# Patent

## Title
Coronavirus, nucleic acid, protein, and methods for the generation of vaccine, medicaments and diagnostics

## Abstract

A new coronavirus is disclosed herein with a tropism that includes humans. Means and methods are provided for diagnosing subjects (previously) infected with the virus. Also provided are among others vaccines, medicaments, nucleic acids and specific binding members.

## Background
The invention relates to the fields of virology and medicine. More in particular the invention relates to the identification of a new coronavirus and to means and methods associated with a virus such as means and methods for typing the virus in various samples and diagnosing of disease, means and methods for developing vaccines and medicaments for the treatment of infected subjects or of subjects at risk thereof.
Coronaviruses, a genus in the family of Coronaviridae, are large enveloped plus strand RNA viruses. The genomic RNA is 27 to 32 kb in size, capped and polyadenylated. Three serologically distinct groups of coronaviruses have been identified. Within each group, viruses are identified by hosts range and genome sequence. Coronaviruses have been identified in mice, rats, chickens, turkeys, swine, dogs, cats, rabbits, horses, cattle and humans (39, 40). Most coronaviruses infect only one host species and can cause severe disease including gastroenteritis, and respiratory tract diseases. In humans, 3 coronaviruses have been studied in detail. HCoV-229E and HCoV-OC43 have been identified in the mid sixties and are known to cause common cold (13-17, 19, 41, 42). Besides common cold it has been suggested that the HCoV-229E may cause a more serious disease in infants as HCoV-229E virus has been isolated from infants suffering from lower respiratory tract disease(28). The third and most recently identified coronavirus: SARS-CoV, is, with its ability to cause a life threatening pneumonia (43), the most pathogenic human coronavirus identified thus far. It has been suggested that SARS-CoV is the first member of a fourth group of coronaviruses, or that the virus is an outlier of the group 2 coronaviruses (27, 44).
The genome of coronaviruses encodes four structural proteins: the spike protein, the membrane protein, the envelope protein and the nucleocapsid protein. Several non-structural proteins are involved in replication and transcription, which are encoded by two long overlapping open reading frames (ORFs) at the 5′ end of the genome (1A and 1B). These 2 ORFs are connected via a ribosomal frame shift. The polypeptides encoded by ORF 1A and 1B are post-translationally processed by viral encoded proteases. Furthermore, additional non-structural proteins are encoded between the S and E gene, or between the M and N gene or downstream of the N gene. Some of these “accessory non-structural protein genes” have been found to be not essential for virus reproduction(45, 46). The coronavirus gene products of 1A and 1B are translated from the genomic RNA but the remaining viral proteins are translated from subgenomic mRNAs (sg mRNA), each with a 5′ end derived from the 5′ part of the genome. The sg mRNA are derived via a discontinuous transcription process that most probably occurs during negative strand synthesis (47). Discontinuous transcription requires base-pairing between cis-acting elements, the transcription associated sequences (TRSs), one located at the 5′ part of the genome (the leader TRS) and others located upstream of the ORFs (the body TRSs)(48)).
The novel coronavirus that we present here was isolated from a child suffering from bronchiolitis. Infection by this virus was not an isolated case since we found 7 more persons suffering from respiratory tract disease carrying the virus. In addition, we show here the complete genome sequence providing critical information concerning the genome structure of the new coronavirus.
To date there is a range of human diseases with unknown etiology. For many of these a viral origin has been suggested, emphasizing the importance of a continuous search for new viruses22, 23, 24. Major difficulties are encountered when searching for new viruses. First, some viruses do not replicate in vitro, at least not in the cells that are commonly used in viral diagnostics. Second, for those viruses that do replicate in vitro and that cause a cytopathic effect (CPE), the subsequent virus-identification methods may fail. Antibodies raised against known viruses may not recognize the cultured virus and virus specific PCR methods may not amplify the new viral genome. We have developed a method for virus discovery based on the cDNA amplified restriction fragment length polymorphism technique (cDNA-AFLP). With this technique, RNA or DNA is reproducibly amplified. There is no need to have prior knowledge of the sequence of the target gene1. Generally the cDNA-AFLP method is used to monitor differential gene expression, however, we modified this method such that it can amplify viral sequences either directly from patient blood-plasma/serum samples or indirectly from CPE-positive virus culture (FIG. 1). In the modified Virus-Discovery-cDNA-AFLP (VIDISCA) method the mRNA isolation step prior to amplification is replaced by a treatment to selectively enrich for viral nucleic acid. Of relevance to the purification is a centrifugation step to remove residual cells and mitochondria. In addition, a DNAse treatment can be used to remove interfering chromosomal and mitochondrial DNA from degraded cells whereas viral nucleic acid is protected within the viral particle. Finally, by choosing frequently cutting restriction enzymes, the method can be fine-tuned such that most viruses will be amplified.
In January 2003 a 7-month-old child appeared in the hospital with coryza, conjunctivitis and fever. Chest radiography showed typical features of bronchiolitis and a nasopharyngeal aspirate specimen was collected (sample nr: NL63) five days after the onset of disease. All diagnostic tests on this sample for respiratory syncytial virus (RSV), adenovirus, influenza A and B virus, parainfluenza virus type 1, 2 and 3, rhinovirus, enterovirus, HCoV-229E and HCoV-OC43 were negative. Immunofluorescent assays to detect RSV, adenovirus, influenza A and B virus, and parainfluenza virus type 1, 2 and 3 in cultures of the virus remained negative. Acid lability and chloroform sensitivity tests demonstrated that the virus was most likely enveloped and not a member of the Picornavirus group. In fact it was a new coronavirus.
