Title

Development of cell-based systems for examination of cis- and trans-functions in replication of positive strand RNA viruses

Date of Award

2004

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Asit K. Pattnaik, Committee Chair

Abstract

Many positive strand RNA viruses, including West Nile virus and SARS coronavirus, share analogous cis- and trans-elements for replication. We developed a cell-based system to analyze these elements in replication of hepatitis C virus (HCV). We detected intermediates indicative of replication by HCV polymerase in transient transfection of HepG2 cells. However, further analysis resulted in the detection of nonspecific background, which could not be eliminated. Therefore, we developed a replication system to analyze another positive strand RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV).The consensus nucleotide sequence of the genome of a pathogenic PRRSV isolate from Iowa was determined. The genome contains 15,413 nt, a poly(A) tail with 60--80 residues and shares more than 90% similarity with other North American isolates. A full-length cDNA clone (pFL12) of the viral genome was assembled in pBR322 vector. Transcripts, derived from the viral cDNA, contained the full-length viral genome flanked by a 5' cap and a precise 3' terminus, which included a poly(A) tail with 43 adenosine residues. Transfection of in vitro transcripts into MARC-145 cells resulted in recovery of infectious virus. The cloned and parental viruses shared similar growth kinetics in cell culture. In inoculated pigs, the growth and pathogenic properties of the cloned virus were indistinguishable from the parental virus in terms of induction of viremia, seroconversion and hyperthermia. The cloned virus retained genetic stability through multiple rounds of replication in cultured cells and in inoculated and sentinel pigs. Retention of genetic stability and natural virulence properties makes this infectious cDNA clone ideal for use in the study of PRRSV gene functions and vaccine development. Furthermore, the sequence stability of the cDNA during several rounds of propagation in E. coli makes it highly amenable to reverse genetic manipulation.Recovered vFL12 genome contained a poly(A) tail extended from 43 to 60--80 adenosine residues, reflecting the parental virus genome. Analysis of mutants of pFL12 with varying lengths of poly(A) tails, from 1 to 70 adenosines, indicated that mutants containing 5 or more residues could be rescued. The infectivity of the mutants in transfected cells correlated directly with the length of the poly(A) tail. In several attempts, virus could not be rescued from a mutant containing a poly(A) tail of one adenosine residue, indicating that more than one adenosine is required for infectivity of the template.

Keywords

Biology, Molecular; Biology, Cell; Biology, Microbiology; Health Sciences, Immunology

Link to Full Text

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