# Patent

## Title
Enterovirus, vaccines, medicaments and diagnostic kits

## Abstract

The invention provides a new enteroviral species call EV79, and functional parts, derivatives and analogues of said virus. Proteinaceous molecules capable of specifically binding EV79, such as isolated or recombinant antibodies, are also herewith provided. A virus and/or proteinaceous molecule of the invention is particularly suitable for diagnosis of an EV79-related disease. Vaccines, pharmaceutical compositions and diagnostic kits comprising said virus or proteinaceous molecule are also provided, as well as methods for producing same. A diagnostic kit of the invention may as well comprise a primer/probe capable of amplifying and/or hybridizing the genome or part thereof of EV79.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of PCT International Patent Application No. PCT/NL2004/000184 filed on Mar. 12, 2004, designating the United States of America, and published in English, as PCT International Publication No. WO 2004/104189 A1 on Dec. 2, 2004 the contents of the entirety of which are incorporated herein by this reference.


BACKGROUND
The invention relates to the field of medicine. The invention particularly relates to the isolation of a new enterovirus and methods for detection, identification, diagnosis, treatment and prevention of the enterovirus.
According to the most recent classification of viruses, the family of the Picornaviridae is divided in six genera, among which are the human enteroviruses, rhinoviruses, parechoviruses, aphthoviruses, cardioviruses, and hepatoviruses. The genus enterovirus includes the following human virus species: polioviruses and human enterovirus groups A to D. Poliovirus (PV) consists of three serotypes: poliovirus 1 (PV-1), PV-2 and PV-3. Human enterovirus A (HEV-A) consists of 12 serotypes, including coxsackievirus A2, A16 and enterovirus 71 (more details concerning these viral strains are listed in Table 1). Human enterovirus B (HEV-B) consists of 37 serotypes; human enterovirus C (HEV-C) consists of 11 serotypes and human enterovirus D (HEV-D) consists of 2 serotypes. Enteroviruses are single strand RNA viruses. Their genomes comprise approximately 7500 nt.
Enteroviruses cause a wide spectrum of clinical syndromes ranging from mild fever to respiratory infections, meningitis, encephalitis, paralytic poliomyelitis and myocarditis. An estimated 10 to 30 million enteroviral infections occur annually in America, causing significant short-term mobility and economic impact. Life-threatening enteroviral infections may occur, especially in high-risk individuals such as immunocompromised patients.
Hence, enteroviruses cause a wide spectrum of clinical syndromes. Enteroviral infections often result in “general” symptoms, such as diarrhoea and fever, which are involved in many different kinds of disease. Therefore, symptoms of an enteroviral infection cannot be directly correlated to an enteroviral genus-related disease. Characterization of the causative agent is therefore often necessary in order to provide adequate treatment. Once a causative agent is known, efficient diagnosis, prevention and/or treatment becomes possible.
DISCLOSURE OF THE INVENTION
The newly found enterovirus, (designated EV79) was characterized and sequenced. A nucleic acid sequence of a prototype EV79 is provided in FIG. 19 and parts thereof in FIG. 2B. In one aspect, the invention therefore provides an isolated and/or recombinant nucleic acid comprising a sequence as depicted in FIG. 19 and/or FIG. 2B, or a functional part, derivative and/or analogue thereof. The virus EV79 is characterized by the prototype, however, many natural variants of the prototype EV79 virus exist. The existence of such natural variants is normal for RNA viruses that undergo frequent mutation through, for instance, the introduction of mistakes by the polymerases that copy the genome.
EV79 viruses that have a slightly divergent nucleic acid sequence are thus also provided by the present invention. Such viruses are considered to be a derivative of the nucleic acid having the prototype nucleic acid sequence. The variant does not necessarily have to be a natural variant. It is very well possible to generate variants through recombinant means. For instance, many parts of the virus can be altered through nucleotide substitution to make use of the redundancy in the triplet genetic code for particular amino acids, thus, without altering the amino acid sequence of the encoded proteins. However, even amino acid alterations can typically be introduced without affecting the replicating and coding potential of the viruses. For instance, conservative amino acid substitutions are often tolerated.
Alterations in the prototype virus may be up to 70% of the nucleic acid sequence without altering the replicating potential of the virus. Thus, in one embodiment, the invention provides an isolated and/or recombinant nucleic acid that is at least 70% homologous to a nucleic acid of the prototype EV79. Most of the viable variants however are at least 95% homologous and more preferably at least 99% to a nucleic acid according to the prototype EV79. The homology between different enteroviruses in the UTR regions is typically high, for this reason, the homology in this application is measured in a region outside the UTR regions, preferably in a protein coding region. Thus, the invention provides a derivative of EV79 virus comprising at least 95% homology and preferably at least 99% homology (on the nucleic acid level or on the protein level) in at least one protein coding region depicted in FIG. 20, FIG. 3, Table 10 or Table 11.
The nucleic acid of the virus or parts thereof can be cloned and used as a probe to detect the virus in samples. Thus the present invention further provides an isolated and/or recombinant nucleic acid comprising a stretch of 100 consecutive nucleotides of a nucleic acid of the prototype virus, or a region that is at least 95% and preferably at least 99% homologous to 100 consecutive nucleotides, wherein the 100 nucleotides are outside the UTR region. In a preferred embodiment, the part essentially does not overlap with a UTR region. In a preferred embodiment, the part is in a protein coding domain. A stretch of 100 consecutive nucleotides is considered to be a functional part of the virus of the present invention.
A preferred part of EV79 is a nucleic acid encoding a protein as depicted in FIG. 20, Tables 10 and 11, or FIG. 3. Further provided is a bacterial vector comprising a nucleic acid of EV79 or a functional part, derivative and/or analogue thereof. Further provided is a bacterium comprising the bacterial vector. Considering the divergence with other known enteroviruses, the sequence of EV79 or a part thereof can be used to generate a primer that is specific for EV79 and thus capable of specifically replicating EV79 nucleic acid. Similarly, a probe can be generated that specifically hybridizes to EV79 nucleic acid under stringent conditions. Thus, the invention further provides a primer and/or probe, capable of specifically hybridizing to a nucleic acid of an EV79 virus or functional part, derivative or analogue thereof. Preferably, the primer or probe is capable of hybridizing to the nucleic acid under stringent conditions.
In a preferred embodiment, the primer comprises a consecutive stretch of between 10 and 50 nucleotides of a sequence as depicted in FIG. 19, preferably between 15 and 25 nucleotides. The person skilled in the art is well capable of determining the conditions needed for specific hybridization of a nucleic acid to a nucleic acid of EV79. Typically, the specificity increases with increasing size of overlap with and homology with the nucleic acid depicted in FIG. 19. A typical probe specific for a nucleic acid depicted in FIG. 19, comprises at least 20 consecutive nucleotides. A probe may essentially comprise the entire EV79 sequence. However, a probe specific for EV79 has a size of between 20 and 2000 nucleotides and a homology of at least 95, and preferably at least 99% with the nucleic acid depicted in FIG. 19. Probes may be labeled directly or indirectly via an intermediate. Primers may be generated for sequences throughout the genome of EV79, however when a specific amplification of EV79 sequences (or variants thereof) is desired preferably at least one primer is chosen outside the UTR region.
In a particularly preferred embodiment, both primers are chosen outside the UTR region. In a preferred embodiment, a primer comprises sufficient sequence divergence with related enteroviruses, in this way specificity for EV79 sequences or variants thereof is warranted. Typically this is achieved by having at least one and preferably at least between 2-5 nucleotide differences in the primer compared to the sequence it intends to discriminate from, typically a related human enterovirus.
In a particularly preferred embodiment, the primer and/or probe comprises a sequence as depicted in FIG. 2B or Table 4.
The nucleic acid of the prototype virus encodes various proteins and polyproteins. These proteins are expressed, for instance, in cells producing the virus or transformed with a nucleic acid encoding the (poly)protein. The invention thus further provides an isolated and/or recombinant proteinaceous molecule comprising a sequence as depicted in FIG. 20, Table 10, Table 11, or FIG. 3, or a functional part, derivative and/or analogue thereof. Many different variants of the proteins having the same function in kind, not necessarily in amount are, as mentioned above, present in nature and can be generated artificially, thus the invention further provides an isolated and/or recombinant proteinaceous molecule that is at least 70% homologous to a proteinaceous molecule mentioned above. Such homologous proteins are considered derivatives of a protein encoded by the prototype. Preferably, a derivative protein comprises at least 95% and more preferably at least 99% homology with a protein encoded by the prototype EV79.
Fragments and parts of a proteinaceous molecule encoded by the prototype virus can be generated, such parts are therefore also provided by the present invention. In a preferred embodiment, provided is an isolated and/or recombinant proteinaceous molecule comprising a stretch of at least 30 consecutive amino acids of a proteinaceous molecule encoded by the prototype virus. A protein encoded by the prototype virus can be encoded through a variety of different nucleic acid sequences using the redundancy of the genetic code. Thus the invention further provides a nucleic acid encoding a protein depicted in FIG. 20, Table 10, Table 11, or FIG. 3.
The invention thus further provides an isolated or recombinant virus comprising an EV79 nucleic acid sequence or a functional part, derivative and/or analogue thereof. Also provided is an isolated or recombinant virus comprising a proteinaceous molecule as depicted in FIG. 20, Table 10, Table 11, or FIG. 3, or a functional part, derivative and/or analogue thereof. Subjects that have become infected with EV79 can display a number of different clinical and/or subclinical symptoms. Thus further provided is an isolated or recombinant virus or a functional part, derivative or analogue thereof capable of inducing an EV79-related disease, particularly diarrhea. The virus comprises substances that can be used to generate specific binding partners that are able to specifically bind the substance of the virus. Binding partners can, for instance, be generated by means of injection of the virus into an immuno-competent subject.
As a result of the immunization, the serum obtained from the subject will typically contain a number of different antibodies specific for the virus or an immunogenic part, derivative and/or analogue thereof. Specific binding partners (specific binding molecules) can of course be generated through a large variety of different technologies. For instance, phage display technologies could be generated. The method of producing the specific binding partner is not limited herein. The binding is typically specific for a proteinaceous part of the virus, but can, of course, also be specific for a virus specific post-translation modification of a protein contained in the virus.
Thus, the present invention further provides an isolated binding molecule capable of specifically binding a substance (preferably a proteinaceous molecule) of an EV79 virus, preferably against a proteinaceous molecule encoded by a nucleic acid of the prototype EV79. Preferably, a proteinaceous molecule comprising an amino acid sequence as depicted in FIG. 20, Table 10, Table 11, or FIG. 3, or a functional part, derivative and/or analogue thereof. The binding molecule can be capable of specifically binding a nucleic acid sequence of an EV79, preferably of FIG. 19 or FIG. 2B. The binding molecule is preferably a proteinaceous molecule. However, other binding molecules are also within the scope of the present invention. For instance, it is possible to generate protein mimetics or analogues having the same binding quality as a protein in kind not necessarily in amount.
Provided is a further method for producing a binding molecule according to the invention comprising—producing molecules capable of binding an EV79 virus or functional part, derivative or analogue thereof or an isolated and/or recombinant proteinaceous molecule encoded by a prototype nucleic acid of EV79, and—selecting a proteinaceous binding molecule that is specific for this virus and/or this proteinaceous molecule.
The overall homology of EV79 virus with other human enteroviruses is not very high. Thus many different binding molecules capable of specifically binding to EV79 virus can be generated. Such binding molecules can be used to detect EV79 virus in a sample. The invention thus further provides an isolated or recombinant virus, which is immune-reactive with a binding molecule capable of specifically binding EV79 virus. Similarly, the invention provides the use of an isolated and/or recombinant proteinaceous molecule as depicted in FIG. 20, Table 10, Table 11 or FIG. 3, or a functional part, derivative and/or analogue thereof, for detecting a binding molecule capable of specifically binding EV79 virus, or functional part, derivative and/or analogue of the virus in a sample. Vise versa, EV79 virus can be used to detect a molecule capable of specifically binding the virus in a sample.
Binding of EV79 virus to a susceptible target cell occurs via a specific receptor. This receptor can be used as a binding molecule of the invention. Preferably, the binding molecule comprises an antibody or functional equivalent thereof. The detection methods can be used to diagnose EV79-related disease in a subject. In one embodiment is provided a method for detecting an EV79 virus or functional part, derivative or analogue thereof in a sample, comprising hybridizing and/or amplifying a nucleic acid of the virus or functional part, derivative or analogue with an EV79 specific primer and/or probe and detecting hybridized and/or amplified product. Further provided is a kit, preferably a diagnostic kit comprising an EV79 virus or functional part, derivative or analogue thereof, a binding molecule according to the invention, and/or an EV79 virus specific primer/probe according to invention.
In a particular preferred embodiment is provided the use of a primer or probe capable of specifically hybridizing to a nucleic acid of an EV79 virus or functional part, derivative or analogue thereof, or a binding molecule capable of specifically binding a proteinaceous molecule depicted in FIG. 20, Table 10, Table 11 or FIG. 3, or an EV79 virus and/or a nucleic acid or functional part, derivative or analogue of a prototype EV79 for detecting and/or identifying an EV79 enterovirus in a sample. Preferably, the nucleic acid comprises a sequence as depicted in FIG. 19 or FIG. 2B.
The invention further provides a vaccine comprising EV79 virus or functional part, derivative or analogue thereof. Further provided is a vaccine comprising a proteinaceous molecule depicted in FIG. 20, Table 10, Table 11, or FIG. 3, or functional part, derivative and/or analogue of such a proteinaceous molecule. A proteinaceous molecule of the invention may be provided in the vaccine by itself or as a part of the protein or as derivatives or analogues thereof. A suitable analogue is a nucleic acid encoding an EV79 virus proteinaceous molecule or a functional part or derivative thereof. The nucleic acid may be used in a DNA vaccine approach, which is also provided in the present invention.
Further provided is a composition for immunizing a subject against at least one EV79 protein, this composition comprising a nucleic acid encoding an EV70 prototype protein, or a protein of a variant of EV79, or an immunogenic part, derivative and/or analogue of the EV79 prototype. As carrier for the DNA vaccine it is often suitable to incorporate an expressible EV79 virus nucleic acid in a viral replicon allowing replication of the EV79 virus nucleic acid in the target cell and thereby allowing amplification of the provided immune response. EV79 virus encoded proteins that are particularly suited for such a DNA vaccine approach are the VP1, VP2 and VP3 proteins depicted in FIG. 20, Table 10, Table 11, or FIG. 3, or a functional part, derivative and/or analogue thereof. A part of a VP protein preferably comprises an immunogenic part of the VP protein.
Other suitable candidates are the VP4, 2a, 2b and 3C protein or a functional part, derivative and/or analogue thereof. Typically a vaccine includes an appropriate adjuvant. Apart from the use in a vaccine the mentioned virus and/or proteinaceous molecules can also be used to generate and/or boost an EV79 virus specific immune response in a subject. The immune response can be either cellular or humoral. Thus, further provided is an isolated T-cell comprising a T-cell receptor that is specific for EV79 virus or a proteinaceous molecule encoded by a prototype EV79 virus. Further provided is an isolated B-cell producing an antibody specific for EV79 virus or a proteinaceous molecule encoded by an EV79 virus. The antibody or T-cell receptor can be cloned whereupon a cell line can be provided with an expression cassette comprising the nucleic acid encoding the cloned receptor or antibody. Thus, the invention further provides a cell producing such a receptor or antibody. Such a cell is preferably a cell that is suitable for large scale production of the mentioned proteins such as CHO cells.
It is also possible to provide a subject with passive immunity to EV79 virus. To this end the subject can be provided with an EV79 specific binding molecule of the invention. Such immunity can be used to provide a barrier for (further) infection with EV79 virus in the subject, thus further provided is a vaccine comprising an EV79 virus specific binding molecule according to the invention. In a preferred embodiment, passive immunity is provided by a human or humanized antibody capable of specifically binding an EV79 virus of the invention. Preferably, the antibody is produced by a human cell line, preferably the cell line comprises an adenovirus E1 region, preferably at least an E1A region of adenovirus 2 or adenovirus 5. The barrier does not have to be perfect.
The presence of a binding molecule at least reduces the spread of the virus to other target cells in the subject. The passive immunity may be administered to a subject as prophylactic to at least reduce the spread of EV79 virus in the subject when exposed to the virus. Alternatively, the passive immunity may be provided to a subject already infected with the virus. In the latter case, one or more EV79 virus specific binding molecules of the invention are used as a medicament to at least reduce the spread of the virus in the subject and thereby at least in part combat the virus infection. The invention thus further provides a medicament comprising an EV79 virus specific binding molecule according to the invention.
Further provided is the use of a virus of the invention or functional part, derivative or analogue thereof or a proteinaceous molecule of the invention or an EV79 virus specific binding molecule of the invention, for the preparation of a vaccine against an enterovirus-related disease.
Further provided is a method for treating an individual suffering from, or at risk of suffering from, an EV79-related disease, comprising administering to the individual a vaccine or medicament according to the invention. In yet another embodiment, provided is a method for determining whether an individual suffers from an EV79-related disease, comprising obtaining a sample from the individual and detecting an EV79 virus or functional part, derivative or analogue thereof in the sample.
In yet another embodiment, provided is an isolated cell, or recombinant or cell line comprising EV79 virus, or a functional part, derivative and/or analogue thereof. Preferably, the cell is a primate cell, preferably a monkey cell. In a preferred embodiment, the cell is a cell that replicates the EV79 virus of the invention. In a particular embodiment, the cell is a kidney cell. The cell can be used to produce the EV79 virus of the invention or to attenuate EV79 such that it becomes less pathogenic. Virus attenuation is spontaneous upon continued culture of the virus on the mentioned preferred cell lines. Attenuated EV79 virus can be used as a vaccine.
EV79 virus encodes endoproteases called protease 2a and protease 3C. Sequences in the prototype EV79 virus are depicted in FIG. 20. Proteases are important for the processing of the polyproteins encoded by EV79. The action of the protease is at least in part inhibited by a viral protease inhibitor as further described herein. Thus, the invention further provides a compound for at least in part inhibiting EV79 virus replication.
Of these compounds, the protease inhibitors are particularly preferred. Since the 2a and 3C enzymes recognize peptide substrates with a glutamine residue at the P(1) site, ketone-containing glutamine analogs (e.g., azaglutamine) can be used as inhibitors. Additionally, nitric oxide (NO)-releasing compounds like S-nitroso-N-acetyl-penicillamine (SNAP), glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN) or glycerrhizin can be used to inhibit 3C activity. Also 5-(3,4-dichlorophenyl)methylhydantoin interferes with the post-translational processing of the polyprotein.
The vinylogous ethyl ester (AG7088, see structural formula 1 depicted below,) that inhibits human rhinovirus (HRV) 3C protease activity.




