DETECTION OF RHINOVIRUS INFECTION OF THE NASAL-MUCOSA BY OLIGONUCLEOTIDE IN-SITU HYBRIDIZATION
DETECTION OF RHINOVIRUS INFECTION OF THE NASAL-MUCOSA BY OLIGONUCLEOTIDE IN-SITU HYBRIDIZATION
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DOI：
10.1165/ajrcmb.10.2.8110476
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发表时间：
1994-02-01
影响因子：
6.4
通讯作者：
HOLGATE, ST
中科院分区：
文献类型：
--
作者：
BARDIN, PG;JOHNSTON, SL;HOLGATE, ST
人鼻病毒（HRV）引起普通感冒，并经常引起下呼吸道症状，如咳嗽和喘息。尽管假定HRV感染主要涉及纤毛上皮细胞，但这尚未在体内得到证实，并且感染的细胞分布和传播以及感冒相关的鼻和胸部症状的发病机制仍然是推测性的。我们已经开发了原位杂交（ISH），探索本地化的病毒气道组织，采用HRV 16衍生的寡核苷酸探针测序后，该血清型的基因组的一部分。逆转录-聚合酶链反应用于从HRV 16产生DNA用于测序;这产生了305个核苷酸碱基，其显示出与其他HRV相当大的同源性。HRV 16序列用于设计用作反义和正义探针的寡核苷酸。将这些探针以及随机序列和病原体对照寡核苷酸应用于HRV感染的细胞-凝块复合物，并最终应用于在HRV证实的感冒之前、期间或之后获得的六对鼻活检的切片。通过在未感染的组织中、在被其他病毒感染的细胞中、在RNase预处理后以及在应用对照探针的情况下不存在信号来建立杂交体的特异性。在感冒期间获得的6份活检标本中，有3份在上皮细胞中观察到杂交信号，使用病毒（+）链探针;中间（-）链，意味着病毒复制，存在于1份活检标本中。非上皮细胞感染的证据尚不确定。HRV在感冒期间引起鼻上皮的生产性感染，并且它们的细胞内定位可能产生炎症介质和细胞因子谱的扰动。ISH的使用将允许研究探索HRV相关症状的发病机制，并阐明下呼吸道受累的机制。
Human rhinoviruses (HRVs) cause the common cold and often induce lower airway symptoms such as cough and wheezing. Although HRV infection is presumed to involve primarily ciliated epithelial cells, this has not been confirmed in vivo, and the cellular distribution and spread of infection as well as the pathogenesis of cold related nasal and chest symptoms remain speculative. We have developed in situ hybridization (ISH) to explore localization of the virus to airway tissues, employing HRV 16-derived oligonucleotide probes after sequencing part of the genome of this serotype. A reverse transcription-polymerase chain reaction was used to generate DNA from HRV 16 for sequencing; this yielded 305 nucleotide bases that showed considerable homology to other HRVs. The HRV 16 sequence was used to design oligonucleotides functioning as antisense and sense probes. These probes as well as random sequence and pathogen control oligonucleotides were applied to HRV-infected cell-clot complexes and finally to sections from six paired nasal biopsies obtained before, during, or after HRV-proven colds. Specificity of hybrids was established by the absence of signal in uninfected tissue, in cells infected with other viruses, after RNase pretreatment, and with application of control probes. Hybridization signals were observed in epithelial cells in three of six biopsies obtained during a cold, using probes to viral (+) strand; intermediate (-) strand, implying viral replication, was present in one biopsy. Evidence for infection of nonepithelial cells was inconclusive. HRVs cause productive infection of nasal epithelium during a cold and their intracellular localization may produce perturbation of inflammatory mediators and cytokine profiles. Use of ISH will permit studies exploring the pathogenesis of HRV-related symptoms and clarification of the mechanisms of lower airway involvement.