PRRSV HEB-2013株的分离与全基因序列分析

2013年9月从河北某发病猪场分离到1株猪繁殖与呼吸综合征病毒(PRRSV),将该毒株命名为HEB-2013株,在进行全基因组测序后发现,HEB-2013株基因组全长为15336 nt,包含9个开放阅读框,其中5’UTR长189 nt,3’URT长165 nt;随后将该毒株的全基因序列与NCBI上可查的历年华北地区PRRSV基因组序列进行了比对分析,发现其与2006年分离的TJ株同源性最高,为99.80%,而与2000年分离的BJ-4株同源性最低,仅为88.39%,进一步综合NSP2、GP3和GP5的氨基酸序列比对结果,系统分析了潜在毒力位点与病毒致病性的关系,并且提出了5个新的有可能与病毒致病性相关的潜在毒力位点。     

2株蛋鸡源基因Ⅸ型新城疫病毒全基因序列测定及遗传进化分析

为分析鸡源基因Ⅸ型新城疫病毒的分子特征及遗传进化关系,对两株蛋鸡源NDV进行部分生物学特性测定及全基因测序分析。参考基因Ⅸ型NDV毒株的序列设计10对引物,采用RT-PCR的方法对NDV分离株Layer/China/Yulin/10和Layer/China/Changan/10分段进行扩增及测序,拼接获得全基因序列。结果表明,2株病毒的MDT分别为37.2和56.5h,ICPI分别为1.838和1.603,F基因的裂解位点序列均为112 R-R-Q-R-R-F117,HN基因均编码571个氨基酸,均符合NDV强毒株的特征;2株病毒的基因组全长均为15 192bp,同源性高达99.9%;F基因和全基因系统进化树显示2株NDV均属于ClassⅡ中的基因Ⅸ型,和其他基因Ⅸ型和基因Ⅲ型毒株的亲缘性较高,与野鸟源NDV同源性为99.9%,遗传距离为0.001 1,以上结果表明,这两株鸡源NDV与野鸟源NDV的亲缘关系较近。提示野鸟可能在基因Ⅸ型NDV的扩散与传播中有重要的作用。     

全基因组测序技术在猪源沙门氏菌血清分型和耐药性分析中的应用

【目的】为研究全基因组测序技术在猪源沙门氏菌血清型和耐药性检测方面的应用能力。【方法】以60株猪源沙门氏菌为研究对象,应用传统玻片凝集法和微量肉汤稀释法对其进行血清分型和耐药性检测,并提取所有菌株核酸进行全基因组测序组装,利用SeqSero2和ResFinder4在线预测每株菌的血清型和耐药基因,然后与传统方法进行比较分析。【结果】血清凝集试验共鉴定出8种沙门氏菌血清型,而全基因组测序鉴定出9种血清型,二者分型结果的符合率为90.0%;对11种抗菌药物的药敏试验显示：沙门氏菌对四环素（73.3%）的耐药率最高,其次为氨苄西林（66.7%）、磺胺异噁唑（66.7%）和复方新诺明（53.3%）,基于全基因组预测的恩诺沙星、美罗培南、头孢噻呋和阿奇霉素的耐药性与药敏试验结果完全一致,对氨苄西林、磺胺异噁唑、四环素预测的结果符合率也均大于90.0%。【结论】全基因组测序分析的沙门氏菌血清型和耐药性与常规检测方法的符合率较高,且具有操作简便的优势,可以作为分析沙门氏菌血清型和耐药性的有效方法。     

Complete AV block in a neonatal foal suffering from nutritional myodegeneration

This case report concerns a 1-day-old, full-term Quarter Horse colt presented due to a progressive inability to rise and nurse. Besides the general weakness, a marked bradycardia was evident, which was caused by a third-degree atrioventricular (AV) block. Subsequently, the diagnosis of acute nutritional myodegeneration (NMD) was made based on clinical signs, elevated muscle enzymes, ECG findings, as well as low serum selenium and vitamin E values. Despite intensive care and supportive therapy, the foal´s condition deteriorated, and euthanasia was elected. Necropsy confirmed the diagnosis of NMD and revealed diffuse necrosis and inflammatory infiltration of the skeletal and myocardial musculature, as well as the cardiac conduction system which might have resulted in complete AV block.     

