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Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown that bacteria promote oral infection with a variety of enteric viruses. However, it is unknown whether antibiotics have microbiota-independent antiviral effects for enteric viruses or whether antibiotics influence extra-intestinal, systemic infection. Here, we examined the effects of antibiotics on systemic enteric virus infection by performing intraperitoneal injections of either coxsackievirus B3 (CVB3) or poliovirus followed by quantification of viral titers. We found that antibiotic treatment reduced systemic infection for both viruses. Interestingly, antibiotics reduced CVB3 titers in germ-free mice, suggesting that antibiotic treatment alters CVB3 infection through a microbiota-independent mechanism. Overall, these data provide further evidence that antibiotics can have noncanonical effects on viral infection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Globally, the use of single DTaP-IPV/Hib vaccines that combine DTaP-IPV and Hib is widespread, but in Japan vaccination is usually concomitant at separate sites. The immunogenicity and safety of a primary vaccination series and booster of a combined pentavalent DTaP-IPV/Hib vaccine were evaluated and compared to separate administration of DTaP-IPV and Hib in Japanese infants., Methods: Healthy Japanese infants were administered DTaP-IPV/Hib (Group A: N = 207) or DTaP-IPV + Hib (Group B: N = 207) by the subcutaneous (SC) or DTaP-IPV/Hib by the intramuscular (IM) route (Group C: N = 10). All subjects received a 3-dose primary vaccination series and a booster. Non-inferiority (Group A versus Group B) was tested post-primary series and subsequent post hoc analyses were performed for anti-Hib. Safety was assessed by parental reports., Results: Non-inferiority for SC administration of Group A versus Group B for the primary series was demonstrated for antibody responses to all antigens except Hib using the threshold of 1.0 μg/mL. Post hoc analyses for anti-Hib demonstrated non-inferiority for the primary series response using 0.15 μg/mL, and for pre-booster antibody persistence and the booster response using 0.15 μg/mL and 1.0 μg/mL. The immune response was similar for each antigen following SC or IM administration. There were no safety concerns in any group, and a lower incidence of injection sites for the IM route was observed as expected., Conclusions: These data show the good immunogenicity and safety profile of the DTaP-IPV/Hib vaccine as a 3-dose infant primary series followed by a booster in the second year of life in Japan., Competing Interests: Declaration of Competing Interest TN is a consultant to Daiichi Sankyo and Kitasato Daiichi Sankyo Vaccines and received a research grant from Daiichi Sankyo and Kitasato Daiichi Sankyo Vaccines; EV is an employee of Sanofi Pasteur, DT, SN, TS, YI, and HM are employees of Sanofi; HT is an employee of Daiichi Sankyo., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objective: To determine some virulent trait-related properties of poliovirus isolates from children with acute flaccid paralysis following vaccination with oral polio vaccine (OPV)., Design: Six polioviruses earlier characterised into wild, vaccine-derived and OPV-like were studied using the plaque morphology and growth kinetics at supra-optimal temperature., Setting: Department of Virology, University of Ibadan, Nigeria., Subjects: Polio isolates from six children who developed acute flaccid paralysis following vaccinations with various doses of OPV were used. All the children were located in the Northern part of the country where poliovirus is still circulating., Main Outcome Measures: The two vaccine-derived polioviruses acquired wild type characteristics., Results: All the six poliovirus isolates developed different forms of plaques ranging from tiny, small and large. The plaque formed could however not be used to identify the different isolates. Growth of the different isolates at supra-optimal temperature showed that the three wild polioviruses grew to a higher titre when compared with the Sabin 2 control. The two vaccine derived isolates behaved like the wild poliovirus while the OPV-like virus acquired an intermediate characteristics between wild and sabin., Conclusion: The wild polioviruses represented in this study are among the last vestiges of the circulating polioviruses found in the world. It is possible that the observed biological properties of wild types 1 and 3 described in the study are typical of the West African polioviruses. These properties will provide useful previews to the final identification of some important clinical isolates especially type 1 which may grow rapidly in cell culture.

