并提出预防措施。方法 对18例后颅窝肿瘤术后早期脑积水患者的临床资料进行回顾性分析。结果 本组18例术后24h内一般情况均良好,之后随即出现神志淡漠、嗜睡、头痛、呕吐等,CT检查见脑积水。在施行包括侧脑室外引流术在内的综合治疗后,16例痊愈,2例死亡。结论 术后小脑水肿、颅内血肿、肿瘤残留等可能是导致该并发症的主要原因。预防措施包括术中规范的显微神经外科操作以尽量保护脑组织、严密止血、术后综合治疗等。     

肩胛骨骨折的手术治疗

目的探讨肩胛骨骨折的手术治疗方法、优点及术中应注意的事项。方法全麻侧卧,Judet后入路,切口自肩峰背侧偏内1 cm,沿肩胛冈向内侧延伸,下至肩胛体下角,离断三角肌,沿骨面剖离,将三角肌和冈下肌翻向外侧;骨折复位,用钢板和螺钉固定。结果术后随访8个月~2年,16例骨折全部愈合,愈合时间为1.5~4.5个月,平均2.5个月。按Dolfi评分系统评定:优9例,良5例,可1例,差1例。优良率87.5%。结论手术治疗肩胛骨折能及时清除积血,使骨折能达到良好复位、固定,减少粘连和畸形愈合。     

81例老年肠癌患者的手术治疗分析与观察

目的 为进一步对做好老年肠癌患者的手术治疗工作提供科学依据.方法 针对性治疗与观察81例老年肠癌手术患者,采用回顾性方法研究分析临床资料.结果 本组81例患者均于入院确诊后12～72 h内手术,均获得根治切除术的手术治疗,手术时间平均(176.3±35.4)min,术中出血平均(142.6±13.1) ml,术后住院天数平均(10.1±3.4)d,术后出现切口感染2例,对出院患者进行随访6～12个月,有严重并发症出现的27例,本组总共出现死亡病例10例,死亡率为10.29%.结论 老年肠癌患者手术治疗是具有一定困难和风险的,只有熟练掌握老年肠癌手术治疗方法和相关技能,才能取得较好的手术效果.     

Isolation and genomic characterization of a classical Muscovy duck reovirus isolated in Zhejiang,China

A classical Muscovy reovirus was isolated from a sick Muscovy duck with white necrotic foci in its liver in Zhejiang, China, in 2000. This classical reovirus was propagated in a chicken fibroblast cell line (DF-1) with obvious cytopathic effects. Its genome was 22,967 bp in length, with approximately 51.41% G + C content and 10 dsRNA segments encoding 11 proteins, which formed a 3/3/4 electrophoretic PAGE profile pattern. The length of the genomic segments was similar to those of avian orthoreoviruses (ARV and N-MDRV), ranging from 3959 nt (L1) to 1191 nt (S4). All of the segments have the conserved terminal sequences 5′-GCUUUU——UUCAUC-3′, and with the exception of the S4 segment, all the genome segments apparently encode one single primary translation product. The genome analysis revealed that the S4 segment of classical MDRV is a bicistronic gene, encoding the overlapping ORFs for p10 and σC but distinct from ARV and N-MDRV/N-GRV, which codes for p10, p18 and σC via the tricistronic S1 segment. A comparative sequence analysis provided evidence indicating extensive sequence divergence between classical MDRV and other avian orthoreoviruses. A phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) and the major outer capsid proteins σC was performed. Members of the DRVs in the Avian orthoreovirus species were clustered into two genetic groups (classical MDRV and N-MDRV genotype), and the classical MDRV isolates formed distinct lineages (China and Europe lineages), suggesting that the classical MDRVs isolated in restricted geographical region are evolving by different and independent pathways.     

红花栝楼鲨烯合酶基因的克隆及其序列分析

目的克隆红花栝楼鲨烯合酶(squalene synthase,SS)的基因并进行序列分析,为进一步研究葫芦科植物三萜合成通路关键酶SS的正选择位点与功能的关联性分析奠定基础。方法根据绞股蓝与罗汉果SS基因c DNA比对分析的结果,设计SS的5’端简并引物,采用3’RACE扩增红花栝楼SS全长c DNA基因。结果获得红花栝楼SS c DNA全长共1 466个核苷酸,其中包括一个含有1 254个核苷酸的开放读码框,编码417个氨基酸残基。通过NCBI的Blast比对,发现红花栝楼SS基因编码的氨基酸序列与已知植物SS基因编码的氨基酸序列同源性为78%~94%,核苷酸的同源性为74%~93%。结论成功克隆了红花栝楼SS的全长c DNA,为进一步研究红花栝楼SS基因结构、基因表达、基因突变提供了基础,并为葫芦科植物三萜合成通路关键酶SS的正选择位点与功能的关联性分析提供了数据支持。