多重耐药胸膜肺炎放线杆菌GD2107株全基因组测序及生物信息学分析

【目的】通过对多重耐药胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)GD2107株进行全基因组测序及生物信息学分析,丰富APP基因组数据库信息;构建基于ApxⅣ基因的系统进化树,分析该菌株的进化关系,为探索APP致病机制和临床防控猪传染性胸膜肺炎(porcine contagious pleuropneumia,PCP)提供参考。【方法】通过药敏试验测定分离菌株的耐药谱;采用全基因组测序技术(whole genome sequencing,WGS)对细菌DNA进行全基因组测序,分别利用Illumina NovaSeq、PacBio SequeI测序平台对全基因组测序结果进行基因功能注释及生物信息学分析(包括基因组基本信息、功能元件分析及亚系统分析等);基于ApxⅣ基因构建系统进化树。【结果】药敏试验结果显示,GD2107菌株对青霉素、头孢拉定(先锋Ⅵ)、卡那霉素等14种抗菌药均耐药。对GD2107株全基因组测序得到1条大小为2 271 987 bp的环状染色体(GC含量为41.21%)和2个大小分别为5 027和3 497 bp的环状质粒,共预测到2 290个编码基因,包含19个rRNA (7个5S rRNA、6个16S rRNA、6个23S rRNA)、21个tRNA基因、20个ncRNA;23个基因岛、4个原噬菌体和2组CRISPR相关序列;分别有2 112、1 549和1 866个基因在COG、KEGG和GO数据库中得到注释,且相关蛋白集中分布于APP的代谢过程;另外在毒力因子(VFDB)和耐药因子(CARD)数据库中还注释到48个毒力基因和22个耐药基因(仅floR基因位于质粒上)。绘制该菌株的全基因组圈图,将基因组信息提交至NCBI,获得染色体GenBank登录号为CP097377,质粒登录号分别为CP097378和CP097379。系统进化树分析发现,该菌株与来自中国的APP菌株(CP063424.1)进化关系最近。【结论】本研究完成了对多重耐药菌株GD2107的全基因组测序与生物信息学分析,全面认识了该菌株基因组的结构和功能并探究了耐药和致病机制中的相关基因,进化关系显示该菌株具有一定的地域流行性,为预防PCP的流行和探索APP的致病机制提供了参考。     

Multiple genotyping in bovine pre-implantation embryos with whole genome amplification

This study examined genetic diagnosis using whole genome amplification (WGA) in bovine embryos. The first experiment was conducted to compare the WGA efficiency of primer extension preamplification-PCR (PEP-PCR) and multiple displacement amplification (MDA), and to optimize the DNA extraction method. The sensitivity of SRY -specific PCR from MDA products increased when DNA of fibroblasts was extracted by a NaOH treatment instead of the conventional method (heat treatment). The detectability of SRY from PEP-PCR products was lower than that in MDA regardless of the DNA extraction method (proteinase K or NaOH treatment). Sexing and genotyping were performed using MDA products from embryo biopsy. The accuracy of PCR-based and LAMP-based sexing was 100% regardless of the amounts of biopsy. Genotyping of CL16 , BND3 , SCD and F11 in MDA products from 10 to 20% of trophectoderm was successful 97, 97, 95 and 95% of the time, respectively, but reduced biopsy amount (<10% of trophectoderm) decreased the accuracies (33–83%). Microsatellite markers were analyzed using MDA products from 10 to 20% of trophectoderm. In eight out of 16 microsatellites, genotypes were not contradictory among the dam, sire and embryos. In the other eight microsatellites, the inconsistency rates were 17–83%. These results indicate that MDA is useful for multiple genetic diagnoses in bovine embryos.     

