key: cord-010187-ymhcfyxx
authors: Gromeier, Matthias; Lu, Hui-Hua; Wimmer, Eckard
title: Mouse neuropathogenic poliovirus strains cause damage in the central nervous system distinct from poliomyelitis
date: 2005-03-25
journal: Microb Pathog
DOI: 10.1016/s0882-4010(05)80002-6
sha: 
doc_id: 10187
cord_uid: ymhcfyxx

Poliomyelitis as a consequence of poliovirus infection is observed only in primates. Despitea host range restricted to primates, experimental infection of rodents with certain genetically well defined poliovirus strains produces neurological disease. The outcome of infection of mice with mouse-adapted poliovirus strains has been described previously mainly in terms of paralysis and death, and it was generally assumed that these strains produce the same disease syndromes in normal mice and in mice transgenic for the human poliovirus receptor (hPVR-tg mice). We report a comparison of the clinical course and the histopathological features of neurological disease resulting from intracerebral virus inoculation in normal micewith those of murine poliomyelitis in hPVR-tg mice. The consistent pattern of clinical deficits in poliomyelitic transgenic mice contrasted with highly variable neurologic disease that developed in mice infected with different mouse-adapted polioviruses. Histopathological analysis showed a diffuse encephalomyelitis induced by specific poliovirus serotype 2 isolates in normal mice, that affected neuronal cell populations without discrimination, whereas in hPVR-tg animals, damage was restricted to spinal motor neurons. Mouse neurovirulent strains of poliovirus type 2 differed from mouse neurovirulent poliovirus type 1 derivatives in their ability to induce CNS lesions. Our findings indicate that the characteristic clinical appearance and highly specific histopathological features of poliomyelitis are mediated by the hPVR. Our data lead us to conclude that the tissue tropism of mouse-adapted poliovirus strains in normal mice is fundamentally different from that of poliovirus in hPVR-tg mice and primates, and that this is indicative of an as yet unknown mechanism of adsorption and uptake of the virus into cells of the murine CNS.

Poliomyelitis is a rare neurological complication of primate infection with poliovirus (PV), a member of the Picornavirus family, genus Enterovirus. Ingestion of virulent particles results in intestinal uptake, presumably through M-cells, 1 initial viral replication in subjacent lymphatic tissue, spread to deeper cervical and mesenteric *Author to whom correspondence should be addressed. 0882-4010/95/040253+15 $08.00/0 O 1995 Academic Press Limited lymph nodes (lymphatic phase), and ultimately entry of the virus into the bloodstream (viremic phase). 2 The latter is a prerequisite for passage of the blood brain barrier 3 and subsequent lytic infection of motor neurons. Only a small proportion of PV infections lead to a distinctive neurological syndrome that ranges in severity from transient flaccid monoparesis to progressive flaccid paraplegia with respirato.ry impairment and sometimes bulbar involvement. 4 The hallmark of poliomyelitis histopathology is selective damage to anterior horn motor neurons along the entire spinal cord. Spinal neurons outside the motor neuron system are characteristically spared, 5 in spite of their close anatomical relationship to the target of polioviral attack.

The predominant molecular determinant of PV tropism is the hPVRY The nucleotide sequence of hPVR identified it to be a member of the immunoglobulin superfamily, whose four mRNA isoforms are the result of alternative splicing events, and give rise to different receptor molecules. 6'8 hPVRc~ and hPVR& are integral membrane proteins with divergent cytoplasmatic domains, whereas hPVR/~ and hPVR7 are secreted molecules lacking the putative transmembrane domain. 6 The hPVR is a highly glycosylated protein with an apparent molecular weight of 80kDa2 The animal model for poliomyelitis in hPVR-tg mice showed PV-induced damage of comparable anatomical distribution as in primates, 1°'11 an observation confirming views of the hPVR as the critical determinant conferring PV susceptibility.