Analogues of such protease inhibitors that comprise the same activity in kind not necessarily in amount are also provided by the present invention. Such analogues include, compounds comprising a peptide with the preferred sequence, wherein the peptide comprises a modification. Other analogues include compounds having protein mimetic activity that mimic the preferred amino-acid sequence.
Enteroviral RNAs are not capped instead they carry a small protein called VPg (encoded by the 3B gene) covalently attached to their 5′ end. The so-called WIN-compounds insert themselves in the hydrophobic pocket of the picornaviral nucleocapsid, preventing the virus from uncoating its genomic RNA are preferred. Preferred compounds are: Pleconaril, 3-methylthio-5-aryl-4-isothiazolecarbonitriles or pyridyl imidazolidinones. Also nucleotide analogs like Ribavirin, mycophenolic acid, 6-azauridine and pyrazofurin or other RNA synthesis inhibitors like 3-methylkaempferol inhibit the replication of Picornaviruses. The invention thus further provides the use of a WIN-compound for the (preparation of a medicament for the) treatment of an EV79 infection.
The invention further provides a method for the treatment of a subject suffering from or at risk of suffering from an EV79 infection comprising administering to the subject a compound selected from the group consisting of a peptide containing a ketone-containing glutamine analog (e.g., azaglutamine), a nitric oxide (NO)-releasing compounds like S-nitroso-N-acetyl-penicillamine (SNAP), glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN) or glycerrhizin, 5-(3,4-dichlorophenyl)methylhydantoin, a vinylogous ethyl ester (AG7088, having structural formula 1 depicted below);




