Intraspinal Inoculation Research Articles

Use of hyperattenuated poliovirus as a replacement for Sabin or wild-type strains for laboratory assays in a post eradication world

Poliovirus serotype 2 (PV2) has been declared eradicated and was removed from the live vaccine programme in 2016. WHO have developed the Global Action Plan (GAPIII) to describe containment conditions for laboratory work with poliovirus after eradication, and in the UK, all PV2 has been reclassified to hazard group 3 (HG3). Laboratory work involving live poliovirus will continue to be required years after eradication for the purposes of: vaccine testing; environmental surveillance; immunoglobulin testing; and for the development of new public health interventions. Compliance with GAPIII and HG3 containment conditions would impose financial burden and failure would lead to disruption to essential activities. Development of safer poliovirus strains for use outside of containment could overcome these issues. The S19 poliovirus strain, designed to be hyperattenuated and extremely genetically stable, was used as a cassette for the introduction of capsid proteins of other strains and serotypes (Knowlson et al. 2015), originally for vaccine production. S19 viruses are unable to infect transgenic mice (by intraspinal inoculation) or non-human primates (by mouth) at very high doses and are unlikely to infect humans at biological temperature. PV2 S19 strains have recently been approved for use outside of GAPIII containment requirements by the Containment Advisory Group. Validation of strains for laboratory assays and a highly sensitive QC assay based on NGS will be described.

Characterization of Inflammatory Changes, Viral Spread and Demyelination after Intraspinal Inoculation of Theiler’s Murine Encephalomyelitis Virus in a Resistant Mouse Strain

Characterization of Inflammatory Changes, Viral Spread and Demyelination after Intraspinal Inoculation of Theiler’s Murine Encephalomyelitis Virus in a Resistant Mouse Strain

Development and biological properties of a new live attenuated mumps vaccine

To develop a new live attenuated mumps vaccine, a wild mumps Y7 strain isolated from a patient who developed mild parotitis was treated with nitrosoguanidine and ultraviolet, followed by selection of a temperature-sensitive clone. The selected clone, Y125, showed stable temperature-sensitivity in Vero cells. Intraspinal inoculation of marmosets with the Y125 produced only minimal histopathological changes, while intracerebral inoculation of neonatal rats revealed that the Y125 did not cause hydrocephalus. Both these effects of the Y125 were similar to those of the non-neurovirulent Jeryl Lynn strain. Furthermore, subcutaneous inoculation of the Y125 induced high levels of neutralizing antibodies in all Cercopithecus monkeys examined. Although the safety and immunogenicity should be confirmed in further field trials in humans, the present results indicate that the Y125 could be a promising vaccine candidate.

Treatment of CNS Malignancies with a Recombinant Oncolytic Poliovirus

Spread to the central nervous system (CNS) and the leptomeninges is a frequent complication of systemic cancers that is associated with serious morbidity and high mortality. We have developed a novel therapeutic approach against primary CNS tumors and secondary CNS malignancy complicating systemic cancer based on the human neuropathogen poliovirus. Susceptibility to poliovirus infection and ensuing rapid cell lysis are mediated by the cellular poliovirus receptor CD155, as well as cell-internal factors involved in control of viral gene expression and genome replication. We observed that CD155, a putative cell adhesion molecule of the immunoglobulin superfamily physiologically expressed in the developing CNS, is ectopically expressed on numerous primary and secondary CNS neoplasms. An association with malignant cells renders CD155 a molecular target for therapeutic intervention with poliovirus. However, any therapeutic application of poliovirus must contend with its inherent neuropathogenic potential. Poliovirus gene expression is driven by the viral internal ribosomal entry site (IRES), which mediates translation initiation in a 5- end, cap-independent manner. We discovered that IRES function is subject to potent cell type-specific restrictions that can be exploited to selectively drive viral gene expression and propagation in cancerous cells. Polioviruses replicating under control of a heterologous IRES derived from their relative human rhinovirus type 2, displayed rapid growth and cell killing in cancerous cells. However, these recombinants failed to efficiently translate their genome and propagate in cells of neuronal lineage. Consequently, polio-/rhinovirus recombinants are non-pathogenic after intraspinal inoculation into mice transgenic for the poliovirus receptor CD155 and in non-human primates. Oncolytic poliovirus recombinants have shown promise in preclinical investigations against primary tumor explant cultures and in xenograft animal studies and are currently being prepared for phase-I clinical trials against glioblastoma multiforme.

