MicrobiologyBytes

It was my privilege to work with Phil Minor during my PhD. 25 years later (gulp), Phil looks back and forward to the polio endgame.

The Polio-Eradication programme and issues of the end game. J Gen Virol. Nov 29 2011
Poliovirus causes paralytic poliomyelitis, an ancient disease of humans that became a major public health issue in the 20th century. The primary site of infection is the gut where virus replication is entirely harmless; the two very effective vaccines developed in the 1950s (Oral Polio Vaccine, or OPV and Inactivated Polio Vaccine, or IPV) induce humoral immunity which prevents viraemic spread and disease. The success of vaccination in developing countries and in middle income countries encouraged the World Health Organization to commit itself to an eradication programme which has made great advances. The features of the infection including its largely silent nature and the ability of the live vaccine (OPV) to evolve and change in vaccine recipients and their contacts make eradication particularly challenging. Understanding the pathogenesis and virology of the infections is of major significance as the programme reaches its conclusion.

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Studies of virus particles and the steps in their life cycle have spearheaded our understanding of biological systems at the molecular level. These studies, however, relied on virus specimens isolated from nature. This dependency changed forever in 2002 when the chemical synthesis of poliovirus, in the absence of any natural template, was published. The work caused a shock wave because it led to excitement as well as revulsion, reflecting the new reality that, for better or worse, all of the more than 2,000 viruses whose genome sequences are deposited by the National Center for Biotechnology Information can be recreated in the laboratory in the absence of natural isolates. So what have we learned?

Synthetic poliovirus and other designer viruses: what have we learned from them? (2011) Annu Rev Microbiol. 65:583-609
Owing to known genome sequences, modern strategies of DNA synthesis have made it possible to recreate in principle all known viruses independent of natural templates. We describe the first synthesis of a virus (poliovirus) in 2002 that was accomplished outside living cells. We comment on the reaction of laypeople and scientists to the work, which shaped the response to de novo syntheses of other viruses. We discuss those viruses that have been synthesized since 2002, among them viruses whose precise genome sequence had to be established by painstakingly stitching together pieces of sequence information, and viruses involved in zoonosis. Synthesizing viral genomes provides a powerful tool for studying gene function and the pathogenic potential of these organisms. It also allows modification of viral genomes to an extent hitherto unthinkable. Recoding of poliovirus and influenza virus to develop new vaccine candidates and refactoring the phage T7 DNA genome are discussed as examples.

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In 1979 a young researcher blundered into a laboratory and began working towards a PhD. Four years later, he emergerd blinking into the sunlight and headed off to California for a while. This is the story of those years, which has lain buried under a layer of dust ever since until it was recently published online. Along the way, we had some laughs. For example, this may amuse some people:

Data Handling
Nucleotide sequence data was entered and analysed on a PDP 11/4 computer using a package of programs obtained from R. Staden, MRC Laboratory of Molecular Biology, Cambridge, UK (Staden, 1980).

At any rate, the truth can now be told:

Title: Studies on the Genome Structure of Neurovirulent and Attenuated Polioviruses
Author: Cann, Alan James
Date: 1984
Publisher: University of Leicester
Description: Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester, 1984.
Abstract: The RNA genomes of neurovirulent and attenuated type 3 polioviruses have been cloned in E. coli using an efficient RNA.cDNA hybrid technique. The complete nucleotide sequence of the vaccine-associated neurovirulent revertant P3/119 and, in collaboration with others, the attenuated vaccine strain P3/Leon 12 a1b, have been determined. These have been compared with that of the neurovirulent parent strain P3/Leon/37. Ten nucleotide sequence differences were observed between the parent P3/Leon/37 and the vaccine P3/Leon 12 a1b, three of which resulted in amino acid substitutions. Between the vaccine and the revertant P3/119, seven nucleotide sequence differences were observed. Three of these resulted in amino acid substitutions. The possible significance of individual nucleotide sequence differences to the attenuation of and reversion to neurovirulence in poliovirus type 3 is discussed. The nucleotide sequence of P3/Leon 12 a1b was the first to be determined for a type 3 poliovirus. Comparison of this sequence with published type 1 sequences has demonstrated the extent of the molecular homology between them.