A so-called WIN compound such as a Pleconaril, 3-methylthio-5-aryl-4-isothiazolecarbonitrile or a pyridyl imidazolidinone, a nucleotide analogue such as ribavirin, mycophenolic acid, 6-azauridine or pyrazofurin, or an RNA synthesis inhibitor such as 3-methylkaempferol.
The invention further provides a proteinaceous molecule encoded by EV79 nucleic acid, wherein the proteinaceous molecule is a 2a or 3C protease or a functional equivalent thereof. Functional equivalents include a proteolytically active part and/or derivative having one or more conservative amino acid substitutions. There are many methods known in the art to determine whether a compound has anti-enteroviral activity, preferably anti-proteolytic activity of an enterovirus. The invention thus further provides a method for determining whether a compound comprises anti-enterovirus replication activity characterized in that the method utilizes EV79-virus or an EV79 protein involved in replication of EV79 or a functional part, derivative and/or analogue thereof. Preferably, the invention provides a method for determining whether a compound is capable of at least in part inhibiting a viral protease characterized in that the protease is a 2a and/or 3C protease of EV79 or a functional part, derivative and/or analogue thereof. Preferred compounds that can be tested for 3CL inhibiting quality are peptides (preferably hexapeptides) located N-terminally of 2a and/or 3C pro-cleavage sites, preferably a hexapeptide comprises a sequence that is immediately N-terminal of the 2a or 3C pro-cleavage site, preferably the (hexa)peptide comprises a modification for additional stability. Compounds effective in at least in part inhibiting 2a and/or 3C proteolytic activity can be used for the preparation of a medicament for the treatment of an individual suffering or at risk of suffering from-an EV79 virus infection.
One or more of the preferred anti-enteroviral replication compounds can be used as a medicament for the treatment of a subject suffering from or at risk of suffering from an EV79 virus infection. The invention thus further provides a medicament for the treatment of an individual suffering from an enterovirus infection or an individual at risk of suffering therefrom comprising wherein the enterovirus comprises a nucleic acid sequence of an EV79 prototype virus or a functional part, derivative and/or analogue thereof.
In the present invention several different recombinant viruses are produced using EV79 virus nucleic acid as a backbone. Such replication competent or replication defective recombinant virus can be used, for instance, as gene delivery vehicles. On the other hand, parts of an EV79 virus can be used in gene delivery vehicles that are based on other means for delivering genetic material to a cell. Thus the invention further provides a gene delivery vehicle comprising at least part of an EV79 virus nucleic acid, preferably of the prototype virus. Preferably, the gene delivery vehicle comprises a nucleic acid encoding a protein of EV79 virus or a functional part, derivative and/or analogue thereof.
The invention also shows chimeric enteroviruses comprising nucleic acid derived from at least two enteroviruses wherein at least one of these parts is derived from an EV79 virus. The EV79 virus-derived part comprises preferably at least 50 nucleotides of a protein coding domain. More preferably, the EV79-derived part comprises at least 500 and more preferably at least 1000 nucleotides of the sequence as depicted in FIG. 19 or a functional derivative thereof. In a preferred embodiment, the invention provides a chimeric enterovirus comprising at least 1000 nucleotides of a sequence as depicted in FIG. 19 and at least 1000 nucleotides of another enterovirus wherein the latter 1000 nucleotides comprise a sequence that is more than 5% sequence divergent with a sequence as depicted in FIG. 19.
The sequences of a number of EV79 virus-fragments are depicted in FIG. 2B and FIG. 3. The location of the fragments in the large genomic RNA is depicted in FIG. 3. The invention therefore, in one aspect, provides an isolated or recombinant virus comprising a nucleic acid sequence as depicted in Tables 2B and/or 3, or a functional part, derivative or analogue of the virus. With the aid of the identifying prototype fragments it is possible to further sequence the genome. One way of doing this is by primer walking on the genome. A primer is directed to a region of which the sequence is known and this primer is used to sequence a flanking region that is as yet unknown. A subsequent primer can be generated against the newly identified sequence and a further region can be sequenced.
The present invention provides a new enteroviral species called EV79. The EV79 virus was isolated from an immunocompromised patient suffering from severe diarrhea. Of course, now that the existence of this EV79 virus is known, it is possible to screen individuals and non-human animals for the presence of EV79. In FIG. 2A, a 5′-UTR region of a virus of the invention is depicted. FIG. 2B depicts a part of the genome of the virus adjacent to the 5′-UTR. This sequence is capable of distinguishing EV79 from other enteroviruses.
In one aspect, the invention thus provides an isolated or recombinant virus comprising a nucleic acid sequence as depicted in FIG. 2B, or a functional part, derivative or analogue of the virus. Now that a nucleic acid sequence as depicted in FIG. 2B is provided, it is easily possible to sequence other parts of an EV79 genome using current methods in the art. For instance, a probe can be generated based on the sequence of FIG. 2B. The probe can be bound to a solid support, after which the support can be incubated with a sample comprising EV79. After EV79 virus is bound, the virus can be isolated and its genome can subsequently be amplified. By aligning genomic sequences of closely related viruses, conserved sequence domains can be identified allowing the design of synthetic oligonucleotides. These oligonucleotides can be used to amplify additional and overlapping parts of the EV79 genome.
Alternatively, the EV79 virus can be amplified as described above from a pool of total nucleic acid isolated from a clinical sample or in vitro cultured cells.
It is preferred to use a primer based on the sequence of FIG. 2B and/or a “universal primer” set comprising random primers, preferably as described in patent application EP1276901 owned by the present applicant (incorporated herein by reference). In one preferred embodiment, a combination of a primer based on the sequence of FIG. 2B and a universal primer set is used. The universal primers are capable of binding an upstream region, including unidentified parts of an EV79 genome, enabling amplification of (part of) the genome extending beyond the nucleic acid sequence of FIG. 2B. After amplification, obtained product can be sequenced using, for instance, the dideoxy method.23 Of course many other methods for sequencing the remaining part of the viral nucleic acid are available to the skilled person and these may be used as well.
The invention also provides a composition of matter comprising isolated EV79, and/or a virus essentially corresponding to EV79.
The term “a virus essentially corresponding to EV79” refers to EV79 viruses which are either identical to the EV79 strain identifiable by a sequence as depicted in FIG. 2B or which comprise one or more mutations as compared to the EV79 strain. These mutations may include naturally occurring variations, since RNA viruses are prone to mutations. Moreover, the mutations may include artificially made mutations. Preferably, the mutations still allow for detection of the EV79 variants using common detection methods such as hybridization, NASBA, RT-PCR and ELISA with EV79 specific nucleic acid and/or antibody.
The composition of matter may comprise live, attenuated and/or killed EV79 virus. The composition may as well comprise a functional part, derivative and/or analogue of the virus. In one embodiment, the composition comprises a recombinant vector derived from EV79 virus.
The sequence of FIG. 2B comprises at least part of an open reading frame encoding an amino acid sequence as depicted in FIG. 3. This amino acid sequence is also part of the EV79 virus strain obtained by the present inventors. The sequence comprises the VP4 region and at least part of the VP2 region of EV79. Hence, in another aspect, the invention provides an isolated or recombinant virus comprising an amino acid sequence as depicted in FIG. 3, or a functional part, derivative or analogue of the virus. Phylogenetic analysis demonstrates that EV79 is distantly related to some HEV-A enteroviruses including human enterovirus 71, coxsackievirus A2 and A16 with approximately 70% identity (FIG. 1).
An amino acid sequence as depicted in FIG. 20, Table 10, Table 11, and/or FIG. 3 can be encoded by different nucleic acid sequences. It is, for instance, possible to replace the third nucleotide of a codon by another nucleotide without changing the encoded amino acid residue. For instance, serine can be encoded by codons TCT, TCC, TCA, and TCG. Hence, the last nucleotide in the codons can be replaced by any other nucleotide, according to the “wobble” theory. As different organisms often utilize different codon usage, a nucleic acid encoding (part of) an EV79 virus can be artificially adapted to a certain organism that is used for producing the virus. Moreover, since viruses, especially RNA viruses, are prone to mutations, naturally occurring EV79 virus strains may comprise changes in their genomes that do not significantly affect encoded amino acid product. In one embodiment, the invention therefore provides an isolated or recombinant virus comprising a nucleic acid encoding an amino acid sequence as depicted in FIG. 20, Table 10, Table 11 and/or FIG. 3, or a functional part, derivative or analogue of the virus.
Additionally, a virus of the invention is provided comprising an amino acid sequence that differs to some extent to an amino acid sequence as depicted in FIG. 20, Table 10, Table 11 and/or FIG. 3. The amino acid sequence may be either naturally or artificially mutated. Conservative amino acid substitution may be applied: substitution of one or more amino acid residue(s) with another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is likely not to be seriously affected. Alternatively, mutations may be present that do affect at least one property of a virus of the invention. For instance, a certain desired characteristic may be artificially improved. However, mutations affecting functioning of a virus of the invention also occur in nature. In one embodiment, the amino acid sequence is more than 45 percent homologous to at least part of an amino acid sequence as depicted in FIG. 20, Table 10, Table 11 and/or FIG. 3. The part preferably comprises at least 20, more preferably at least 30, most preferably at least 50 amino acid residues.
Preferably, the amino acid sequence is at least (85 percent, more preferably 95 percent, most preferably 98 percent homologous to at least part of an amino acid sequence as depicted in FIG. 3. One embodiment of the invention thus provides an isolated or recombinant virus comprising an amino acid sequence which is more than 45% homologous to at least part of an amino acid sequence as depicted in FIG. 3, the part having at least 20, preferably at least 30, amino acid residues.
In one aspect, the invention provides an isolated or recombinant virus comprising an amino acid sequence which is more than 43% homologous to at least part of an amino acid sequence position 31-60 as depicted in FIG. 3, this part having at least 20, preferably at least 30, amino acid residues. In another aspect, the invention provides an isolated or recombinant virus comprising an amino acid sequence which is more than 80% homologous to at least part of an amino acid sequence position 91-150 as depicted in FIG. 3, the part having at least 20, preferably at least 30, amino acid residues. In yet another aspect, the invention provides an isolated or recombinant virus comprising an amino acid sequence which is more than 86% homologous to at least part of an amino acid sequence position 61-90 as depicted in FIG. 3, this part having at least 20, preferably at least 30, amino acid residues. In yet another aspect, the invention provides an isolated or recombinant virus comprising an amino acid sequence which is more than 90% homologous to at least part of an amino acid sequence position 1-30 as depicted in FIG. 3, the part having at least 20, preferably at least 30, amino acid residues.
In one aspect, a virus of the invention is provided comprising a nucleic acid sequence that differs to some extent to a nucleic acid sequence as depicted in FIG. 2B. As indicated before, RNA viruses are prone to mutations. Therefore, many EV79 strains exist in nature that differ to some extent from the strain obtained by the inventors. Such strains, comprising variations as compared to the sequence as depicted in FIG. 2B are, for instance, depicted in FIG. 4. Since these strains belong to the same EV79 species, they can be isolated, amplified and/or identified with a nucleic acid sequence based on a nucleic acid sequence as depicted in FIG. 2B.
A nucleic acid “based on” a sequence as depicted in FIG. 2B means herein that the nucleic acid may comprise the whole sequence, or an essential part of the sequence, which sequence may be modified to some extent. Preferably, the sequence is at least 45 percent homologous, more preferably 55 percent, more preferably 65 percent, even more preferably 75 percent, most preferably 90 percent homologous to a sequence as depicted in FIG. 2B.