Unexpectedly, hPVR mRNA and hPVR-related proteins were shown to be present in a wide variety of tissue homogenates not known to be sites of PV replication, ~'1° an observation suggesting additional limiting factors of PV susceptibility, mRNAs specifying the simian 12 and murine ~3 homologues to hPVR have been isolated, but only the monkey-specific PVR can promote PV infection. ~2 Attempts to use rodents as possible models of PV-induced disease resulted in the isolation of mouse-neurovirulent (ran) strains. A 1937 PV serotype 2 field isolate from Lansing, Michigan, [PV2(L)], which caused a syndrome described as 'polioencephalitis' upon intracerebral injection in the cotton rat, TM provided the first system of PV encephalitogenesis in the wild type (wt) mouse. ~s Histopathological analyses of murine infection with the rodent-passaged PV2(L) isolate described a pattern of damage in accordance with concepts of primate poliomyelitis. ~6 A structural element conferring mouse neurovirulence to PV2(L) was determined to map in the capsid region. ~7 Sufficient in causing this effect was a segment within the BC-Ioop of VP1 since mouse avirulent PV type 1 (Mahoney) [PVI(M)], after transposition of the Lansing BC-Ioop, caused neurological disease in mice. 18' 19 Surprisingly, point mutations in distant regions of VP1 and VP2 could be shown to exert a mn phenotype. 2°.2~ The histopathological features of neurological disease caused by these genetically well defined mn PV strains have not been reported thus far.

In addition to the selection of wt PV strains expressing a mn phenotype in mice, attenuated PV strains with a mouse host range phenotype but reduced neurovirulence have also been described [PV type 2, strain W222]. The attenuated (att) phenotype of PV2(W2) is reminiscent of the Sabin strains of PV that express an att phenotype for primates. 23 The genetic determinants of viral neurotropism have been studied in several viral systems. Most frequently, these determinants mapped to the envelope gene; examples are the alphaviruses Sindbis virus 24 We have infected Swiss-Webster-, and ICR-mice with a variety of mn virus isolates of different serotypical origin. Type 2 strains PV2(L), PV2(MEF-1), a recent PV2 isolate from India (IND), and PV2(W2) caused a fatal diffuse encephalomyelitis in these mice with considerable differences in intensity between individual viral strains. PVl(LSa), a mouse-adapted derivative of PVl(M), 37 caused a characteristic non-progressive panmyelitic syndrome. We have also studied the histopathology of murine infection with PVI(M), carrying a single mutation in position 54 of capsid protein VP1 that we constructed according to a published report. 2~

Infections with each of these strains did not follow the stereotypic course of predictable progression seen in PV-induced poliomyelitis in hPVR-tg ICR-mice. Syndromes with distinct and consistent features followed intracerebral infection with each group of strains. These syndromes could be separately characterized and distinguished from hPVR-mediated poliomyelitis, on both clinical and histopathological grounds.

Poliovirus isolates and experimental animals. PV Transgenic ICR-mice expressing the hPVR ~° were a generous gift from A. Nomoto (The University of Tokyo, Japan). Swiss-Webster-and normal ICR-mice were obtained from Taconic (Germantown, NY, U.S.A).

Ultraviolet (UV) irradiation conditions. UV irradiation was performed in a UV-stratalinker (Stratagene, LaJolla, CA, U.S.A) A suspension of PV was irradiated at a distance of ca. 10 cm with an intensity of ca. 3.5 J/m z. Virus was exposed to the irradiation source in ice-cooled plastic dishes at a solution depth of ca. 1ram. The loss of infectivity was confirmed in a plaque assay.

Poliovirus intracerebral infections. Virus preparations were used to infect tg or normal mice with the desired input titer by intracerebral inoculation. Mice were anesthetized, and a 25 gauge hypodermic needle was used to inject maximally 30 pl of virus suspension in Dulbecco's minimal essential medium (DMEM). The point of injection was the middle on the median between the ear pinnacle and the eye. Infected mice were regularly observed for symptoms. None of the treated animals showed any external signs of damage or neurological disturbances in 24 h following injection.

Tissue processing, sectioning, and staining. Affected animals in the final stage of their disease were sacrificed according to approved protocols, and their bodies were immediately perfused with 15 ml of phosphate buffered saline (PBS), followed by 15 ml of 4% neutral buffered paraformaldehyde. The brain and spinal cord were removed and fixed for 2 h at room temperature in the fixative used for perfusion. Tissue specimens were rinsed for 30 rain in icecold PBS, then placed in 70% ethanol over night. Dehydration was achieved through a scheme of gradually increasing ethanol concentration, followed by clearing in toluene for 2 h and infiltration with paraffin at 57.5°C for 2 h. Neural tissues were embedded and cut on a rotary microtome at a thickness of 10/~m. Tissue sections were placed on microscopic slides treated 

Transgenic mice expressing the PV receptor TM were infected intracerebrally with PVI(M) with an amount of virus ranging from 10 4 to 5x 10 6 plaque forming units (PFU). All infected mice developed a characteristic neurological syndrome displaying stereotypic clinical features irrespective of input titer of virus and differing only with regard to onset of symptoms (Table 1) . Once visible signs of functional impairment were apparent, the progression of disease followed a predictable course. Two to five days after intracerebral injection, initial signs were invariably a flaccid paralysis of the lower extremities and tail (Fig. 1A) . The condition was rapidly progressive leading to complete immobilization and respiratory difficulty within 48 h. Respiratory distress caused visible signs of bobbing of the head, strenuous respiratory effort, insufficient thoracic excursions, and nasal flaring. Animals in the final stage of the disease were killed and their CNS tissues processed according to standard procedures.

