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Vaccination remains our main defence against influenza, which causes substantial annual mortality and poses a serious pandemic threat. Influenza virus evades immunity by rapidly changing its surface antigens but, even when the vaccine is well matched to the current circulating virus strains, influenza vaccines are not as effective as many other vaccines. Influenza vaccine development has traditionally focused on the induction of protective antibodies, but there is mounting evidence that T cell responses are also protective against influenza. Thus, future vaccines designed to promote both broad T cell effector functions and antibodies may provide enhanced protection. As we discuss, such vaccines present several challenges that require new strategic and economic considerations. Vaccine-induced T cells relevant to protection may reside in the lungs or lymphoid tissues, requiring more invasive assays to assess the immunogenicity of vaccine candidates. T cell functions may contain and resolve infection rather than completely prevent infection and early illness, requiring vaccine effectiveness to be assessed based on the prevention of severe disease and death rather than symptomatic infection. It can be complex and costly to measure T cell responses and infrequent clinical outcomes, and thus innovations in clinical trial design are needed for economic reasons. Nevertheless, the goal of more effective influenza vaccines justifies renewed and intensive efforts., (© 2024. Springer Nature Limited.)

Aims/hypothesis: This case-control study was nested in a prospective birth cohort to evaluate whether the presence of enteroviruses in stools was associated with the appearance of islet autoimmunity in the Type 1 Diabetes Prediction and Prevention study in Finland., Methods: Altogether, 1673 longitudinal stool samples from 129 case children who turned positive for multiple islet autoantibodies and 3108 stool samples from 282 matched control children were screened for the presence of enterovirus RNA using RT-PCR. Viral genotype was detected by sequencing., Results: Case children had more enterovirus infections than control children (0.8 vs 0.6 infections per child). Time-dependent analysis indicated that this excess of infections occurred more than 1 year before the first detection of islet autoantibodies (6.3 vs 2.1 infections per 10 follow-up years). No such difference was seen in infections occurring less than 1 year before islet autoantibody seroconversion or after seroconversion. The most frequent enterovirus types included coxsackievirus A4 (28% of genotyped viruses), coxsackievirus A2 (14%) and coxsackievirus A16 (11%)., Conclusions/interpretation: The results suggest that enterovirus infections diagnosed by detecting viral RNA in stools are associated with the development of islet autoimmunity with a time lag of several months.

The rapidly increasing incidence of type 1 diabetes implies that environmental factors are involved in the pathogenesis. Enteroviruses are among the suspected environmental triggers of the disease, and the interest in exploring the possibilities to develop vaccines against these viruses has increased. Our objective was to identify enterovirus serotypes that could be involved in the initiation of the disease process by screening neutralizing antibodies against 41 different enterovirus types in a unique longitudinal sample series from a large prospective birth-cohort study. The study participants comprised 183 case children testing persistently positive for at least two diabetes-predictive autoantibodies and 366 autoantibody-negative matched control children. Coxsackievirus B1 was associated with an increased risk of β-cell autoimmunity. This risk was strongest when infection occurred a few months before autoantibodies appeared and was attenuated by the presence of maternal antibodies against the virus. Two other coxsackieviruses, B3 and B6, were associated with a reduced risk, with an interaction pattern, suggesting immunological cross-protection against coxsackievirus B1. These results support previous observations suggesting that the group B coxsackieviruses are associated with the risk of type 1 diabetes. The clustering of the risk and protective viruses to this narrow phylogenetic lineage supports the biological plausibility of this phenomenon.

Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2) capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

For human vaccines to be available on a global scale, complex production methods, meticulous quality control, and reliable distribution channels are needed to ensure that the products are potent and effective at the point of use. The technologies used to manufacture different types of vaccines can strongly affect vaccine cost, ease of industrial scale-up, stability, and, ultimately, worldwide availability. The complexity of manufacturing is compounded by the need for different formulations in different countries and age-groups. Reliable vaccine production in appropriate quantities and at affordable prices is the cornerstone of developing global vaccination policies. However, to ensure optimum access and uptake, strong partnerships are needed between private manufacturers, regulatory authorities, and national and international public health services. For vaccines whose supply is insufficient to meet demand, prioritisation of target groups can increase the effect of these vaccines. In this report, we draw from our experience of vaccine development and focus on influenza vaccines as an example to consider production, distribution, access, and other factors that affect vaccine uptake and population-level effectiveness., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The increased incidence and extended geographical reach of Dengue virus over the past two decades have made the development of an effective vaccine an international urgency. Various strategies are being pursued, including live, vectored and killed/recombinant preparations. For all approaches, the challenge is to induce a broad durable immune response against all four serotypes of Dengue virus simultaneously whilst avoiding the possible exacerbation of risk of developing the severe forms of disease through incomplete or modified responses. This review presents the current state of knowledge and discusses the challenges of further clinical development.