The minimal size of the “essential part” of course varies with varying functions. If a part of a nucleic acid sequence is to be used as a primer, the minimal size preferably comprises about 5 nucleotides, more preferably about 8 nucleotides, even more preferably about 14 nucleotides, most preferably about 20 nucleotides. If a part of the nucleic acid sequence is to be used as a probe, the minimal size preferably comprises about 15 nucleotides, more preferably about 20 nucleotides.
An EV79 virus comprising mutations in its genome can as well be artificially made, for instance, to adapt codon usage to an expression system of choice or to attenuate the virus for vaccination purposes. Preferably, an isolated or recombinant virus is provided comprising a nucleic acid sequence that is at least 45% homologous to a nucleic acid sequence as depicted in FIG. 2B, or a functional part thereof. In one embodiment, a virus of the invention is provided comprising a nucleic acid sequence that is at least 55 percent, preferably at least 65 percent, more preferably at least 75 percent, more preferably at least 85 percent, even more preferably at least 95 percent, most preferably at least 98 percent, homologous to a nucleic acid sequence as depicted in FIG. 2B, or a functional part thereof. In yet another embodiment, an isolated or recombinant virus is provided comprising a nucleic acid sequence that is at least 45 percent, preferably at least 55 percent, more preferably at least 65 percent, more preferably at least 75 percent, more preferably at least 85 percent, even more preferably at least 95 percent, most preferably at least 98 percent homologous to at least part of a nucleic acid sequence as depicted in FIG. 2B, the part preferably having at least 30, more preferably at least 40, more preferably at least 50, most preferably at least 70 nucleotides.
In a preferred embodiment, an isolated or recombinant virus is provided comprising a nucleic acid sequence that is at least 45% homologous to at least part of a nucleic acid sequence position 500 to 1134 as depicted in FIGS. 2A and 2B, the part preferably having at least 30, more preferably at least 40, more preferably at least 50, most preferably at least 70 nucleotides. More preferably, the nucleic acid sequence is at least 45% homologous to at least part of a nucleic acid sequence position 500 to 1748 as depicted in FIGS. 2A and 2B, the part having at least 30, more preferably at least 40, more preferably at least 50, most preferably at least 70 nucleotides. In a more preferred embodiment, an isolated or recombinant virus is provided comprising a nucleic acid sequence that is at least 45% homologous to at least part of a nucleic acid sequence position 683 to 1134 as depicted in FIG. 2B, the part having at least 30, more preferably at least 40, more preferably at least 50, most preferably at least 70 nucleotides. In yet another preferred embodiment, the nucleic acid sequence is at least 45% homologous to at least part of a nucleic acid sequence position 683 to 1748 as depicted in FIG. 2B, the part having at least 30, more preferably at least 40, more preferably at least 50, most preferably at least 70 nucleotides.
The invention also provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 42% homologous to at least part of a nucleic acid sequence position 651-700 as depicted in FIGS. 2A and 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 58% homologous to at least part of a nucleic acid sequence position 701-750 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In yet another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 88% homologous to at least part of a nucleic acid sequence position 751-800 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 72% homologous to at least part of a nucleic acid sequence position 801-850 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 54% homologous to at least part of a nucleic acid sequence position 901-950 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 64% homologous to at least part of a nucleic acid sequence position 951-1000 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another in another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 48% homologous to at least part of a nucleic acid sequence position 1001-1050 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 56% homologous to at least part of a nucleic acid sequence position 1051-1100 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
In another aspect, the invention provides an isolated or recombinant virus comprising a nucleic acid sequence which is more than 53% homologous to at least part of a nucleic acid sequence position 1101-1134 as depicted in FIG. 2B, the part having at least 20, preferably at least 30, more preferably at least 40, most preferably at least 50 nucleotides.
With the teaching of the present invention it has become possible to ascribe “general” symptoms such as, for instance, diarrhea to an EV79-related disease. Hence, an EV79-related disease can now be diagnosed, for instance, by demonstrating the presence of EV79 virus, or antibodies against the virus, in a sample.
Now that a virus of the invention has been provided, a person skilled in the art is well capable of generating a functional part, derivative or analogue of the virus. A functional part of an EV79 virus is defined as a part that has the same EV79-specific properties in kind, although not necessarily in amount. Which properties need to be compared depends from a particular application. For instance, if the EV79 infection properties are concerned, a property to infect a cell should be considered, in which case a functional part of EV79 should also be capable of infecting a cell, eventually to a different extent. If however a capability of inducing an immune response in a host is considered, a single EV79-specific epitope of an EV79 virus can be considered to be a functional part.
A functional derivative of a virus is defined as a viral compound, which has been altered such that the properties of the compound are essentially the same in kind, but not necessarily in amount. A derivative can be provided in many ways, for instance, through nucleotide substitution (preferably “wobble” based), through (conservative) amino acid substitution, subsequent modification, etc.
Analogous compounds of a virus can also be generated using methods in the art. For instance, a chimeric virus can be produced, or an EV79 virus with a chimeric protein. For instance, EV79 can be rendered more immunogenic by generating a cell surface associated fusion protein comprising at least part of an EV79 cell surface protein and a non-EV79 immunogenic part. EV79 virus comprising such chimeric protein can be used for inducing an enhanced immune response in a host, for instance, for vaccination purposes.
As used herein, the term “a virus of the invention” is meant to also comprise a functional part, derivative and/or analogue of the virus.
A functional part of a protein is defined as a part that has the same kind of properties in kind, but not necessarily in amount. The properties may comprise immunogenic properties. By immunogenic properties is meant the capability to induce an immune response in a host. A functional derivative of a protein is defined as a protein, which has been altered such that the properties of the molecule are essentially the same in kind, but not necessarily in amount. A derivative can be provided in many ways, for instance, through conservative amino acid substitution.
A person skilled in the art is well able to generate analogous compounds of a protein. This can, for instance, be done through screening of a peptide library. Such an analogue has essentially the same properties of the protein in kind, but not necessarily in amount.
Now that an EV79 virus is provided, primers and/or probes capable of specifically hybridizing to a nucleic acid of a virus of the invention can be generated. By “specifically hybridizing” is meant herein that the primer and/or probe is capable of hybridizing to the nucleic acid because the primer and/or probe comprises a sequence of at least 3, preferably at least 5, nucleotides that is complementary to the nucleic acid of the virus. In a preferred embodiment, the primer and/or probe consists of a sequence that is complementary to the nucleic acid of the virus. Preferably, the primer and/or probe is capable of hybridizing with a nucleic acid of a virus of the invention under stringent conditions. A primer/probe of the invention is particularly suitable for amplifying/detecting at least part of the genome of an EV79 virus.
One embodiment provides a primer and/or probe capable of specifically hybridizing to a nucleic acid sequence as depicted in FIG. 2B. In Table 2, sequences of primers of the invention are provided.
The invention also provides an isolated molecule capable of specifically binding a virus or functional part, derivative and/or analogue of the invention. The isolated molecule preferably comprises a proteinaceous molecule. The molecule may be capable of specifically binding a nucleic acid of the virus, such as at least part of a sequence as depicted in FIG. 2B. The molecule is however preferably capable of specifically binding an amino acid sequence of the virus. The amino acid sequence may be located inside an EV79 virus particle, either partly or entirely. However, the amino acid sequence preferably comprises (part of) a protein associated with the surface of the virus, such as an epitope. By “associated with the surface” is meant a protein that is at least partly exposed to the surface of the virus. Such protein, for instance, comprises a viral capsid protein. In one embodiment, the amino acid sequence comprises at least part of an amino acid sequence as shown in FIG. 3. The part preferably comprises at least 5, more preferably at least 10 amino acid residues.
By a molecule capable of specifically binding a nucleic acid sequence is meant herein a molecule that is capable of distinguishing between related nucleic acid sequences under stringent conditions. A molecule capable of specifically binding a proteinaceous molecule is defined as a molecule that is capable of distinguishing between different proteinaceous molecules because it has more affinity for a specific amino acid sequence, a specific conformation of the proteinaceous molecule, etc. Non-specific “sticking” of a molecule is not considered specific binding.
A molecule of the invention preferably comprises an antibody or a functional part, derivative and/or analogue thereof. A functional part of an antibody has essentially the same properties of the antibody in kind, but not necessarily in amount. The functional part is preferably capable of specifically binding an antigen of EV79. However, the functional part may bind such antigen to a different extent as compared to the whole antibody. A functional part or derivative of an antibody, for instance, comprises a FAB fragment or a single chain antibody. An analogue of an antibody, for instance, comprises a chimeric antibody. As used herein, the term “antibody” is also meant to comprise a functional part, derivative and/or analogue of the antibody.
A molecule capable of specifically binding an EV79 virus can be obtained using current methods in the art. For instance, an EV79-directed antibody may be obtained from an infected individual. The antibody can, for instance, be isolated from a sample, such as, for instance, a blood, serum, sputum, saliva or tissue sample, obtained from the infected individual. Alternatively, a non-human animal can be inoculated with EV79 in order to induce an immune response. Produced antibodies can subsequently be collected from the animal. Collected antibodies often need to be purified, for instance, by affinity chromatography.
Furthermore, the antibody can be recombinantly produced, for instance, by micro-organisms, cell lines (i.e., monoclonal antibodies) or transgenic animals. In that case, codon optimization may be required. An EV79-directed antibody may as well be synthesized using common techniques such as solid phase synthesis.
Once a molecule of the invention is obtained, a desired property, such as its binding capacity, can be improved. For proteinaceous molecules this can, for instance, be done by an Ala-scan and/or replacement net mapping method. With these methods, many different proteinaceous molecules are generated, based on an original amino acid sequence but each molecule containing a substitution of at least one amino acid residue. The amino acid residue may either be replaced by Alanine (Ala-scan) or by any other amino acid residue (replacement net mapping). Each variant is subsequently screened for the desired property. Generated data are used to design an improved proteinaceous molecule.
Once a molecule of the invention, such as an antibody capable of specifically binding Prima-7, has been obtained, it may be necessary to test for EV79 specificity, because antibodies may cross react with other enteroviruses if they are directed against a conserved region of enteroviral species region. Specificity can, for instance, be tested by coating wells with the molecules, subsequently incubating the wells with different enteroviral species, and determining whether the molecules appear to specifically bind EV79 only, to a significant extent. Of course many alternative methods for testing specificity are available in the art. In one aspect, the invention thus provides a method for producing a molecule capable of specifically binding a virus or functional part, derivative or analogue of the invention comprising:

    
    
        producing molecules capable of binding a virus or functional part, derivative or analogue of the invention, and
        selecting a molecule that is specific for the virus.
    
    


An antibody or functional part, derivative and/or analogue of the invention can be used to detect the presence of an EV79 virus in a sample. The sample may comprise a blood, serum, or tissue sample. Preferably, however, the sample comprises a feces, sputum or saliva sample. The antibody or functional part, derivative and/or analogue can, for instance, be used in an affinity column. The antibody or functional part, derivative and/or analogue may as well be coated on ELISA microtiter plates, after which the plate can be incubated with a sample from a human or non-human animal. If the antibody appears to specifically bind to some component of the sample, it is indicative for the presence of EV79 in the sample. Binding of an EV79 component can, for instance, be demonstrated by a second, labeled, antibody directed against EV79.
As an alternative for ELISA, optical biosensors (e.g., BiaCore 3000 or the Affinity Sensors IAsys) can be used to detect the interaction of EV79 encoded proteins and a ligand (e.g., antibody or Fab). These optical systems are equally sensitive as an ELISA but allow detection in real-time.
A typical biosensor experimental cycle is illustrated in FIG. 7. The first step is usually the immobilization of a given ligand and a control to the biosensor cuvette surface. Several surfaces are commercially available, which include, for instance, biotin, carboxylate, hydrophobic and amine surface types. The next step can be the addition of a sample wherein the operator would like to study the interaction of a specific ligate to the immobilized ligand. This step is termed the binding step. Buffer can then be added to dissociate any weakly and non-specific bound molecules. A fourth step may be a regeneration step, whereby the surface is brought back to its original state. After regeneration, the cuvette can either be re-used for another binding experiment or stored usually at 4° C.7 27).
A molecule of the invention is suitable for isolating EV79 present in a sample. The sample preferably comprises a feces sample from an individual suffering from diarrhea. A high throughput automated assay can be established wherein test samples are incubated with a molecule of the invention. Bound virus can, for instance, be detected and/or identified by staining of virus particles or detection of EV79 nucleic acid using a primer and/or probe of the invention. Such assay provides for easy and fast diagnosis. Moreover, different strains of EV79 can be obtained. The strains may be purified, after which they can be further investigated, for instance, in order to improve diagnostic protocols. In one aspect, the invention thus provides an isolated or recombinant virus that is immunoreactive with a (preferably proteinaceous) molecule of the invention capable of specifically binding EV79.
A primer and/or probe of the invention can also be used for detection and/or identification of a virus of the invention. After nucleic acid isolation, for instance, with the Boom method, a PCR reaction can be set up using at least one primer of the invention. In one embodiment, one (specific) primer of the invention is used in combination of one “universal” primer. In another embodiment, two specific primers of the invention are used. Subsequently, a probe of the invention can be used in order to determine whether amplified EV79 viral product is obtained. Alternatively, a nucleic acid of a virus of the invention can be used for detecting the presence of nucleic acid capable of hybridizing therewith. In one embodiment, the nucleic acid comprises a sequence as depicted in FIG. 2B. It will be acknowledged that an antisense strand should be used in order to detect a sense EV79 nucleic acid by way of hybridization.
It has become possible to detect a virus of the invention in a sample, for instance, using a molecule, primer and/or probe of the invention. Alternatively, it is possible to screen a sample for the presence of a molecule capable of specifically binding a virus of the invention, such as a specific ligand or an antibody directed against a virus of the invention. This is often desirable for diagnostic purposes because after viral infection of an individual, immunopathogenesis often changes over time. During the first stage an initial immune response of the cell mediated type is often invoked, which may switch into humoral responses over time. Because of the change, one kind of immunological diagnostic procedure may not be suitable for the whole period of infection. Alternating positive and negative results may be obtained.
Therefore, in addition to measuring the presence of EV79 virus, it is advantageous to determine the presence of antibodies against the virus in a sample of an individual as well. This can be performed by incubating a sample of the individual with a virus of the invention (or functional part, derivative and/or analogue thereof) and detecting bound antibodies using common methods in the art. A use of a virus or functional part, derivative and/or analogue of the invention for detecting a molecule capable of specifically binding a virus of the invention, such as a specific ligand or an antibody against the virus, in a sample is therefore also herewith provided. Of course, it is not necessary to use whole virus particles since a nucleic acid sequence and/or an amino acid sequence of the virus can also be used to detect antibodies. The amino acid sequence preferably comprises an epitope of a surface-associated protein of a virus of the invention, because an individual's immune system is directly exposed to such surface-associated proteins after viral infection. Therefore, antibodies against the surface-associated proteins are most likely to be present. In one embodiment, a sequence as depicted in FIG. 3 or a functional part, derivative or analogue thereof is used for detecting a molecule capable of specifically binding a virus of the invention, such as a specific ligand or an antibody against a virus of the invention.
A virus of the invention, or a functional part, derivative or analogue thereof, is particularly suitable for use as a vaccine, preferably against an EV79-related disease. In a preferred embodiment, an attenuated virus or a functional part such as a surface associated protein, an epitope thereof or a glycosylation bond is used. The vaccine preferably comprises a suitable adjuvant such as, for instance, Specol or a double oil emulsion. The virus or functional part may furthermore be coupled to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or an immunogenic conjugate of a protein such as ovalbumin. Methods for generating a vaccine are known in the art. Since an EV79 virus of the invention is an enterovirus, a vaccine against EV79 may be generated by analogy with currently used vaccines against other enteroviruses, such as poliovirus. Methods for production of vaccines known to persons skilled in the art encompass, for instance, dead virus, live attenuated virus, viral subunits, DNA vaccines, DNA vaccines in suitable vaccine vectors like adenovirus or AAV and gene therapy approaches through integrating viral vectors. Administration of such vaccine to an individual provides protection against EV79 by way of activation of the individual's immune system.
A virus of the invention, or functional part, derivative and/or analogue thereof, is also suitable for use as a medicament. For instance, the virus may be provided with a nucleic acid encoding a proteinaceous molecule with beneficial properties for local treatment of the gastrointestinal tract.
In another embodiment, a molecule of the invention, preferably a proteinaceous molecule of the invention, is used as a vaccine or medicament. The vaccine or medicament is preferably used for protection and/or treatment of an EV79-related disease. The molecule can be administered to an individual, preferably in combination with a suitable adjuvant and/or carrier as described above, before an EV79 infection has taken place. Such passive immunization provides (often temporary) protection against subsequent infection. The molecule may also be administered as a medicament after an EV79 infection. The molecule, capable of specifically binding EV79, will at least in part counteract EV79 infection. A medicament comprising a molecule of the invention can of course be combined with one or more other medicaments, such as inflammation inhibitors. In one embodiment, several medicaments are separately administered to an individual. However, a molecule of the invention can also be combined with another pharmaceutically active compound in one pharmaceutical preparation.
The invention thus provides a vaccine comprising a virus or functional part, derivative or analogue and/or a molecule of the invention.
A medicament comprising a molecule of the invention is also herewith provided. The medicament preferably comprises a proteinaceous molecule of the invention. A vaccine or medicament of the invention is preferably used for at least in part preventing and/or treating an EV79-related disease.
Enteroviruses often induce symptoms, such as diarrhea, respiratory problems and fever that are involved with many kinds of diseases. Hence, these symptoms cannot directly be ascribed to an enteroviral infection. Now that a virus of the invention is provided, it has become possible to determine whether an individual suffers from an enteroviral genus-related disease. More specifically, it can be determined whether an EV79-related disease is involved. A virus or functional part, derivative or analogue of the invention can be used to determine whether a sample from an individual comprises antibodies against the enterovirus. Moreover, the presence of enterovirus in a sample can be determined using a proteinaceous molecule of the invention, for instance, using affinity chromatography or ELISA. The presence of enterovirus in a sample can as well be determined by subjecting nucleic acid of the sample to an amplification reaction such as PCR, RT-PCR, TMA or NASBA and determining whether amplified enteroviral nucleic acid is present, using at least one primer or probe of the invention. Once an enteroviral genus-related disease has been diagnosed, it is possible to provide adequate treatment, such as, for instance, antibodies specifically directed against the enterovirus. Alternatively, or in addition, general antiviral medicaments may be used.
A use of a virus, molecule and/or a primer/probe of the invention for diagnosis of an enteroviral genus-related disease is, therefore, also provided. Preferably, the enteroviral genus comprises an EV79 enterovirus.
The invention also provides a diagnostic kit comprising a virus or functional part, derivative or analogue, and/or a primer/probe of the invention. With a kit of the invention, an enterovirus-related disease can be diagnosed, for instance, by way of demonstrating the presence of enterovirus, or enterovirus-directed antibodies, in a sample. In one embodiment, the kit also comprises suitable means for nucleic acid isolation and/or amplification.
A diagnostic kit comprising a molecule, preferably a proteinaceous molecule, of the invention capable of specifically binding EV79 is also herewith provided. The molecule is preferably present on a suitable solid support, such as a microtiter plate. The kit, particularly suitable for demonstrating the presence of enterovirus in a sample, preferably comprises suitable means for staining enterovirus as well. Such means are commonly available in the art.
In one aspect, the invention provides a method for determining whether an individual suffers from an EV79-related disease, comprising obtaining a sample from the individual and detecting an EV79 virus or functional part, derivative or analogue thereof in the sample by hybridizing and/or amplifying a nucleic acid of the virus or functional part, derivative or analogue with a primer and/or probe of the invention and detecting hybridized and/or amplified product.
In another aspect, the invention provides a method for determining whether an individual suffers from an EV79-related disease, comprising obtaining a sample from the individual and detecting an EV79 virus or functional part, derivative or analogue thereof in the sample by specifically binding of the virus or functional part, derivative or analogue with a (proteinaceous) molecule of the invention and detecting bound product.
In yet another aspect, the invention provides a method for determining whether an individual suffers from an EV79-related disease, comprising obtaining a sample from the individual and detecting EV79-specific antibodies in the sample by specifically binding the antibodies with an EV79 virus or functional part, derivative or analogue of the invention and detecting bound product.
Once an EV79-related disease has been diagnosed, it can be treated with a medicament of the invention. Additionally, an individual at risk of obtaining an EV79 infection can be vaccinated with a vaccine of the invention in order to prevent an EV79-related disease. For instance, immunocompromised individuals can be provided with a vaccine comprising molecules of the invention capable of specifically binding EV79 virus.
A method for treating an individual suffering from, or at risk of suffering from, an EV79-related disease, comprising administering to the individual a vaccine or medicament of the invention is therefore also provided.
The invention is further illustrated by the following examples. The examples do not limit the scope of the invention in any way.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
In the drawings, which illustrate what is currently considered to be the best mode for carrying out the invention:
FIG. 1: The NJ tree of phylogenetic analysis based on the 150 aa peptide sequence of EV79. Distance: p-distance; bootstrap test: 500.
FIG. 2A: 5′-UTR region of EV79 (SEQ ID NO:61).
FIG. 2B: VP4 and VP2 region of EV79 (SEQ ID NO:61).
FIG. 3: Amino acid sequence of VP4 and VP2 region translated from EV79 (SEQ ID NO:63).
FIG. 4: Screening feces samples with specific RT-PCR, alignment of PCR product of strain 07-1787 (SEQ ID NO:64), PCR product of strain F26kol03 (SEQ ID NO:65), PCR product of strain F26kol06 (SEQ ID NO:66), PCR product of strain F26kol10 (SEQ ID NO:67), PCR product of strain F27kol20 (SEQ ID NO:68), PCR product of strain F27kol21 (SEQ ID NO:69), PCR product of strain F35kol32 (SEQ ID NO:70), and PCR product of strain F35kol36 (SEQ ID NO:71) are shown.
FIG. 5: Restriction map of partial Prima 7 genome showing the location of the oligonucleotides and the open reading frame.
FIG. 6: Nucleic acid and amino acid sequence homology between EV79 and human enterovirus.
FIG. 7: Biosensor experimental cycle.
FIG. 8: The Enterovirus Prima 7 genome comprises 7.419 KB and exhibits the typical organization of a positive sense single stranded RNA virus. Depicted is a cDNA derivative of the genomic RNA with added restriction sites. The genome posses a single open reading (ORF) of 2193 amino acids (AA), flanked by 5′ and 3′ untranslated regions (UTR). Within the deduced amino acid sequence of the ORF, domains encoding capsid proteins VP1 to 4, proteases 2a and 3C, a helicase (2C), a RNA dependant polymerase (POL) and non-structural proteins 2b and 3A, were identified on the basis of homology. Between 3A and protease 3C, domain 3B encoding a 22 AA VPg that is covalently attached to the 5′ end of the genomic RNA. Predicted 2a and 3C proteolytic cleavage sites separate all these domains, except VP4 and VP2. The cleavage between these two domains is predicted to occur autocatalyticly.2 
FIG. 9: The similarity of the GenBank reference sequences for Coxsackie A16 (NC—001612) human enterovirus A, Coxsackie B1 (NC—001472) human enterovirus B, Coxsackie A21 (NC—001428) human enterovirus C, Enterovirus 70 (NC—001430) human enterovirus D, Plaque A2 virus (NC—003988) human enterovirus E and Enterovirus 71 (AF302996) to PRIMA 7 was determined in a window of 200 nt sliding along the Prima 7 genome in steps on 20 nt and plotted against its position in Prima 7.
FIG. 10: A similar analysis was performed with an alignment all available full-length genome sequences of group A human enteroviruses. Coxsackie A16 Cox (NC—001612), Coxsackie A16 (CAU05876), Coxsackie A16 (AF177911), Enterovirus 71 strain TW/2272198 (AF119795), Enterovirus71 (AF302996), Enterovirus 5666/sin/002209 (AF352027), and Enterovirus 71 (ETU22522). It is clear from this plot that even in a comparison of the most closely related group A enteroviruses, EV79 exhibits the low sequence identity throughout its genome.
FIG. 11: The first chimeric genome (FIG. 11) was generated by a reciprocal recombination in the VP3, fusing nucleotide 2187 of Coxsackie virus A16 (NC—001612) to nucleotide 2129 of EV79. Recombination occurred at the conserved sequence TGGGA, retaining the large open reading frame and generating a hybrid polyprotein. The Coxsackie A16 derived A16-derived part is indicated with a single line, whereas the EV79 derived EV79-derived part is indicated with grey boxes. Below is depicted the recombination site with the Coxsackie part in bold capitals and the EV79 derived EV79-derived part is underlined. ACTGCAATGCTGGGAACCCATGTGATATGGGATTTTGGTTTACAATCATC (SEQ ID NO:72)
FIG. 12: A second chimeric genome was generated by an imaginary reciprocal recombination fusing nucleotide 3773 of Enterovirus 71 (U22522) to nucleotide 3713 of EV79 again creating a hybrid 2b-encoding domain within polyprotein. Recombination could occur within the conserved sequence ATGGA. The Enterovirus 71 derived 71-derived part is indicated by a single line and the EV79 derived EV79-derived part is indicated by grey boxes. Below is depicted the crossover site where the EV71 sequences are in bold print and the EV79 sequences are underlined. TCTTATGGTTAGATGAAGAAGCCATGGAACAGGGGGTCACGGATTACATC (SEQ ID NO:73)
FIG. 13: Hybrid 1 is the result of a crossover between Coxsackie A16 (NC—001612) and EV79. Hybrid 2 is a chimera of Enterovirus 71 (U022522) and EV79. The actual recombination sites can be deduced from the SimPlot graph.
FIG. 14: The predicted secondary structure of nucleotides 1 and 752 (ATG). Five major stem loops are predicted of which nucleotides 456 to 564 correspond to Poliovirus loop V. This particular stem loop confers to Poliovirus the ability to translate efficiency in neuronal cells. This region folds in slightly different secondary structures, differing slightly in enthalpy. Obviously, the presence of RNA-binding proteins could stabilize certain secondary structures in a manner Mfold is unable to predict.
FIG. 15: The strongest sequence conservation is encountered in the 5′-untranslated region (5′-UTR), probably because this region plays a pivotal role in the replication and initiation of protein synthesis.22 Inverted repeats in the 5′-UTR allow extensive internal base pairing and the resulting secondary structure can be predicted using the M-Fold program.14 The putative initiation codon resides immediately downstream of a CT rich tract (position 643-660) which is part of a putative Internal Ribosome Entry Site (IRES) or ribosome landing pad. This ATG corresponds to the one we identified as initiation codon on the basis of homology. All seven other ATGs preceding the putative initiation codon are out of frame and, according to the M-fold prediction, sequestered in hairpin loops. The region encompassing the fifth stem-loop in the 5′-UTRs from Poliovinises 1 (AY056702), 2 (D00625), 3 (AJ29398), Enterovirus 70 (D00820), Enterovirus 71 (U022521), Coxsackie A16 (U05876), Coxsackie A21 (D00538) and Enterovirus 79 were aligned with ClustalW. Specific mutations in the stem-loop V (indicated in bold print above the alignment) of polioviruses, preventing proper base pairing in the stem-loop, reduce transcription20 and decrease neurovirulence.10 Such attenuated polio strains are used for vaccine production. The implicated nucleotides (marker in yellow) have been conserved in EV79, hence could also be used to create attenuated strains. Alignment of 5′-UTR sequence of human enterovirus D00538CXA21 (SEQ ID NO:74), 5′-UTR sequence of human enterovirus D00820EV70 (SEQ ID NO:75), 5′-UTR sequence of human enterovirus D00625PV2 (SEQ ID NO:76), 5′-UTR sequence of human enterovirus AJ293918PV3 (SEQ ID NO:77), 5′-UTR sequence of human enterovirus U05876CXA16 (SEQ ID NO:78), 5′-UTR sequence of human enterovirus U22521EN71 (SEQ ID NO: 79), 5′-UTR sequence of human enterovirus AY056702PV1 (SEQ ID NO:80), and 5′-UTR sequence of human EV79 (SEQ ID NO:81) are shown.
FIG. 16: Upon introduction into a suitable E. coli host, production of a HisTrx-StrepII bearing VP1 or VP2 fusion protein can be induced by addition of IPTG to the culture medium. After cell lysis, the expressed proteins can be detected and purified with the appropriate affinity matrices by virtue of their His, Thioredoxin and StrepII affinity tags.
FIG. 17: The deduced amino acid sequences of Coxsackie A16 (NP—0042242 and AAD55085), Coxsackie B1 (NP—040958), Coxsackie A21 (NP—040759), Coxsackie B6 (Q9QL88), Plaque A2 (NP—653149), Polio 1 (AAM09804), Enterovirus 70 (NP—040760), Enterovirus 5666 (AAB30618) and Enterovirus 5865 (AAK13008) were aligned with ClustalW. The local protein similarity to EV79 to the group A enteroviruses varies from 61 to 92%.
FIG. 18: Sequences were aligned using Clustal-W included in the Vector NTI suite 7 software package (InforMax, Bethesda, U.S.A.). Phylogenetic analyses were performed by the Neighbor-Joining (NJ) method, as implemented in the MEGA 2.1 software package.12 Kimura's 2-parameter distances11 were estimated for the nucleotide sequences, and P-distances were used for amino acid sequences. Five hundred bootstrap replicates were analyzed. The distance data deduced from the polyprotein alignment was used to construct a phylogenetic tree. Bootstrap values are indicated at the nodes. The distance bar indicates 5% sequence divergence.
FIG. 19: Prima 7 (EV79) nucleotide sequence (SEQ ID NO:82).
FIG. 20: Deduced amino acid sequence EV79 (SEQ ID NO:83).
FIG. 21: Restriction maps of pLXRN and its derivative pEV79.
FIG. 22: VPI orthologs were retrieved from Genbank, aligned using ClustalX and the corresponding phylogenetic tree was constructed with Mega 2.1. The protein accession numbers are indicated following the species name. Several representatives of Enterovirus 71 (EV71) and Coxsackie A16 (Cox A16) were used to account for the intra-species sequence variation. To facilitate correlation with serological data additional Group A human enterovirus VP1 (Cox A2, A3, A5 A8, A10 and A14) were added. For the Human Enteroviruses (HEV) A, B, C, D and E the reference genomes were used (worldwideweb.ncbi.nlm.nih.gov/genomes/VIRUSES/10239.html). Clusters in VP1 derived VP1-derived phylogenetic trees have been demonstrated to correlate with serotype clusters. These serotype clusters correspond to currently recognized enteroviral species. In the VP1 phylogenetic tree, EV79 occupies a distinct branch strongly suggesting that it represents a new serotype/enteroviral species. Because of their high scores in an EV79 VP1 blast search, a number of Simian Picornaviruses (SPV 1 to 45) were included in the analysis. Surprisingly, the EV79 VP1 clusters with a group of Simian Picornavirus VP1 orthologs, exhibiting 72 to 70% sequence similarity to Simian enterovirus 13 and 18 respectively. This suggests a simian origin for EV79 and hence could be the first example of zoonotic potential among enteroviruses.



DETAILED DESCRIPTION OF THE INVENTION
Examples
Isolation of Prima 7
In an alignment of full-length genomic sequences of 55 Picornavirus isolates, three groups could be distinguished. For the group encompassing most of the human enteroviruses, primers and a nested RT-PCR assay were designed capable of amplifying all members of the group. This assay was used to screen clinical stool samples of 201 HIV-positive subjects, 17 of which yielded a PCR product of the predicted (300 bp) size. Four of these samples showed a band of 450 bp after the first amplification, indicative of high virus titers. Cloned fragments of two PCR products exhibited 89 and 91% homology to the 5′-UTR of human enterovirus 71 and poliovirus respectively.