Mutations frequently occur in oral poliovirus vaccine (OPV) strains upon replication in the human intestine. These strains occasionally revert to being neurovirulent. The more prolonged the excretion of OPV, the higher the risk of reversion. OPV strains can be secreted for several months in humans presenting humoral immune system deficiencies. The duration of excretion of OPV strains or other enteroviruses in individuals infected with the human immunodeficiency virus (HIV) is unknown. We investigated whether HIV infection, which is very prevalent in the Central African Republic, causes prolonged excretion of enteroviruses and, in particular, of OPV strains in adults. We studied 28 HIV-infected adults living with children who were immunized with OPV during national immunization days (NIDs). Blood samples were collected to confirm HIV status and to evaluate immunodeficiency before the NIDs. Stool samples for enterovirus isolation were also collected before the NIDs, between the two rounds of immunization and 2, 4 and 6 months after the second round of immunization. No poliovirus was isolated from any stool sample. Eight enteroviruses were isolated from eight adults (maximum one strain per patient). Enteroviruses were not more frequently isolated from severely immunodeficient patients. Thus, HIV-infected adults do not appear to be at high risk of infection with OPV strains and the excretion of enteroviruses (and thus of polioviruses) does not seem to be prolonged in HIV-infected adults.

Recombination in vaccine strains of polioviruses is a very frequent phenomenon. In the present report, 12 strains isolated from patients after OPV administration and healthy vaccinees were investigated for the identification of recombinant strains as well as for the further analysis of their recombination types and the localization of the recombination sites. The identification of these strains was achieved through reverse transcription, polymerase chain reaction and restriction fragment length polymorphism assays applied to four sequences of the viral genome, which are located in 5'UTR, VP3-VP1, 2C and 3C-D regions. The exact recombination sites were determined by sequencing. Four of the 12 included strains were recombinant in one or more regions. Two of the recombinant strains had an S2/S1 recombination, localized in 3C-D region, one had an S3/S2 localized in 2C and finally a birecombinant strain was identified having an S3/S2/S1 recombination type, with the two recombinations localized in 2C and 3D regions, respectively. The significance of the recombination types and sites observed concerning the viral viability are discussed.

Poliovirus isolates were screened for recombinants by combined analysis of two distant polymorphic segments of the poliovirus genome (one in the capsid and the other in the polymerase-coding region). Using a restriction fragment length polymorphism (RFLP) assay, a high number of recombinant genomes was found among vaccine-derived strains excreted by poliovirus vaccine vaccinees or vaccine-associated paralytic poliomyelitis cases. Some of these subjects carried a wild-type poliovirus (non-vaccine-specific) nucleotide sequence in the 3' part of the genome. Using a similar approach, a collection of wild-type poliovirus strains isolated in South India between 1985 and 1993 was screened for recombinants. Genotypes were defined by the parallel application of RFLP assays and genomic sequencing of the capsid protein VP1 and the 3D polymerase polypeptide. Analyses revealed several instances where the position of an isolate on the phylogenic tree for the capsid protein-coding segment did not agree with its position on the tree for the polymerase-coding region. In this way, several wild-type/wild-type and wild-type/vaccine recombinants could be identified, indicating that recombination is encountered commonly in the natural evolution of poliovirus strains.

In 1988, the Regional Committee of the Eastern Mediterranean Region (EMR) of the World Health Organization (WHO) resolved to eliminate poliomyelitis by 2000. Substantial progress toward polio eradication has been achieved in the region. Surveillance for cases of acute flaccid paralysis (AFP) and examination of stool specimens from AFP cases for the presence of poliovirus provide critical data to target supplemental vaccination activities. This report summarizes the progress in AFP and poliovirus surveillance in EMR from 1995 through September 1998 and highlights the importance of virologic investigations to determine whether viruses isolated represent indigenous transmission, importations, or laboratory contamination.