Differential localization of neurons susceptible to enterovirus 71 and poliovirus type 1 in the central nervous system of cynomolgus monkeys after intravenous inoculation.

Poliovirus and enterovirus 71 (EV71) are both neurotropic enteroviruses that cause serious neurological diseases, such as poliomyelitis and encephalitis. The neurovirulence of EV71 in cynomolgus monkeys was demonstrated previously by intraspinal inoculation. In this study, an improved simian model of EV71 infection was established by using intravenous inoculation, which revealed clinical and neuropathological similarities between this model and human cases of encephalitis. Experimental EV71 infection induced direct neurological manifestations, such as tremor, ataxia and brain oedema, but not non-neurological complications, such as pulmonary oedema and cardiac failure. Using this model of EV71 infection, the neurotropic characteristics of the prototype strains of EV71 and poliovirus type 1 (PV1) were compared. Three monkeys were inoculated intravenously with 10(5.5) TCID50 EV71 and all developed neurological disease signs within 4-6 days of inoculation. However, after inoculation with 10(5.5) TCID50 PV1 strain OM1 (PV1-OM1), the major manifestation was flaccid paralysis, starting from the lower limbs 6-9 days post-inoculation. Histopathological and virological analyses of moribund monkeys revealed that disseminated EV71 infection was characterized by severe panencephalitis involving both the pyramidal and extrapyramidal systems. In contrast, the lesions induced by PV1-OM1 were mainly restricted to the pyramidal tract, particularly the spinal motor neurons, thalamus and motor cortex. In conclusion, neuropathological involvement in this model correlated well with the apparent differences in neurological disease induced by EV71 and PV1-OM1. Thus, intravenous inoculation with EV71 is an excellent model to study the neuropathology of EV71 and to evaluate candidate vaccines and potential antiviral agents.

Neurovirulence of mumps virus: intraspinal inoculation test in marmosets

An intraspinal inoculation test of mumps virus using marmosets was performed in order to develop a neurovirulence test of mumps vaccines. In the group inoculated with non-neurovirulent Jeryl Lynn vaccine strain at 10 2.0 pfu/dose, there were only minimal histopathological changes in 3 of the 5 marmosets. In contrast, all marmosets inoculated with neurovirulent Urabe and NK-M46 vaccine strains developed extensive encephalitis and meningitis. Thus, this marmoset model, which can distinguish between non-neurovirulent and neurovirulent vaccine strains, is useful for evaluating neurovirulence of vaccine strains and elucidating the molecular pathogenesis of mumps.

Pyramidal and extrapyramidal involvement in experimental infection of cynomolgus monkeys with enterovirus 71.

Among the enteroviruses, polioviruses and enterovirus 71 (EV71) are two major neurotropic viruses causing serious neurological manifestations. While polioviruses are being eradicated globally by vaccination, EV71 still has the potential to cause a large outbreak such as that in Taiwan in 1998, in which there were many fatalities. In this study, we determined the neurovirulence of EV71 by neuropathological analysis of cynomolgus monkeys after experimental infection with five EV71 strains, which were isolated from individual patients with fatal encephalitis; meningitis; and hand, foot, and mouth disease. After intraspinal inoculation, the monkeys developed neurological manifestations within 1-6 days post-inoculation, irrespective of the inoculated strains. These manifestations included not only pyramidal tract signs such as flaccid paralysis, but also extrapyramidal tract signs such as tremor and ataxia. Histological and viral examinations confirmed virus replication in the spinal cord, brainstem, cerebellar cortex, and dentate nuclei, and cerebrum. The strains isolated during the 1970s and 1990s showed no particular differences with respect to neurotropism. Thus, it is clear that EV71 has a wider neurotropism than that of polioviruses.

Molecular genetic analysis of revertants from a poliovirus mutant that is specifically adapted to the mouse spinal cord.

