扬州蚕豆病毒病的鉴定及野豌豆潜隐病毒M扬州分离物基因组克隆及其序列分析

 对田间疑似病毒侵染的蚕豆样品,提取总RNA,分离siRNA,建库并通过高通量测序,其结果经velvet拼接组装, 经Blastn与NCBI比对分析,发现该样品感染4种病毒:野豌豆潜隐病毒M (vicia cryptic virus M,VCV-M)、野豌豆潜隐病毒 (vicia cryptic virus, VCV)、紫云英矮宿病毒 (milk vetch dwarf virus, MDV) 和三叶草黄脉病毒 (clover yellow vein virus, ClYVV)。其中,有14条VCV-M相关的contigs。RT-PCR扩增并拼接获得VCV-M-YZ基因组,其长度为3 434 nt, 其5′-UTR和3′-UTR 分别为142 nt和117 nt,各自形成与该属代表病毒南方番茄病毒 (southern tomato virus, STV) 5′-UTR和3′-UTR相似的高级结构,且A+U含量均较高。 VCV-M-YZ与已报道的VCV-M基因组序列一致性在98%以上。系统进化分析表明,VCV-M-YZ与已报道的VCV-M属于一个种,无明显的地理分布差异。本研究结果丰富了VCV-M群体的基因组遗传信息,为VCV-M的监测及深入研究提供了基础。     

Is there evidence for post-epidemic attenuation in the Dutch elm disease pathogen Ophiostoma novo-ulmi?

The first pandemic of Dutch elm disease (DED) in Europe and North America caused by the introduced Ophiostoma ulmi began in the early 1900s but declined unexpectedly in Europe from the 1930s onwards after killing 30–40% of the elms. Later a second pandemic caused by the much more aggressive Ophiostoma novo-ulmi spread across the same areas and by 1990 most of Britain's c. 30 million mature elms had died. During the second pandemic, O. novo-ulmi acquired debilitating viruses and changed from being largely clonal to highly heterogeneous through horizontal transfer of novel, sometimes deleterious, genes from O. ulmi. In the post-epidemic period a new disease dynamic has emerged, with millions of small recruitment elms repeatedly attacked by sequential cycles of disease. This study investigated the possibility of pathogenic attenuation in O. novo-ulmi as the epidemic has progressed. Isolates collected from the three original outbreak areas in Britain in 1982–3 (early post-epidemic period) and 2011 (advanced post-epidemic) were compared for two fitness components: pathogenic aggressiveness and in vitro growth rate. Mean aggressiveness of the 2011 isolates to clonal English elm (Ulmus procera) proved not to differ significantly from that of the 1982 isolates, either overall or within each outbreak area. Similarly, the mean growth rates of the 1982 and 2011 samples showed no differences. The implications of these findings are discussed in relation to the future of the current DED pandemic in Europe, the decline of the first pandemic, and the difficulty of drawing parallels between different tree–pathogen systems.     

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.     

Use of S1 nuclease in deep sequencing for detection of double-stranded RNA viruses

Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5–28.4- and 10.1–208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses.     

猪繁殖与呼吸综合征病毒柳州地方株NSP2和ORF5基因变异分析

为了给广西柳州预防和控制猪繁殖与呼吸综合症提供理论数据,本研究对利用RT-PCR方法扩增了该地区流行株LZ5和LZ6的NSP2和ORF5基因并进行测序分析,然后与GenBank中已发表的PRRSV毒株的NSP2和ORF5基因序列进行比较。结果显示,2株柳州地方流行株均属于美洲型,在NSP2基因第483位和535~563位氨基酸均存在30个氨基酸的不连续缺失,其NSP2和ORF5基因的核苷酸同源性分别为99.5%和99.7%,推导的氨基酸同源性分别为100%和99.5%;与近几年国内流行的高致病性蓝耳病代表性毒株之间的NSP2、ORF5基因核苷酸同源性分别为94.7%~96.2%和97.2%~98.0%,其推导的氨基酸同源性分别为92.0%~95.5%和94.5%~97.0%。序列分析结果表明此次柳州流行的PRRSV与2006年夏高热病引起的基因序列高度同源,在基因序列上并未显示出新的特性,在国内也缺乏明显的地域性差异。     

Plant pararetroviral sequences in wild Dahlia species in their natural habitats in Mexican mountain ranges

Selected wild Dahlia species in their natural habitats from west‐central Mexico were tested for the presence of three caulimoviruses known to be associated with cultivated dahlia (Dahlia variabilis), viz. Dahlia mosaic virus (DMV), DMV‐D10 and Dahlia common mosaic virus. Virus species‐specific primers and PCR were used followed by cloning and sequencing of the amplicons. Results showed that the wild dahlia species in their natural habitat contained DMV‐D10, which is an endogenous plant pararetrovirus. Viral sequences were found in 91% of the samples (n = 56) representing four different wild species. The gene coding for the movement protein of DMV‐D10 from Dahlia coccinea and all other species was cloned and sequenced. Sequence comparisons showed divergence of this gene when compared to that of DMV‐D10 from cultivated dahlias. The discovery of plant pararetroviruses in wild dahlia species in their natural habitats suggests a possible emergence, co‐existence and co‐evolution of pararetroviruses and their host plants.     

老鹳草鞣质的提取工艺及其免疫作用的试验研究

为确定提取老鹳草鞣质的合理生产工艺,及为药物复配提供药理学依据,采用水回流法、水温浸法、丙酮回流法、冷浸搅拌法和乙醇回流法进行了老鹳草鞣质的提取试验;采用MTT方法检测了不同工艺生产的老鹳草鞣质对ConA和LPS刺激鸡外周血中淋巴细胞增殖反应的影响。试验结果表明：水温浸法、丙酮回流法和乙醇回流法鞣质得率和质量分数均较高,考虑经济因素则水温浸法为好。除乙醇回流法外的4种方法提取的老鹳草鞣质在质量浓度较低时对ConA诱导的鸡外周血T淋巴细胞及LPS诱导的B淋巴细胞的增殖均有显著的促进作用。     

山西苹果褪绿叶斑病毒的分布及遗传多样性分析

苹果褪绿叶斑病毒Apple chlorotic leaf spot virus (ACLSV) 是侵染苹果的主要潜隐性病毒之一, 在我国苹果植株上发生普遍, 严重威胁我国苹果的品质与产量。本研究从山西省12个苹果主产区随机采集360份表现褪绿和斑驳等症状的苹果叶片作为研究样本, 通过RT-PCR检测, 360份样本中有209份样本为ACLSV阳性, 对209份阳性样本的外壳蛋白(coat protein, CP)基因进行分离、测序、克隆, 得到12个新的ACLSV分离物(分别命名为 Shanxi 1~Shanxi 12)。选择17个来自不同国家的分离物与12个新的ACLSV分离物在核苷酸和氨基酸层面上进行序列一致性和系统发育分析。结果显示, 29个ACLSV分离物被划分为2个不同进化群体。进一步对2个不同ACLSV群体进行选择压分析和中性检验, 结果表明, 组Ⅰ与组Ⅱ的ACLSV群体之间存在明显的遗传差异, 其中负向选择可能是ACLSV遗传变异的原因之一。本研究较全面地分析了ACLSV的发生、危害, 并对山西苹果的ACLSV分离物进行了遗传结构分析, 为山西苹果褪绿叶斑病毒病的防治提供了理论指导。