We addressed the contributions of persistent virus infection and immune cells to the pathogenesis of Theiler's virus-induced demyelination, a model for human multiple sclerosis. We developed a model involving the transfer of spleen cells into immunodeficient C.B-17-scid (SCID) mice, which normally die of overwhelming virus encephalitis without demyelination when infected with Theiler's virus. Adoptive transfer of nonimmune spleen cells from BALB/c mice into SCID mice resulted in the survival of all mice. However, these mice developed extensive demyelination and virus Ag/RNA persistence in the spinal cord white matter. The most demyelination was observed when mice received an intermediate number of spleen cells (1.8-7.5 x 10(6)), whereas too few cells (0.5 x 10(6)) did not ameliorate the SCID phenotype, and too many cells (30 x 10(6)) resulted in almost complete viral clearance with minimal demyelination. Adoptive transfer of spleen cells depleted of either CD4+ or CD8+ T cells produced vacuolar demyelination associated with virus persistence. In contrast, reconstitution with both CD4+ and CD8+ T cells produced less severe demyelination and partial clearance of virus. These experiments support the hypothesis that demyelination is the result of a balance between persistent virus infection and immune injury mediated by either CD4+ or CD8+ T cells.

To understand the mechanism of post-poliomyelitis muscular atrophy (PPMA) and the post-polio syndrome (PPS) in general, we performed the following studies: (1) histopathology in spinal cord sections from patients who died 9 days to 44 years after acute paralytic poliomyelitis; (2) enzyme histochemistry, immunocytochemistry (for lymphocyte subsets, MHC antigens and N-CAM) and polymerase chain reaction (PCR) for poliovirus RNA in the muscle biopsies from symptomatic or asymptomatic muscles of post-polio patients; (3) determination of lymphocyte subsets and circulating IgG or IgM antibodies against GM1 and poliovirus; (4) virological studies in the spinal fluid for oligoclonal bands and search for poliovirus genome with PCR; (5) electrophysiological studies including single fiber EMG, fiber density and macro-EMG; and (6) [31P] exercise MRS spectroscopy on previously affected muscles to search for a metabolic correlate of fatigue. These studies concluded that in PPS a continuing dysfunction is present in the spinal cord motor neurons, resulting in ongoing muscle denervation and reinnervation first evident at the axonal branch points. Symptoms are related to attrition of the oversprouting motor neurons which after a period of time cannot support all their axonal sprouts, resulting in failure of re-reinnervation. In some patients with PPS there is also an ongoing immune activation and presence of defective viral particles in the spinal fluid. However, their role in the pathogenesis of PPS is presently unknown.

Intravenous infection with Theiler's virus strain GD VII causes acute encephalomyelitis in mice. Endogenous IFN-gamma produced in the spinal cord is important to protect the tissue in mice infected with this virus. Neither CD4+ cells nor CD8+ cells infiltrated the spinal cords of infected mice until Day 9 postinfection. However, the number of CD3+/TCR-gamma delta+ cells increased in the spinal cords of mice infected with the virus. These cells resided in the spinal cords of normal mice, and produced IFN-gamma as a result of stimulation by immobilized anti-CD3 mAb. Elimination of CD3+ cells by the administration of a specific mAb augmented viral replication and suppressed production of endogenous IFN-gamma. Depletion of TCR-alpha beta+ cells and ASGM1+ cells did not affect the viral replication, and did not alter the production of IFN-gamma. Therefore, CD3+/TCR-alpha beta- cells producing IFN-gamma play an important role in the protection of the spinal cord against Theiler's virus infection. These results suggest that CD3+/TCR-alpha beta- cells might be identical to TCR-gamma delta+ cells.

The last confirmed case of poliomyelitis in Namibia had been reported in 1988. However, between Nov 8, 1993, and Jan 7, 1994, 27 cases of paralytic poliomyelitis were confirmed in the country. The outbreak was limited to the south health region; at least 80% of infants in this region have received four doses of oral poliovaccine (OPV) by the age of 1 year. Acute flaccid paralysis (AFP) was the predominant clinical presentation during the outbreak. The patients' ages ranged from 13 months to 12 years; 24 were younger than 5 years. Of the 26 patients whose vaccine status was known, 14 had received four doses of OPV, 6 had one or two doses, and 6 no vaccine. Genotypic analysis showed 86% homology of outbreak isolates with a 1982 Namibian isolate and west African isolates. Factors that may have had a role in the outbreak include establishment of a pool of susceptible people, rapid urbanisation, inadequate sanitation, poor water supply, and possible endemicity of poliovirus in neighbouring areas. Epidemics can occur in areas of high vaccine coverage. Our findings emphasise the need to improve AFP surveillance activities and the estimation of vaccine coverage to identify areas of potential susceptibility for outbreaks.

