Evidence for a coiled-coil structure in the spike proteins of coronaviruses.

The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 30% was found, while there was no detectable sequence conservation in the N-terminal regions. Hydrophobic "heptad" repeat patterns indicated the presence of two alpha-helices with predicted lengths of 100 and 50 A, respectively. It is suggested that, in the spike oligomer, these alpha-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

Regulation of transcription of coronaviruses.

To study factors involved in regulation of transcription of coronaviruses, we constructed defective interfering (DI) RNAs containing sg RNA promoters at multiple positions. Analysis of the amounts of sg DI RNA produced by these DIs resulted in the following observations: (i) a downstream promoter downregulates an upstream promoter; (ii) an upstream promoter has little or no effect on the activity of a downstream promoter. Our data suggest that attenuation of upstream promoter activities by downstream promoter sequences plays an important role in regulating the amounts of sg RNAs produced by coronaviruses. Our observations are in accordance with the models proposed by Konings et al. and Sawicki and Sawicki.

RSV vaccine in development: assessing the potential cost-effectiveness in the Dutch elderly population.

OBJECTIVES: Respiratory syncytial virus (RSV) is increasingly recognized as an important cause of morbidity, mortality and health-care utilization in the elderly population. A theoretical model was built to assess the levels of vaccine effectiveness and vaccine costs for which a hypothetical RSV-vaccine for Dutch elderly could be cost-effective. METHODS: Different vaccination strategies were evaluated by changing the levels of vaccine effectiveness and the willingness to pay per quality-adjusted life year gained (QALY). Outcome measures included direct medical costs, QALYs, life years gained, incremental cost-effectiveness ratios (ICERs) and the maximum total vaccination costs per individual (i.e. (vaccine price+administration costs)×nr of doses) while remaining cost-effective. RESULTS: Using base-case assumptions, it was estimated that vaccination of all persons 60 years and older would prevent 3402GP visits, 2989 antibiotic prescriptions, 535 hospitalizations and 249 deaths and would cost €73,261 per QALY, for a vaccine effectiveness of 70%. Vaccinating only the high risk population of 60 years and older would reduce the estimates to 2042GP visits, 2009 antibiotic prescriptions, 179 hospitalizations and 209 deaths and this reduced the cost per QALY to €34,796 per QALY. Using the same assumptions, the maximum total vaccination costs per individual ranged from €26 when vaccinating all persons 60 and older to €68 when vaccinating only persons aged 85 and above, using a willingness to pay threshold of €50,000 per QALY. For the high risk population aged 60 years and older the estimated maximum total vaccination costs ranged from €52 to €99. CONCLUSION: Vaccination of Dutch elderly against RSV was found cost-effective for several scenarios. As expected, vaccination is more likely to be cost-effective when only including persons who are at increased risk for contracting RSV or the potential complications of RSV. This theoretical study shows that based on the disease burden in the Dutch population aged 60yrs and older there is potential to develop a vaccine that might be considered cost-effective in the Netherlands.

Cost-effectiveness of potential infant vaccination against respiratory syncytial virus infection in The Netherlands.

INTRODUCTION: Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory illness in infants, infecting virtually every child before the age of 2 years. Currently, several Phase 1 trials with RSV vaccines in infants are ongoing or have been completed. As yet, no efficacy estimates are available for these vaccine candidates. Nevertheless, cost-effectiveness estimates might be informative to enable preliminary positioning of an RSV vaccine. METHODS: A decision analysis model was developed in which a Dutch birth cohort was followed for 12 months. A number of potential vaccination strategies were reviewed such as vaccination at specific ages, a two- or three-dosing scheme and seasonal vaccination versus year-round vaccination. The impact of the assumptions made was explored in various sensitivity analyses, including probabilistic analysis. Outcome measures included the number of GP visits, hospitalizations and deaths, costs, quality-adjusted life years and incremental cost-effectiveness ratios (ICERs). RESULTS: Currently, without vaccination, an annual number of 28,738 of RSV-related GP visits, 1623 hospitalizations, and 4.5 deaths are estimated in children in the age of 0-1 year. The total annual cost to society of RSV in the non-vaccination scenario is €7.7 million (95%CI: 1.7-16.7) and the annual disease burden is estimated at 597 QALYs (95%CI: 133-1319). In case all infants would be offered a potentially safe and effective 3-dose RSV vaccination scheme at the age of 0, 1 and 3 months, the total annual net costs were estimated to increase to €21.2 million, but 544 hospitalizations and 1.5 deaths would be averted. The ICER was estimated at €34,142 (95%CI: € 21,652-€ 87,766) per QALY gained. A reduced dose schedule, seasonal vaccination, and consideration of out-of-pocket expenses all resulted in more favorable ICER values, whereas a reduced vaccine efficacy or a delay in the timing of vaccination resulted in less favorable ICERs. DISCUSSION: Our model used recently updated estimates on the burden of RSV disease in children and it included plausible utilities. However, due to the absence of clinical trial data, a number of crucial assumptions had to be made related to the characteristics of potential RSV vaccine. The outcomes of our modeling exercise show that vaccination of infants against RSV might be cost-effective. However, clinical trial data are warranted.