In the present invention we present a detailed description of a novel human coronavirus. Coronaviruses are characterized by a very long non-segmented, single-stranded, (+) sense RNA of approximately 27-31 kb. This is the longest genome of any known RNA virus. The genome has a 5′ methylated cap and 3′ poly-A and functions directly as mRNA. Thus far only 3 human coronaviruses have been characterized, therefore sorting out the characteristics of a fourth human coronavirus supplies attractive information on the variation among the human coronaviruses. The novel virus is a member of the group 1 coronaviruses and is most related to HCoV-229E, yet the differences are prominent. The similarity is not larger than 85% at the nucleotide level, at the position of the 4A and 4B gene of HCoV-229E only one ORF is present in HCoV-NL63 (ORF 3), and the 5′ region of the S gene of HCoV-NL63 contains a unique in frame insertion of 537 nucleotides. Since binding of the receptor has been mapped to the N-terminal part of the protein, the 179 amino acids encoded by the insertion are most likely involved in receptor binding. This unique part at the N-terminus of the spike protein might explain the expanded host range of the virus in cell culture. Where HCoV-229E is fastidious in cell culture with a narrow host range, HCoV-NL63 replicates efficiently in monkey kidney cells. Besides HCoV-NL63 also SARS-CoV is able to replicate in monkey kidney cells (Vero-E6 cells and NCI-H292 cells for SARS-CoV (21)). Yet, comparing the predicted Spike genes did not identify a protein region that is shared by both viruses to clarify the common host range of the viruses in vitro. Also the insertion in the S gene of HCoV-NL63 was not present in the SARS S gene. Alternatively, other viral proteins may be involved in the cell tropism of a virus, however we did not identify any gene of HCoV-NL63 that had more similarity at the protein level to the SARS-CoV than to the similarity to HCoV-229E.
The 2 major differences between HCoV-229E and HCoV-NL63: the insertion in the S gene and the altered non-structural accessory proteins genes, are comparably to the differences that are noted between the porcine coronaviruses PRCoV and TGEV. Although these 2 porcine viruses are antigenically and genetically related their pathogenicity is very different. TGEV causes severe diarrhea with a high mortality in neonatal swine. It replicates and destroys the enterocytes in the small intestine whereas PRCoV has a selective tropism for respiratory tissue with very little to no replication in intestinal tissue. The genome differences in the S, 3A and 3B genes between TGEV and PRCoV are comparable with the differences between HCoV-NL63 and HCoV-229E. Alike HCoV-NL63, TGEV has a unique in frame insertion at the 5′ part of the S gene ranging from 672 to 681nt (53). Furthermore, the accessory protein genes 3A and 3B that are intact in TGEV, are often mutated or inactive in the PRCoV. Extrapolating these data to the human coronaviruses one can speculate that HCoV-NL63 might be a more pathogenic human virus in comparison with HCoV-229E. However there are no epidemiological data supporting this. Based on our data it seems likely that HCoV-NL63 and HCoV-229E share the same pathogenicity. The common cold virus HCoV-229E can cause a more serious disease in infants (28), comparable to our data that suggest that HCoV-NL63 is causing a respiratory disease only in infants and immuno-compromised patients.
To date, a viral pathogen cannot be identified in a substantial portion of respiratory disease cases in humans (on average 20%59), our data indicate that in a part of these cases HCoV-NL63 is involved. The frequency with which HCoV-NL63 was detected in patients suffering from respiratory disease was up to 5% in January 2003. The virus was not detected in any of the samples collected in the spring or summer of 2003, which is in harmony with the epidemiology of human coronaviruses that have a tendency to spread predominantly in the winter season (15). The primers for our diagnostic PCR were located in the 1B gene and the genomic RNA can be used as template. Using primers that anneal in the nucleocapsid gene or 3′UTR supplies more template in the PCR because besides the genomic RNA also all sg mRNA in infected cells are template for amplification. It might be that the number of persons that we found positive for HCoV-NL63 is an underestimation of the correct number of persons carrying HCoV-NL63.
The newly found coronavirus, (designated HCoV-NL63) was characterized and sequenced. A sequence of a prototype HCoV-NL63 is provided in FIG. 19 and parts thereof in table 3. In one aspect the invention therefore provides an isolated and/or recombinant nucleic acid comprising a sequence as depicted in FIG. 19 and/or table 3, or a functional part, derivative and/or analogue thereof. The virus HCoV-NL63 is characterized by the prototype, however, many natural variants exist as for instance shown in FIG. 16 for polymorphisms in the ORF 1a region. 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. HCoV-NL63 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 HCoV-NL63. 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 HCoV-NL63. The homology between different coronaviruses 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 HCoV-NL63 virus comprising at least 95% homology and preferably at least 99% homology (on the nucleic acid level) in at least one protein coding region depicted FIG. 20, 21, 22, 23, or table 3. 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 said 100 consecutive nucleotides (when measured on the nucleic acid level outside a UTR region). A stretch of 100 consecutive nucleotides is considered to be a functional part of the virus of the present invention. Further provided is a bacterial vector comprising a nucleic acid of HCoV-NL63 or a functional part, derivative and/or analogue thereof. Further provided is a bacterium comprising said bacterial vector. The sequence of HCoV-NL63 or a part thereof can be used to generate a primer that is specific for HCoV-NL63 and thus capable of specifically replicating HCoV-NL63 nucleic acid. Similarly, a probe can be generated that specifically hybridizes to HCoV-NL63 nucleic acid under stringent conditions. Thus the invention further provides a primer and/or probe, capable of specifically hybridizing to a nucleic acid of a HCoV-NL63 virus or functional part, derivative or analogue thereof. Preferably, said primer or probe is capable of hybridizing to said nucleic acid under stringent conditions. In a particularly preferred embodiment said primer and/or probe comprises a sequence as depicted in table 3, table 7, table 10 or FIGS. 16 to 18.
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, 21, 22, 23 or table 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% homologues 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 HCoV-NL63. 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 is provided 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, 21, 22, 23 or table 3. The HCoV-NL63 virus can be replicated using in vitro growing cell lines. The virus can be harvested from such cultures and used in a variety of different application including but not limited to the generation of an immune response in a subject. The invention thus further provides an isolated or recombinant virus comprising a HCoV-NL63 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, 21, 22, 23 or table 3, or a functional part, derivative and/or analogue thereof. Subjects that have become infected with HCoV-NL63 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 a HCoV-NL63-related disease.