SA virus, a mutant of the Mahoney strain of type 1 poliovirus (PV1/Mahoney), replicates specifically in the spinal cords of mice and causes paralysis, although the PV1/Mahoney strain does not show any mouse neurovirulence (Q. Jia, S. Ohka, K. Iwasaki, K. Tohyama, and A. Nomoto, J. Virol. 73:6041-6047, 1999). The key mutation site for the mouse neurovirulence of SA was mapped to nucleotide (nt) 928 of the genome (A to G), resulting in the amino acid substitution of Met for Ile at residue 62 within the capsid protein VP4 (VP4062). A small-plaque phenotype of SA appears to be indicative of its mouse-neurovirulent phenotype. To identify additional amino acid residues involved in the host range determination of PV, a total of 14 large-plaque (LP) variants were isolated from a single point mutant, Mah/I4062M, that showed the SA phenotype. All the LP variants no longer showed any mouse neurovirulence when delivered via an intraspinal inoculation route. Of these, 11 isolates had a back mutation at nt 928 (G to A) that restored the nucleotide of the PV1/Mahoney type. The reversions of the remaining three isolates (LP8, LP9, and LP14) were mediated by a second site mutation. Molecular genetic analysis involving recombinants between Mah/I4062M and the LP variants revealed that the mere substitution of an amino acid residue at position 107 in VP1 (Val to Leu) (LP9), position 33 in VP2 (Val to Ile) (LP14), or position 231 in VP3 (Ile to Thr) (LP8) was sufficient to restore the PV1/Mahoney phenotype. These amino acid residues are located either on the surface or inside of the virus particle. Our results indicate that the mouse neurovirulence of PV is determined by the virion surface structure, which is formed by all four capsid proteins.

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.

Targeted foreign gene expression in spinal cord neurons using poliovirus replicons.

A hallmark of poliovirus is the propensity to infect and replicate in spinal cord neurons of the central nervous system. Previously, we characterized a poliovirus self-replicating RNA genome (replicon), which encodes firefly luciferase in place of the capsid genes. This replicon is encapsidated into an authentic poliovirion by providing the poliovirus capsid protein in trans. The amount of enzymatically active luciferase in cells infected with this replicon correlated with the infectious dose. To begin to characterize the in vivo infectious potential of replicons, we have inoculated mice transgenic for the human receptor for poliovirus (PVR), either intracranially or intraspinally, with the replicon encoding luciferase. Wild-type poliovirus delivered to PVR mice via intracranial or intraspinal routes resulted in paralysis and death. Replicon preparations were shown by a sensitive biological assay to be free of infectious poliovirus. Neither intracranial nor intraspinal inoculation of the replicon encoding luciferase resulted in any obvious paralysis or disease symptoms. Following intraspinal inoculation with replicons encoding luciferase, luciferase enzyme activity was detected at 4 h post-inoculation, with peak activity at approximately 8 h post-inoculation; by 48 - 72 h, the luciferase activity had returned to background levels. Luciferase activity was detected in spinal cord predominantly near the site of inoculation, although activity was detected anterior and posterior to the site of inoculation, indicating that replicons undergo limited movement within the CNS presumably via the cerebrospinal fluid. In stark contrast to poliovirus though, inoculation of replicons into the spinal cords of PVR mice did not result in noticeable pathogenesis. Using immunofluorescence with antibodies to double-stain for replicons and neurons, we determined that replicons exclusively infect the neurons of the spinal cord, with the expression of the luciferase and replicon proteins confined to the cytoplasm of the infected cells. Replicons, then, possess the identical capacity for infection of spinal cord neurons in vivo as poliovirus. The lack of discernible neuronal destruction following replicon inoculation into the spinal cord suggests that some of the pathogenesis observed during a poliovirus infection might not be due entirely to primary infection of neurons. Finally, the results of this study point to future use of replicons as a means to target recombinant protein expression to neurons in the spinal cord.

Isolation and molecular characterization of a poliovirus type 1 mutant that replicates in the spinal cords of mice.

The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.

Dual stem loops within the poliovirus internal ribosomal entry site control neurovirulence.

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5' nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.