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

Vaccines are one of the most useful and cost-effective tools for reducing the morbidity and mortality that are associated with infectious diseases. Here, Jeffrey Almond discusses the selection of articles in this Focus issue, in the context of the challenges and opportunities facing vaccine developers today.

The global eradication of poliomyelitis caused by wild-type virus is likely to be completed within the next few years, despite immense logistic and political difficulties, and may ultimately be followed by the cessation of vaccination. However, the existing live-attenuated vaccines have the potential to revert to virulence, causing occasional disease, and viruses can be shed by immunocompromised individuals for prolonged periods of time. Moreover, several outbreaks of poliomyelitis have been shown to be caused by viruses derived from the Sabin vaccine strains. The appearance of such strains depends on the prevailing circumstances but poses a severe obstacle to strategies for stopping vaccination. Vaccine strains that are incapable of reversion at a measurable rate would provide a possible solution. Here, we describe the constructions of strains of type 3 poliovirus that are stabilized by the introduction of four mutations in the 5' noncoding region compared to the present vaccine. The strains are genetically and phenotypically stable under conditions where the present vaccine loses the attenuating mutation in the 5' noncoding region completely. Type 1 and type 2 strains in which the entire 5' noncoding regions of Sabin 1 and Sabin 2 were replaced exactly with that of one of the type 3 strains were also constructed. The genetic stability of 5' noncoding regions of these viruses matched that of the type 3 strains, but significant phenotypic reversion occurred, illustrating the potential limitations of a rational approach to the genetic stabilization of live RNA virus vaccines.

We evaluated the protective role of passively transferred circulating antibodies in protecting non-human primates against experimental rotavirus infection. Pooled sera with rotavirus-specific IgG titers that were either high (1:10,000), intermediate (1:300), or negative (< 1:25) were infused i.v. into naive pigtailed macaques (ages 3-6 months). Rotavirus-specific IgG could be detected in the sera at 18 h in all animals infused with antibody-containing serum, and fecal IgG titers could be detected only in animals given high-titer pooled sera. When orally challenged with 10(6) fluorescent-forming units of a simian rotavirus strain, YK-1, at 18 h after serum transfer, control animals shed virus starting 1-3 days after challenge and continued to shed virus at high titers for 6-8 days, whereas passively immunized macaques did not shed virus or had delayed shedding at low titers for only a limited time. The observation that passively transferred antibodies can suppress or delay viral infection in rotavirus-challenged pigtailed macaques has important implications for the design and testing of parenteral candidate rotavirus vaccines.

The current live-attenuated vaccine strains of poliovirus are genetically unstable and capable of rapid evolution in human hosts, resulting in reversion to neurovirulence and, occasionally, disease. They can also be shed by recipients for a considerable time after vaccination. This raises questions about how and when to stop vaccination after wild-type viruses have been eliminated. Persistence of vaccine revertant viruses in the population would present a risk to new cohorts of unvaccinated children and threaten the success of the eradication programme. A number of Sabin vaccine strain derivatives have been described that are, in theory, genetically more stable than the present vaccines and therefore less likely to revert to virulence. The approaches used in their derivation are outlined here and data presented for two strains showing a significant improvement in genetic stability. These strains were designed according to our understanding of the molecular basis of attenuation and incorporate changes in the sequence of an RNA structural domain that plays a key role in attenuation. They may also be less transmissible than the current type 3 vaccine strain and are potentially useful in the strategically difficult final stages of poliomyelitis eradication.