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 be generated by means of injection of the virus into in 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 can of course be generated through a large variety of different technologies. For instance phage display technologies. 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 proteinaceous molecule of a HCoV-NL63 virus, preferably against encoded by a nucleic acid of the prototype HCoV-NL63. Preferably, a proteinaceous molecule as depicted in FIG. 20, 21, 22, 23 or table 3, or a functional part, derivative and/or analogue thereof. The binding molecule can be capable of specifically binding a nucleic acid sequence of a HCoV-NL63, preferably of FIG. 19 or table 3. 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 further a method for producing a binding molecule according to the invention comprising

    
    
        producing molecules capable of binding a HCoV-NL63 virus or functional part, derivative or analogue thereof or an isolated and/or recombinant proteinaceous molecule encoded by a prototype nucleic acid of HCoV-NL63, and
        selecting a proteinaceous binding molecule that is specific for said virus and/or said proteinaceous molecule.
    
    


The overall homology of HCoV-NL63 virus with other human coronaviruses is not very high. Thus many different binding molecules capable of specifically binding to HCoV-NL63 virus can be generated. Such binding molecules can be used to detect HCoV-NL63 virus in a sample. The invention thus further provides an isolated or recombinant virus which is immunoreactive with a binding molecule capable of specifically binding HCoV-NL63 virus. Similarly, the invention provides the use of an isolated and/or recombinant proteinaceous molecule as depicted in FIG. 20, 21, 22, 23 or table 3, or a functional part, derivative and/or analogue thereof, for detecting a binding molecule capable of specifically binding HCoV-NL63 virus, or functional part, derivative and/or analogue of said virus in a sample Vise versa, HCoV-NL63 virus can be used to detect a molecule capable of specifically binding said virus in a sample. Binding of HCoV-NL63 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 HCoV-NL63 related disease in a subject. Thus provided is a method for detecting a HCoV-NL63 virus or functional part, derivative or analogue thereof in a sample, comprising hybridizing and/or amplifying a nucleic acid of said virus or functional part, derivative or analogue with a HCoV-NL63 specific primer and/or probe and detecting hybridized and/or amplified product. Further provided is a kit, preferably a diagnostic kit comprising a HCoV-NL63 virus or functional part, derivative or analogue thereof, a binding molecule according to the invention, and/or a HCoV-NL63 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 a HCoV-NL63 virus or functional part, derivative or analogue thereof or a binding molecule capable of specifically binding a proteinaceous molecule depicted in FIG. 20, 21, 22, 23 or table 3 or an HCoV-NL63 virus and/or a nucleic acid or functional part, derivative or analogue of a prototype HCoV-NL63 for detecting and/or identifying a HCoV-NL63 coronavirus in a sample. Preferably said nucleic acid comprises a sequence as depicted in table 3.
The invention further provides a vaccine comprising HCoV-NL63 virus or functional part, derivative or analogue thereof. Further provided is a vaccine comprising a proteinaceous molecule depicted in FIG. 20, 21, 22, 23 or table 3 or functional part, derivative and/or analogue of such a proteinaceous molecule. A proteinaceous molecule of the invention may be provided as a vaccine by itself or as a part of the protein or as derivatives or analogues thereof. A suitable analogue is a nucleic acid encoding a HCoV-NL63 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. As carrier for the DNA vaccine it is often suitable to incorporate an expressible HCoV-NL63 virus nucleic acid in a viral replicon allowing replication of the HCoV-NL63 virus nucleic acid in the target cell and thereby allowing boosting of the provided immune response. A HCoV-NL63 virus encoded protein that is suited for such a DNA vaccine approach is the S protein depicted in FIG. 22 or a functional part, derivative and/or analogue thereof. A part of an S protein preferably comprises an immunogenic part of the 537 in frame insertion as compared with HCoV-229E virus. Preferably said part comprises essentially said 537 insertion. With the 537 insertion is meant a sequence corresponding to sequences 20472 to 21009 of FIG. 19. Other suitable candidates are the M and or the N 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 a HCoV-NL63 virus specific immune response in a subject. The immune response can be both cellular or humoral. Thus further provided is an isolated T-cell comprising a T-cell receptor that is specific for HCoV-NL63 virus or a proteinaceous molecule encoded by a prototype HCoV-NL63 virus. Further provided is an isolated B-cell producing an antibody specific for HCoV-NL63 virus or a proteinaceous molecule encoded by a HCoV-NL63 virus. The antibody or T-cell receptor can be cloned whereupon a cell line can be provided with an expression cassette comprising 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 HCoV-NL63 virus. To this end the subject can be provided with a HCoV-NL63 specific binding molecule of the invention. Such immunity can be used to provide a barrier for (further) infection with HCoV-NL63 virus in the subject, thus further provided is a vaccine comprising a HCoV-NL63 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 a HCoV-NL63 virus of the invention. 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 HCoV-NL63 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 HCoV-NL63 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 a HCoV-NL63 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 a HCoV-NL63 virus specific binding molecule of the invention, for the preparation of a vaccine against a coronaviral genus related disease. Further provided is a method for treating an individual suffering from, or at risk of suffering from, an HCoV-NL63 related disease, comprising administering to said individual a vaccine or medicament according to the invention. In yet another embodiment is provided a method for determining whether an individual suffers from an HCoV-NL63 related disease, comprising obtaining a sample from said individual and detecting a HCoV-NL63 virus or functional part, derivative or analogue thereof in said sample.