TABLE 1A







Picorna group 2 amplification primers












Prime



Length of



Name
Target virus
Sequence (5′-3′)
Remarks
Amplicon





PICG2U1
Picorna group 2
GGTACCTTTGTRCGCCTGT
5′, first PCR
450 bp





(SEQ ID NO:1)


PICG2D1
Picorna group 2
GACACCCAAAGTAGTCGG
3′, first PCR




(SEQ ID NO:2)


 


PICG2U1′
Picorna group 2
CAAGCACTTCTGTTTCCCC
5′, nested PCR
300 bp




(SEQ ID NO:3)


PICG2D1′
Picorna group 2
CATCGRCCTGATCTACAC
3′, nested PCR




(SEQ ID NO:4)









Viral RNA Isolation

RNA extraction was performed by using the method described by Boom et al. Briefly, 100 μl of clinical specimen or reconstructed specimen from enterovirus panels was mixed with 900 μl of L6 lysis buffer, 50 μl of silica and then incubated for 10 minutes at room temperature. After washing silica particles bound with nucleic acid for several steps, RNA was eluted in 50 μl of nuclease-free water.
Nested RT-PCR Amplification for Discovery
After viral RNA isolation, 10 μl of elution containing isolated RNA was used for nested RT-PCR amplification tests. A downstream primer, named PICG2D1 (5′-GACACCCAAAGTAGTCGG-3′) (SEQ ID NO:2) and derived from 5′-untranslated region (5′-UTR) region of enteroviruses, were used for the cDNA synthesis. First and second strand cDNA synthesis was performed as described previously (De Wolf et al., 1994). Briefly, after incubation for 45 minutes at 42° C. to synthesize the first strand cDNA, the corresponding generic upstream primer of the first PCR amplification, named PICG2U1 (5′-GGTACCTTTGTRCGCCTGT3′) (SEQ ID NO:1), PCR buffer, deoxynucleotide triphosphates, 2.5 mM MgCl2, and 2 U of Taq polymerase (Perkin-Elmer Cetus) were added. After an incubation for 5 minutes at 95° C., the reaction mixture was subjected to 35 cycles of amplification with the following profile: 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes. After the first PCR amplification, 5 μl of PCR product was added to the nested PCR reaction mixture consisting of a nested upstream primer, named PICG2U1′ (5′-CAAGCACTTCTGTTTCCCC-3′) (SEQ ID NO:3) and a nested generic downstream primer, named PICG2D1′ (5′-CATCGRCCTGATCTACAC-3′) (SEQ ID NO:4). The reaction mixture was subjected to 25 cycles of amplification with the same profile as the first PCR amplification. The target fragments, which were approximately 450 bp in length for the first PCR product or 300 bp in length for the nested PCR product, were cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, Calif.) for plasmid preparation and subsequently gene sequencing reactions.
DNA Sequencing and Analysis
Enterovirus-PCR product containing plasmids were sequenced with the BigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.), using its −21 M13 and M13 reverse primers. Electrophoresis of sequencing reaction mixtures was performed with an Applied Biosystems 377 automated sequencer, following the manufacturer's protocols. The Vector NTI suite 7 software package was used to analyze all sequencing data.
Determining the Nucleotide Sequence of the Complete Prima 7 Genome
With a third oligonucleotide (07VP4D1, AGCTTCCACCACCACCC) (SEQ ID NO:5), located in a conserved domain of the Picorna VP4 region, the sequence for clone 07 was extended to 1034 nucleotides (see FIG. 5).
The resulting deduced amino acid sequence of the putative VP4 region occupies a distinct branch within the phylogenetic tree and clusters in the enterovirus 70 group, identifying it as a novel enterovirus. Using a combination of primers targeting conserved domains and the proprietary PALM method, we are in the process of determining the full-length genomic sequence for this new enterovirus.
Sequence Extension with Deduced Oligonucleotides
By alignment of the deduced amino acid sequences of the related enteroviruses 70, 71 and Coxsackie A16, conserved domains were identified. After back-translation of these conserved protein domains (Table 2), taking into account the appropriate codon bias, degenerate primers could be designed and used for RT-PCR. By selecting appropriate combinations and orientations of the oligos from Table 2, overlapping fragments of the Prima 7 genomic RNA can be amplified by RT-PCR. From these overlapping fragments the complete genome sequence can be assembled.
Palm
Alternatively, using a combination of 5′-oligonucleotides located in the analyzed part of the Prima 7 genome (Table 3, 07UVP01 to 07UVP07) and a 3′ tagged random primer (JZH2R) additional fragments of the EV79 genome were amplified using a nested RT-PCR protocol similar to the one mentioned previously.








TABLE 2







Primers targeting conserved domains within the



Prima 7 open reading frame














Position
Position
Position



Conserved

Cox
Entero
Entero


domain
Deduced Oligonucleotide
A16
71
70





GYDASLSPVWF
GARATYCAYGARWSBATYMGVTGGACNAARGAY1
7132
7128
ND



1
(SEQ ID NO:13)
7164
7161









(SEQ ID
GARATYCAYGARTCWATWMGATGGACMAARGAY2
Consensus


NO:6)
(SEQ ID NO:14)


 











AGQCGGVVT
GCNGGNCARTGYGGNGGNGTSGTSACN1
5824
5820
5818


2
(SEQ ID NO:15)
5850
5846
5844









(SEQ ID
GCIGGRCARTGTGGWGGIGTGGTSACM2
Consensus


NO:7)
(SEQ ID NO:16)


 











NEKFRDI
AAYGARAARTTYMGVGAYATY1
5632
5628
5626


3
(SEQ ID NO:17)
5652
5648
5646









(SEQ ID
AATGARAARTTYAGRGAYATCAS2
Consensus


NO:8)
(SEQ ID NO:18)


 











QMVSTVD
CARATGGTNWSNACNGTNGAY1
4657
4653
4639


4
(SEQ ID NO:19)
4677
4673
4659









(SEQ ID
CARATGGTRTCYACYGTRGAY2
Consensus


NO:9)
(SEQ ID NO:20)


 











FDGYKQQ
TTYGAYGGNTAYAARCARCAR1
4573
4569
4555


5
(SEQ ID NO:21)
4593
4589
4575









(SEQ ID
TTTGAYGGRTAYAAACARCAR2
Consensus


NO:10)
(SEQ ID NO:22)


 











GSPGTGK
GGNWSNCCNGGNACNGGNAAR1
4468
4464
4450


6
(SEQ ID NO:23)
4488
4484
4470









(SEQ ID
GGMTCWCCAGGYACTGGRAARTC2
Consensus


NO:11)
(SEQ ID NO:24)


 











AFGTGFT
GCNTTYGGNACNGGNTTYACN1
3820
3816
3802


7
(SEQ ID NO:25)
3840
3836
3822









(SEQ ID
GCITTYGGRACWGGITTYACTGA2
Consensus


NO:12)
(SEQ ID NO:26)





1Using maximum codon degeneracy


2Using sequence preference in the aligned viral genomes














TABLE 3







Oligonucleotides for PALM extension of the



Prima 7 Sequence










Oligonucleotide
Applica-




name
tion
Sequence 5′-3′





JZH2R
1st PCR
GCTATCATCACAATGGACNNNNNG



(SEQ ID NO:27)


 


JZH1
1st PCR
GCTATCATCACAATGGAC


(SEQ ID NO:28)


 


07UVP01
1st PCR
TTCACTCAACCTGTTGTGG


(SEQ ID NO:29)


 


07UVP02
2nd PCR
CACTCAAATCACCATCAGC


(SEQ ID NO:30)


 


07UVP03
1st PCR
GCCAGATCGAGTAGACACTA


(SEQ ID NO:31)


 


07UVF04
2nd PCR
ATGCAACACAACCTGG


(SEQ ID NO:32)


 


07UVP05
1st PCR
TGAAGGAGCCACTACAGCTATACC


(SEQ ID NO:33)


 


07UVF06
2nd PCR
ACTGCAGATGATGGTGTCTCAGCA


(SEQ ID NO:34)









Propagation of Prima 7 in Cell Culture

Clinical feces samples were resuspended (30% v/v) in medium B (25 g/L Nutrient Broth No. 2 Oxoid, 250 u/mL Penicillin, 250 μg/mL Streptomycin 1.5 μg/mL Amphotericin B). The growth medium was removed from tubes containing cultured cells (Vero-, human embryonic lung fibroblasts or monkey kidney-cells) with a heat-sterilized platinum needle medium-sucking device. Feces, stored in medium B, were centrifuged at 2500 RPM for 10 minutes and 1 ml of the resulting supernatant was filtered through a 45 μM syringe-mounted filter. 3-4 droplets of the filtrate were added to the cultured cells. The closed tubes were gently inverted to ensure the patient material is in contact with the cells attached to the glass wall and the tubes were incubated for 30 minutes at 37° C. MEM Hanks 8% FCS (Gibco) solution was equilibrated at room temperature and 0.375 μg/mL Amphotericin B and 10 mg/mL Neomycin (Gibco) were added. To each tube 1 ml MEM Hanks 8% FCS was added and the tubes were incubated at 37° C. Twice a week for at least two weeks, the cells are microscopically observed and a virus-specific cytopathic effect (CPE) is scored.
Method to Detect Prima 7 in Stool Samples
Based on the Prima 7 sequence (Table 4), two sets of nested oligonucleotides were designed and RNA extraction was performed as previously described. After viral RNA isolation, 10 μl of elution containing isolated RNA was used for nested RT-PCR amplification tests. A downstream primer, named EV07D1 was used for the cDNA synthesis.







TABLE 4







Prima 7 detection oligonucleotides









Primer name 
Application
Sequence 5′-3′













EV07U1
1st PCR
CAGAGATCTTGCATACCTGT



(SEQ ID NO:35)


 


EV07D1
1st PCR
AGTATCCTTGGAGTATTCGGGC


(SEQ ID NO:36)


 


EV07U2
2nd PCR
GAACCAGAACATAGCTGCTAGC


(SEQ ID NO:37)


 


EV07D2
2nd PCR
CACCATATCCGACTGTGATG


(SEQ ID NO:38)