Histopathological analysis of the CNS of PVl(M)-infected hPVR-tg mice revealed the spinal pathology of poliomyelitis described in earlier reports. 1°' 11 Selective loss of anterior horn motor neurons along the entire spinal cord was invariably present (Fig. 2B) . Foci of virally-induced damage in the higher cervical cord extended into the brain stem and were accompanied by minor signs of inflammation in a predominantly perivascular distribution. Characteristically, apart from a clearly defined lesion in the pyramidal cell layer of the hippocampal formation, lesions above the brain stem were absent in all cases analyzed. The appearance of initial lesions in the lumbar spine, distant from the injection site without evidence of damage to cortical motor neurons or descending tracts, indicated that virus had been disseminated via the hematogenous or cerebrospinal fluid route.

Twenty eight day old Swiss-Webster-and ICR-mice were injected intracerebrally with PVI(M), PV2(L), PV2(MEF-1), PV2(IND), or PV2(W2), with amounts of virus ranging from 104 to 5x 106. Three groups of four animals each with different genetic backgrounds, Swiss-Webster and ICR, were injected with the same viral strain. It is important to note that we were unableto detect clinical or histological evidence for differences in susceptibility towards PV infection between both outbred mouse strains used. Therefore, in the following text, the term 'normal mouse' will refer to Swiss-Webster mice. All mn PV strains assayed caused poliomyelitis when injected into hPVR-tg mice (clinical and histopathological details are described later). None of the normal mice injected with PVI(M) showed clinical signs of neurological damage, whereas inoculation of type 2 PV strains produced signs of CNS infection ( Table 2) .

Mice infected with PV2(MEF-1) and PV2(IND) were most severely affected. Initial symptoms appeared 2-5 days post infection (p.i.) in all animals but they were inconsistent regarding the sites of manifestation and quality of functional impairment. Motor symptoms consisted predominantly of spastic weakness involving the lower or upper extremities in a random fashion ( Table 2) . Paraplegic animals had a characteristic posture marked by kyphoscoliotic deformity (Fig. 1B) . The progression of motor symptoms did not follow any apparent topographical scheme. Pareses frequently were accompanied by gait ataxia or motor incoordination. Clinical signs of respiratory involvement as described above usually did not appear during the course of the disease ( Table 2 ). The clinical course proceeded to a terminal stage within 4 days after onset of symptoms. Preterminal animals were severely emaciated once functional impairment supervened, and refused intake of fluid and food offered within their reach.

Compared to PV2(MEF-1) and PV2(IND) a proportionally lower number of PV2(L)infected animals died (Table 1) . Their clinical syndrome was less variable, but also diverged from the regular pattern of disease localization and progression seen in poliomyelitic hPVR-tg mice. Motor symptoms similar to those in PV2(MEF-1) infected littermates dominated the clinical picture. Progression of motor involvement did not follow any recognizable pattern and it only rarely included respiratory function ( Table  2 ). Eventually immobilization led to quick deterioration of the starving animals. PV2(W2) was previously reported to be of attenuated neuropathogenicity with respect to PV2(L) in mice. 44 Accordingly, the proportion of infected animals that developed disease, and the severity and pace of progression of symptoms, were less than in animals infected with other PV type 2 isolates (Table 1 ). This observation confirms the attenuated nature of the strain." The spectrum of functional deficits observed was identical to PV2(L)-induced disease. Likewise, there was considerable variability in the sites involved in production of symptoms, course of progression, 