In yet another embodiment is provided an isolated cell, or recombinant or cell line comprising HCoV-NL63 virus, or a functional part, derivative and/or analogue thereof. Preferably said cell is a primate cell, preferably a monkey cell. In a preferred embodiment, said cell is a cell that replicates the HCoV-NL63 virus of the invention. In a particular embodiment the cell is a kidney cell. The cell can be used to produce the HCoV-NL63 virus of the invention or to attenuate HCoV-NL63 such that it becomes less pathogenic. Virus attenuation is spontaneous upon continued culture of the virus on the mentioned preferred cell lines. Attenuated HCoV-NL63 virus can be used as a vaccine.
HCoV-NL63 virus encodes an endoprotease. A sequence for the protease in the prototype HCoV-NL63 virus is depicted in FIG. 21). The protease is important for the processing of the polyproteins encoded by HCoV-NL63. 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 HCoV-NL63 virus replication. Preferred compounds are inhibitors of inosine monophosphate dehydrogenase (55) (e.g. Ribavirin(54) and mycophenolic acid), orotidine-5′-phosphate decarboxylase inhibitors (e.g. 6-azauridine and pyrazofurin), 3CL-protease inhibitors(56) (e.g. the VNSTLQ-AG7088 ester, see below), cap-methylase inhibitors(58) (carboxylic adenosine analogs e.g. Neoplanocin A and 3-deazaneoplancin A), nitrous oxide synthase inducing compounds (e.g. glycyrrhizin) and Interferons (57). Of these the protease inhibitors are particularly preferred. The sequence VNSTLQ is the N-terminal proteolytic processing site of SARS-3CLpro that is used in the 3Clpro inhibitor VNSTLQ-AG7088 (56). In this compound the hexapeptide VNSTLQ is C-terminally linked to the vinylogous ethyl ester (AG7088, see structural formula I depicted below,) that inhibits SARS 3CLpro activity.




The hexapeptide VNSTLQ corresponds to YNSTLQ in HCoV-NL63. Therefore YNSTLQ-AG7088 inhibits the HCoV-NL63 3CLpro orthologs. Thus in a preferred embodiment the protease inhibitor comprises the amino acid sequence VNSTLQ more preferably YNSTLQ. 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.
S-adenosylmethionine-dependant ribose 2′-orthomethyltransferase Plays a role in the methylation of cap structure (GpppNm) at the 5′ end of the viral RNA. Antiviral compounds inhibiting this transfer of methyl groups to reaction (carboxylic adenosine analogs e.g. Neoplanocin A and 3-deazaneoplancin A) interfere with expression of viral proteins.
The invention further provides a proteinaceous molecule encoded by HCoV-NL63 nucleic acid, wherein said proteinaceous molecule is a 3CL protease or a functional equivalent thereof. Functional equivalents include an 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 anticoronaviral activity, preferably antiproteolytic activity of a coronavirus. The invention thus further provides a method for determining whether a compound comprises anticoronavirus replication activity characterized in that said method utilizes HCoV-NL63-virus or a HCoV-NL63 protein involved in replication of HCoV-NL63 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 said protease is a 3CL protease of HCoV-NL63 or a functional part, derivative and/or analogue thereof. Preferred compounds that can be tested for 3CL inhibiting quality are hexapeptides located N-terminally of 3Clpro cleavage sites. Compounds effective in at least in part inhibiting 3Cl proteolytic activity can be used for the preparation of a medicament for the treatment of an individual suffering or at risk of suffering from a HCoV-NL63 virus infection.
One or more of the preferred anticoronaviral replication compounds can be used as a medicament for the treatment of a subject suffering from or at risk of suffering from a HCoV-NL63 virus infection. The invention thus further provides a medicament for the treatment of an individual suffering from an coronavirus infection or an individual at risk of suffering there from comprising wherein said coronavirus comprises a nucleic acid sequence of a HCoV-NL63 prototype virus or a functional part, derivative and/or analogue thereof.
In the present invention several different recombinant viruses are produced using HCoV-NL63 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 a HCoV-NL63 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 a HCoV-NL63 virus nucleic acid. Preferably of the prototype virus. Preferably comprising a nucleic acid encoding a protein of HCoV-NL63 virus or a functional part, derivative and/or analogue thereof. The invention also shows chimearic coronaviruses comprising nucleic acid derived from at least two coronaviruses wherein at least one of said parts is derived from a HCoV-NL63 virus. Said HCoV-NL63 virus derived part comprises preferably at least 50 nucleotides of a protein coding domain. More preferably said HCoV-NL63 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 chimearic coronavirus comprising at least 1000 nucleotides of a sequence as depicted in FIG. 19 and at least 1000 nucleotides of another coronavirus wherein said 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 HCoV-NL63 virus fragments are depicted in table 3. The location of the fragments in the large genomic RNA is depicted in FIG. 5. The invention therefore, in one aspect, provides an isolated or recombinant virus comprising a nucleic acid sequence as depicted in table 3, or a functional part, derivative or analogue of said virus. With the aid of the identifying prototype fragments it is possible to further sequence the genome. One way of doing this 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. This procedure can be repeated until the entire sequence of the virus is elucidated. As a source of the virus one may turn to Dr. C. van der Hoek, Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Alignments of the determined nucleic acid sequences revealed the reading frame used in the sequences found, accordingly the invention further provides an isolated or recombinant virus comprising an amino acid sequence as depicted in (table 3). or a functional part, derivative or analogue of said virus. A particular amino acid sequence can be produced from a variety of nucleic acids depending on the codons used. Thus the invention further provides a nucleic acid encoding an amino acid sequence as depicted in (table 3). Further provided is an isolated or recombinant virus comprising a nucleic acid sequence encoding an amino acid sequence as depicted in (table 3), or a functional part, derivative or analogue of said virus.