RECENT ADVANCES IN SAFETY TESTING OF LIVE ORAL POLIOVIRUS VACCINES. • 979

In studies with polioviruses (PV) isolated from infected children, Sabin, seeking suitable strains to attenuate for live oral PV vaccine (OPV), derived substrains with exceptionally low virulence for monkeys inoculated intraspinally-the species and route of inoculation most sensitive to residual neurovirulence. Because OPV strains tend to undergo partial genetic reversion, it is important to verify that live vaccine preparations retain all markers of attenuation, especially lack of neurovirulence. For many years US federal regulations have required that each monovalent virus pool be tested for neurovirulence by injection into the spinal cords of monkeys before incorporation into trivalent OPV. Monkey neurovirulence testing (MNVT) is expensive and uses very large numbers of animals-testing for a single lot of trivalent OPV requiring 88 monkeys or more. We have studied three tests that might eventually reduce the reliance on MNVT if not replace it: 1) A molecular assay to detect increased content in PV vaccine of those virions with revertant mutations at sites in the viral genome thought to be responsible for neurovirulence (“mutant analysis by polymerase chain reaction and restriction enzyme cleavage”/MAPREC). 2) Intraspinal inoculation of transgenic (Tg) mice expressing the human PV-receptor gene and susceptible to infection with PV. 3) Increased replication of neurovirulent strains of type-3 PV in cultures of interferon-treated human neuroblastoma cells. MAPREC and Tg-mouse tests successfully detected preparations of type-3 OPV (the least stable type of OPV most often incriminated in vaccine-associated paralytic poliomyelitis) that had failed MNVT. The neuroblastoma test recently showed promise in detecting revertant type-3 OPV. Both MAPREC and Tg-mouse tests are currently subjects of multi-center studies coordinated through the World Health Organization.

Studies on Neurovirulence in Poliovirus-Sensitive Transgenic Mice and Cynomolgus Monkeys for the Different Temperature-Sensitive Viruses Derived from the Sabin Type 3 Virus

We have studied methods for testing the neurovirulence of live poliovaccine viruses by intraspinal inoculation into mice carrying the human poliovirus receptor gene (Tg mice). A comparison of the neurovirulence of Sabin type 3 vaccine virus and related viruses using the 50% paralysis dose determined after intraspinal inoculation into the Tg mice as an index revealed a close correlation between the results of the paralysis dose in Tg mice, the neurovirulence expressed by the histopathological lesion score In monkeys, and the temperature sensitivity of the viruses. The results of experiments in the Tg mice also showed a good correlation with the number of mutations at position 472 from U to C in the 5′ noncoding region in the genomes of the viruses tested. These results strongly suggest that the neurovirulence test for oral poliomyelitis vaccine using the Tg mice is an excellent method and may be used in place of the test using monkeys.

Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine.

Production of live attenuated oral poliomyelitis vaccine (OPV) requires rigorous neurovirulence safety testing of each vaccine lot, currently carried out in monkeys. It has been reported that a change from 472-U to 472-C in the type 3 OPV RNA is associated with an increased histologic lesion score produced upon intraspinal inoculation of the mutant virus in monkeys. We have developed a method, based on polymerase chain reaction, for measuring the relative abundance of these mutant sequences directly in vaccine preparations and used this method to evaluate the proportion of 472-C in 40 different lots of type 3 OPV. Six vaccine lots that had failed the intraspinal monkey neurovirulence test contained a higher proportion of 472-C than all other lots that had passed this test. OPV type 3 virus containing 472-C was rapidly selected during serial passages in African green monkey kidney cells that are used for manufacturing of the vaccine. We have also found that the wild-type poliovirus type 3 strain Leon/37, from which the vaccine strain was originally derived, contained a mixture of 472-U and 472-C sequences. No other mutations in OPV type 3 RNA have been detected by similar assays at position 2034, also associated with attenuation, or at several other positions reported to be altered in some vaccine preparations. Our results suggest that molecular diagnostics may provide a supplement or a potential alternative to animal testing of live attenuated vaccines.

Ultrastructural changes of motoneurons in monkeys infected with enterovirus 71. Brief report.

Tissues of the central nervous system (CNS) of cynomolgus monkeys were examined by electron microscopy after intraspinal inoculation of enterovirus 71 (E71). A characteristic finding was the appearance of numerous membrane-bound vesicles (Mbvs) in affected motoneurons. Similar Mbvs were also present in E71-infected cynomolgus monkey kidney (CMK) cells in culture. Virus-like particles were found within or around Mbvs in motoneurons as well as in infected CMK cells in culture. Neither Mbvs nor E71 antigens as detected by immunofluorescence were found in supporting tissues, vascular endothelium, or inflammatory cells. Quite similar findings had been seen in poliovirus infections. It was suggested that the appearance of Mbvs in the cytoplasm of motoneuron is the direct results of virus multiplication in E71 infection as in poliovirus infection.

Pathogenesis of human poliovirus infection in mice. II. Age-dependency of paralysis.

