Strains Of Poliovirus Type Research Articles

Разработка рекомбинантного онколитического штамма полиовируса 3-го типа с измененным клеточным тропизмом

Diffuse gliomas are incurable, prevalent, and aggressive central nervous system tumors. Therefore, the development of selective oncolytic viral strains for malignant neoplasms is highly relevant. This study aimed to create an oncolytic virus based on a vaccine strain of poliovirus type 3 with natural antitumor activity. To achieve this goal, we replaced the internal ribosome entry site (IRES) of poliovirus with the corresponding fragment of human rhinovirus 30. The resulting recombinant oncolytic strain RVP3 retained the serotype of poliovirus type 3, as confirmed by virus neutralization micro-test with specific antiserum. In addition, the oncolytic efficacy of RVP3 was assessed in vitro on a broad panel of cell cultures. According to the results, RVP3 has changed its tropism, losing the ability to replicate in conditionally normal cell lines of embryonic astrocytes and embryonic fibroblasts while retaining the ability to replicate in tumor cells.

Cambios genéticos en cepas de poliovirus tipo 2 aisladas de pacientes con poliomielitis paralítica asociada a la vacuna

Poliomyelitis is currently a rare disease in developed countries, where only vaccinal strains seem to be in circulation, which replace wild poliovirus. Nevertheless, it is still a serious disease for children in underdeveloped countries of Asia and Africa. We analysed nine strains of poliovirus type 2 isolated from the faecal matter of patients with vaccine associated paralytic poliomyelitis (VAPP), from the beginning of anti polio vaccination campaigns in our country. These strains were submitted to sequencing of a fragment of 114 base pairs from the 5 NTR (non traductional region), where one of the main determinants of attenuation/reversion to the neurovirulence of poliovirus lies in the position of nucleotide 481. In this position it was observed how guanine had been replaced by adenine in all the strains that were sequenced, so that it coincided with the homologous sequence of the wild strain, as well as with that of strains obtained from healthy children immunised with the live vaccine. This presupposes that other changes must occur or that other factors must be involved for VAPP to occur or not, and we therefore suggest the sequencing of other regions of the genome in search of other possible differential changes in nucleotides.

Sabin attenuated LSc/2ab strain of poliovirus spreads to the spinal cord from a peripheral nerve in bonnet monkeys (Macaca radiata).

Vaccine-associated paralytic poliomyelitis is a serious concern while using the live attenuated oral polio vaccine for the eradication of poliomyelitis. The bonnet monkey model of poliovirus central nervous system (CNS) infection following experimental inoculation into the ulnar nerve allows the comparative study of wild-type and attenuated poliovirus invasiveness. Dosages >/=10(4) TCID(50) of Mahoney strain of poliovirus type 1 [PV1(M)] result in paralysis. In contrast, even with 10(7) TCID(50) of Sabin attenuated strain of poliovirus type 1 (LSc/2ab), no paralysis occurs, but virus spreads into the CNS where viral RNA is found in spinal cord neurons. While wild-type PV1(M) viral RNA replicates in neurons (and possibly in glial cells) and in cells around vessel walls, which may be mononuclear or endothelial cells, attenuated viral RNA is detected only in neurons. Systemic viraemia and gastrointestinal virus shedding occurs only in PV1(M)-infected animals. While a systemic serologic response is detected in both groups of animals, cerebrospinal fluid antibodies are detected only in animals infected with PV1(M). Both the PV1(M) and LSc/2ab strains spread to the cervical spinal cord and then to the lumbar spinal cord following ulnar nerve inoculation. Neuronophagia and neuronal loss are only seen in PV1(M)-infected monkeys in whom clinical paralysis is observed. Infection with LSc/2ab does not result in neuronophagia, neuronal loss or clinical paralysis. Spread of attenuated poliovirus in spinal cord neurons without causing paralysis following inoculation into the ulnar nerve is an important finding.