The clinical discrepancies between wt PV-induced poliomyelitis in hPVR-tg mice, and mouse-adapted PV2 infections in normal mice, were confirmed by histopathological differences. The severity and variability of clinical signs secondary to infection with the PV2 strains in normal mice was consistent with the histopathological findings. Extent, morphology and localization of viral lesions in Swiss-Webster-and ICR-mice were comparable. The range of vitally-induced damage encompassed a variety of lesions indiscriminately affecting structures within the cerebral hemispheres and the entire spinal cord. Characteristically, spinal pathology was patchy and irregular, and it did not follow the consistent pattern of damage to the entire cord seen in PVinfected hPVR-tg mice. Unexpectedly, anterior horn motor neurons remained frequently unaffected in normal mice infected with mn PV2, although severe pathological changes within the spinal cord were apparent (Fig. 2C,D) . In contrast, routine poliomyelitis in hPVR-tg mice invariably led to complete and exclusive elimination of the motor neuron population in the spinal cord with only minor infiltrative changes ( Fig. 2B) . Unlike the limitation of vitally-induced lesions in PV infections of hPVR-tg mice to the spinal cord, PV2-induced disease involved the entire CNS. Microglial nodules accompanied by mixed lymphocytic and neutrophilic infiltrates were scattered throughout the brain and were found within the insular, piriform and temporal cortex (Fig. 2F) , and the basal ganglia and thalamus (Fig. 2H) . These structures were never affected in infected hPVR-tg mice (Fig. 2E,G) . Perivascular cuffing and dense infiltration of perivascular and periventricular parenchyma were distributed ubiquitously in the CNS. The cerebral white matter was a frequent site of viral lesions. Lymphocytic infiltrates within the cerebral or cerebellar peduncles, the internal capsule, or the long descending tracts and posterior columns within the upper cervical cord, occasionally caused rarefaction necrosis with secondary demyelination (Fig. 21) . Immunohistochemical staining for viral proteins with a polyclonal anti-PV2 antiserum revealed positive signals in those structures frequently involved by virally- Fig. 3 .

Signs of widespread lesions and diffuse encephalomyelitis were most commonly associated with the isolates PV2(MEF-1) and PV2(IND), whereas in PV2(L) cases a myelitic pattern of disease predominated over encephalitis.

Parallel to the clinical findings, overall CNS damage induced by PV2(W2) was moderate in comparison with the other serotype 2 isolates. Neurological damage focused on the spinal cord with rare cerebral involvement. Spinal pathology qualitatively resembled the PV2 panmyelitis described above but the extent and number of lesions were reduced (data not shown).

To analyze the clinical course of murine infection with PV2 in mice expressing the hPVR, tg mice were inoculated intracerebrally with PV2(MEF-1) or PV2(L). The resulting clinical picture featured signs typically seen in murine poliomyelitis. The onset of disease was marked by a flaccid paraparesis, followed by ascending progressive symptoms as in poliomyelitis. All affected animals developed a preterminal dyspneic stage before they were killed.

Associated histopathology showed elements characteristic of hPVR-mediated poliomyelitis, as well as PV2-induced encephalomyelitis. Anterior horn motor neurons had changes typical for poliomyelitis. In addition, there was almost always severe hemispheric involvement, never associated with hPVR-tg murine poliomyelitis resulting from infection with PVI(M). Lesions equivalent to those seen in PV2-induced encephalomyelitis were distributed in the same widespread indiscriminate manner throughout the CNS. Gray matter structures were affected as well as cerebral white matter (data not shown).

Two groups of ten 28-day-old Swiss-Webster-and ICR-mice were each infected intracerebrally with PVI(LS-a) at a range of 105 to 5 x 106PFU. As in previous assays, there was no notable difference in clinical signs and histopathological features in mice with different genetic background. All infected animals developed a specific neurological syndrome with stereotypical onset, clinical course, functional deficits, and pattern of progression. Four days p.i., mice showed the first signs of an insidious spastic paraparesis (Fig. 1C) . No ascending motor deficits occurred, respiratory function remained unaffected, and there were no fatalities. Intracerebral inoculation of PVl(LS-a) was required to produce symptoms, since intravenous injection of virus with concomitant intracerebral needle puncture produced no neurological impairment. Intracerebral inoculation of equal particle numbers of PVI(LS-a) virus, inactivated by UV-irradiation, also caused no neurological signs of CNS damage. This finding indicated viral replication to be a prerequisite for PVI(LS-a) neuropathogenicity. Histopathology of the neurological syndrome caused by this variant centered exclusively on the spinal cord. Extensive infiltrates, originating from spinal blood vessels, invaded both the gray and white matter. Pathologic changes involved the entire spinal cord, but did increase in extent of damage caudally (Fig. 4A,B) . There was no apparent predilection for any cell subtype. Motor neuron damage was seen within areas of destructive necrotic tissue change secondary to the inflammation. At no time during the infection could damage to motor neurons within the thoracic or cervical cord be distinguished (Fig. 4B) , despite prominent inflammation affecting these regions. Spinal sections taken from animals which underwent partial reversal of neurological defects, 4 weeks p.i., revealed complete regression of infiltrative changes and preservation of motor neuron populations (Fig. 4C ).

