Molecular mechanisms of attenuation of the Sabin strain of poliovirus type 3

Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES). We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on the Sabin3 IRES than on the PV3 IRES. Translation mediated by the PV3 and Sabin3 IRESs in neurons of the chicken embryo spinal cord demonstrated a translation deficit for the Sabin3 IRES that could be rescued by increasing PTB expression in the CNS. These data suggest that the low levels of PTB available in the CNS, coupled to a reduced binding of PTB on the Sabin3 IRES, leads to its CNS-specific attenuation. This study also demonstrates the use of the chicken embryo to easily investigate translation of RNA within a neuron in the CNS of an intact living organism.     

Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus.

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.     

Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles.

Treatment of the Sabin strain of type 1 poliovirus with trypsin produced two stable fragments of capsid protein VP1 which remained associated with the virions. Trypsinized virus was fully infectious and was neutralized by type-specific antisera. The susceptible site in the Sabin 1 strain was between the lysine at position 99 and the asparagine at position 100. A similar tryptic cleavage occurred in the Leon and Sabin strains of type 3 poliovirus, probably at the arginine at position 100, but not in the type 1 Mahoney strain, which lacks a basic residue at either position 99 or position 100. Tryptic treatment of heat-treated virus and 14S assembly intermediates produced unique stable fragments which were different from those produced in virions. The implications of our results for future characterization of the surface structures of these particles and structural rearrangements in the poliovirus capsid are discussed.     

A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.     

Reversion to neurovirulence of the live-attenuated Sabin type 3 oral poliovirus vaccine

The complete nucleotide sequence has been determined of a strain of poliovirus type 3, P3/119, isolated from the central nervous system of a victim of fatal vaccine-associated poliomyelitis. Comparison of this sequence with those obtained previously for the Sabin type 3 vaccine, P3/Leon 12a1b and its neurovirulent progenitor, P3/Leon/37, reveals that these three strains are on a direct geneaological lineage and therefore that P3/119 is a bona fide revertant of the vaccine. P3/119 differs in sequence from its attenuated vaccine parent at just seven positions. Only one of these differences, a mutation from U to C at position 472 in the presumed noncoding region of the genome, is a back mutation to the wild type sequence. Of the six other differences, three give rise to coding changes in virus structural proteins, two are silent changes in the major open reading frame of the genome and one affects the 3'-terminus just prior to the poly A tract. These differences indicate that there are three possible types of molecular change which could, singly or collectively, result in attenuation and reversion to neurovirulence of the Sabin type 3 vaccine.     

Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine.

The poliovirus type 3 Sabin oral poliovirus vaccine strain P3/Leon/12a1b differs in nucleotide sequence from its neurovirulent progenitor P3/Leon/37 by just 10 point mutations. The contribution of each mutation to the attenuation phenotype of the vaccine strain was determined by the construction of a series of recombinant viruses from infectious cDNA clones. The neurovirulence testing of recombinant viruses indicated that the attenuation phenotype is determined by just two point mutations: a C to U in the noncoding region at position 472 and a C to U at nucleotide 2034 which results in a serine-to-phenylalanine amino acid substitution in the structural protein VP3.     

Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion

A 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.     

Characterization of bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (PIV-3) has a buoyant density of 1.197. The RNA of PIV-3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self-anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 X 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 X 10(6)). PIV-3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein 1, protein 2, protein 3, glycoprotein 1, and glycoprotein 2 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein 2 was suggested to be nucleocapsid protein.     

ATP binding to bovine serum albumin.

Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307,000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44,400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417,000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1% (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321,000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307 000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44 400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417 000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1 % (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Complete genomic characterization of an intertypic Sabin 3/Sabin 2 capsid recombinant

The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.