Coronaviruses as many other types of viruses acquire a plurality of spontaneous and selected mutations upon spreading of the virus through the subject population and/or during culturing ex vivo. Moreover, artificial mutations having no recognized counterpart in nature can be introduced into the sequence of the prototype virus or a derivative thereof, without altering the viral- and/or disease causing properties of the virus. Having characterized the prototype of the newly discovered subtype gives access to this group of viruses belonging to the same subtype. Thus the invention further provides an isolated or recombinant virus comprising a nucleic acid sequence that is approximately 80% homologous to a sequence as depicted in table 3, or 80% homologous to an amino acid sequence depicted in Table 3 (. Preferably the homology is at least 90%, more preferably at least 95% and even more preferably at least 99%.
The respective prototype fragments were compared with a database of viral sequences and hits having a particularly high homology are mentioned in the tables 5 and 6. It may be noted that the compared fragments do not share extensive homology with any of the currently known Coronaviruses. The invention thus provides an isolated and/or recombinant virus comprising an amino acid sequence which is more than 89% homologous to 163-2 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 60% homologous to 163-4 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising a nucleic acid sequence which is more than 85% homologous to 163-9 nucleic acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 94% homologous to 163-10 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 50% homologous to 163-11 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%.
Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 87% homologous to 163-14 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 83% homologous to 163-15 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 78% homologous to 163-18 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising a nucleic acid sequence which is at least 50% homologous to a nucleic acid sequence as depicted in Table 3. Preferably said homology is at least 80%, more preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
The invention also provides a functional part, derivative and/or analogue of an isolated and/or recombinant HCoV-NL63 virus. A part of a virus can be a membrane containing part, a nucleocapsid containing part, a proteinaceous fragment and/or a nucleic acid containing part. The functionality of the part varies with the application chosen for the part, for instance, part of the virus may be used for immunization purposes. In this embodiment the functionality comprises similar immunogenic properties in kind as the entire virus not necessarily in amount. Another use of the virus is the infectivity of the virus, for instance, for in vitro (or in vivo) culture, in this embodiment the functionality comprises a similar infectivity in kind not necessarily in amount. Many other functionalities may be defined, as there are many different uses for viruses, non-limiting examples are the generation of chimeric viruses, (i.e. with one or more other (corona) viruses, and the generation of viral vectors for vaccination and/or gene therapeutic purposes. Such viruses and/or vectors also contain a functional part of HCoV-NL63 and are thus also encompassed in the present invention. A functional derivative of a virus of the invention is defined as a virus that has been altered such that the properties of said compound are essentially the same in kind, 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, etcetera.
Analogous compounds of a virus can also be generated using methods in the art. For instance, a chimeric virus can be produced, or an HCoV-NL63 virus having a chimeric protein. For instance, HCoV-NL63 can be rendered more immunogenic by generating a cell surface associated fusion protein comprising at least part of an HCoV-NL63 surface protein and a non-HCoV-NL63 immunogenic part. HCoV-NL63 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 said virus.
The three groups of coronaviruses are associated with a variety of diseases of humans and domestic animals, including gastroenteritis and upper and lower respiratory tract disease. The human coronaviruses HCoV-229E and HCoV-OC43 are associated with mild disease (the common cold) but more severe disease is observed in children16, albeit at a very low incidence. Several coronaviruses cause a severe disease in animals and SARS-CoV is the first example of a coronavirus that causes severe disease in humans. However, it should be emphasized that a substantial part of respiratory disease cases in humans remains undiagnosed. For instance, a recent survey of respiratory viruses in hospitalized children with bronchiolitis in Canada could not reveal a viral pathogen in about 20% of the patients17. The fact that we identified the new coronavirus in a child with bronchiolitis shows that HCoV-NL63 is a pathogenic respiratory virus.
When considering that the HCoV-NL63 is a pathogenic respiratory virus able to cause bronchiolitis in infected children, the interesting question remains why HCoV-NL63 was not recognized previously by cell culture. We found that the virus can be cultured in monkey kidney cells (tMK or LLC-MK2 cells), cells that are often used in a routine diagnostic setting and one might therefore speculate that HCoV-NL63, like SARS-CoV, was newly introduced from an animal reservoir into the human population or that this is a human virus that recently broadened its host cell range. Clearly it is of importance to study the prevalence of HCoV-NL63 infection, and screening specimens from patients with respiratory tract disease using the HCoV-NL63 diagnostic RT-PCR will shed light on this issue.
It is remarkable that the new human coronavirus was harvested from tMK cells and LLC-MK2 cells since coronaviruses are typically fastidious in cell culture with a narrow host range. However, both SARS-CoV and HCoV-NL63 seem to replicate efficiently in monkey kidney cells (Vero-E6 cells and NCI-H292 cells for SARS-CoV). The recently described genome of SARS-CoV has several exclusive features, including some unique open reading frames that are probably of biological significance15, 18. We will therefore analyze the complete genome sequence of HCoV-NL63 to screen for similarities and differences with SARS-CoV that may determine the expanded host cell range and enhanced pathogenicity of these viruses.
HCoV-NL63 is associated with a particular phenotype in infected subjects. The phenotype can encompass bronchiolitis, coryza, conjunctivitis and fever and may further encompass other respiratory problems and diarrhea. In one embodiment the invention thus further provides an isolated and or recombinant virus of the invention (having one or more of the above mentioned homology) wherein said virus or functional part, derivative and/or analogue further comprises the capability to induce an HCoV-NL63 related disease or symptom in a subject. In another embodiment the invention provides an isolated and/or recombinant virus of the invention further comprising the property to cause CPE in tertiary monkey kidney cells (tMK; Cynomolgus monkey37) and/or upon passage onto the monkey cell line LLC-MK2 (ECCAC 85062804, ATCC CCL-7). In a preferred embodiment said virus does not produce CPE in Vero-cells (ATCC CRL-1586)34.
