[Evaluation of the efficiency and reactogenicity of emulsion-based adjuvant systems in the manufacture of polyclonal enteroviral diagnostic sera].

Whether various adjuvants might be used in the manufacture of commercial enteroviral diagnostic sera (EDS) was studied. The following adjuvants: Ribi, SAF-1, and TiterMax were compared; vaseline-lanoline emulsion used to prepare EDSs, as well as modified Freund's complete adjuvant served as controls. Chinchilla rabbits were intramuscularly injected enterovirus antigens (enterovirus 70 and ECHO 2) together with the adjuvant emulsions. TiterMax showed the highest efficiency comparable with the activity of Freund's adjuvant. The activities of Ribi, SAF-1, and vaseline-lanoline emulsion were 3-4 times lower. The neutralizing activity of the sera obtained after 2-3 (TiterMax) or 4-5 (Ribi, SAF-1) immunizations was maximal. Further immunizations resulted in a reduction in the titers of neutralizing antibodies. TiterMax and vaseline-lanoline emulsion caused minimal complications at the site of inoculation whereas SAF-1 and Ribi gave rise to severer inflammatory responses.

[Cytotoxic properties of diagnostic rabbit sera to enteroviruses specific features and localization].

Hyperimmune rabbit enterovirus-neutralizing diagnostic sera have a pronounced cytotoxic effect on the test tissue culture RD, HEp-2, and Vero cells and primary renal cells from African green monkeys (Cercopithecus aethiops). The specific features of this developing toxic effect, such as reduced proliferative activity and partial cytolysis, were revealed. The time course of changes in the generation of serum cytotoxicity during an immune response to repeated antigenic stimulation of donor animals is described. There is experimental evidence for no association of serum toxicity with specific neutralizing antibodies and with antibodies to cell culture antigens. Affinity chromatography is one of the possible techniques for purification of cytotoxic sera.

[Moraprenylphosphates suppress reproduction of Taylor murine encephalomyelitis virus and accumulation of VP3 viral protein in susceptible cell cultures BHK-21 and P388D1].

Influence of moraprenylphosphates (phosphorylated polyprenol of plant origin) upon the accumulation of Taylor murine encephalomyelitis virus VP3 protein in the susceptible cell cultures was studied. It has been shown that moraprenylphosphates inhibited the accumulation of VP3 at early stages of infectious process. Moraprenylphosphates were found to decrease infectivity of the virus as well as virus-induced cellular apoptosis. Mechanisms of immunomodulating and antiviral activity of moraprenylphosphates and prospects of their use as antiviral drugs have been discussed.

Cell-specific proteins regulate viral RNA translation and virus-induced disease.

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.

Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus.

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.

An attenuated variant of the GDVII strain of Theiler's virus does not persist and does not infect the white matter of the central nervous system.

The DA strain of Theiler's virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5' noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid controls persistence by allowing the virus to infect cells in the white matter of the spinal cord.

Poliovirus neurovirulence correlates with the presence of a cryptic AUG upstream of the initiator codon.

Poliovirus mutants with extended (> 150-nt) deletions in the 5'-untranslated region between the internal ribosome entry site and the initiator codon have been selected previously (Pilipenko et al., Cell 68, 119-131, 1992; Gmyl et al., J. Virol. 67, 6309-6316, 1993). These deletions were transferred into the genome of a mouse-pathogenic poliovirus strain and found to be strongly attenuating. The deletions can be considered as covering three structural elements, a stem-loop (domain E) with a conserved cryptic AUG and two spacers, upstream and downstream of it. In an attempt to identify putative essential determinants of neurovirulence in these individual structural elements, appropriate mutants were engineered. The results demonstrated that neither of the above elements is essential for neurovirulence. The results strongly suggested that the presence of a cryptic AUG in the oligopyrimidine/AUG tandem followed, at a sufficient distance, by the initiator codon was necessary to ensure the neurovirulent phenotype of our constructs. On the other hand, the attenuated phenotype appeared to correlate with the occurrence of the initiator AUG as a moiety of the oligopyrimidine/AUG tandem. Possible mechanisms underlying these effects are discussed. Identification of the cryptic AUG as an essential determinant for neurovirulence provides a rational basis for the design of genetically stable attenuated poliovirus variants.

Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants.

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.

The primary structure of crossover regions of intertypic poliovirus recombinants: a model of recombination between RNA genomes.