Comparison of neuropathogenicity of poliovirus type 3 in transgenic mice bearing the poliovirus receptor gene and cynomolgus monkeys

To clarify the similarities of poliovirus infection in cynomolgus monkeys and transgenic mice bearing the poliovirus receptor, TgPVR21, we compared the pathological changes of these animals following intraspinal inoculation of two strains of poliovirus type 3 using immunohistochemical detection of the capsid antigen. All of the monkeys inoculated with 106 TCID50 viruses showed flaccid paralysis 2 or 3 days post-inoculation (p.i.). TgPVR21 mice showed paralysis starting from 2 to 3 days p.i. Histologically, neurons having pyknotic nuclei and eosinophilic cytoplasm and neuronophagia were characteristically observed in both animals, but central chromatolysis was not observed in infected TgPVR21. The median lesion scores in the monkeys and TgPVR21 were well correlated, though the distribution of poliovirus-infected lesions in the central nervous system was different. In both animals the motor neurons and the brainstem nuclei responsible for flaccid paralysis were infected by the virus, while the cerebral cortex and thalamus were infected in the monkeys but not in TgPVR21. These results confirmed the reliability of neurovirulence tests using TgPVR21 as a substitute for monkeys, in respect to the spinal and brainstem lesions of poliovirus type 3.

Isolation of polioviruses and other enteroviruses in south Greece between 1994 and 1998.

During the five-year period between 1994 and 1998, a total of 217 clinical samples were assessed for the isolation of enteroviruses at the Enterovirus Reference Centre for South Greece. Fourteen enterovirus strains belonging to different serotypes were isolated. These field strains were detected by cell culture in appropriate cell lines. They were subsequently identified by neutralizing antibodies with the LBM (Lim-Benyesh Melnick) mixed antisera pools up to 1995 and RIVM (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) pools from 1996 onwards. The isolated viruses included two strains of poliovirus type 2 Sabin-like, three strains of poliovirus type 1 non-Sabin-like, one Coxsackie B2 (CBV2) strain, one Coxsackie B5 (CBV5) strain, one Echo 5 (ECV5) strain, one Echo 7 (ECV7) strain, three Coxsackie A16 (CAV16) strains, and two currently enteroviral strains unidentified by RIVM pools. Reverse transcription-polymerase chain reaction (RT-PCR) using poliovirus-specific primers or poliovirus type-specific primers and enterovirus specific primers from the highly conserved 5'-UTR, the latter followed by RFLP, was also applied in 6 clinical isolates (3 strains of poliovirus type 1 non-Sabin-like, 1 polio type 2 Sabin-like, and 2 non-identified by RIVM pools enteroviruses). The advantages and the drawbacks of these assays against the conventional ones are discussed here. The isolations and the subsequent identification of the strains were carried out from fecal samples of clinical cases that included hand-foot-and-mouth disease, meningitis, and acute flaccid paralysis. The reappearance of non-Sabin-like poliovirus strains in Greece in 1996 after 14 years is considered to have an important medical and clinical value.

Circulation of poliovirus among risk groups in Ibadan, Nigeria

Faecal samples collected from 114 fully vaccinated pre-school children and 32 unvaccinated infants in Ibadan, Nigeria, were assayed for poliovirus in Hep-2 and RD cell cultures. 8 strains of poliovirus type 1 were isolated from 146 samples—3 from 32 unvaccinated children aged less than 40 d and 5 from 114 fully vaccinated children aged between 9 and 60 months. Studies using Sabin and wild monoclonal antibodies and the polymerase chain reaction confirmed 7 of the 8 isolates to be of the wild type, a possible source of infection among vaccinated children.

