Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling oligonucleotides of plus and minus strand polarity. The synthetic poliovirus cDNA was transcribed by RNA polymerase into viral RNA, which translated and replicated in a cell-free extract, resulting in the de novo synthesis of infectious poliovirus. Experiments in tissue culture using neutralizing antibodies and CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice confirmed that the synthetic virus had biochemical and pathogenic characteristics of poliovirus. Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence.

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Neuroblastoma is one of the most common solid tumors in children. Treatment is of limited utility for high-risk neuroblastoma and prognosis is poor. Resistance of neuroblastoma to conventional therapies has prompted us to search for a novel therapeutic approach based on genetically modified polioviruses. Poliovirus targets motor neurons leading to irreversible paralysis. Neurovirulence can be attenuated by point mutations or by exchange of genetic elements between different picornaviruses. We have developed a novel and stable attenuated poliovirus, replicating in neuroblastoma cells, by engineering an indigenous replication element (cre), copied from a genome-internal site, into the 5 ¶-nontranslated genomic region (mono-crePV). An additional host range mutation (A 133 G) conferred replication in mouse neuroblastoma cells (Neuro-2a CD155 ) expressing CD155, the poliovirus receptor. Crossing immunocompetent transgenic mice susceptible to poliovirus (CD155 tg mice) with A/J mice generated CD155 tgA/J mice, which we immunized against poliovirus. Neuro-2a CD155 cells were then transplanted into these animals, leading to lethal tumors. Despite preexisting high titers of anti-poliovirus antibodies, established lethal s.c. Neuro2a CD155 tumors in CD155 tgA/J mice were eliminated by intratumoral administrations of A 133 Gmono-crePV. No signs of paralysis were observed. Interestingly, no tumor growth was observed in mice cured of neuroblastoma that were reinoculated s.c. with CD155 . This result indicates that the destruction of neuroblastoma cells by A 133 Gmono-crePV may lead to a robust antitumor immune response. We suggest that our novel attenuated oncolytic poliovirus is a promising candidate for effective oncolytic treatment of human neuroblastoma or other cancer even in the presence of present or induced antipolio immunity. [Cancer Res 2007;67(6):2857-64]

Although the chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles has received much attention (7), the phenotypic properties of the synthetic virus have been largely ignored. The published sequence guiding the synthesis of the synthetic virus was that of a highly neurovirulent wild-type (wt) strain, poliovirus type 1 (Mahoney) [wt PV1(M)] (24, 43). To distinguish the synthetic virus [sPV1(M)] from PV1(M), we engineered 27 nucleotide changes into the sPV1(M) genome as genetic markers (7). Compared to the wt progenitor PV1(M) strain, the sPV1 derivative was, surprisingly, highly attenuated in transgenic mice (7) expressing the poliovirus receptor, CD155 tg mice, which have been constructed by Koike et al. (25). It was plausible that one or several of the nucleotide changes that had been introduced into the sPM1(M) genome altered the neurovirulence of sPV1(M).PV is a neurovirulent member virus of the genus Enterovirus in the family Picornaviridae. It is not yet known where the virus replicates in the gastrointestinal (GI) tract after enteric infection, but secondary lymphatic tissues most likely play a major role (21,30,33). Invasion of PV into the central nervous system is rare and altogether not necessary for viral dissemination in the population (33). Indeed, the ratio of infection to neurological complications in PV infections is very low (10 Ϫ2 to 10 Ϫ3 , depending upon the virus type). Upon invasion of the central nervous system, PV targets predominantly motor neurons for destruction, which leads to paralysis and even death (33). Only humans and nonhuman primates can be infected with PV, although humans are the only natural hosts of the virus. This host range restriction is related to CD155, the only known PV receptor (33). Construction of CD155 tg mice, however, has allowed studies of PV pathogenesis (25, 45) in these animals. We are using CD155 tg mice originally constructed by A. Nomoto and his colleagues (PVRTg21) (25). The CD155 tg mice can be infected by the intramuscular, intravenous, or intracerebral route, but they are resistant to oral infection. The reason for this restriction is that the CD155 gene is not expressed under the control of the human promoter in the GI tract of these animals (21, 61). The unexplained silence of the human CD155 promoter in the mouse GI tract prevents studies of the first crucial steps in PV enteric infection in the CD155 transgenic animals. The genome of PV, which is of plus-strand polarity, is ϳ7,441 nucleotides (nt) long. It carries a small protein, VPg, covalently attached to its 5Ј end (10,29) and is polyadenylated at its 3Ј end (60).The genome consists of a long 5Ј-nontranslated region (NTR), a single large open reading frame, and a short 3ЈNTR (24, 58). Functionally, the 5ЈNTR can be divided into two regions: the 5Ј-terminal cloverleaf (nt 1 to 89) and the internal ribosomal entry site (IRES; nt 123 to 602) (58). Polyprotein synthesis is initiated 164 nt downstream of the IRES element at nt 743 (9, 58). The clove...

The cell composition of early hepatic lesions of experimental murine tularemia has not been characterized with specific markers. The appearance of multiple granulomatous-necrotic lesions in the liver correlates with a marked increase in the levels of serum alanine transferase and lactate dehydrogenase.

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The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.

Forty-five children who had been hospitalized with bronchiolitis caused by respiratory syncytial virus (RSV) at a mean age of 4 months, and 90 matched control children, were tested for occurrence of RSV antibodies at one year of age. Of the children who had suffered from bronchiolitis, forty had demonstrable IgG antibodies, whereas the remaining five only had IgA antibodies against RSV. In the control group, 42% were RSV seropositive. The anti-RSV IgA antibody titres tended to be higher in patients with bronchiolitis than in controls and a larger proportion of the seropositive children in the former than in the latter group had demonstrable IgG antibodies. These findings suggest that RSV infections causing bronchiolitis are more often associated with a strong antibody response than are mild cases of the infection. Follow-up of the children at 3 years of age showed that allergic sensitization and development of asthma had occurred much more frequently in children with past RSV bronchiolitis than in controls. Children with past RSV bronchiolitis who later developed allergic sensitization had elevated RSV IgA antibody titres at one year of age more frequently than children with past RSV-bronchiolitis, who were not sensitized (p = 0.015). No significant differences regarding IgG antibody titres were observed. Since IgA, similarly as IgE, antibody formation is strongly Th2 cell dependent, the results are compatible with other findings suggesting that RSV has an unusual propensity to activate the Th2 cell system. This may contribute to the pathological picture of bronchiolitis in small children and at the same time render the infected child predisposed for later development of allergic sensitization. RSV bronchiolitis may thus be an important risk factor for later development of atopic disease although it cannot be excluded that the bronchiolitis simply serves as a marker that predict later development of atopy.