Mutants consistently accumulating in Sabin 1 poliovirus during serial passaging in vitro were identified by sequence heterogeneity assay and quantitated using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Only four unstable genomic sites were identified in virus passaged 10 times in African green monkey kidney (AGMK) cells, and eight sites in virus passaged in Vero cells. Mutations accumulated both in untranslated regions of RNA (nucleotides 480, 525 and 7441) and in coding sequences, as missense (nucleotides 1449, 4944, and 6203) or silent (nucleotides 1123 and 1141) mutations. The most prominent selectable mutations were found at complementary nucleotides 480 and 525 of the 5'-untranslated region (5'-UTR) of the Sabin strain, changing the G:U pair in F-domain to either A:U or G:C variants. These two variants have been shown previously to have an increased neurovirulence in monkeys. The G:C variant accumulated during passage in Vero cells, while A:U variant accumulated in CV-1 cells. Virus passaged in AGMK cells accumulated both variants. Higher temperature (37 instead of 34 degrees) strongly favored selection of mutants in Vero cells, had a smaller effect on mutant accumulation in AGMK cells, and had no effect in CV-1 cells. Monopools of type 1 oral poliovirus vaccine (OPV) made by seven manufacturers were found to contain both 480-A and 525-C revertants at a combined level of 1.1-2.7%. Viral samples with increased amounts of these revertants had higher neurovirulence in monkeys. Our results suggest that quantitation of these reversions by MAPREC may be prognostic for results of the monkey neurovirulence test (MNVT) and can be used for monitoring type 1 OPV consistency.

Background: Although the risk of vaccine-associated paralytic poliomyelitis (VAPP) has remained relatively constant during the past 30 years, estimates of VAPP depend largely on the completeness of reporting to the existing passive surveillance system. The National Vaccine Injury Compensation Program constitutes an alternative system for reporting VAPP, and data available from this system permitted us to evaluate the completeness of the national poliomyelitis surveillance system., Methods: We compared cases of paralytic poliomyelitis reported to the national surveillance system (maintained by the Centers for Disease Control and Prevention, Atlanta, Ga) with cases recommended for compensation by the National Vaccine Injury Compensation Program, Rockville, Md, and we calculated the observed completeness of reporting to the national system for 1980 through 1991. A capture-recapture method was also used to estimate completeness of reporting, ie, to account for cases potentially missed by both systems. In addition, we reviewed the epidemiology and updated the risk of VAPP based on the most current information on cases of VAPP., Results: From 1980 through 1991, 105 cases of paralytic poliomyelitis were identified by the Centers for Disease Control and Prevention and National Vaccine Injury Compensation Program systems, 98 (93%) of which were VAPP (average, 8.2 cases per year). The observed completeness of reporting to the Centers for Disease Control and Prevention was 94%, and the estimated completeness of reporting (capture-recapture method) was 81%. The overall risk of VAPP was one case per 2.5 million doses of oral poliovirus vaccine distributed. In the sensitivity analysis, the risk estimates of VAPP remained relatively stable throughout a wide range of assumptions regarding underreporting and specificity of the case definition for paralytic poliomyelitis., Conclusion: The risk of VAPP remains virtually unchanged from previous estimates despite the inclusion of previously unidentified VAPP cases. Despite the potential for both underreporting and misclassification of cases, our risk estimates were relatively insensitive to either of these biases. Since both of these biases were in opposite directions, and both probably occurred with low frequency, the risk estimates provided in this report appear valid and approximate the "true" risk of VAPP in the United States.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.