The invention further provides a nucleic acid as depicted in table 3, and an amino acid sequence as depicted in Table 3, or a functional part and/or equivalent of such a nucleic acid and/or amino acid sequence. A functional equivalent of said nucleic acid comprises the same hybridization properties in kind, not necessarily in amount, as said nucleic acid (or part thereof). A functional equivalent of an amino acid sequence of the invention comprises the same immunogenic properties in kind, not necessarily in amount, as said amino acid sequence (or part thereof). A part of a nucleic acid of the invention comprises at least 15 nucleotides, preferably at least 20, more preferably at least 30 nucleotides. A part of an amino acid sequence comprises at least 5 amino acids in peptidic linkage with each other, more preferably at least 8, and more preferably at least 12, more preferably at least 16 amino acids. In a preferred embodiment said nucleotides and/or amino acids are at least semi-consecutive, more preferably, said nucleotides and/or amino acids are consecutive. An equivalent of a nucleic acid and/or amino acid sequence of the invention or part thereof comprises at least 80% homology to a nucleic acid and/or amino acid sequence of the invention, preferably at least 90% homology, more preferably at least 95% and even more preferably at least 99% homology to a nucleic acid and/or amino acid sequence of the invention or a part thereof.
The invention further provides a primer and/or probe, capable of specifically hybridizing to a nucleic acid of a virus or functional part, derivative or analogue according to the invention, preferably a primer and/or probe, capable of specifically hybridizing to a nucleic acid sequence as depicted in Table 3. More preferably, a primer and/or probe, which is capable of hybridizing to said nucleic acid under stringent conditions. In a particular preferred embodiment is provided a primer and/or probe, comprising a sequence as depicted in Table 7.
The art knows many ways in which a specific binding member can be generated against an identified nucleic acid, lipid and/or amino acid sequence. Such specific binding members may be of any nature but are typically of a nucleic acid and/or proteinaceous nature. The invention thus further provides an isolated molecule capable of specifically binding a virus, nucleic acid and/or amino acid or functional part, derivative or analogue thereof according to the invention. Said isolated molecule is also referred to as specific binding member. Preferably said specific binding member is capable of specifically binding at least part of a nucleic acid sequence as depicted in table 3 and/or at least part of an amino acid sequence as depicted in Table 3. In a preferred embodiment said binding member is a proteinaceous molecule. Preferably an antibody or a functional part, derivative and/or analogue thereof. A specific binding member preferably comprises a significantly better binding property for the HCoV-NL63 virus compared to unrelated control. However, for instance for antibodies, it is possible that the epitope specifically recognized in HCoV-NL63 is also present in a limited number of other molecules. Thus though the binding of the binding member may be specific, it may recognize also other molecules than those present in HCoV-NL63. This cross-reactivity is to be separated from a-specific binding and is a general property of antibodies. Cross-reactivity does not usually hinder the selection of suitable specific binding members for particular purposes. For instance a specific binding member that also recognized a protein in liver cells can be used in many applications even in the presence of liver cells, where additional information such as location in the cell can often be used to discriminate.
One source of an antibody of the invention is the blood of the infected subjects screened for the virus of the present invention. One may further characterize B-cells obtained from said subject. A suitable B-cell may be cultured and the antibody collected. Alternatively, the antibody may be sequenced from this B-cell and generated artificially. Another source of an antibody of the invention can be generated by immunisation of test animals or using artificial libraries to screen a purified fraction of virus. A functional part of an antibody has essentially the same properties of said antibody in kind, not necessarily in amount. Said functional part is preferably capable of specifically binding an antigen of HCoV-NL63. However, said functional part may bind such antigen to a different extend as compared to said 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 said antibody.
Once antibody of the invention is obtained, a desired property, such as its binding capacity, can be improved. 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. Said 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 said desired property. Generated data are used to design an improved proteinaceous molecule.
There are many different ways in which a specific binding member can be generated. In a preferred embodiment the invention provides a method for producing a specific proteinaceous binding member comprising producing proteinaceous molecules capable of binding a virus according to the invention or to a functional part, derivative or analogue, and selecting a proteinaceous molecule that is specific for said virus. If need be, the method may be used to generate a collection of proteinaceous molecules capable of binding to said virus or functional part, derivative and/or analogue thereof and selecting from said collection one or more binding members capable of specifically binding said virus or functional part, derivative and/or analogue thereof.
Any specific binding member is characteristic for the HCoV-NL63virus of the invention. Thus a virus that is specifically reactive with such binding member is an HCoV-NL63 virus and thus provided by the invention. Thus the invention provides an isolated and/or recombinant virus that is immunoreactive with specific binding member of the invention, preferably a proteinaceous binding member. The invention further provides a composition of matter comprising isolated HCoV-NL63 virus, and/or a virus essentially corresponding to HCoV-NL63. The term, a virus “essentially corresponding to HCoV-NL63” refers to HCoV-NL63 viruses which are either identical to the HCoV-NL63 strain described hereinabove, or which comprises one or more mutations compared to the said HCoV-NL63strain. These mutations may include natural mutations or artificial mutations. Said mutations of course should allow detection with a specific binding member of HCoV-NL63, not necessarily with all of the specific binding members). Said mutations should allow the detection of the variants using common detection methods such as antibody interaction, amplification and/or hybridization.
Considering that specific binding members are important molecules for instance for diagnostic purposes, the invention further provides the use of a virus of the invention or functional part, derivative and/or analogue thereof, for detecting a molecule capable of specifically binding said virus in a sample. Further provided is the use of a nucleic acid and/or amino acid sequence of a virus or functional part, derivative or analogue as defined by the invention, for detecting a molecule capable of specifically binding said virus or functional part, derivative and/or analogue in a sample. Preferably said nucleic acid and/or amino acid sequence comprises a sequence as depicted in table 3 or Table 3 or a functional part, derivative or analogue thereof. Preferably said part is at least 30 nucleotides and/or amino acids long wherein said part preferably comprises more than 95% sequence identity, preferably more than 99%. In a preferred aspect said specific binding member comprises a specific ligand and/or antibody of said virus.
Further provided is a primer and/or probe according to the invention, a specific binding member of the invention, and/or a nucleic acid of a virus or functional part, derivative or analogue according to the invention, for detecting and/or identifying a HCoV-NL63 coronavirus or part thereof in a sample. Preferably, said nucleic acid comprises a sequence as depicted in table 3.