Hepatitis B vaccination strategies tailored to different endemicity levels: some considerations.

Hepatitis B is a serious public health problem. Worldwide three different levels of hepatitis B endemicity (high, intermediate and low) can be distinguished. Areas with different levels of endemicity require tailored vaccination strategies to fit the needs for individuals at risk and/or countries, depending on the infection risk per age group, vaccination rate, duration of protection after vaccination, cost effectiveness of vaccination strategies and ease of implementation in the national immunization schedules.This opinion paper evaluates these factors and proposes a combination of infant risk group and universal adolescent vaccination for low endemic countries thus targeting the different groups at risk. A universal infant vaccination schedule starting with a newborn vaccination within 24h after birth is more appropriate in intermediate- and high-endemic regions.

The use of health economics to guide drug development decisions: Determining optimal values for an RSV-vaccine in a model-based scenario-analytic approach.

Health-economic modelling is useful for assessing the clinical requirements and impact of new vaccines. In this study, we estimate the impact of potential vaccination for respiratory syncytial virus (RSV) of infants in the Netherlands. A decision analysis model was employed using seasonal data from a cohort of children (1996-1997 through 1999-2000) to assess hospitalisation, costs and impact of vaccination. Yearly, an estimated 3670 infants are hospitalised with RSV-infection in the Netherlands, vaccination protecting infants from 3 months of life onwards could prevent approximately 1000-3000 hospitalisations, depending on the effectiveness of the potential vaccine. Additionally, vaccination could prevent a major share of RSV-related costs. Comparison of the calculated break-even prices with the average price of recently introduced vaccines indicates that pricing for a potential RSV-vaccine most likely allows for only a single dose vaccination or several doses at a relatively low price per dose in order to achieve cost savings. However, if evidence on relevant RSV-related mortality would become available, higher pricing would be justified, while still remaining below accepted thresholds for cost-effectiveness.

On the design of national vaccination programmes.

The decision to include a vaccine in a national vaccination programme (or not) is usually evidence-based. Thereby, it is essential that the target disease causes a high burden of disease and that vaccination reduces this burden considerably. Furthermore, vaccination should be considered to be cost-effective by a government. Vaccines are usually administered according to standard vaccination schedules, which have been established on historical grounds. We argue and demonstrate with examples (meningococci C, Haemophilus influenzae, pneumococci and Bordetella pertussis) that adaptation of these standard vaccination schedules can be cost-saving and lead to better protection. To facilitate the improvement of vaccination programmes, a better understanding of protective immune responses (correlates of protection) and immunologic memory are required.

The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure.

Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of influenza virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'- and 3'-terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.

Recombinant genomic RNA of coronavirus MHV-A59 after coreplication with a DI RNA containing the MHV-RI spike gene.

A strategy for targeted RNA recombination between the spike gene on the genomic RNA of MHV-A59 and a synthetic DI RNA containing the MHV-RI spike gene is described. The MHV-RI spike gene contains several nucleotide differences from the MHV-A59 spike gene that could be used as genetic markers, including a stretch of 156 additional nucleotides starting at nucleotide 1497. The MHV-RI S gene cDNA (from nucleotide 277-termination codon) was inserted in frame into pMIDI, a full-length cDNA clone of an MHV-A59 DI, yielding pDPRIS. Using the vaccinia vTF7.3 system, RNA was transcribed from pDPRIS upon transfection into MHV-A59-infected L cells. DPRIS RNA was shown to be replicated and passaged efficiently. MHV-A59 and the DPRIS DI particle were copassaged several times. Using a highly specific and sensitive RT-PCR, recombinant genomic RNA was detected in intracellular RNA from total lysates of pDPRIS-transfected and MHV-A59-infected cells and among genomic RNA that was agarose gel-purified from these lysates. More significantly, specific PCR products were found in virion RNA from progeny virus. PCR products were absent in control mixes of intracellular RNA from MHV-A59-infected cells and in vitro-transcribed DPRIS RNA. PCR products from intracellular RNA and virion RNA were cloned and 11 independent clones were sequenced. Crossovers between A59 and RI RNA were found upstream of nucleotide 1497 and had occurred between 106 nucleotides from the 5'-border and 73 nucleotides from the 3'-border of sequence homologous between A59 and RI S genes. We conclude that homologous RNA recombination took place between the genomic RNA template and the synthetic DI RNA template at different locations, generating a series of MHV recombinant genomes with chimeric S genes.

Replication of synthetic defective interfering RNAs derived from coronavirus mouse hepatitis virus-A59.

