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Applications Of Single-cell Technologies In Researches On Positive-stranded RNA Viruses

Posted on:2011-08-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:X HuangFull Text:PDF
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The genome of positive-stranded RNA viruses is plus strand RNA, which can be used as mRNA to generate viral proteins. Many of the members are causative agents of highly contagious diseases in humans, plants and animals, e.g., HCV, poliovirus (PV), SARS virus, dengue virus, foot and mouth disease virus (FMDV), bovine viral diarrhea virus (BVDV). So far, there are no effective and rapid methods for prevention and cure of these harmful viruses. Viral genome replication is the basis of virus survival and multiplication, which is an indispensable process in virus life cycle. Studies on positive strand RNA viral replication play important roles in avoiding the spread of pathogens.Foot-and-mouth disease virus is a positive-sense, single-stranded RNA virus with a negative strand as its replication intermediate, which can cause severe acute infection in sensitive cell lines (BHK-21 cells and PK-15 cells). To investigate better the actual state of virus infection, there is a need to measure the amount of FMDV RNA in a single acutely infected cell rather than in a large number of cells. Therefore, in the present study, a strand-specific single-cell quantitative real-time RT-PCR was developed to analyze the RNA of FMDV. This new method includes three techniques: a technique for isolating single cells with micromanipulators, lyses of single-cell samples and sensitive quantitative real-time RT-PCR. In the assay of acute infection, 185 of 224 (82.6%) single-cell samples were positive and contained viral genome copies ranging from several to thousands, and up to 1000000 copies. However, not all cells were infected and there were differences in the number of viral RNA copies among cells. Virus replication as well as the relationship bewteen virus and host cell can be studied in single cells basing on the technique, which helps to clarify the mechnism of virus replication and virus evolution and also provides as a new method for further research on infectious cell biology. Besides, the methods for isolation and pre-treatment of single cells are universal and can be used in other cell lines (both suspended cells and adherent cells) whether inoculated with viruses or not, which are the basis of other analyses in single cell level.There are two mechanisms by which viral replication can be initiated:primer independent or de novo, and primer dependent. FMDV and PV belong to primer dependent while BVDV belongs to primer independent. In this study, single cell quantitative real-time RT-PCR assays were carried out for quantitation of both positive strands and negative strands of FMDV, PV and BVDV to investigate the replication of positive-stranded RNA viruses. Results showed that experimental data (Log10+RNA and Log10-RNA) of Vero cells infected with PV and BHK-21 cells/PK-15 cells infected with FMDV had good linear correlations. Mathematical modeling analysis of our data indicated that one-RNA may be used to generate one +RNA and initial+RNA copies as well as the rate of+RNAs for replication were different in two viruses (FMDV and PV). However, quantitation data of BKC cells infected with BVDV showed almost no linear correlations. Results of mathematical modeling analysis indicated that one-RNA may be used to generate one+RNA and initial+RNA copies as well as the rate of+RNAs for replication were totally different from the other two viruses.The weak bases (ammonium chloride), which diffuse into acidic endosomes and raise the pH of endocytic organelles, prevent the required low pH-dependent proteins conformational changes leading to virus genome release, can reduce the yield of FMDV after infection. In this study, baby hamster kidney cell line (BHK-21) infected with FMDV serotype O was maintained in medium with NH4Cl weak base. Survived cells were isolated using micromanipulator to form single-cell clones,17 positive-cloned cell strains were obtained. One cell strain (BHK-Op) was selected randomly and cultured as usual for analysis with quantitative real-time RT-PCR, western blotting, transmission electron microscope. FMDV particles were observed successfully in BHK-Op48 cells, which dispersed in the cytoplasm and loacted in some small endosomes (not more than 10, usually 2 or 3) with a diameter of 20-25nm. Virions in acutely infected cells mainly located in endosomes close to the nucleus in large quantities. Infection of the persistent viruses could not form plaques in host cells but virulence was enhanced. Basing on analysis and comparison of cDNA sequences of resident viruses and wild type viruses,15 amine acid mutations were found, all of which located in nonstructural proteins rather than in structural proteins, which was inconsistent with previous studies. Two conservative amino acid mutations in 3C protein located exactly in the RNA binding domain and were relevant to virus replication, which is likely to be one of the reasons for lost plaques. The establishment of persistent infections in vitro provides an effect model for studying coevolutions of virus and host cell as well as interactions of virus and receptors. Besides, microarray experiments are underway to attempt to search host cell proteins or factors pertaining to FMDV persistence in BHK-21 cells.
Keywords/Search Tags:Foot-and-mouth disease virus, Poliovirus, Bovine viral diarrhea virus, Viral genome replication, Persistent infection, Amonium chloride, Single-cell quantitative real-time RT-PCR
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