HCoV-NL63 virus may be used to generate an immune response in a subject. This can be useful for instance in vaccination strategies. Thus the invention further HCoV-NL63 provides HCoV-NL63 virus or functional part, derivative or analogue thereof for use as a vaccine or medicament. The medicament use is typically when the subject is already infected with the virus and the immunogen is used to augment the immune response against the virus. The invention further provides a specific binding member of the invention for use as a vaccine or medicament. This use is particularly favorable for when the specific binding member comprises a proteinaceous molecule, preferably an antibody or functional part, derivative and/or analogue thereof. Such an antibody can provide passive immunity but may also have active components such as proteases attached to it. The medicament use may again be the case wherein a subject infected with an HCoV-NL63 virus is treated with the specific binding member.
Vaccines may be generated in a variety of ways. One way is to culture the HCoV-NL63 virus for example on the mentioned monkey cell line(s) and to use inactivated virus harvested from the culture. Alternatively, attenuated virus may be used either inactivated or as a live vaccine. Methods for the generation of coronavirus vaccines may be adapted to produce vaccines for the HCoV-NL63 of the invention. The invention thus further provides the use of an HCoV-NL63 virus or functional part, derivative or analogue thereof for the preparation of a vaccine against a coronaviral genus related disease. The invention further provides the use of a specific binding member of the invention for the preparation of a vaccine or medicament against a coronaviral genus related disease. Further provided is the use of an HCoV-NL63 virus or functional part, derivative or analogue thereof, a specific binding member of the invention, a nucleic acid of the invention or a primer and/or probe of the invention for diagnosis of a coronaviral genus related disease. Preferably said coronaviral genus related disease comprises a HCoV-NL63coronavirus related disease.
Further provided is a vaccine comprising an HCoV-NL63 virus or functional part, derivative or analogue thereof and/or a specific binding member of the invention. Also provided is a medicament comprising an HCoV-NL63virus or functional part, derivative or analogue thereof and/or a specific binding member of the invention. Preferably said vaccine or medicament is used for at least in part preventing and/or treating a HCoV-NL63 related disease.
An important use of the present invention is the generation of a diagnostic tool for determining whether a subject is suffering from an HCoV-NL63 virus infection or has been exposed to an HCoV-NL63 virus infection. Many different diagnostic applications can be envisioned. They typically contain an identifying component allowing the typing of the virus that is or was present in the subject. One diagnostic tool for HCoV-NL63 makes use of the particular proliferation characteristics of the virus in various cell lines. It replicates in the mentioned preferred monkey cell lines but does not replicate in Vero-cells. This property can be used to discriminate HCoV-NL63 from other known coronaviruses. Thus in one aspect the invention provides a diagnostic kit comprising at least one of the preferred monkey cell lines, preferably the tertiary monkey kidney cells (tMK; Cynomolgus monkey or the monkey cell line LLC-MK2.
Many modern diagnostic kits comprise a specific binding member (to detect the virus or virus infected cells) and/or an HCoV-NL63 virus or a functional part, derivative and/or analogue thereof and/or amino acid of the invention or a functional part, derivative and/or analogue thereof (for detecting antibodies in blood components of the diagnosed subject). Many other current diagnostic kits rely on identification of HCoV-NL63 virus specific nucleic acid in a sample. There are various ways in which such an assay may be implemented one is a method for detecting an HCoV-NL63 virus or functional part, derivative or analogue thereof in a sample, comprising hybridizing and/or amplifying a nucleic acid of said virus or functional part, derivative or analogue with a primer and/or probe according to the invention and detecting hybridized and/or amplified product. The invention thus also provides a diagnostic kit comprising an HCoV-NL63 virus or functional part, derivative or analogue thereof, a specific binding member according to the invention and/or a primer/probe according to the invention.
Further provided is a method for treating an individual suffering from, or at risk of suffering from, a HCoV-NL63 related disease, comprising administering to said individual a vaccine or medicament according to the invention. Also provided is a method for determining whether an individual suffers from a HCoV-NL63 related disease, comprising obtaining a sample from said individual and detecting a HCoV-NL63 virus or functional part, derivative or analogue thereof in said sample with a method and/or diagnostic kit of the invention.
Further provided is an isolated or recombinant nucleic acid encoding a virus or functional part, derivative and/or analogue according to the invention and a nucleic acid according to the invention, comprising at least a functional part of a sequence as depicted in Table 3. Further provided is an amino acid sequence encoded by a nucleic acid according to the invention, and an amino acid sequence according to the invention, comprising at least a functional part of a sequence as depicted in Table 3. Further provided is a proteinaceous molecule capable of specifically binding HCoV-NL63, obtainable by a method according to the invention and, the use of such a proteinaceous molecule in a vaccine or a diagnostic method for the detection of HCoV-NL63.
EXAMPLES
Example 1
cDNA-AFLP for Virus Discovery
We modified the cDNA-AFLP technique such that it can amplify viral sequences from blood-plasma/serum samples or from CPE-positive culture supernatants (FIG. 1). In the adjusted method the mRNA isolation step prior to amplification is replaced by a treatment to purify viral nucleic acid. Of importance to the purification is a centrifugation step to remove residual cells and mitochondria. In addition, a single DNAse treatment is sufficient to get rid of interfering chromosomal DNA and mitochondrial DNA from broken down cells and finally, by choosing frequent cutting restriction enzymes, the method is fine-tuned such that the majority of viruses will be amplified. With this so-called Virus Discovery cDNA-AFLP (VIDISCA) we were able to amplify viral nucleic acids from EDTA-plasma of a person with hepatitis B virus infection and a person with an acute Parvo B19 infection (results not shown). The technique can also detect HIV-1 in a positive culture supernatant demonstrating its capacity to identify both RNA and DNA viruses (results not shown).