Sixty-eight laboratory strains representing 49 enterovirus, 10 adenovirus, and 3 reovirus serotypes were inoculated in a recombinant murine cell line expressing the human poliovirus receptor gene (L alpha cells). Only polioviruses caused cytopathic effect over a 10-day period. Likewise, only polioviruses were isolated, by use of L alpha cells, from 168 fecal specimens from children from developing countries. These results suggest that the recombinant L alpha cells can be used for selective isolation of poliovirus from clinical specimens.

For some microorganisms, virulence may be an inadvertent consequence of mutation and selection in the parasite population, occurring within a host during the course of an infection. This type of virulence is short-sighted, in that it engenders no advantage to the pathogen beyond the afflicted host. Bacterial meningitis, poliomyelitis and AIDS are three candidates for this model of the evolution of virulence.

Paralytic poliomyelitis is caused by the destruction of neurons in the central nervous system during the lytic replication of poliovirus. The human gene for the poliovirus receptor has been cloned recently, and transgenic mice generated that are susceptible to this virus. This has allowed the critical amino acids that determine the specificity of poliovirus to be identified.

We constructed a Theiler's virus mutant designated DA3304, in which the amino acid at position 101 of VP1 was changed from a threonine to an alanine. Because of this single amino acid change, DA3304 could still produce a biphasic central nervous system disease similar to that produced by the wild-type DA virus. However, DA3304 was significantly attenuated in both the acute and the chronic phases and induced smaller demyelinating lesions than the wild-type DA virus. The data are most compatible with the attenuated phenotype in DA3304 being due to the change of binding efficiency between the virus and receptor resulting from the physical alteration at the mutation site.

Poliomyelitis remains an important public health problem in China. Most cases and outbreaks are associated with wild type 1 polioviruses. To obtain an overview of type 1 poliovirus transmission in China, partial genomic sequences were compared for 24 case isolates from 12 provinces. Because polioviruses evolve rapidly during infection of humans, the genetic relationships among isolates provide a measure of the extent of epidemiologic linkage among cases. The observed genetic relationships were complex: six different genotypes, apparently derived from five separate endemic origins, were found. One genotype was recombinant, having noncapsid sequences derived from the Sabin type 1 vaccine strain and capsid sequences derived from a genotype indigenous to several northern and eastern provinces. Some isolates from geographically separate provinces were closely related; other isolates were related to wild polioviruses found in neighboring countries. The combination of epidemiologic and virologic analyses may facilitate the development of more effective strategies for poliomyelitis eradication.

Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), a model for multiple sclerosis, is a chronic T cell-mediated disease. Development of clinical symptoms in susceptible mouse strains generally correlates with TMEV-specific delayed-type hypersensitivity (DTH) responses. These responses, minimal or absent in resistant mouse strains, have been proposed as the pathogenic basis for the central nervous system inflammation and demyelination characterizing the disease. We demonstrate here that normally resistant (C57BL/6 x DBA/2)F1 hybrid mice develop clinical symptoms and DTH responses against TMEV after low doses of gamma-irradiation. Parental C57BL/6 animals remain resistant after similar pretreatment. Thus low-dose irradiation elicits a "latent" susceptibility to TMEV-IDD in some, but not all, resistant mice. Adoptively transferred spleen cells from syngeneic, unirradiated donors reconfer resistance on irradiated, infected B6D2F1 hybrids and reduce DTH responsiveness against TMEV, suggesting a protective role for a radiation-sensitive splenic population(s). The closely related C57BL/6 and C57BL/10 strains differ with respect to intrinsic and latent susceptibility.

The history of the enteroviruses is described, and how poliovirus came to be recognized as the prototype species of the genus, a subdivision of the family Picornaviridae. Albert Sabin was one of the main contributors. He isolated several enterovirus types and established them as causative agents of human disease. The enteroviruses were discovered only after new methods were introduced for working with viruses. They are now recognized as constituting one of the genera of the picornavirus family. Pico-rna-virus stands for viruses which are small (pico), and have an RNA genome. The enterovirus genus includes the polioviruses, the coxsackieviruses and the echoviruses of humans, plus a number of enteroviruses of lower animals (e.g., monkeys, cattle, pigs, mice). Over 100 serotypes are now recognized, the first having been the polioviruses.

