MOLECULAR AND GENETIC STUDIES OF FLANDERS VIRUS

该提案的主要目的是表征分子 哈特病毒成员之一的弗兰德斯病毒（FV）的遗传组成 弹状病毒科Park血清群。 FV已被证明有8个 病毒特异性蛋白和至少6种mRNA。 将从FV特异性聚腺苷酸化RNA制备cDNA文库， 通过正反交选择FV特异性克隆并分组 杂交和北方印迹。 选定的克隆将用于 杂交选择研究，以确定 每一个独特的mRNA。 FV基因将被定位，基因间区域 利用可用的通读RNA以及通过引物鉴定 直接从基因组中延伸RNA， 寡脱氧核苷酸引物。 将使用两种技术进行测序 基因组3 '端：第一，二维寡核苷酸指纹图谱 第二，通过3 '端的多聚腺苷酸化和 寡聚（dT）引物插入3 '端。 受感染细胞的RNA将被 通过RNA酶保护测定分析以确定正链前导序列是否 RNA被制造出来。 选择的基因，如任何独特的基因， 将对水疱病毒或狂犬病毒中代表的病毒进行测序。 大量的"子弹形"病毒从节肢动物身上回收， 脊椎动物被归为弹状病毒科， 未表征及其作为人类和/或动物病原体的潜力 未知 哈特帕克血清群病毒已从蚊子中分离出来 已知以人类为食的物种和两种成员的抗体 在非洲的人类中发现了这种病毒。 弹状病毒家族 包括许多已知为疾病病原体的病毒 在人类和动物中，但基本上只有水泡病毒， 已经对狂犬病毒进行了深入研究。 最近的发现更多 特定弹状病毒中的5种以上遗传元件或基因产物， 包括FV，强调需要详细的分子和遗传 其他弹状病毒的研究。 如果事实上哈特帕克血清组 病毒在6或7个mRNA上编码8种蛋白质， 转录、翻译或蛋白质加工可以提供有价值的 负链基因表达调控的新见解 RNA病毒，并形成进一步评估的基础， 它们之间的相互关系，特别是弹状病毒， 副粘病毒和丝状病毒。

The primary objective of this proposal is to characterize the molecular and genetic composition of Flanders virus (FV), a member of the Hart Park Serogroup of the Rhabdoviridae. FV has been shown to have 8 virus-specific proteins and at least 6mRNAs. A cDNA library will be prepared from FV-specific polyadenylated RNA and FV-specific clones selected and grouped by reciprocal-cross hybridization and Northern blots. Selected clones will be utilized in hybrid-selection studies to determine the protein-coding assignment of each unique mRNA. FV genes will be mapped and the intergenic regions identified utilizing available readthrough RNAs as well as by primer extension directly from the genome RNA from synthetic oligodeoxynucleotide primers. Two techniques will be used to sequence the genome 3'end; first, two-dimensional oligonucleotide fingerprint analysis and second, by polyadenylation of the 3'-end and extension of an oligo (dT) primer into the 3'end. RNA from infected cells will be analyzed by RNase protection assay to determine if a plus strand leader RNA is produced. Selected genes such as any unique genes not represented in the vesiculovirus or lyssavirus will be sequenced. A large number of "bullet-shaped" viruses recovered from arthropods and vertebrates have been assigned to the Rhabdoviridae yet remain uncharacterized and their potential as human and/or animal pathogens unknown. Hart Park Serogroup viruses have been isolated from mosquito species known to feed on humans and antibodies to two members of the group have been detected in humans in Africa. The Rhabdovirus family includes a number of viruses known to be etiological agents of disease in humans and animals yet essentially only the Vesiculoviruses and Lyssaviruses have been intensively studied. Recent findings of more than 5 genetic elements or gene products in specific rhabdoviruses, including FV, emphasize the need for detailed molecular and genetic studies of other Rhabdoviruses. If, in fact, Hart Park Serogroup viruses encode 8 proteins on 6 or 7 mRNAs the mechanism(s) of transcription, translation, or protein processing could provide valuable new insights into the regulation of gene expression in negative-stranded RNA viruses and form a basis for further assessment of the interrelatedness among them, particularly the Rhabdoviruses, Paramyxoviruses, and Filoviruses.