Type 3 poliovirus/Finland/1984 is genetically related to common Mediterranean strains

Strains of poliovirus type 3 isolated in Finland in 1984 and 1985 (P3/Fin/84) are known to differ considerably from the type 3 vaccine strains in both nucleotide sequence and antigenic properties. In the search for the origin of the outbreak we first tested 80 type 3 strains that had been isolated elsewhere in the world during the years 1953 to 1986. An oligonucleotide probe complementary to a highly variable 17 nucleotide interval in the 5' non-coding region of the genomic RNA of P3/Fin/84 reacted with five strains. Also it was revealed that two of the latter five strains were related to the P3/Fin/84 strains in two separate genomic regions compared after partial RNA sequencing. One of them was isolated in Switzerland in 1980 and the other in Turkey in 1981. The Swiss strain was from a patient who had recently returned from a journey to various Mediterranean countries. Consequently, 16 other strains isolated in the late 1970s and early 1980s in Europe or in the Mediterranean countries were studied in detail by partial genomic sequencing and with neutralizing monoclonal antibodies. Two separate regions of the genome were compared by sequencing and corresponding dendrograms were constructed. The Switzerland and Turkey strains were found to be the strains most closely related to the viruses of the 1984 Finland epidemic. These results indicate that type 3 poliovirus strains related to P3/Fin/84 had been circulating in Mediterranean countries since the late 1970s.

Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.

Molecular pathogenesis of type 2 poliovirus in mice

Lansing strain of poliovirus type 2 (PV-2) produces a fatal paralytic disease in mice after intracerebral inoculation. To identify virus-containing nerve cells in poliovirus-infected mice, we developed a technique of in situ hybridization for viral RNA with a poliovirus-specific riboprobe. Large numbers of genomes were found in motoneurons and their processes, as well as in ghosts of neurons with or without inflammatory cells. This indicates that the death of motoneurons is due to a direct effect of viral replication. The presence of viral RNA in neuronal processes suggests an axonal dissemination of poliovirus within the central nervous system (CNS). Detection of viral genomes in small cells located in anterior and in posterior horns shows that motoneurons are not the only cells susceptible to poliovirus in the CNS. To see if PV-2/Lansing still exits in nature, PV-2 isolated from the CNS and from the gut of two human cases of paralytic poliomyelitis were characterized by determining epitope maps and phenotypic markers and by sequencing regions of the genome coding for antigenic sites and the 5' non-coding region. Comparison of virus isolated from the intestine and from the CNS of the same subject revealed a high degree of homology, suggesting that virus isolated from the gut is representative of virus in the neuronal lesions. The isolate from a case had more homology to PV-2/Lansing, whereas the other isolate showed more homology to PV-2/Sabin. These results show that, despite intensive vaccination with oral polio vaccine for 25 years, wild PV-2/Lansing-like viruses, pathogenic for mice, still circulate in nature ad induce human paralytic cases.

Synthesis of Immunogenic, but Non-infectious, Poliovirus Particles in Insect Cells by a Baculovirus Expression Vector

A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses.

Production and characterization of neutralizing monoclonal antibodies against poliovirus type 1, 2, and 3

Neutralizing monoclonal antibodies were produced against a reference vaccine or a reference wild strain of poliovirus type 1, 2, and 3. After 26 fusions, 55 monoclonal antibodies were obtained with serotype 1 as the immunizing antigen, 180 with serotype 2, and 115 with serotype 3. The neutralizing activity of these monoclonal antibodies was tested first with the two reference strains and then if reactive, against a panel of 10 well-characterized strains of each serotype, 5 vaccinelike (VL) and 5 nonvaccinelike (NVL). All monoclonal antibodies were type specific without reactivity with any of the heterologous strains. There was a wide range of reactivity within the strains of each serotype. Several monoclonal antibodies to serotype 1 reacted with all type 1 strains, while several neutralized strongly all VL strains and weakly one or more of the NVL strains. Most of the 180 monoclonal antibodies to serotype 2 neutralized to various degrees all strains of this serotype and about half reacted very strongly with all homologous strains whether VL or NVL. None could differentiate all VL and NVL homologous strains. Of the 115 monoclonal antibodies to serotype 3, several monoclonal antibodies neutralize to various levels all homologous strains and some can differentiate VL and NVL strains.