Cardiovirus specific sequences located in the 5' NTR are used to amplify viral RNA by PCR. General primers were selected for the amplification of cardioviruses, including encephalomyocarditis virus (EMCV), mengovirus and Theiler's murine encephalomyelitis virus (TMEV. Additionally, ECHO virus type 22, an atypical enterovirus, could also be detected with the general cardiovirus primers. An internal encephalomyocarditis virus specific probe and a general cardiovirus probe are used to discriminate EMCV from other cardioviruses. The sensitivity of the PCR was determined at 10 pfu after hybridization with an internal oligonucleotide probe. Specificity was tested with a broad range of picornaviruses and other nonrelated RNA and DNA viruses. The applicability of the assay was demonstrated by the detection of TMEV RNA in tissue samples from experimentally-infected mice.

The three poliovirus serotypes are very stable. Breakthrough of the serotype barrier has never been observed in the natural evolution of poliovirus. This serotype stability contrasts with the high level of genomic and phenotypic variability that occurs within the bounds of serotype. The efficient control of poliomyelitis by immunization is based upon type-specific immunity and serotype stability. The development of attenuated strains by Albert Sabin was possible because of the high variability of poliovirus genomes. The three Sabin strains, one for each serotype, were selected as variants of non-attenuated wild polioviruses, and each represents a unique poliovirus genotype. A consequence of poliovirus variability is the polymorphic character of its genome. This polymorphism makes possible the identification of poliovirus genotypes upon which studies on poliovirus evolution, virologic surveillance, and poliomyelitis diagnostics are based. The antigenic and genomic peculiarities of the Sabin strains are used to distinguish them from wild polioviruses among field isolates. The mechanisms of poliovirus variation and their significance to the evolution of both wild and vaccine poliovirus strains are the subjects of this article. The natural evolution of polioviruses is discussed in the context of the global initiative to eradicate poliomyelitis, which relies on the worldwide use of Sabin's vaccine.

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus which causes a biphasic central nervous system (CNS) disease in certain strains of mice. Lytic virus replication within the CNS causes acute damage at early times post-infection, with the surviving animals developing a chronic CNS demyelinating disease. This damage is thought to result both from direct viral damage and from an immunopathological CD4+ T-cell mediated delayed-type hypersensitivity response to virus. By contrast, CD4+ T cells have a vital protective role at early times post-infection, as mice specifically depleted of CD4+ T cells of this subset prior to infection with TMEV die within 3-5 weeks. In an investigation of how CD4+ T cells act to mediate protection in TMEV-infected mice, we show that CD4+ cell-depleted animals, which fail to make a significant antiviral antibody response, could be protected by passive transfer of neutralizing antibodies. However, surviving animals had high levels of persisting virus in the CNS and they developed very severe symptoms of chronic demyelinating disease. The appearance of infectious virus was not due to selection of neutralizing antibody-resistant viral variants. These results demonstrate that the key protective role of CD4+ T cells in TMEV-infected mice is to provide help for antibody production by B cells at early times post-infection, but that other CD4+ cell-dependent mechanisms must contribute to control of virus replication, and are of importance in determining the levels of virus subsequently persisting in the CNS, and hence the severity of the chronic demyelinating disease.