First and second strand cDNA synthesis was performed as described previously.6 Briefly, after an incubation for 45 minutes at 42° C. to synthesize the first strand cDNA, the corresponding generic upstream primer of the first PCR amplification, named EV07U1, PCR buffer, deoxynucleotide triphosphates, 2.5 mM MgCl2, and 2 U of Taq polymerase (Perkin-Elmer Cetus) were added. After an incubation of 5 minutes at 95° C., the reaction mixture was subjected to 35 cycles of amplification with the following profile: 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes. After the first PCR amplification, 5 μl of PCR product was added to the nested PCR reaction mixture consisting of a nested upstream primer, named EV07U2 and downstream primer, named EV07D2. The reaction mixture was subjected to 25 cycles of amplification with the same profile as the first PCR amplification.
Method of Raising Polyclonal Antibodies
Appropriate domains within the Prima 7 polyprotein (e.g., VP1 or VP4) can be selected and amplified with suitable oligonucleotides and RT-PCR. The corresponding purified viral antigens can be obtained by expression in a suitable host (e.g., Yarrowia lipolytica as described in Nicaud et al.16 Female NZW rabbits (approx 4 kg) are primed with 0.5 to 5.0 mg of viral protein antigen preparation. The antigen is suspended in 0.5 ml. of phosphate buffered saline (pH 7.3) and emulsified in an equal volume of complete Freund's adjuvant (CFA). Freund's Adjuvant is a well-established adjuvant system that is appropriate for use in these experiments where small amounts of antigen are used, and where immunogenicity of the antigen (although likely) is unknown. Published guidelines for use will be followed, including limiting injection to 0.1 ml at each site, using CFA only for initial immunization dose. This antigen preparation (1 ml total volume) is injected subdermally in the loose skin on the backside of the rabbit's neck. This injection route is immunologically effective and minimizes the possibility of local inflammation associated with unilateral or bilateral flank injection (such ensuing flank inflammation can impair animal mobility). After resting for 3 weeks, one ml of blood will be removed from the ear artery for a test bleed. Antibodies will be boosted if titers of the desirable antibodies are judged to be too low. Rabbits with adequate antibody levels will be boosted subdermally 1.0 mg of antigen contained in CFA. Boosted animals will be bled after two weeks; i.e., 15 ml of blood will be taken from the ear artery using a heat lamp to dilate the blood vessel. The rabbit will be placed in a commercial restraint, tranquilized with xylazine not more than seven times in total after which the rabbit will be exsanguinated by cardiac puncture following anesthesia using xylazine/ketamine.
Method for Vaccine Production
For the production of a subunit vaccine, the VP1 domain, perhaps combined with the VP2 and VP3 proteins, could be expressed in a suitable eukaryotic host (e.g., Y. lipolytica or Vero cells) and purified using preferentially two small affinity tags (e.g., His-tag or the StrepII tag). After appropriate purification, the resulting viral proteins can be used as a subunit vaccine.
Alternatively, the Prima 7 virus can be propagated in Vero-cells as described above and subsequently treated as described by Wu et al.28 Briefly the virus is precipitated from culture medium with 20% polyethylene glycol 6000 and purified by ultracentrifugation at 80,000×g for 4 hours through a discontinuous 40-65% sucrose gradient followed by a linear 5 to 40% CsCl gradient for 4 hours at 120,000×g. The resulting virus preparation can be inactivated by heating for 30 minutes at 65° C. as described by Blondel et al.3 
Analysis of VP1 or any of the Prima 7 Viral Proteins Binding to an Immobilized Ligand (e.g., Antibody) in an Optical Biosensor
Binding reactions were carried out in an IAsys two-channel resonant mirror biosensor at 20° C. (Affinity Sensors, Saxon Hill, Cambridge, United Kingdom) with minor modifications. Planar biotin surfaces, with which a signal of 600 arc s corresponds to 1 ng of bound protein/mm2, were derivatized with streptavidin according to the manufacturer's instructions. Controls showed that the viral proteins did not bind to streptavidin-derivatized biotin surfaces (result not shown). Biotinylated antibody was immobilized on planar streptavidin-derivatized surfaces, which were then washed with PBS. The distribution of the immobilized ligand and of the bound VP1 on the surface of the biosensor cuvette was inspected by the resonance scan, which showed that at all times these molecules were distributed uniformly on the sensor surface and therefore were not micro-aggregated. Binding assays were conducted in a final volume of 30 μl of PBS at 20±0.1° C. The ligate was added at a known concentration in 1 μl to 5 μl of PBS to the cuvette to give a final concentration of VP1 ranging from 14 to 70 nM. To remove residual bound ligate after the dissociation phase, and thus regenerate the immobilized ligand, the cuvette was washed three times with 50 μl of 2 M NaCl-10 mM Na2HPO4, pH 7.2, and three times with 50 μl of 20 mM HCl. Data were pooled from experiments carried out with different amounts of immobilized antibody (0.2, 0.6, and 1.2 ng/mm2). For the calculation of kon, low concentrations of ligate (VP1) were used, whereas for the measurement of koff, higher concentrations of ligate were employed (1 μM) to avoid any rebinding artifacts. The binding parameters kon and koff were calculated from the association and dissociation phases of the binding reactions, respectively, using the non-linear curve-fitting FastFit software (Affinity Sensors) provided with the instrument. The dissociation constant (Kd) was calculated from the association and dissociation rate constants and from the extent of binding observed near equilibrium.
Method to Detect Prima 7 in Stool Samples
Based on the Prima 7 sequence (Table 5), two sets of nested oligonucleotides were designed and RNA extraction was performed as previously described.4 After viral RNA isolation, 10 μl of elution containing isolated RNA was used for nested RT-PCR amplification tests. A downstream primer, named EV07D1 was used for the cDNA synthesis.







TABLE 5







Prima 7 detection oligonucleotides









Primer name
Application
Sequence 5′-3′













EV07U1
1st PCR
CAGAGATCTTGCATACCTGT



(SEQ ID NO:35)


 


EV07D1
1st PCR
AGTATCCTTGGAGTATTCGGGC


(SEQ ID NO:36)


 


EV07U2
2nd PCR
GAACCAGAACATAGCTGCTAGC


(SEQ ID NO:37)


 


EV07D2
2nd PCR
CACCATATCCGACTGTGATG


(SEQ ID NO:38)









First and second strand cDNA synthesis was performed as described previously (De Wolf et al., 1994). Briefly, after an incubation for 45 minutes at 42° C. to synthesize the first strand cDNA, the corresponding generic upstream primer of the first PCR amplification, named EV07U1, PCR buffer, deoxynucleotide triphosphates, 2.5 mM MgCl2, and 2 U of Taq polymerase (Perkin-Elmer Cetus) were added. After an incubation for 5 minutes at 95° C., the reaction mixture was subjected to 35 cycles of amplification with the following profile: 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes. After the first PCR amplification, 5 μl of PCR product was added to the nested PCR reaction mixture consisting of a nested upstream primer, named EV07U2 and downstream primer, named EV07D2. The reaction mixture was subjected to 25 cycles of amplification with the same profile as the first PCR amplification. The PCR products were size fractionated on a 1% agarose gel, excised, purified using the Qiaquick columns according to the protocol supplied by the manufacturer (Qiagen, Westburg), cloned into pCRII (Invitrogen) via topoisomerase 1-mediated ligation. For each PCR product, plasmids from three randomly picked colonies were isolated using a Qiagen Spin Miniprep kit and analyzed using the Big-Dye (Applied Biosystems) sequencing protocol. Results are depicted in FIG. 4.
Nucleic Acid Sequence
By a combination of the PALM method, long range RT-PCR and 5′-RACE the complete nucleotide sequence of Enterovirus Prima 7 was obtained.
Phylogenetic analysis revealed that Prima 7 clusters with group A of human enteroviruses. The most closely related group A members are Enterovirus 71 and Coxsackie A16 which exhibit 68% overall sequence identity at the nucleic acid level (Table 4).
To exclude the possibility that Prima 7 (EV79 in all Figures) occupies a separate branch in the phylogenetic tree because it is an inter-group recombinant, we performed a SimPlot analysis13 with reference sequences of human enterovirus groups A, B, C, D and E. Throughout its genome Prima 7 maintained the strongest similarity to group A enteroviruses, suggesting Prima 7 is not an inter-group recombinant.
When the Prima 7 sequence was used as query in a SimPlot analysis comparing it to all available full-length human group A Enteroviruses, it appeared to be equidistant to all of them. If however, any other group A enterovirus is used as query sequence, Prima 7 exhibits the lowest similarity throughout its genome. This suggests strongly that Prima 7 is a new group A enterovirus and not merely a recombinant from two distantly related group A enteroviruses.
Hybrid Genomes
The first chimeric genome was generated by a reciprocal recombination in the VP3, fusing nucleotide 2187 of Coxsackie virus A16 (NC—001612) to nucleotide 2129 of EV79. Recombination occurred at the conserved sequence TGGGA, retaining the large open reading frame and generating a hybrid polyprotein.
A second chimeric genome was generated by a reciprocal recombination fusing nucleotide 3773 of Enterovirus 71 (U22522) to nucleotide 3713 of EV79 again creating a hybrid 2b-encoding domain within polyprotein. Recombination has occurred within the conserved sequence ATGGA.
Untranslated Regions
The strongest sequence conservation is encountered in the 5′ untranslated region (5′-UTR), probably because this region plays a pivotal role in the replication and initiation of protein synthesis.22 Inverted repeats in the 5′-UTR allow extensive internal base pairing and the resulting secondary structure can be predicted using the M-Fold program.14 The initiation codon resides immediately downstream of a CT rich tract (position 643-660) which is part of a putative Internal Ribosome Entry Site (IRES) or ribosome landing pad. This ATG corresponds to the one we identified as initiation codon on the basis of homology. All seven other ATGs preceding the putative initiation codon are out of frame and, according to the M-fold prediction, sequestered in hairpin loops.
Open Reading Frame Analysis
Assuming the ATG in position 683 is the initiation codon we obtain an ORF of 2193 AA. Blast searches were used to tentatively identify the proteolytic cleavage products of the encoded polyprotein.







TABLE 6







Prima 7 encoded proteins












Protein
Position
Number AAs
Deduced Mw
















 VP4*
686-889
 68
7.282



VP2
 890-1654
255
27.912



VP3
1655-2386
244
26.578



VP1
2387-3271
295
32.471



2A
3272-3721
150
16.460



2B
3722-4018
 99
10.730



2C
4019-5002
329
36.974



3A
5003-5257
 85
9.602



3B
5258-5323
 22
2.274



3C
5324-5872
183
20.153



POL
5873-7258
462
52.746



Total
 683-7258
2193 
243.141







*It is assumed that the N-terminal methionine residue is removed, prior to myristylation of VP4






Table 10

VP4 capsid protein (PFAM02226) (SEQ ID NO:39) (M) GAQVSTQKTGSHENQNIAASGSTINYTTINYYKDSYAASAAKQDFSQDPSKFTQPVVDA LKETAPPLK
The VP4 capsid protein contains an N-terminal myristylation consensus site GXXXS/T19 (SEQ ID NO:84)
VP2 capsid protein (CD00205, PFAM00073) (SEQ ID NO:40)







SPSAEACGYSDRVAQLTLGNSTITTQEAANITVGYGEWPEYSKDTEATA


 


VDKPTRPDVSVNRFYTLPAKLWANNSKGWYWKFPDVLCELGVFGQNAQY


 


HYLYRSGFCIHVQCNASKFHQGTLLVAAIPELMLARSSNDTNPATAPHP


 


PYNATQPGEAGKEFAYPYILDSGIPLSQALIFPHQWINLRTNNCATIVM


 


PYINCLPFDSALNHCNFSLVVIPVAPLAYNEGATTAIPITVTVAPMCSE


 


FSGLRQAVVQ






VP3 capsid protein (CD00205, PFAM00073) (SEQ ID NO:41)







GLPAELKPGTNQFLTTDDGVSAPILPGFHPTPEMHIPGEVKNLLEICQV


 


ESILEVNNLTTNKAASQLMTRLLIPVEAQTAVDALCAAFKVDPGRDGPW


 


QSTLVGQICRYYTQWSGSLEVTFMFTGSFMATGKMLIAYTPPGAPQPAN


 


RRIAMLGTHVIWDFGLQSSVTLVIPWISNTHYRAMGSNDYFDYYSAGIV


 


TIWYQTNFVVPSGAPTSAYIIALAAAQKNFTLRLPKDTGDISQTAILQ






VP1 capsid protein (CD00205) (SEQ ID NO:42)







GDPIEEAINNTVAGTLNRALGSASHTTAQNTQQSSHQIGTGEVPALQAA


 


ETGATSNTSDENMLETRCVINSHSVAETSISHFFSRAGLVGMLDLLTSG


 


DTDIGFTSWDIDIMGFVQQRRKLEMFTYMRFDAEFTFLTVGATGAAPAT


 


VIQYMYVPPGAPKPTQRDSFEWQTSTNPSIFVKVSDPPAQVSVPFMSPA


 


AAYQWFYDGYPTFGNHPTNQDFRYGICPNNLMGTFCVRVLGSEKLTEAL


 


RVRIYMRIKHVRAWIPRPLRSQKYLLKNYPNFDGADVTPTSASRANITT


 


A






Capsid Proteins as Vaccine

Neutralizing antibodies against poliovirus target epitopes in VP2, VP3 and VP115 and for EV71 VP1 has been proposed as primary target for vaccine production.24,28 
Additionally, detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection.24 
The elements determining pathogenicity and tissue tropism have been localized in VP126 and the 5′-UTR. For Coxsackievirus A9, modification of the conserved sequence motif PALTAVETGHT (SEQ ID NO:43) results in a reduced capacity to produce infectious progeny virus.1 The Enterovirus 79 VP1 contains a similar sequence motif (in red print, yellow background), so we could claim modification of conserved sequence motifs (indicated in red) as a tool to obtain attenuated strains. Additionally, the three dimensional structure of VP1 harbors an apolar pocket, usually containing a small hydrophobic molecule (pocket factor).22 New antivirals target this apolar pocket, displacing the pocket factor and interfering with the uncoating of the viral genome. We could claim recombinant VP1 as a screening tool for such antiviral compounds.
VP1 could be expressed in a suitable microbial host (e.g., Escherichia coli) as an affinity tagged protein (e.g., His tag and or StrepII tag).
The VP2, VP3 and VP1 protein encoding cDNAs can be amplified by RT-PCR using oligonucleotide pairs VP2-VP2R, VP3F-VP3R and VP1F-VP1R respectively. The PCR primers add attB1 and attB2 sites to respectively the 5′ and 3′ end of all three amplicons. The resulting PCR products of 818, 785 and 938 bp can be inserted into pDONR201 (Invitrogen) by Topoisomerase-mediated ligation generating entry clones for the Gateway system (Invitrogen). The Gateway system allows directional transfer of any sequence located between attB1 and attB2 sites through an efficient in-vitro recombination reaction. Invitrogen supplies Gateway compatible expression vectors for a number of hosts like Escherichia coli (e.g., pBAD-DEST49, pDEST17) Saccharomyces cerevisiae (e.g., pYES2-DEST52), Baculovirus (e.g., pDEST20) as well as insect-(pMT-DEST48) and mammalian cell lines (e.g pT-REX-DEST31).
As an example, the EV79-derived PCR cDNA fragments for VP2, VP3 and VP1 were ligated into pDONR201 and transferred to the pThio-HisB (Invitrogen) Gateway derivative pGP7 to give pEV2, pEV3 and pEV4. The expression construct pEV1 to 3 direct production of VP fusion proteins in E. coli bearing an N-terminal His-tagged thioredoxin domain, which acts as a solubility-enhancing cassette, and a C-terminal StrepII affinity tag. The His- and StrepII affinity tag facilitate detection and recovery. The first step in protein recovery is the lysis of the host cells in buffer containing a suitable cocktail of protease inhibitors. Any inclusion bodies are solubilized by sonication in the presence of 8 M urea. The cleared lysates were loaded on columns containing NiNTA resin (Qiagen) and subjected to metal affinity purification according to the manufacturer's protocol. The eluates of the metal affinity purification were diluted with a suitable buffer and transferred to Streptactin columns as prescribed by the manufacturer (IBA GmbH). In this way, purified proteins can be produced in E. coli.