A mutant PVI(M) virus, which had previously been reported to exert neuropathogenic potential in normal mice 21 was tested in a similar manner as the above mentioned strains. PVl(VP1-54) carried a point mutation at position 54 (P1054S) within capsid protein VP1. Intracerebral inoculation of each four Swiss-Webster-and ICR-mice with at least I x 109PFU resulted in visible signs of neurological damage in 50% of affected animals in both groups. With the genetic variant constructed in this laboratory, this large inoculum was required to induce clinical signs. The nature of the neurological syndrome was partly obfuscated by the vigorous host response to the application of excess viral antigen, and the resulting clinical deterioration. Sick mice showed prominent systemic signs of disease such as ruffled fur, reduced activity, and emaciation. Symptoms of minor motor impairment were present in half of the diseased mice, but the limb pareses were difficult to assess in quality due to the general weakened state of the animal. Mild limb weakness did not progress with any apparent disease pattern. Severely weakened, emaciated animals succumbed to the effects of infection, without the clear causal relationship of neurological disease and fatal outcome seen in poliomyelitis, hPVR-tg mice injected with PV1(VP1-54) developed a neurological condition indistinguishable from PVI(M) related poliomyelitis (data not shown). Similar to clinical findings, the histopathologic features induced by PVl(VP1-54) were less defined than in the other described infections. There was no supraspinal involvement, and spinal cord sections at all levels displayed widespread parenchymal invasion and reactive migroglial proliferation. No targeted attack against specific cell populations was apparent (Fig. 4D) . Histopathologic evidence for a primary thoracic and cervical cord affection was not present.

With the rare exception of certain serotype 2 PV strains, e.g. PV2(MEF-1) and PV2(L), naturally occurring PV strains can only infect primates, and even PV2(MEF-1) and PV2(L) are mouse neurovirulent only when artificially inoculated by intracerebral injection. The narrow host range of PV results from its stringent dependence on the express exactly the same cell tropism in the murine CNS as they do in the primate CNS unlikely and, hence, that mn PVs would cause the same specific disease syndrome (poliomyelitis) in normal mice as they do in primates. To shed some light on these complex problems we have compared the disease syndromes caused by a variety of different PV strains in hPVR-tg and normal mice. We have come to the conclusion that mn PV strains do not produce poliomyelitis in normal mice.

The occurrence of poliomyelitis in hPVR-tg mice corroborates the function of the hPVR as a major determining factor in the pathogenesis of the peculiar histopathological and clinical features of this disease. 1°'11 Poliomyelitis in tg mice followed a stereotypic course: initial flaccid pareses of the lower limbs, followed by cranial progression, involving respiratory function, and fatal outcome. In contrast, the clinical symptoms resulting from mn PV infections in normal Swiss-Webster-and ICR-mice could not be explained by classical terms of the syndrome poliomyelitis. Infection of normal mice with mn PV strains caused a diffuse encephalomyelitis with highly variable neurological symptoms appearing in random order, at topographically unrelated sites. Mouse neuropathogenicity was shared by several serotype 2 field isolates, the mouse adapted attenuated strain PV2(W2), the PVI(M) derivative PVI(LS-a), and PVI(M) with a mutation in residue 54 of capsid protein VPI. Interestingly, the type 2 PV field isolates PV2(MEF-1) and PV2(IND), have been reported to be mn without a lengthy process of adaptation, whereas other type 2 [PV2(L)] and type 1 strains [PVI(LS-a)] were passaged in the rodent CNS before they acquired the mn phenotype. Mn type 1 or type 3 PV field isolates have not been described. The molecular basis underlying this peculiar property of type 2 PV strains is not understood.