猪繁殖与呼吸综合症病毒生物学特性

猪繁殖与呼吸综合征(PRRS)为当前危害世界养猪业的重要疾病之一.本文根据当前各国学者对此病原的研究成果从病毒的分类地位,形态、理化特性,组织分布,培养特性,免疫学特性,病毒的基因组成、表达及其功能,病毒蛋白及功能,基因组的遗传和变异作一个系统的综述.     

Interploid crossing overcomes plastome-nuclear genome incompatibility in intersubgeneric hybridization between evergreen and deciduous azaleas

Appearance of albino seedlings caused by plastome-nuclear genome incompatibility was avoided by using polyploid parents in intersubgeneric hybridization between evergreen azaleas and deciduous Rhododendron japonicum f. flavum. Intra- and interploid crosses of 2x or 4x evergreen azaleas with 2xR. japonicum f. flavum showed high capsule set, but their reciprocal crosses failed to set capsules. Green and albino hybrids were obtained from 2x evergreen azaleas × 2xR. japonicum f. flavum crosses where ptDNAs of the green and albino plants were derived from R. japonicum f. flavum (paternal parent) and evergreen azaleas (maternal parents), respectively. All the progenies from the crosses of 4x evergreen azaleas × 2xR. japonicum f. flavum were green triploids with evergreen azalea-specific ptDNA. The efficiency of obtaining green hybrids in interploid crosses was higher than that in intraploid crosses. These results suggest that interploid crosses of 4x evergreen azaleas × 2xR. japonicum f. flavum can overcome plastome-nuclear genome incompatibility between evergreen azalea-specific plastome and the nuclear genome in the resulting hybrid.     

黄瓜与酸黄瓜异源多倍体3种同工酶表达基因组剂量效应

 以甜瓜属（Cucumis）黄瓜与酸黄瓜的3种不同倍性异源多倍体 - 双单倍体种间杂种F₁（2n = 2x = 19,HC）、异源三倍体黄瓜（2n = 3x = 26,HCC）和异源四倍体C. hytivus (2n = 4x = 38,HHCC)及其双亲为试材,比较研究过氧化物酶（POD）、谷氨酸脱氢酶（GDH）和天冬氨酸转氨酶（AAT）在不同倍性异源多倍体中的酶谱表达特性,探讨基因组剂量对酶谱表达的影响。结果表明：不同倍性异源多倍体的酶带染色力表现出明显的差异,但酶带数和酶带分布基本一致。在AAT同工酶中,酶带的表达随基因组剂量的提高而增强,在POD和GDH同工酶中,大部分酶带的表达随基因组剂量的提高而减弱。这些结果表明甜瓜属异源多倍体在不同的酶系统中,酶谱表达可能会表现出截然相反的基因组剂量效应。本研究结果可为     

一株Asia 1型口蹄疫病毒的全基因组序列测定及比较分析

参考GenBank上已发表的口蹄疫病毒（FMDV）全长基因组序列,设计了覆盖基因组全长的数对引物,通过RT-PCR方法对1株Asia1型（简称As01）FMDV进行分段克隆及序列测定,将测序结果利用软件进行拼接。结果表明,该毒株的FMDV基因组[不合poly（C）]全长8180nt,其中编码区为6987nt,5’和3’非编码区（UTR）分别为1078nt和95nt,3＇UTR之后为20nt的poly（A）尾巴。将As01株与其他FMDV参考毒株利用分子生物学软件进行多序列比对分析表明,As01株与Asia 1／Jiangsu／China／2005、Asia 1／Isr 1／3／63、ZB／CHA／58等Asia 1型FMDV遗传进化关系较为密切,而与O型和A型FMDV遗传关系较远。