The mechanism of resistance of newborn mice to poliovirus-induced paralysis was studied by comparing regional virus replication in the adult and in the newborn central nervous systems (CNS) after intracerebral (ic) and intraspinal inoculation. Initial virus replication in the brains was similar in both age groups. Paralysis correlated with replication of virus in the spinal cord to a constant threshold, and this replication in newborns was delayed. Intraspinal inoculation of newborns eliminated the delay, indicating that neonatal anterior horn motor neurons were fully susceptible to infection. Cordectomy prevented the spread of virus, despite patent cerebrospinal fluid (CSF) pathways. Thus, poliovirus appeared to spread within the CNS via an axonal transport system. Known maturational changes in the fast transport system may explain the relative resistance of immature mice to poliovirus-induced paralysis.

Experimental infections with enteroviruses

Ninety-six cynomolgus monkeys were inoculated by 4 different routes with ECHO viruses types 2, 7, 9, 14, 16, and 18. These serotypes differed in parameters such as infectivity, tissue invasion and neurovirulence. Only types 2, 14, and 18 were infective following alimentary inoculation. Following extraneural parenteral routes viremia developed chiefly for types 9, 14, and 16, and antibodies developed for all serotypes. After intraspinal inoculation a large fraction of monkeys developed paralysis of limb segments; both homologous viruses and inflammatory lesions were present in CNS tissues. ECHO types 7 and 14 were more virulent (by any route) than other serotypes.

Presence of two interrferring enterovirral agents (ECHO virus type 9 and poliovirus type 2) in the human central nervous system.

A cytopathogenic and suckling-mouse pathogenic agent isolated from the CNS of an infant appeared to consist essentially of two different interfering viruses. The major component was ECHO-9 virus, the minor component was polio virus type 2. Both viruses were segregated by various methods: (1) cultivation in human papilloma cells, in which only the poliovirus multiplied, (2) plating of high dilutions of monkey kidney culture fluid, which yielded ECHO-9 single plaque progeny, (3) suckling-mouse passage. Poliomyelitis-like lesions developed after intracerebral and intraspinal inoculation of monkey kidney culture fluid containing both components, but only in monkeys that had been treated with cortisone. The CNS of these monkeys yielded mostly ECHO-9 virus, and occasionally poliovirus type 2. ECHO-9 virus was the interfering agent, which considerably suppressed the multiplication of poliovirus type 2, not only through a number of monkey kidney culture passages, but also in the CNS of monkeys. Since the high concentration of virus found in the CNS of the patient was mainly a result of a high concentration of the ECHO-9 component, the latter has apparently interfered with the poliovirus component also in the human CNS. A slight interfering effect of the poliovirus on the multiplication of ECHO-9 virus has been observed in suckling-mice. Although the ECHO-9 virus has entered the human CNS accompanied by poliovirus type 2, the neuroinvasiveness of the former, under certain conditions, is evident. Likewise, there is evidence of multiplication of this particular strain in the human and monkey CNS. The problem as to whether this virus is able to produce poliomyelitis-like lesions in the human CNS, however, remains to be solved.

岡山地方の健康小児のマウス馴化性ポリオウイルス3型に対する血清中和抗体分布に就て

1) The results of mouse neutralisation tests with intraspinal inoculation technic were compared with those obtained by HeLa cell tissue culture neutralisation method, using three_ types of poliomyelitis virus vs. diluted sera (1:10) from 12 typical cases of paralytic poliomyelitis. Only one serum specimen gave a clear cut positive by tissue culture neutralisation test, but negative results by mouse neutralisation test, and the remainder showed complete agreement between the results of both methods.This preliminary study indicated that the neutralisation tests in mice with mouse-adapted polioviruses were utilized effectivly in circumstances which tissue culture neutralisation test could not be done.2) In 1955, 111 sera were collected from inhabitants of Okayama area. The percentage positive of neutralizing antibodies examined in mice with these diluted sera (1:10) against 100 PD50 of type I and II, and 20 PD50 of type III were followed: in placenta cord serum, against type I 83.3%, against type II 66.7%, but type III 33.3%; thereafter these percentage decrease rapidly, reached the lowest level in 6 months to 1 year group. Then the positive percentage against type I and II sharply rose up from 2-4 years group and reached the adult level in 10-14 years group against type I and in 5-9 years group against type II. But during 2-19 years old, the positive percentage against type III were still remained about 20-29%. These pattern of antibody-level illustrated that the distribution of type I and II of poliomyelitis virus in Okayama were resemble to those of Tokyo and Osaka, but the one of type III showed endemic nature and its infections seemed not to be prevalent in our area.