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沙发
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发表于 2015-2-3 11:03:21
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2# wwwkkk83
关于如何鉴定一个全新 的病毒 http://bbs.virology.com.cn/thread-4671-1-1.html
by freecell
新病毒的鉴定是个贯穿多学科的过程。
首先是重视临床鉴别诊断。例如2003年的SARS发现，就是通过与常见呼吸道病毒或其它病原微生物的症状进行鉴别，从而得到部分线索。
其次是血清学检验方法的运用。利用抗原捕捉ELISA，筛选和排除可疑病原体；采用双份血清IgG、IgM进行检测，分析抗体产生规律，摸清感染进程；
其三是经典病毒学方法的运用。接种细胞，产生CPE。但是HCV的细胞模型直到去年才建立起来。一般认为，在电镜下观察到病毒颗粒，是病毒鉴定的金标准。
其四是运用分子生物学方法，分别进行扩增，排除可疑病原体。在得到序列的情况下，进行BLAST，得到病毒的大致分类位置；
最后当然是功能基因组学和蛋白质组学方法了，例如基因芯片、2D等。有趣的是，轮状病毒不同型的鉴别，是通过SDS-PAGE实现的，而且至今尚在应用。
值得一提的是，上述方法并非截然分开的，需要进行综合运用。鉴定一个全新的病毒，需要扎实的理论、丰富的经验、睿智的实验设计、大胆的推测演绎。
原文是 freecell的帖子。
3#bigben
呵呵，今天刚讲了一个文献，在2000年science发表了一篇report，发现1999年在印尼和新加坡流行的Nipah virus是一个新的副粘病毒科成员。大体思路如下：
1 EM studies of Nipah virus, demonstrated features characteristic of a virus belonging to the family Paramyxoviridae.
电镜发现Nipah virus（在印尼一个村落首次发现）有副粘病毒科形态特征
2 Serologic studies suggested that Nipah virus was the principal etiologic agent of the outbreak in Malaysia and Singapore.
血清学实验表明，所分离到的病毒是导致在印尼和新加坡流行的病原体
3 Sequence studies of Nipah virus clearly placed it in the family Paramyxoviridae and Nipah and Hendra viruses are closely related members of a new genus.
利用副粘病毒设计引物，将Nipah virusPCR扩增，测序，并进行进化树分析，表明属于副粘病毒科
感觉最重要是将病毒分离并培养，还是Koch原则
全文：
Nipah Virus: A Recently Emergent Deadly Paramyxovirus
K. B. Chua, et al.Science 288, 1432 (2000)
背景：
尼帕病毒感染
Nipah Virus Infection
类别:畜禽传染病·人畜主要共患传染病
尼帕（Nipah）病毒感染是一种新发现的以脑炎为特征的病毒性传染病，在感染猪群和与猪接触的人中流行，可引起人和猪的死亡。猪发病后主要症状为发热、呼吸困难、持续而剧烈的咳嗽，最特殊的现象是腹部肌肉发生阵发性痉挛性收缩，直至死亡。目前尚无有效治疗方法。
原文：Nipah virus: a recently emergent deadly paramyxovirus.
4# feynman
据说老美在研究SARS初，由取样到疑是冠状病毒只用了几个小时，都是数据库的功劳
5# wwwkkk83
Discovery of New Viral Agents（from FIELDS VIROLOGY 5e）
In recent years, numerous new viral agents have been discovered. In some instances, conventional viral culture
techniques have yielded new viruses. For example, detection of Hendra virus, a new paramyxovirus that causes
infection of horses and humans, was based on growth of the virus in cell cultures in use in diagnostic virology
laboratories (124). In other cases, powerful nucleic acid-based techniques have been applied to detect viruses
that could not be cultivated. Application of these techniques is likely to yield additional new viruses in coming
years. Short descriptions of the culture-independent discovery of three human pathogenic viruses follow.
The first outstanding example of culture-independent virus discovery is HCV. This virus was discovered through
molecular cloning of plasma known to be infectious for chimpanzees. Serum from a person with non-A, non-B
hepatitis was used to screen an expression library of approximately one million candidate clones, resulting in
discovery of one clone expressing an HCV gene product. Nucleic acid sequencing provided the identification of the
new virus as a flavivirus and led to the development of a blood test used for screening of donated blood units (27).
Cultivation of HCV has still not been achieved.
The discovery of Sin Nombre virus, the cause of hantavirus pulmonary syndrome, began with the detection of
antibodies in the serum of affected individuals that cross-reacted with known human hantaviruses. Based on that
clue, an RT-PCR assay was designed using primers based on sequences conserved in the four hantaviruses
previously known to be human pathogens. Sequencing of a product amplified from specimens obtained from
human cases revealed the presence of a new hantavirus (126). That discovery has led to the discovery of
numerous additional hantaviruses (45). Laboratory cultivation of Sin Nombre virus and related viruses remains
extremely difficult.
Kaposi's sarcoma virus (HHV-8) was discovered as the cause of Kaposi's sarcoma through use of the technique of
representational difference analysis (107). This technique involved the use of DNA extracted from Kaposi's
sarcoma tissue, which was used along with DNA extracted from normal tissue. Unique sequences were detected
that were homologous to known gamma herpesviruses (24). The new virus was subsequently shown to be highly
associated with Kaposi's sarcoma as well as with multicentric Castleman's disease and AIDS-related body cavitybased
lymphoma. Cultivation of the virus in cell culture has subsequently been achieved (60).
More recently, microarrays that employ conserved sequences of large numbers of known viruses have been
created and used successfully in virus discovery (192).
Several new respiratory viruses have been discovered using a combination of conventional testing and innovative molecular methods.
HMPV (187), the SARS coronavirus (46,96,132), and HCoV-NL (61,189) were discovered by careful and persistent conventional
techniques, and careful observation for CPE. A technique called VIDISCA was also used to discover HCo-NL63
(189). This technique involves enrichment of viral nucleic acid, digestion with frequent cutting restriction
endonucleases, ligation of linkers containing PCR primer binding sites, amplification by PCR, and sequencing of
PCR products. A new parvovirus called human bocavirus was discovered in nasopharyngeal secretions from
children with respiratory illness using a technique for direct sequencing of nucleic acids present in the original
specimen followed by assembly of sequenced fragments (2). |
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发表于 2015-2-3 10:55:37
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