A number of echoviruses use decay accelerating factor (DAF) as a cellular receptor or attachment protein for cell infection. Binding of echovirus 7 to DAF at the cell surface, but not to soluble DAF in solution, triggers the formation of virus particles exhibiting an altered sedimentation coefficient ('A' particles) which are considered indicative of the particle uncoating process. We have previously demonstrated that antibodies to beta(2)-microglobulin block cell infection at a stage prior to 'A' particle formation and suggested that this reflects the involvement of beta(2)-microglobulin (or the associated MHC-I) in a virus-receptor complex that forms at the cell surface. We demonstrate here that antiserum to CD59 specifically blocks infection of rhabdomyosarcoma cells by a range of echoviruses, including viruses that bind DAF (e. g. echovirus 7) and those that use currently unidentified receptors other than DAF. The block occurs prior to 'A' particle formation and is cell-type specific. The potential role of CD59 as an active member, or passive participant, in the virus-receptor complex is discussed.

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

Picornavirus infection requires virus uncoating, associated with the production of 135S "A" particles and 80S empty particles from 160S mature virions, to release the RNA genome into the cell cytoplasm. Normal albumin inhibits this process. We now show that when depleted of fatty acids, albumin induces the formation of echovirus A particles.

Many serotypes of echovirus (EV) and Coxsackie B virus (CBV) bind human decay-accelerating factor (DAF) and use it as a receptor for infection. Analogues for DAF have been isolated from mice and rats and characterized; these analogues have amino acid identities to human DAF of approximately 60%. EV serotypes 3, 6', 7, 11-13, and 29 and CBV serotypes 1, 3, and 5 caused hemagglutination of human erythrocytes but not rat or mouse erythrocytes, suggesting failure to bind rodent DAF. To confirm this evidence, radiolabeled viruses were incubated with transfected Chinese hamster ovary (CHO) cells that were abundantly expressing each type of DAF. Only cells that expressed human DAF bound virus. Although binding of EV and CBV was specific for human DAF, complement inhibition by DAF expressed in CHO cells was similar for each analogue.

We showed previously that a human rhinovirus 14 (HRV14) 3' untranslated region (3' UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J. Virol 69:7835-7844, 1995). This enabled the HRV14 single 3' UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3' UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3' UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3' UTR of rhinovirus with both poliovirus and rhinovirus polymerases.

Decay accelerating factor (DAF) functions as a cell attachment receptor for a wide range of human enteroviruses, the interaction accounting for the haemagglutination phenotype exhibited by many members of this family. Haemagglutination inhibition assays using purified truncated soluble DAF (sDAF) receptors and short consensus repeat (SCR) domain-specific antibodies have been used to determine the domain(s) of DAF to which the viruses bind. Further sDAF-mediated virus neutralization and biosensor analysis have been used to confirm the virus-binding domains of DAF. Of the four distinct clusters of human enteroviruses, three contain representatives that bind DAF. The majority of DAF-binding enteroviruses occupy the 'CBV-like' cluster, and require SCR domains 2-4 for DAF binding. In contrast, the DAF-binding representatives of the 'ENV70-like' and 'PV-like' clusters require SCR1 for DAF interaction. These studies confirm that DAF binding is a widespread characteristic amongst phylogenetically divergent clusters within the enteroviruses and suggest that the ability to bind DAF may have evolved more than once within this group of viruses.

Echoviruses induce a wide spectrum of diseases in man, the most severe being meningitis. In neonates, however, a severe systemic infection can be observed, leading to death. Serum albumin is the most abundant protein in plasma and most interstitial fluids, and its functions include osmoregulation and transport and delivery of hydrophobic molecules such as fatty acids and steroids. The results of cold-synchronized one-step growth analysis of echovirus 7 infection and sucrose-gradient analysis of A-particles suggest that physiological concentrations of albumin block echovirus 7 infection by inhibiting uncoating. The blockage was reversible and was still effective when albumin was added 30 min after virus adsorption. Inhibition of uncoating was confirmed by using rhodanine, a known specific inhibitor of echovirus uncoating. After removal of the albumin blockage, addition of rhodanine perpetuated the inhibition. Serum and interstitial albumin concentrations may limit echovirus infection in vivo and thereby act as an extracellular determinant for echovirus tropism.

Several echoviruses (EVs) have previously been shown to use decay accelerating factor (DAF) as a cellular receptor. Since DAF is expressed on erythrocytes, EVs that use this receptor cause haemagglutination. Here we show that all EVs that haemagglutinate do so via attachment to DAF and that this interaction can be inhibited by a monoclonal antibody (MAb) specific for DAF domain SCR3. Although the viruses haemagglutinate via DAF some can bind to rhabdomyosarcoma cells from which DAF has been removed and infect in the presence of a MAb against DAF. This suggests that some EVs have the capacity to interact with more than one cellular receptor.