Summary:
The major objective of this study was to identify the nucleotide sequence differences which account for the neurovirulent or attenuated phenotype of three closely related strains of poliovirus type 3. To achieve this, an efficient RNA.cDNA hybrid cloning method was devised. Although this was not the first report of hybrid cloning, previous unfavourable comments on its efficiency (Wood and Lee, 1976; Zain et al., 1979) and suggestions that the method given rise to cloning artefacts (Okayama and Berg, 1982) have almost certainly discouraged widespread use. Thorough investigation of each of the manipulative steps involved has shown that these problems can be overcome (Cann et al, 1983). Comparable in efficiency to the more used double-stranded cDNA cloning technique, the hybrid method has the advantages of experimental simplicity and that cDNA clones corresponding to entire virus genome can be obtained from a single experiment. The method has proved to be ideally suited to the molecular cloning of picornavirus genomes. It is possible that the transformation efficiency of RNA.cDNA hybrids could be further increased by treatment with E. coli DNA ligase and DNA polymerase I before ligation, thus carrying out repair of the hybrid molecule in vitro, as in the method of Okayama and Berg (1982). This modification has not yet been tested.
Together with the work of Dr G. Stanway on the neurovirulent strains P3/Leon/37 and P3/119, hundreds of cDNA clones were examined and more than 22 kbp of nucleotide sequence determined. These experiments resulted in the identification of a mall number of mutations in the genomes of the strains studied which must be responsible for their differences in neurovirulence. However, it has not yet been possible to identify the individual mutations involved in attenuation and reversion and further experiments are currently in progress. These experiments represent a number of different approaches. Firstly, the sequence of other neurovirulent vaccine revertants are being determined, to ascertain whether the mutations observed in P3/119 are shared by other strains. Secondly, the work of Racaniello and Baltimore (1981a) has demonstrated that the construction of recombinant virus genomes in vitro, at the level of cloned cDNA, is possible. Transfection of susceptible cells with these recombinant genomes gives rise to new, viable viruses with a defined set of mutations which can then be examined phenotypically. Initial experiments with recombinants between the neurovirulent strain P3/Leon/37 and the vaccine strain P3/Leon 12 a1b and also between the vaccine strain and the neurovirulent revertant P3/119 are in progress. Final proof that the mutations involved in attenuation and reversion have been accurately identified could be provided by the construction of an attenuated strain by recombination between P3/Leon/37 and P3/119. Parallel studies on neurovirulent and attenuated type 1 strains should help to explain the different stabilities of the type 1 and type 3 vaccines and may suggest how the type 3 vaccine can be modified to improve Stability. Finally, it is hoped to use site-directed mutagenesis of cloned virus genomes ln vitro to produce strains with specific biological properties.
Although the main aim of the work presented here has been the investigation of the molecular basis of attenuation in poliovirus, the information obtained has wider significance. The complete nucleotide sequence of P3/Leon 12 a1b was the first to be determined from a type 3 poliovirus. This has been compares with that of type 1 and the extent of the molecular homology between the demonstrated (Stanway et al, 1983a). As part of a larger study based on the analysis of monoclonal antibody resistant mutants, the major neutralizing antibody binding site of poliovirus type 3 has been identified (Minor et al, 1983). The nucleotide sequence information obtained is also being used currently in the design of synthetic antigenic peptides, a development which may hold many advantages for the prevention and perhaps treatment of poliomyelitis and other related plcornaviral infections. At the outset of the work described in this dissertation, it was difficult to envisage that improved alternativen to the Sabin vaccines would ever be a realistic proposition. It now seems that the immediuate future holds just such a prospect.

I started my scientific career working on vaccine-derived polioviruses. We’ve come a long way since those distant days and we’re slowly getting closer to eradicating polio worldwide, but it’s not all over yet:

In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide. Subsequently, the Global Polio Eradication Initiative of the World Health Organization (WHO) reduced the global incidence of polio associated with wild polioviruses (WPVs) from an estimated 350,000 cases in 1988 to 1,998 reported cases in 2006 and reduced the number of countries that have never succeeded in interrupting WPV transmission to four (Afghanistan, India, Nigeria, and Pakistan). However, because vaccine-derived polioviruses (VDPVs) can produce polio outbreaks in areas with low rates of Sabin oral poliovirus vaccine (OPV) coverage and can replicate for years in immunodeficient persons, enhanced strategies are needed to limit emergence of VDPVs and stop all use of OPV once WPV transmission is eliminated. This report updates a summary of VDPV activity published in 2006 and describes VDPVs detected during January 2006-August 2007.

VDPVs can cause paralytic polio in humans and the potential for sustained circulation of poliovirus. VDPVs resemble WPVs biologically and differ from the majority of Sabin vaccine-related poliovirus isolates by having genetic properties consistent with prolonged replication or transmission. Because poliovirus genomes evolve at a rate of approximately 1% per year, Sabin vaccine-related isolates that differ from the corresponding OPV strain by more than 1% of nucleotide positions (usually determined by sequencing the genomic region encoding the major viral surface protein, VP1) are estimated to have replicated for at least 1 year after administration of an OPV dose. This is substantially longer than the normal period of vaccine virus replication of 4-6 weeks.

Poliovirus isolates can be distinguished by their three serotypes: type 1, type 2, and type 3. Isolates also can be divided into three categories, based on the extent of VP1 nucleotide sequence divergence from the corresponding Sabin OPV strain: 1) Sabin vaccine-like viruses (<1% divergent), 2) VDPVs (1%-15% divergent), and 3) WPVs (>15% divergent) (4). VDPVs are further categorized as 1) circulating VDPVs (cVDPVs), which emerge in areas with inadequate OPV coverage; 2) immunodeficient-associated VDPVs (iVDPVs), which are isolated from persons with primary immunodeficiencies who have prolonged VDPV infections after exposure to OPV; and 3) ambiguous VDPVs (aVDPVs), which are either clinical isolates from persons with no known immunodeficiency or environmental isolates whose ultimate source has not been identified.

Update on Vaccine-Derived Polioviruses Worldwide, January 2006-August 2007
MMR Weekly 2007 56: 996-1001

Related:

• Epidemics to eradication: the modern history of poliomyelitis
• Imaging Poliovirus Entry in Live Cells