We have analyzed the replication of deletion mutants of defective interfering (DI) RNAs derived from the coronavirus mouse hepatitis virus (MHV)-A59 in the presence of MHV-A59. Using two parental DI RNAs, MIDI and MIDI delta H, a twin set of deletion mutants was generated with progressively shorter stretches of 5' sequence colinear with the genomic RNA. All deletion mutants contained in-frame ORFs. We show that in transfected cells and after one passage the DI RNAs were detectable and that their accumulation was positively correlated with the length of 5' sequence they contained. However, accumulation of two twin mutants, delta 2, in which sequences from nucleotide position 467 were fused to those from position 801, was undetectable. In passage 4 cells, but not in transfected or in passage 1 cells, recombination with genomic RNA led to the appearance of the parental DI RNAs. The accumulation of these parental RNAs was inversely correlated with the length of 5' sequence on the deletion mutants and was highest in the delta 2 samples. In sharp contrast to the data reported for MHV-JHM-derived DI RNAs, we show that MHV-A59-derived mutant RNAs do not require an internal sequence domain for replication. The data suggest that coronavirus replication involves an RNA superstructure at the 5' end of the genome or one comprising both ends of the genomic RNA. We also conclude from the recombination data that in-frame mutants with impaired replication signals are more fit than out-frame mutants with intact replication signals.

Characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain A59 with maturation defects in the spike protein.

Two temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59, ts43 and ts379, have been described previously to be ts in infectivity but unaffected in RNA synthesis (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). We present a detailed analysis of the protein synthesis of the mutant viruses at the permissive (31 degrees C) and nonpermissive (39.5 degrees C) temperatures. It was found that synthesis of the nucleocapsid protein N and the membrane protein M of both viruses was insensitive to temperature. However, the surface protein S of both viruses was retained in the endoplasmic reticulum at the nonpermissive temperature. This was shown first by analysis of endoglycosidase H-treated and immunoprecipitated labeled S proteins. The mature Golgi form of S was not present at the nonpermissive temperature for the ts viruses, in contrast to wild-type (wt) virus. Second, gradient purification of immunoprecipitated S after pulse-chase labeling showed that only wt virus S was oligomerized. We conclude that the lack of oligomerization causes the retention of the ts S proteins in the endoplasmic reticulum. As a result, ts virus particles that were devoid of S were produced at the nonpermissive temperature. This result could be confirmed by biochemical analysis of purified virus particles and by electron microscopy.

The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity.

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.

Introduction of site-specific mutations into the genome of influenza virus.

We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with defined biological properties.

Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses.

Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37 degrees C, suggesting the presence of a receptor-destroying enzyme. Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaxillary mucin as substrate similar to the enzymatic activity found in influenza C viruses. Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact. In addition, hemagglutination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Clostridium perfringens neuraminidase or by addition of sialic acid-containing gangliosides. These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize O-acetylated sialic acid or a similar derivative as cell receptor.

Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus.

The defective interfering (DI) RNA MIDI of mouse hepatitis virus strain A59 (MHV-A59) contains a large open reading frame (ORF) spanning almost its entire genome. This ORF consists of sequences derived from ORF1a, ORF1b, and the nucleocapsid gene. We have previously demonstrated that mutations that disrupt the ORF decrease the fitness of MIDI and its derivatives (R. J. de Groot, R. G. van der Most, and W. J. M. Spaan, J. Virol. 66:5898-5905, 1992). To determine whether translation of the ORF per se is required or whether the encoded polypeptide or a specific sequence is involved, we analyzed sets of related DI RNAs containing different ORFs. After partial deletion of ORF1b and nucleocapsid gene sequences, disruption of the remaining ORF is still lethal; translation of the entire ORF is not essential, however. When a large fragment of the MHV-A59 spike gene, which is not present in any of the MHV-A59 DI RNAs identified so far, was inserted in-frame into a MIDI derivative, translation across this sequence was vital to DI RNA survival. Thus, the translated sequence is irrelevant, indicating that translation per se plays a crucial role in DI virus propagation. Next, it was examined during which step of the viral life cycle translation plays its role. Since the requirement for translation also exists in DI RNA-transfected and MHV-infected cells, it follows that either the synthesis or degradation of DI RNAs is affected by translation.

Mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion.

The spike (S) protein of murine coronavirus strain A59 (MHV-A59) is a type I membrane protein that induces membrane fusion. In this study we have analyzed the role of two domains in the S protein on fusion. The 180-kDa mature S protein is partially cleaved into two 90-kDa subunits during transport to the plasma membrane. We have identified several amino acids that are important for cleavage of S, and we show that cleavage is not strictly required for fusion. However, the level of cleavage seems to influence the fusion kinetics. After introduction of an arginine at position P2 to mimick the MHV-JHM cleavage site, full cleavage of the spike protein was obtained. Further, we analyzed the effect of mutations in the transmembrane (TM) domain of the S protein. Maturation and cell surface expression of the mutant proteins were not affected, and all proteins became acylated. The mutant in which the predicted transmembrane domain was shortened did not induce syncytia. From a group of mutants in which several conserved cysteines in the TM domain had been replaced by serines, one was unable to induce syncytia, another showed delayed syncytia formation, and the third mutant induced syncytia as did the wild-type protein. The potential role of the transmembrane domain in fusion is discussed.