The presence of cellular factors that bind to the 3' untranslated region (UTR) of picornaviruses was investigated by electrophoretic mobility shift assays (EMSAs). A cellular factor(s) that binds specifically the 3' UTR of polio-, coxsackie- and rhinoviruses was detected. Furthermore, this factor(s) is distinct from those which bind to the 5' terminal 88 nt (the 'cloverleaf') of poliovirus. Mutations within the 3' UTR which decrease the affinity of the RNA for the cellular factor in EMSAs decrease RNA replication and virus viability. Revertants of these mutants display changes which are predicted to stabilize the RNA secondary structure of the 3' UTR. These results indicate that binding of a cellular factor to the UTR plays a role in virus replication and that RNA secondary structure is important for this function.

The inclusion of a foreign marker gene, chloramphenicol acetyltransferase (CAT) gene, into the poliovirus genome allows its replication and encapsidation to be easily monitored using a simple enzyme assay. Such poliovirus replicons require the presence of helper virus for their successful propagation and thus are similar to defective interfering (DI) viruses. In genomes containing the CAT gene, the majority of the P1 virus capsid region of the poliovirus genome could be removed without destroying viability. The smallest replicon was significantly smaller than any naturally occurring DI particle so far reported, yet it retained the ability to replicate and be encapsidated by structural proteins provided by helper virus in trans. The efficiency with which the replicons were encapsidated was investigated using a direct immunostaining technique that allows individual cells infected with either a replicon or helper virus to be quantified. These results were compared to the frequencies of trans-encapsidation of polioviruses and coxsackievirus B4 using a two-stage neutralization assay. Poliovirus types 1, 2 and 3 but not coxsackievirus B4, coxsackievirus A21 or rhinovirus 14 provided efficient trans-encapsidation of poliovirus type 3 or type 3-derived replicons. These results suggest that a specific encapsidation process operates and that it does not involve RNA sequences within the region of the genome encoding the capsid proteins.

A monoclonal antibody (MAb) that blocks most echoviruses (EVs) from infecting rhabdomyosarcoma (RD) cells has been isolated. By using the CELICS cloning method (T. Ward, P. A. Pipkin, N. A. Clarkson, D. M. Stone, P. D. Minor, and J. W. Almond, EMBO J. 13:5070-5074, 1994), the ligand for this antibody has been identified as beta2-microglobulin (beta2m), the 12-kDa protein that associates with class I heavy chains to form class I HLA complexes. A commercial MAb (MAb 1350) against beta2m was also found to block EV7 infection without affecting binding to its receptor, DAF, or replication of EV7 viral RNA inside cells. Entry of EV7 into cells was reduced by only 30% by antibody and cytochalasin D, an inhibitor of endocytosis mediated by caveolae and clathrin-coated pits, but was not significantly reduced by sodium azide. The block to virus entry by cytochalasin D was additive to the block induced by antibody. We suggest that EV7 rapidly enters into a multicomponent receptor complex prior to entry into cells and that this initial entry event requires beta2m or class I HLA for infection to proceed.

Recent studies have identified at least nine distinct receptors used by the piconaviruses for cell entry. Does the evolution of receptor usage correlate with the different tropisms observed in this group of viruses, and does this influence pathogenesis, or is it the consequence of another selection mechanism that favours virus survival?

Bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob Disease (CJD) belong to a group of degenerative neurological disorders collectively known as the transmissible spongiform encephalopathies (TSEs). The group also includes scrapie of sheep and goats, kuru of humans, chronic wasting disease of mule deer and elk and transmissible encephalopathy of mink. These fatal diseases cause behavioural changes, alterations of sensation, changes in mental state and ataxia. The typical pathology is non-inflammatory vacuolation (spongiosis) in neuronal perikarya and in the grey matter neuropil. Occasionally, there may also be amyloid plaque deposition in certain regions of the brain and, less frequently, the spinal cord. All the diseases have long incubation periods which, depending on the host, may range from many months to several decades. Death is inevitable after a slow progressive illness. In this review, I shall cover the recent UK outbreak of BSE and its relationship to new variant Creutzfeldt-Jakob disease.