The nucleotide sequence of crossover sites in the genome of four intertypic (type 3/type 1) poliovirus recombinants has been determined. The approximate boundaries of the crossover regions were first estimated by RNase T1 oligonucleotide mapping of the recombinant genomes; then appropriate regions were sequenced by the chain termination method using oligonucleotide-primed reverse transcription of the recombinant RNAs. The crossover sites (defined as the contiguous sequences shared by the recombinant and both parental genomes flanked, in the recombinant genome, by heterotypic RNA segments) are 5, 5, 7, and 11 nucleotides long, respectively. The recombination was precise and was not accompanied by any other genetic alterations. The recombination sites were found to be located within genome segments having a potential to form secondary structure elements. Based on this observation, a model of recombination between picornaviral RNA genomes has been proposed. The essence of this model consists in bringing together homologous regions of two recombining RNA genomes via formation of intermolecular duplexes, detachment of the nascent 3' end of the newly synthesized complementary RNA from a "parting" site on the first template and its subsequent "jumping" to the identical (or closely related) "anchoring" site on the other template. Features of this model are discussed in some detail.

[Primary structure of the crossover region in the genome of two intertype poliovirus recombinant]. / Pervichnaia struktura oblasti perekresta v genome dvukh mezhtipovykh rekombinantov poliovirusa.

The nucleotide sequence of the crossover region on genomes of two intertypic (type 3/type 1) poliovirus recombinant, has been determined by the primer extension method. No deletions, insertions or rearrangements have been observed. Identical contiguous sequences, 7 or 11 nucleotides in length, respectively, have been found in two regions of the parental genomes, involved in the recombination.

Recombinants between attenuated and virulent strains of poliovirus type 1: derivation and characterization of recombinants with centrally located crossover points.

Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1-6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree.

Neurovirulence of the intertypic poliovirus recombinant v3/a1-25: characterization of strains isolated from the spinal cord of diseased monkeys and evaluation of the contribution of the 3' half of the genome.

A tsRNA- intertypic recombinant, v3/a1-25, which has the 5' and 3' halves of the genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain whether the illness was caused by the recombinant itself or by temperature-resistant trRNA+ mutants that might have arisen in the inoculated monkeys, five independent virus strains have been isolated from the spinal cord of the diseased animals. While two of these isolates exhibited RNA+ and RNA +/- phenotypes, respectively, the other three strains retained the parental RNA- character. Except for the RNA+ strain, the RNase T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal deviation from the parental pattern. These results were interpreted to mean that v3/a1-25 is intrinsically neurovirulent despite the presence of a tsRNA- mutation(s) in the 3' half of its genome. Nevertheless, this mutation, or other peculiarities of the 3' half of the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This notion was inferred from the fact that, when used in a relatively small dose (about 10(3) p.f.u.), v3/a1-25 appeared to exhibit a lower level of neurovirulence compared to either the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the genome 3' half derived from a neurovirulent trRNA +/- type 1 poliovirus strain. The problem of genetic determination of poliovirus neurovirulence and attenuation is briefly discussed.

Construction and properties of intertypic poliovirus recombinants: first approximation mapping of the major determinants of neurovirulence.

An attempt was made to map, in a general way, the region of the poliovirus genome that is responsible for the neurovirulent and attenuated phenotypes of different virus strains. A set of four recombinants was investigated, one described previously (E. A. Tolskaya, L. I. Romanova, M. S. Kolesnikova, and V. I. Agol, 1983, Virology 124, 121-132) and three obtained in the present work with the following genetic structure: a 5' end-adjacent segment of the genome derived from either a virulent strain (452/62 3D), or from an attenuated strain (Leon-2) of poliovirus type 3, the remaining RNA sequences being derived from either a virulent strain (Mgr), or an attenuated strain (LSc-gr3) of poliovirus type 1. The crossover points in the recombinant genomes were centrally located, somewhere between the gene(s) that determines antigenic specificity of the virus and the locus that determines resistance of virus multiplication to low doses of guanidine. The recombinant nature of the newly selected clones was definitively established by mapping RNase T1 oligonucleotides of their genome. The recombinants were characterized with respect to their ability to produce infectious progeny and synthesize viral RNA at an elevated temperature. Neurovirulence of the recombinants was assayed by intracerebral inoculation of monkeys. Irrespective of the origin of the 3' end-adjacent segment of the genome, the recombinants that inherited the 5' end-adjacent segment from the neurovirulent parent were neurovirulent, whereas the recombinants with the 5' end-adjacent segment derived from the attenuated parent were not. The results suggest that the major determinants of neurovirulence of these recombinants (and by inference, of their parental viruses) reside in the 5' end-adjacent segment of poliovirus genome, known to code for capsid proteins.