Replication of poliovirus in human mononuclear phagocyte cell lines is dependent on the stage of cell differentiation

Both wild-type and attenuated strains of poliovirus type 3 were found to replicate in the U-937 cells, an established cell line derived from human histiocytic lymphoma. However, preincubation of the cells with phorbol ester (PMA) that brought about the typical adherent macrophage-like appearance of the cultures, converted the cells to practically nonpermissive for poliovirus, while their ability to replicate vesicular stomatitis virus was not impaired. RC2A, another human cell line derived from monocytic leukemia, showed no detectable progeny virus production within 48 hr. Poliovirus replication in U-937 cells may be a suitable model for detailed studies of poliovirus-leukocyte interactions.

Characterisation of two different types of poliovirus isolated from a patient

A clinical case of paralytic poliomyelitis with almost simultaneous isolation of two different types of poliovirus was investigated. A strain of poliovirus type I was isolated from the cerebrospinal fluid and a type 3 strain of poliovirus was isolated from the patient's faeces. Both strains were characterised by two-dimensional oligonucleotide mapping. The type I virus was shown to be of wild type while the type 3 virus was shown to be vaccine-associated.

A temperature-sensitive mutant of poliovirus type 1 altered in capsid assembly

Summary The temperature-sensitive mutant of the Mahoney strain of poliovirus type 1, is 203, has previously been characterized as a heat-labile, RNA+ mutant [1]. Labelled extracts from HeLa cells infected with is 203 at 39°C were analysed by centrifugation through sucrose gradients: no 150-S virions, no 74-S procapsids and few if any 14-S precursor particles could be detected. Analysis of the viral polypeptides by SDS-PAGE showed a relative increase in the amount of polypeptide P1, the precursor to capsid polypeptides. However, normal cleavage of the polypeptide was observed at the nonpermissive temperature during a subsequent chase experiment. Maturation of is 203 procapsids into virions was not hampered at 39°C. From these data, it was concluded that the is defect of mutant is 203 affects the first morphogenesis step, i.e the assembly of precursor polypeptide P1 monomers into 14 S pentamers. Neither analysis of the is 203 polypeptides by 2D-electrophoresis nor analysis of the RNA by RNase T1-resistant oligonucleotide fingerprinting allowed us to map the is 203 mutation(s). A series of ts+ revertants of is 203 were isolated and analysed. One class of revertants showed an alteration in the pl of polypeptides VPO and VP2. These revertants produced normal size plaques at 39°C, but their virions were still heat-labile. A second class of revertants was characterized by small plaques at 39°C and heatstable virions ; no polypeptide alteration could be evidenced in this case. These results suggest that two distinct mutations are responsible for the is 203 phenotype.

Molecular cloning of the genomes of poliovirus type 3 strains by the cDNA: RNA hybrid method.

The genomes of two neurovirulent strains of poliovirus type 3, wild type P3/Leon/37 and a vaccine revertant P3/119/70, have been cloned in E. coli. The cDNA: RNA hybrid method used was efficient and may have wide applicability for cDNA cloning. Overlapping clones spanning the entire genome were obtained for each strain. These have been used to produce full-length DNA copies of the two genomes each within a single plasmid.

Nucleic acid sequence of the region of the genome encoding capsid protein VP1 of neurovirulent and attenuated type 3 polioviruses.

Three closely related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque-purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA . cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.

Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1

Summary Following chemical mutagenesis, four temperature-sensitive mutants of the Mahoney strain of poliovirus type 1 were isolated. These mutants (clones 035, 203, 221 and 247) had a 4 × 102 to 4 × 103-fold lower plaquing efficiency on HeLa cells at 38.5° C than at 32.5° C. Standard growth curves showed that the viral mutants were restricted at 38.5° C but not at 37° C. The virions of clone 247 showed a greatly increased heat sensitivity: infectivity of the mutant stocks decreased by one log after a 1.5-min incubation at 45° C or after 15 min at 38.5° C. In contrast, equivalent heat inactivation of wild type virus or mutant ts 035 required about 6.5 min at 45° C and both viruses were completely stable for 30 min at 38.5° C. The virions of clones 203 and 221 showed partial heat sensitivity at 45° C but none at 38.5° C. Clones 035, 221 and 247 were unable to synthesize viral RNA at 38.5° C. Shift up of HeLa cells infected by any of these mutants from 32.5° C to 38.5° C, during the first 4 h of the virus growth cycle, resulted in prevention or arrest of viral RNA replication. Viral RNA synthesis continued unabated when the infected cells were shifted to non-permissive temperature at 6 h after infection. The RNA− phenotype at non-permissive temperature was therefore only manifest at early times after infection. Genetic recombination between the four mutants was investigated using both the conventional virus yield test and an infectious center assay. Recombinant plaques were found to occur with a significant frequency between ts 035 and ts 203, whereas no recombination was detected when ts 221 or ts 247 were one of the parents.

Differences in multiplication of virulent and vaccine strains of poliovirus type I, II, and III in laboratory animals

Multiplication of virulent and vaccine strains of poliovirus type I, II and III in laboratory animals of different species was studied comparatively. The main criterion of virus reproduction was the production of the photoresistant virus progeny after inoculation of the animals with proflavin-photosensitized virus strains. On the whole, virulent poliovirus strains were characterized by replication in a wide range of hosts (monkeys, cotton rats, white mice, guinea pigs, rabbits, chickens, chick embryos), a low infective dose, production of the photoresistant progeny to a high titre, clinically overt disease in some animal species. The vaccine strains multiplied in a norrower range of hosts, had a high infective dose, a low titre of virus progeny, and caused no clinical symptoms of infection. These differences may serve as a marker for differentiation between virulent and attenuated strains in vivo. Administration of guanidine before inoculation of newborn cotton rats completely prevented or delayed by several days the production of photoresistant virus progeny. This fact confirms the stability of the proflavin-poliovirus complex under conditions ruling out virus replication.

PRELIMINARY COMMUNICATION ON THE SURVIVAL, THE SITES HARBORING VIRUS AND GENETIC STABILITY OF POLIOVIRUS IN COCKROACHES.

SummarySabin's attenuated strain of poliovirus type 3 persisted in cockroaches (Blaberus discoidalis) after experimental infection for as long as 51 days. No evidence was obtained indicating multip...

The basis for the interaction between attenuated poliovirus and polyions

Thin-layer electrophoresis on Sephadex G-25 of m + and m − strains of poliovirus type 1 showed the isoelectric point of both strains to lie between pH 7.4 and 7.6. The interaction of these virus strains with polyions was studied by chromatography on the anion-exchanger DEAE-Sephadex and the cation-exchanger Sephadex-sulfate using different buffers as eluting agents. The m − strain adsorbed to the cation exchanger at a pH below, but not at or above, the isoelectric point and to the anion exchanger at a pH below and above, but less efficiently at, the isoelectric point. The reason for this effect may be that the positively charged DEAE-Sephadex attached anions, which, when complexed, adsorbed the positively charged virus at a pH below the isoelectric point. The m + strain did not adsorb to the ion exchangers except to DEAE-Sephadex in borate buffers above the isoelectric point. The salt concentration and the type of buffer used influenced the adsorption of the m − strains to the polyions. Both strains of poliovirus could also be quantitatively separated by ultracentrifugation in gradients of DEAE-dextran or Dextran sulfate. The m − strain had a lower rate of sedimentation than the m + strain under conditions favoring adsorption to the polyion. DEAE-dextran abolished the inhibitory effect of acid overlay on the plaque forming capacity of the m − variant in monkey kidney cells and inhibited the plaque formation of both m − and m + variants of the strain L Sc 2ab in HeLa cells, but not in monkey kidney cells. This effect may be due to the bicarbonate buffer.