Soluble forms of decay-accelerating factor (DAF) (CD55), the receptor for echovirus 7, were synthesized in the yeast Pichia pastoris. Purified recombinant protein containing SCR domains 2, 3, and 4, but lacking the serine/threonine rich region, was shown to block infection of susceptible cells by echovirus 7. In contrast to the situation with poliovirus and its receptor, the neutralization of echovirus 7 by soluble DAF was completely reversible and did not lead to the formation of 135S A-particles. Binding of virus to susceptible cells, by contrast, did lead to the formation of A particles, mainly from virus that had been internalized. The data suggest that a secondary factor(s) may contribute to A-particle formation and uncoating of echovirus 7.

The role of the 3' untranslated region (3'UTR) in the replication of enteroviruses has been studied with a series of mutants derived from either poliovirus type 3 (PV3) or a PV3 replicon containing the reporter gene chloramphenicol acetyltransferase. Replication was observed when the PV3 3'UTR was replaced with that of either coxsackie B4 virus, human rhinovirus 14 (HRV14), bovine enterovirus, or hepatitis A virus, despite the lack of sequence and secondary structure homology of the 3'UTRs of these viruses. The levels of replication observed for recombinants containing the 3'UTRs of hepatitis A virus and bovine enterovirus were lower than those for PV3 and the other recombinants. Extensive site-directed mutagenesis of the single stem-loop structure formed by the HRV14 3'UTR indicated the importance of (i) the loop sequence, (ii) the stability of the stem, and (iii) the location of the stem immediately upstream of the poly(A) tail. The role of a 4-bp motif at the base of the HRV14 stem, highly conserved among rhinoviruses, was examined by site-directed mutagenesis of individual base pairs. This analysis did not pinpoint a particular base pair as crucial for function. The requirement for immediate adjacent positioning of the open reading frame and the 3'UTR was examined by insertion of a 1.1-kb heterologous sequence. A replicon containing this insert replicated to about 30% of the level observed for the wild type. However, the corresponding virus consistently deleted most of the inserted fragment, suggesting that its presence was incompatible with a full replication cycle.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.

CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates complement activity by accelerating the decay of C3/C5 convertases. Recently, we and others have established that this molecule acts as a cellular receptor for echovirus 7 and related viruses. DAF consists of five domains: four short consensus repeats (SCRs) and a serine/threonine-rich region, attached to the cell surface by a glycosylphosphatidyl inositol anchor. Chinese hamster ovary cells stably transfected with deletion mutants of DAF or DAF-membrane cofactor protein recombinants were analyzed for virus binding. The results indicate that the binding of echovirus 7 to DAF specifically requires SCR2, SCR3, and SCR4. There is also a nonspecific requirement for the S/T-rich region which probably functions to project the binding region away from the cell membrane. The three nonpeptide modifications of DAF, N-linked glycosylation, O-linked glycosylation, and the glycosylphosphatidyl inositol anchor, are not required for virus binding. The SCRs of membrane cofactor protein, the closest known relative of DAF, cannot substitute for those of DAF with retention of virus binding activity. The monoclonal antibody used to identify DAF as an echovirus receptor, and which inhibits binding of the virus (monoclonal antibody 854), binds to SCR3.

Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.

All three live, attenuated vaccine strains of poliovirus contain important attenuation determinants in a short conserved sequence in the 5' noncoding region. Evidence suggests these act by weakening a secondary-structural element critical for the unusual mechanism of translational initiation of picornaviruses, in which ribosomes bind directly to a site far downstream of the 5' end. Understanding the molecular basis of attenuation may allow novel vaccine strains to be designed.

The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d). The independence of the cloverleaf structure formed by the 5'-terminal 88 nucleotides and the ribosome landing pad or internal ribosome entry site (IRES) was investigated by constructing a 5'UTR composed of the PV3 cloverleaf and the IRES from encephalomyocarditis virus. Chloramphenicol acetyltransferase gene-containing replicons and viruses containing this recombinant 5'UTR showed levels of replication similar to those of the corresponding genomes containing the complete PV3 5'UTR, indicating that the cloverleaf and the IRES may be regarded as functionally independent and nonoverlapping elements.