Earlier reports stressed the similarity between primate poliomyelitis and encephalomyelitis caused by PV2(L) in normal mice. 16 Comparative studies at that time were impeded by the lack of a murine model of hPVR-mediated poliomyelitis. Viral antigen was shown to be expressed in spinal anterior horn motor neurons, ~8 but in situ hybridization revealed the presence of viral RNA in cells in posterior portions of the spinal cord as well as the white matter. "s Previous analyses of PV neurovirulence using mn strains were based on the assumption of a similar pathogenesis of mn PV infections in wt mice and in primates. One parameter to assess the potential of viral neurovirulence is the LDso value, and in earlier studies, animals showing evidence of neurological functional impairment were generally killed without further histopathological analysis (see for example, ref. 17) . Our results suggest that the expression of the mn phenotype of PV strains in mice should not be compared to the expression of PV neurovirulence in primates, the normal host. Indeed, it appears that the expression of a mn phenotype in wt mice can result from very different genetic changes in PV strains, and that each genetic change can produce different syndromes. This is apparent not only from various type 2 mn strains, but also from the type 1 mn strain PVI(LS-a) [a derivative of PVI(M) whose genotype has been recently elucidated38]. Histopathological changes within the CNS should therefore be monitored when murine infections with mn PV strains are studied.

A major determinant for the mn phenotype of PV2(L) is the BC-Ioop of VP1 ~7 a surface protrusion of about 10 amino acids located at the apex of the poliovirion 46 that functions as a neutralization antigenic site. 47 Exchange of the VP1 BC-Ioop of the mouse-inert PVI(M) with the BC-Ioop of PV2(L) produces the mn phenotype, ~8' 19 an observation that prompted speculation that the VP1 BC-Ioop is involved in the recognition of a putative mouse receptor. No evidence for this hypothesis has been obtained as yet.

Strain PVI(LS-a), a mn derivative of PVI(M), accumulated 54 point mutations during passage in non-human tissue, of which 10 mutations led to amino acid exchanges in the capsid region28 These capsid mutations, however, were insufficient to produce the mn phenotype. Instead, the mutations in capsid protein VP1 plus, surprisingly, five mutations in the coding region of the non-structural protein 2A "r°, a proteinase, produced mouse neurovirulence. The syndrome PV1 (LS-a) ir~duced in normal mice, however is distinct from the encephalomyelitis caused by type 2 PV strains.

A pathological entity different from those observed with all other mn poliovirus strains, wes seen with PV1(VP1-54), a virus that we constructed according to a previous report. 21 PV1(VP1-54) is a mn derivative of PVI(M) that carries only a single amino acid exchange in capsid protein VP1 (P1054S). The mutation, located at an interpentameric interface inside the capsid, was suggested to destabilize the virion in the mouse CNS, but the neurological syndrome caused by infection with PV1(VP1-54) was not histopathologically characterized. 21 Infection of mice with PV1(VP1-54) led to neurological damage only after intracerebral inoculation of excess viral antigen. The poorly defined histopathological features of CNS involvement lacked characteristics of specificity observed in hPVR-mediated poliomyelitis.

The genotypic differences between mn PV strains and the clinical syndromes they cause, which differ from hPVR-mediated poliomyelitis, challenge the hypothesis of common determinants of a mn phenotype. Rather, it appears that a multitude of non-related genetic elements affect this phenotypic marker in various ways. As indicated before, the nature of the receptor(s) used by the mn PV strains in the mouse CNS remains to be determined. Available data suggest that the mouse homologue to the hPVR does not serve as surrogate for the mn PV strains. 13 It is more likely that the mn PV strains enter neuronal tissue via cell-surface protein(s) unrelated to hPVR. If so, it is not surprising that the syndromes produced by these viral strains are distinct from poliomyelitis.

In recent years picornaviruses other than poliovirus have been implicated as causative agents of neurological disease. These include enterovirus (EV) 70, 48 the etiologic agent of acute hemorrhagic conjunctivitis, or EV 71. 49 However, it has also been reported that the encephalomyelitis caused by the newly emerging human pathogen EV 71 did not always resemble poliomyelitis. S° The evolution of enterovirus strains with new neuropathogenic properties distinct from previously non-neuropathogenic ancestors might be due to adaptation to new receptor entities. The mouse-neurotropic PV variants with altered cell tropism constitute a precedent for the emergence of non-poliomyelitic picornaviral CNS disease. We thank A. Nomoto for the generous gift of the hPVR-tg mouse strain used in this study and B. Jubelt and O. Kew for kindly providing PV type 2 strains. We are grateful to P. Coyle for critical review of this manuscript. We thank N. Peress for helpful discussion and D. Colflesh for help with microscopic imaging. This work was supported in part by NCI grant CA28146, and NIH grants AI15122 and AI32100. M.G. is a recipient of a grant from the Stipendienprogramm Infektionsforschung, Heidelberg, Germany.

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