TABLE 7







Amplification oligos for expression of VP2,



VP3 and VP1 fusion protein








Oligo
Sequence












P2F
ACAAGTTTGTACAAAAAAGCAGGCTTCTCACCATCAGCTGAAG



(SEQ
CATGTGGC


ID NO:


44)


 


P2R
ACCACTTTGTACAAGAAAGCTGGGTCTTGAACCACAGCTTGC


(SEQ
CTAAGACC


ID NO:


45)


 


P3F
ACAAGTTTGTACAAAAAAGCAGGCTTCGGACTACCGGCAGAAT


(SEQ
TAAAACC


ID NO:


46)


 


P3R
ACCACTTTGTACAAGAAAGCTGGGTCTTGCAGGATGGCGGTTT


(SEQ
GTGAG


ID NO:


47)


 


P1F
ACAAGTTTGTACAAAAAAGCAGGCTTCGGTGACCCTATTGAAG


(SEQ
AGGCCATC


ID NO:


48)


 


P1R
ACCACTTTGTACAAGAAAGCTGGGTCAGCTGTTGTGATATTGG


(SEQ
CTCTAGATGC


ID NO:


49)









The VP2, VP3 and VP1 complementary sequences are indicated in bold print. The remainder of the PCR primers is composed of either in-frame attB1 or attB2 sites








TABLE 11





some EV79 proteins















2A protease (PFAM00947) (SEQ ID NO:50)








GVFGQQSGAVYVGNYKIVNRHLATEADWNSLVWESYNRDLLVTSVNAQGC



DTIARCSCKAGVYFCKSMNKHYPVSFQGPGIVEVQANEFYPHRYQTHVLL


GHGTSIPGDCGGILRCQHGVIGLVTMGGDGLVGFADLRDLFWLDDEAMEQ


 


2B (Pico_2B PFAM01552) permeability enhancing


protein (SEQ ID NO:51)


GVTDYIKGLGDAFGTGFTDSISREIQQLKNYLLGSENVVEKILKALIKVV


SALVIVVRSDYDLVTLTATLALIGCHGSPWAWLKSKVSNLLDIPIAQKQ


 


2C (RNA helicase PFAM00910) (SEQ ID NO:52)


SDSWLKKFTEMANAARGLEWIANKISKFIDWVKEKIVPAAKEKVEFLSNL


KQLPLLESQIANIEQSAASQEDLENLFSNVAYLAHYCRKFQPLYASEAKR


IYAMEKRINNYMQFKSKHRIEPVCLIIRGPPGTGKSLATGIIGRAIAEKY


HSSVYSLPPDPDHFDGYKQQVVTVMDDLCQNPDGKDMSLFCQMVSTVEFI


PPMASLEEKGVSFTSKFVIASTNSSNIIVPTVSDSDAIRRRFYMDCDIEV


PESFRTPQGRLDAARAAKLCSENNTANFKKCSPLVCGKAIQLRDRKSGVR


YGLDSVVSELIREYNNRSAVGNTIEALFQ


 


3A (SEQ ID NO:53)


GPPQFKPIRITLDKPAPDAISDLLASVDSEEVRQYCRHQGWIIPEKPTNI


ERHVNRALMILQSVTTVVAVISLVYVIYKLFAGFQ


 


3B or VPg (SEQ ID NO:54)


GAYSGMPKTAVKKPVLRTAVAQ


 


3C protease (PFAM00548) (SEQ ID NO:55)


GPGLDFALSLLKKNIRKCQTDQGHFTLLGIRDRLAVLPRHASPGDSIWIE


HKQIKILDAVELVDEQQVNLELTLITLDTNEKFRDITKFIPEQIEGTADA


TLVINTEAMPSMFVPVGDVQQYGFLNLSGKPTHRTMMYNFPTKAGQCGGV


VTSVGRIVGIHIGGNGRQGFCAALKRSYFASEQ


 


3D or POL RNA dependant RNA Polymerase


(PFAM00680) (SEQ ID NO:56)


GEIQWMKSNKETGNFNINGPTKTKLEPSVFHDVFEGVKEPAVLHSKDKRL


EVDFETALFSKYIGNKMHEPDEYMIQAANHYADQLKQLDIDTSKMSMEDA


CYGTEFLEGIDLATSAGYPYNALGIKKKDILNPQTRDVTKMKMYLDKYGI


DLPYSTYVKDELRAKDKIKKGKSRLIEASSINDSVYLRMCFGHLYEKFHA


NPGTITGSAVGCNPDTFWSKIPIMLPGSLFAFDYTGYDASLSPAWFRALE


IVLKRLGYDQDAISLIEGINHSHHIYRNQTYCVMGGMPSGCSGTSIFNSM


INNIIIRTLLIRTFKGIDLDELNMIAYGDDVLASYPFPIDCAELAKTGLE


YGLVMTPADKSTCFNEVNWENATFLKRGFKPDEQYPFLIHPTMPMKEIHE


SIRWTKDPRNTQDHVRSLCLLAWHNGRETYEEFVDKIRTVPIGKVLALPN


YDNLRRNWLELF









The 2A protease catalyzes its own release from the polyprotein and the inactivation of the host translational initiation factor eIF4Q9,21 which results in the shut down of host protein synthesis after which the IRES dependant viral translation takes over.25 Any compound interfering with this function could be used as an antiviral.
Expression of Coxsackie 2B gradually enhances membrane permeability thereby disrupting the intracellular Ca2+ homeostasis and ultimately causing the membrane lesions that allow release of virus progeny.26 Any compound interfering with this function could be used as an antiviral.
3B or VPg: This protein is covalently attached to the 5′ end of the viral genomic RNA.







TABLE 8







Overall protein similarity to most closely related human group A Enteroviruses














EV71
CoxA16
EV5865
CoxA16
EV5666




AAD44710
AAD55085
AAK13008
NP0042242
AAK30618
EV79

















EV71
100
 89
96
89
95
77


AAD44710


CoxA16

100
90
95
89
77


AAD55085


EV5865


100
90
99
77


AAK13008


CoxA16



100
89
77


NP0042242


EV5666




100 
77


AAK30618


EV79





100 









Example Use of the EV79 Internal Ribosome Entry Site (IRES).
Expression vector pLXRN (GenBank Accession number AF113968) uses a retroviral Long Terminal Repeat (LTR) to drive expression of any cDNA cloned into its polylinker. LTR activity results in transcription of a bicistronic mRNA of which the first open reading frame encodes the protein to be expressed and the second open reading frame encodes the eukaryotic neomycin selection marker. The first and second ORF are separated by an encephalomyocarditis virus-derived IRES, that assures efficient translation of the neomycin resistance.
To exchange the EMCV IRES by the EV79 IRES, first an NcoI site has to be introduced at the initiation codon of the neomycin resistance gene. The restriction site is generated by site directed mutagenesis employing oligonucleotides pLXRN 1 and 2 with the Quickchange kit according to the instruction of its manufacturer (Stratagene). The resulting pLXRN-derivative is the partially NcoI digested and digested to completion with BamHI. The EV79 IRES (EV79 nucleotide positions 1 to 746, FIG. 19) can be amplified from an EV79-containing clinical sample by RT-PCR with primers IRES5 and IRES3, simultaneously adding a BamHI site to its 5′ end and a BspLU11I site at the polyprotein initiation codon. The resulting 762 bp amplicon is BamHI-BspLU11I digested and ligated into the mutagenized pLXRN. The resulting construct is named pEV79 in which the EMCV IRES is replaced by its EV79 ortholog. The pEV79 can be used to transfect mammalian cell lines as previously described.







TABLE 9







pEV79 construction oligonucleotides









Oligo
Use
Sequence













PLXRN1
Mutagenesis
GGGATAATTCCTGCAGCCACCATGGGATCGG



(SEQ ID
PLXRN
CCATTGAAC


NO:57)


 


PLXRN2
Mutagenesis
GTTCAATGGCCGATCCCATGGTGGCTGCAGG


(SEQ ID
PLXRN
AATTATCCC


NO:58)


 


IRES5
Amplifi-
GGGGATCCTTTAAAACAGCTCTAGGGTTG


(SEQ ID
cation


NO:59)
EV79 IRES


 


IRES3
Amplifi-
CCACATGTTCTCTTTCTACTATTCAACTGTA


(SEQ ID
cation
TG


NO:60)
EV79 IRES









EV79 Could Be of Simian Origin

We have discovered a new species of human enterovirus. EV79 occupies a distinct branch within a VP1-derived phylogenetic tree. Genetic clusters of VP1 sequences, the major picornaviral antigen, have been demonstrated to correlate to serotype.5, 17 Recently, a new simian Picornaviral genus and several new species have been proposed solely based on VP1 protein alignments.18 Therefore, EV79 should also represent a new enteroviral serotype. Also in VP2-based and full-length polyprotein-derived phylogenetic trees, EV79 remains distinct from the most closely related group A enteroviruses (e.g., Coxsackie A16 and Enterovirus 71), further strengthening our claim of having discovered a novel enterovirus. The SimPlot analysis shows that throughout its genome, EV79 differs from all other human enteroviruses, excluding the possibility of EV79 arising by inter-species recombination.
Already the close relationship of the simian viruses A13, SV19, 25, 35, 43 and 46 to group A human enteroviruses (e.g., Coxsackie A16 and EV71) prompted already speculations about their potential to infect humans.18 The even closer relationship of the human isolate EV79 to these simian viruses could be proof of this zoonotic potential.
REFERENCES


    1. Caro V., S. Guillot, F. Delpeyroux, and R. Crainic. 2001. Molecular strategy for “serotyping” of human enteroviruses. J. Gen. Virol. 82:79-91.
    2. Oberste M. S., K. Maher, D. R. Kilpatrick, M. R. Flemister, B. A. Brown, and M. A. Pallansch. 1999. Typing of human enteroviruses by partial sequencing of VP1. J. Clin. Microbiol. 37:1288-1293.
    3. Oberste M. S., K. Maher, and M. A. Pallansch. 2002. Molecular phylogeny and proposed classification of the simian picornaviruses. J. Virol. 76:1244-1251.

## Claims

1. An isolated and/or recombinant nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:82 and SEQ ID NO:61.

2. An isolated and/or recombinant nucleic acid that is at least 95% homologous to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 61.

3. An isolated and/or recombinant nucleic acid comprising a stretch of 100 consecutive nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 61.

4. An isolated and/or recombinant nucleic acid encoding a proteinaceous molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NO:83, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:63.

5. An isolated or recombinant virus comprising a nucleic acid sequence according to claim 1.

6. A vector comprising a nucleic acid according to claim 1.

7. A primer and/or probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38.

8. A diagnostic kit comprising a primer/probe according to claim 7.

9. An isolated cell comprising a nucleic acid according to claim 1.

10. An isolated cell according to claim 9, wherein said nucleic acid comprises an expressible coding region of SEQ ID NO:82, or a part of said coding region coding for at least a protein comprising a stretch of 30 consecutive amino acids.

11. An isolated cell according to claim 9, wherein said cell is a eukaryotic cell.

12. A gene delivery vehicle comprising a sequence according to claim 1.

13. An attenuated virus according to claim 5.

14. A polycistronic messenger RNA comprising an internal ribosomal entry site (IRES) that comprises positions 1 to 748 of SEQ ID NO:82.

15. A chimeric enterovirus comprising at least 1000 consecutive nucleotides of SEQ ID NO: 82 and at least 1000 consecutive nucleotides of another enterovirus wherein said latter 1000 consecutive nucleotides comprise a sequence that is more than 5% sequence divergent with SEQ ID NO: 82.

16. An isolated cell comprising a vector according to claim 6.