A mouse pancreas-adapted variant of coxsackievirus B4 (P-CB4) has been shown to replicate in, and cause an excessive release of insulin from, pancreatic beta cells cultured in vitro. The prototype CB4 strain (JVB Benschoten), from which the adapted variant was derived, although able to replicate in cultured islets does not cause a similar release of insulin from the beta cells. The pancreas-adapted virus has also been shown to cause host cell protein synthesis shut-off in beta cells and to inhibit (pro)insulin biosynthesis. These metabolic changes occur in the absence of cytolytic damage [Szopa et al.: Bioscience Reports 5:63-69, 1985 and Cell Biochemistry and Function 4:181-187, 1986]. To investigate the genetic basis for this beta cell tropism, the complete nucleotide sequence of P-CB4 has been determined and compared to that of the previously published sequence of the prototype CB4 strain (JVB Benschoten) [Jenkins et al.: Journal of General Virology 68:1835-1848, 1987]. Twenty-five nucleotide sequence differences were observed. Of these, six occur in the 5' noncoding region of the genome and 19 in the coding region (resulting in seven amino acid changes). The possible significance of these changes in relation to the beta cell tropism of the pancreas-adapted virus is discussed.

Viral protein synthesis in poliovirus infected cells was found to be influenced by mutations in part of the viral 5'-non-coding region (NCR) in a temperature dependent manner. At elevated temperatures these mutations resulted in virus titre reductions that allowed selection of revertant viruses. Some revertants were found to have retained the 5'-NCR mutations but had compensating mutations in the 2A protease gene that were responsible for the suppression of the temperature sensitive phenotypes. The mutations in 2A enhanced viral protein synthesis at a stage when cap dependent translation was already abolished, suggesting that the virally encoded protein 2A is directly involved in the process of cap independent translation in addition to its role in abolishing cap dependent translation.

Poliovirus (PV) RNA is translated by a cap-independent mechanism involving the internal entry of ribosomes onto the 5' noncoding region (NCR). Using the vaccinia virus-T7 RNA polymerase transient expression system, we showed previously that deletion of certain individual predicted secondary structures within the PV 5' NCR rendered the element defective in directing internal initiation when assayed alone. However, these defective 5' NCRs were functional when coexpressed within cells with full-length PV cDNA (N. Percy, G. J. Belsham, J. K. Brangwyn, M. Sullivan, D. M. Stone, and J. W. Almond, J. Virol. 66:1695-1701, 1992). We have extended the study to demonstrate that when these predicted secondary structures are deleted in combination, the enhanced activity in the presence of the full-length PV cDNA is still observed. Indeed, a poliovirus 5' NCR devoid of all predicted secondary structures is capable of initiating protein synthesis under these conditions. Surprisingly, we also found that this enhancement of activity requires neither any PV protein nor the inhibition of cap-dependent translation. The results indicate that the defective PV 5' NCR elements can be complemented in trans by functional 5' NCRs in a highly sequence specific manner.

The type 2 live-attenuated vaccine strain of poliovirus (P2/Sabin) is associated with rare cases of poliomyelitis in vaccinees or their contacts. Recombinants were generated between infectious clones of a neurovirulent isolate from one such case (P2/117) and P2/Sabin and neurovirulence assays suggested that a maximum of six nucleotide differences between the two strains were responsible for their phenotypic difference. Site-directed mutagenesis of P2/Sabin showed that mutations at just two positions, at 481 in the 5' non-coding region and at VP1-143 in the capsid proteins, resulted in a highly neurovirulent virus. Other nucleotide changes may have weaker phenotypic effects. These results are consistent with those reported in the mouse model by Ren et al. [J. Virol. 65, 1377, (1991)] indicating that, for P2/Sabin at least, the same determinants of attenuation are important in both primates and transgenic mice expressing the poliovirus receptor. Sequence analysis of isolates from other vaccine-associated cases of poliomyelitis and from healthy vaccinees showed that both major determinants of attenuation are unstable on human passage, although selection pressures against an A at 481 are stronger than those against an Ile at 1143.

The World Health Organization has called for improvements to oral polio vaccines (OPV), particularly the type 3 component. Our improved understanding of the genetic basis of attenuation and antigenicity of this strain gives rise to possibilities for constructing new derivatives via mutagenesis of infectious cDNA. This article reviews progress made with various approaches to constructing a new type 3 candidate vaccine strain. These include the construction of antigen chimaeras of poliovirus based on the Sabin type 1 strain, the introduction of new genetically stable attenuation mutations into the 5' non-coding region (NCR) of the type 3 poliovirus genome, and the introduction of mutations which may increase the thermostability of the type 3 vaccine.