To determine how oral poliovirus vaccine (Sabin) strains evolve during replication in humans and to confirm the etiology of vaccine-associated paralytic poliomyelitis (VAPP), we examined 70 vaccine-derived strains isolated from VAPP cases. Two distant sequences of the poliovirus genome were targeted for a double restriction fragment length polymorphism assay (RFLP) of reverse-transcribed genomic segments amplified by PCR, an extension of the method that we described previously (Balanant et al., 1991). One (RFLP-1) was a 480-long nucleotide sequence coding for the N-terminal part of the VP1 capsid polypeptide, situated in the 5' third of the viral genome (nucleotides 2401-2880). The other (RFLP-3D1) was a 291-long nucleotide sequence coding for a part of the viral polymerase, situated near the 3' end of the genome (nucleotides 6086-6376). Strain-specific restriction profiles could be generated for different field isolates by using three restriction enzymes in each case: HaeIII, DdeI, and HpaII for RFLP-1 and HaeIII, DdeI and RsaI for RFLP-3D1. With few exceptions, the vaccine-specific RFLP profiles were found to be conserved in both regions during replication of these viruses in humans. Thus, RFLP could be used as a marker so as to identify the origin of viral isolates at both ends of their genome. Whether viral isolates were vaccine-derived was determined by using strain-specific monoclonal antibodies and RFLP-1. Among the 70 isolates, 21 of the 43 type 2 strains and 15 of the 22 type 3 strains had a recombinant genome. None of the 5 type 1 Sabin-derived isolates was found to be recombinant. Both intertypic vaccine/vaccine and vaccine/non-vaccine recombinants were detected. Partial nucleotide sequencing confirmed the RFLP results in all cases that were investigated. In one case, it was possible to predict the recombination junction site from the restriction profiles. This site was more precisely localized by sequencing. The C6203 > U nucleotide substitution, which is suspected to contribute to the reversion toward neurovirulence of the attenuated Sabin 1 strain, was detected in almost all the recombinant genomes containing Sabin 1-specific sequences at the 3' extremity. This mutation was detected by identification of the modified RsaI profile in the RFLP-3D1. The results presented in this paper suggest that recombination, alone or together with mutation, might be one of the mechanisms of the reversion toward neurovirulence of attenuated vaccine strains and of the natural evolution of poliovirus.

In 1991 in Tajikistan 36 cases of acute poliomyelitis were recorded (spinal form 27, bulbospinal 3, pontospinal 6 cases) in children ranging in ages from 6 months to 6 years. The outcome of the disease was fatal in 4 cases and with crude residual effects in 32 children. Out of 23 children examined, poliomyelitis virus type I was isolated from 13, antibodies to poliovirus type I in titres 1:16 to 1:1024 were detected in 21 children, ECHO 19 virus was isolated from 5 children and antibodies to this virus in titres 1:16 to 1:8192 were demonstrated in 13 children. The fresh isolates of poliomyelitis virus type I had rct+, DS+ and N+ markers. The strains of ECHO 19 virus were highly pathogenic for monkeys causing deaths of the animals within 3-4 days. It is assumed that the main causative agent of the Tajikistan outbreak of poliomyelitis was a virulent "wild" variant of poliovirus type I. The preceding or concomitant infection with ECHO 19 virus (considering the data from the literature and the demonstrated high virulence of this agent for monkeys) could exert an aggravating effect on the course of poliomyelitis or, in some cases, be an independent cause of the disease and, possibly, death of the children.

Inbred strains of mice differ greatly in their susceptibility to the demyelinating disease caused by Theiler's Murine Encephalomyelitis Virus. In this murine disease, which is an animal model for the study of multiple sclerosis, demyelination depends on the persistent infection of the central nervous system. Previous studies identified a locus in the H-2D region of the major histocompatibility complex which controls susceptibility to the persistent infection, and also showed that other loci are involved. In order to identify these loci, we screened the genome of a set of backcross animals with a combination of polymorphic microsatellites and restriction enzymes sites. We now show that viral persistence is also controlled by a locus close to Ifg on chromosome 10 and possibly by a locus near Mbp on chromosome 18.

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.

In five years from 1988 to 1992, 51 polioviruses were isolated from patients with acute flaccid paralysis in Shandong province in China. Of the 51 poliovirus isolates, 17 were type 1, 18 type 2 and 16 type 3. Twelve type 1 viruses isolated during the period from 1988 to 1990 were shown to be wild strains by serology and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. These wild strains were classified into three groups by PCR-RFLP. The other type 1 isolates were Sabin-like strains. No wild strain was isolated in 1991 or 1992. All isolates of types 2 and 3 during the five years were Sabin-like strains. These data suggest that wild strains of polioviruses will soon be eradicated in Shandong province.

