Poliovirus In Cell Cultures Research Articles

Limited genetic changes in the Sabin 1 strain of poliovirus occurring in the central nervous system of monkeys

Replication of attenuated poliovirus strains results in their partial deattenuation. Recently we identified mutations accumulating in the Sabin 1 poliovirus in cell cultures. Here we report genetic changes occurring in this virus during replication in the central nervous system (CNS) of monkeys. Viruses isolated from different parts of the CNS of rhesus monkeys (inoculated into the spinal cord) were screened for sequence heterogeneities and newly identified mutations were independently confirmed and quantified using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). All consistently accumulating mutations identified in this study were located in untranslated regions: GU-->AU or GU-->GC substitution at a complementary pair formed by nucleotides 480 and 525, U-->C substitution at nucleotide 612, and GU-->AU or GU-->GC substitution of a base pair formed by the nucleotides 7427/7441 immediately preceding the poly(A) tract. All these mutations except one (7427) were previously identified in cell culture passages or stool isolates from vaccinees. Sequencing of 11 CNS isolates also identified a few random silent mutations that accumulated as neutral 'passengers', passively co-selected with genuinely selectable mutations present on the same RNA molecule. One isolate also contained the wild-type base at nucleotide 2741 (Ala88-->Thr in VP1). Our results demonstrate a remarkable genetic stability of the Sabin 1 poliovirus in the CNS of monkeys, suggesting that deattenuation is determined by a very limited number of mutations. These mutations can be assayed by MAPREC to monitor the consistency of oral poliovirus vaccine (OPV) production.

Microevolution of Type 3 Sabin Strain of Poliovirus in Cell Cultures and Its Implications for Oral Poliovirus Vaccine Quality Control

Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently, SO-derived samples accumulated more mutations than did RSO derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37°, a U → C transition at nucleotide 5832 occurred faster at 34°, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5786, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production.

Effect of polymyxin B on the replication of poliovirus in cell cultures.

In earlier experiments it was shown that the replication of polio-virus in cell cultures is stimulated by polymyxin B. Data were presented indicating that this effect was due to a hastened release of the virus from the cell rather than to enhanced intracellular multiplication.

Antiviral effect of guanidine.

It has been found that soluble guanidine salts show an antiviral effect on poliovirus and on some other enteroviruses. The activity was first detected in a cell culture screening system when the guanidine salt of hydroxyaminomethylene malononitrile inhibited the cytopathic effect of poliovirus in cell cultures. The studies were extended to various in vivo experiments in which the suggestive therapeutic activity of guanidine salts was again observed in monkeys infected with poliovirus. While these drugs cannot be considered for use in human disease because of severe toxicity, it is significant that potential antiviral compounds detected by the cell culture test show in vivo activity and point to the validity of such direct chemotherapeutic trials.

The Biosynthesis of Poliovirus in Cell Cultures

This chapter considers the recent findings of poliovirus with respect to its biosynthesis. Results of quantitative significance concerning the biosynthesis of poliovirus have come almost exclusively from the use of dispersed mammalian cells, either in primary culture, exemplified by monkey kidney epithelium or in serially propagated cultures, such as the HeLa cell. Either of these cell lines can be used for both the production of virus and its quantitative plaque assay. The particular value of poliovirus is that it is an RNA-containing virus, the replication of which can be studied under experimental conditions approximating those of the coliphage system. The detailed study of the chemical and biophysical properties of the virus particle promises to be rewarding in itself. There is preliminary evidence for free sulfhydryl groups in the protein coat of poliovirus. This should allow attachment of heavy atoms such as mercury to yield a product that would be of aid to the X-ray crystallographer in studies on the structure of the particles.