A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA. A series of mutations in the 5' noncoding region of poliovirus type 3 were introduced into FLC/REP, and their effects were monitored by a simple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential for RNA replication. The CAT-containing poliovirus replicon could also be packaged into poliovirus capsids provided by helper virus and was stable as a subpopulation of virus particles over at least four passages. The location of the CAT gene in FLC/REP excluded the presence of an encapsidation signal in the region of the poliovirus genome comprising nucleotides 756 to 1805.

Part of the 5' noncoding regions of all three Sabin vaccine strains of poliovirus contains determinants of attenuation that are shown here to influence the ability of these strains to grow at elevated temperatures in BGM cells. The predicted RNA secondary structure of this region (nt 464-542 in P3/Sabin) suggests that both phenotypes are due to perturbation of base-paired stems. Ts phenotypes of site-directed mutants with defined changes in this region correlated well with predicted secondary structure stabilities. Reversal of base-pair orientation had little effect whereas stem disruption led to marked increases in temperature sensitivity. Phenotypic revertants of such viruses displayed mutations on either side of the stem. Mutations destabilizing stems led to intermediate phenotypes. These results provided evidence for the biological significance of the predicted RNA secondary structure.

The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.

We have raised antisera and monoclonal antibodies (MAbs) to the C4 region of HIV-1 gp120, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gp120 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gp120. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gp120. Antibodies to these amino acids on recombinant gp120 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle.

We have developed the very safe and efficacious live-attenuated Sabin 1 poliovirus vaccine strain as a vehicle for the presentation of defined epitopes from foreign pathogens (1-3). Precise modification of the poliovirus capsid is made possible by the application of recombinant DNA technology to cloned cDNA copies of the viral genome, which are infectious for mammalian cells in culture (4).

Chimeric polioviruses have been made in which regions of the type 1 Sabin strain corresponding to antigenic sites 2, 3 and 4 have been replaced by the corresponding regions of the type 3 Sabin strain. Manipulation of one site or a component of it generally did not affect the reactions of the others, suggesting that they form independent structural features. The extent to which the inserted site expressed the antigenic properties of type 3 could be assessed by reaction with polyclonal or monoclonal antibodies, or by immunogenicity. Site 2 could be expressed on infectious virus and site 3 on heated non-infectious virus (C antigen), but not on the native virion. The results are consistent with the view that sites consisting of a continuous sequence of amino acids may be presented on chimeras, whereas more complex sites, such as site 4 or site 3 of the native virion, are transferred less readily from type 3 to type 1.

Five poliovirus recombinants containing sequences corresponding to foot-and-mouth disease virus (FMDV) antigenic sites were constructed. Viable virus was recovered from four of these plasmids, in which the VP1 beta B-beta C loop (antigenic site 1) of poliovirus type 1 Sabin had been replaced with sequences derived from the VP1 beta G-beta H loop (antigenic site 1) of FMDV O1 Kaufbeuren (O1K), chimera O1.1 (residues 141 to 154), chimera O1.2 (residues 147 to 156), and chimera O1.3 (residues 140 to 160) or from the beta B-beta C loop of VP1 (antigenic site 3) in chimera O3.1 (residues 40 to 49). One chimera (O1.3) was neutralized by FMDV-specific polyclonal serum and monoclonal antibodies directed against antigenic site 1 of FMDV. Chimeras O1.3 and O3.1 induced site-specific FMDV-neutralizing antibodies in guinea pigs. Chimera O1.3 was capable of inducing a protective response against FMDV challenge in some guinea pigs.

Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been generated in vitro using infectious cDNA clones and used to demonstrate that strong determinants of the attenuation and temperature-sensitive phenotypes of P2/Sabin reside in the 5' 492 nucleotides. In this region of the genome the viruses differ only at nucleotides 437 and 481. The ts phenotype associated with the 5' noncoding region is expressed at different temperatures in different cell lines, suggesting an involvement of cellular factors which may be species specific. Suppression of both the ts and attenuation phenotypes correlates with an A-G mutation at nucleotide 481, although other changes are also involved.