During the 1992 poliovirus type 3 outbreak in the Netherlands virological and serological investigations were conducted. No molecular epidemiological link was traced between poliovirus type 3 that caused the outbreak of poliomyelitis in the Netherlands and isolates from previous epidemics investigated. Serological neutralization assessments indicate that the inactivated poliomyelitis vaccine used in the Dutch national immunization schedule induces immunity to the causative agent.

Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjögren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.

Four-hundred and fourty-six faecal specimens sampled from healthy carriers between 0 and more than 20 year-old age groups in Changle and Wucheng counties of Shandong Province were collected from June to Sept. of 1991. From them, 142 (31.84%) isolates of enteroviruses were obtained. There was no significant difference in the enteroviruses isolation rate between Changle and Wucheng. No poliovirus was isolated. Among other enterovirus isolates, 85 were echoviruses, 6 coxsackieviruses, 9 enterovirus type 70 and 2 enterovirus type 71. Other 40 isolates were positive for cytopathogenic effect (CPE) in cell culture, but they could not be identified by antisera to poliovirus and enteroviruses. The results showed that the infection by non-polio enteroviruses which could cause polio-like paralysis should be paid attention in the poliomyelitis eradication program.

During September 1992-February 1993, 68 cases of poliomyelitis occurred among members of a religious community in the Netherlands. Because members of an affiliated religious community in Alberta, Canada, had direct contact (i.e, travel to and from the Netherlands) with members of the affected community, health authorities in Alberta conducted an investigation during January-February 1993 to determine whether this poliovirus had been imported. This report summarizes the results of this investigation.

The mouse-adapted Lansing strain of poliovirus type 2 PV-2(L) induces fatal poliomyelitis in mice after intracerebral inoculation, while mice inoculated with Mahoney strain of poliovirus type 1 PV-1(M) show no signs of disease. Previous work had indicated that both the 5' non-translated region of the viral genome and the viral capsid protein, neutralization antigenic site I (N-Ag1), were involved in mouse neurovirulence. In order to further explore the role of N-Ag1 in mouse neurovirulence, antigenic chimeras of two poliovirus strains, XF414 and XF324, were constructed. In the two strains, ten amino acids (in XF414) or 16 amino acids (in XF324) in antigenic site I in Vp1 of PV-I (M) were replaced with a PV-2(L)-specific sequences using a mutagenesis cartridge. Mouse neurovirulence tests indicated that mice cerebral inoculated with XF414 and XF324 developed poliomyelitis leading to paralysis or death. The viruses possessing antigenicity of the inoculating viruses were isolated from cerebral tissues of the paralyzed mice. The results demonstrated that N-Ag1 is an important determinant of poliovirus host range, and may be involved in attachment and penetration of poliovirus (in)to cells of the mouse central nervous system.

The Nordic countries, i.e., Denmark, Finland, Iceland, Norway, and Sweden, together with Holland, have all continued to use the killed poliovaccine introduced in the middle of the 1950s and they still use it today. In Denmark, combined vaccination has been practised since 1966, starting with three doses of the killed vaccine and continuing with the oral vaccine. Norway used oral vaccine alone during the 14-year period from 1965 to 1979. In all these countries, the immunizations with the killed vaccine were immediately successful. Poliomyelitis was practically eliminated already by the beginning of the 1960s. After this initial successful period, the different countries experienced different events, from which valuable conclusions can be drawn: 1. Nationwide vaccination with killed vaccine was highly effective. 2. It is of the utmost importance that the potency of the killed vaccine is high. 3. Oral vaccine may cause higher rates of vaccine-associated secondary cases than have been reported in general. 4. In the Nordic countries, the general circulation of wild virus appeared to cease simultaneously with the disease. 5. When virus is reintroduced into the country, unvaccinated groups are vulnerable. Outbreaks in unvaccinated "pockets" have occurred. This phenomenon, however, has also been experienced in countries using oral vaccine. 6. In Stockholm, both wild poliovirus and vaccine-like polio strains were isolated from the sewage water, indicating a constant import of both types of viruses. Virus isolation from thousands of patients with meningitis or diarrhoea have been negative throughout the years.