To eliminate residual cells, 110 μl of virus culture supernatant was spun down for 10 min at maximum speed in an Eppendorf microcentrifuge (13500 rpm). One hundred μl was transferred to a fresh tube and DNAse treated for 45 minutes at 37° C. using 15 μl of DNAse buffer and 20 Units of DNAse I (Ambion). The DNAse treatment was included to get rid of chromosomal DNA from broken down cells. After this 900 μl of L6 lysis buffer and 40 μl of silica suspension was added and nucleic acids were extracted as described by Boom4. The viral nucleic acids were eluted in 40 μl H2O. With 20 μl eluate the reverse transcription was performed using 2.5 μg random hexamers (Amersham Bioscience), 200 U MMLV-RT (InVitrogen) in a buffer containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1% Triton X-100, 4.8 mM MgCl2, and 0.4 mM of each dNTP. The sample was incubated at 37° C. for 90 minutes. Subsequently the second strand DNA synthesis was performed using 26 U Sequenase II (Amersham Bioscience), 7.5 U RNAse H (Amersham Bioscience) in 0.25 mM dNTPs each, 17.5 mM MgCl2 and 35 mM Tris-HCl pH 7.5. After the incubation at 37° C. for 90 minutes a phenol/chloroform extraction was performed followed by an ethanol precipitation. The pellet was dissolved in 30 μl of H2O. The cDNA-AFLP was performed essentially as described by Bachem1 with some modifications. The dsDNA was digested with the HinP I and MseI restriction enzymes (New England Biolabs) according to the manufacturers protocol. After the digestion, MseI adaptor and HinP I adaptor (see below) are added together with 5U ligase enzyme (InVitrogen) and ligase buffer, followed by an additional incubation of 2 hrs at 37° C. The MseI adaptor and HinP I adaptor were prepared previously by mixing a top strand oligo for the MSE and the HinPI adaptors (Table 1) with a bottom strand oligo for the MSE adaptor and for the HinPI adaptor, incubate at 65° C. followed by cooling down to room temperature in the presence of a 1:40 dilution of ligase buffer.
The first PCR was performed with 10 μl of ligation mixture as input, 2.5 U of AmpliTaq polymerase (Perkin-Elmer), 100 ng of HinPI standard primer and 100 ng of MseI standard primer. The PCR reaction was performed according to the profile 5 min 95 C; 20 cycles of: 1 min 95° C.-1 min 55° C.-2 min 72° C.; 10 min 72° C. Five μl of first PCR product was used as input in the second “selective” amplification step containing 100 ng of HinPI-N primer and 100 ng MseI-N (sequence of the standard primers extended with one nucleotide) and 2 U AmpliTaq polymerase. The selective PCRs were amplified according to the profile of the “touch down PCR”: 10 cycles of 60 sec 94° C.-30 sec 65° C.-1 min 72° C. over which the annealing temperature was reduced from 65° C. with 1° C. with each cycle, followed by 23 cycles: 30 sec 94° C.-30 sec 56° C.-1 min 72° C. Finally the sample was incubated for 10 min at 72° C. The PCR products were evaluated on 4% Metaphor® gels (Cambrex, Rockland, USA). If the bands on the gel were very faint the PCR products were concentrated by vacuum drying using 60 μl of the PCR product. The PCR fragments of interest were cut out of gel and DNA was eluted from the gel using the Qiagen gel purification kit according to the manufacturer's protocol. The PCR products were cloned using pCR® 2.1-TOPO plasmid (InVitrogen) and chemically competent One Shot E. coli (InVitrogen). A PCR on the colony was performed and this PCR product was input for sequencing the insert using Big Dye terminator chemistry (Applied Biosystems). The reverse transcription step was excluded, only HinP I digestion and adaptor ligation was performed, the first PCR was performed with 35 cycles instead of 20 and those first PCR fragments were visualized on agarose gel electrophoresis.
DNA Sequencing and Analysis.
Coronavirus-PCR product containing plasmids were sequenced with the BigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.), using the −21 M13RP and T7 primers. Electrophoresis of sequencing reaction mixtures was performed with an Applied Biosystems 377 automated sequencer, following the manufacturer's protocols. The Sequence Navigator (version 1.01) and Auto Assembler (version 2.1) software packages (ABI, California, USA) were used to analyze all sequencing data. Sequences were compared to all sequences in the Genbank database using the BLAST tool of the NCBI. For phylogenetic analysis the sequences were aligned using the ClustalX software package34 with the following settings: Gap opening penalties: 10.00; Gap extension penalty 0.20, Delay divergent sequences switch at 30% and transition weight 0.59. Phylogenetic analysis was carried out using the neighbor-joining method of the MEGA program (9). The nucleotide distance matrix was generated either by Kimura's 2-parameter estimation or by the p-distance estimation (5). Bootstrap resampling (500 replications) was employed to place approximate confidence limits on individual branches.
Determining the Nucleotide Sequence of the Complete HCoV-NL63 Genome.
Using a combination of specific primers, located in the already sequenced domains of the HCoV-NL63 genome, and the proprietary PALM-method (WO 0151661) we are in the process of cloning and determining the full-length genomic sequence for this new coronavirus. Using a combination of 5′-oligonucleotides located in the analyzed part of the HCoV-NL63 genome and a 3′ tagged random primer (JZH2R) additional fragments were amplified using a nested RT-PCR protocol similar to the one mentioned previously.
Isolation of SZ 163
In January 2003 a 7-month-old child appeared in hospital with coryza, conjunctivitis and fever. Chest radiography showed typical features of bronchiolitis and four days after the onset of disease a nasopharyngeal aspirate specimen was collected (sample nr: HCoV-NL63). All routinely used tests on this sample for adenovirus, respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, rhinovirus, HCoV-229E and HCoV-OC43 were negative. The clinical sample was subsequently inoculated onto a variety of cells including human fibroblast lung (HFL) cells, tertiary monkey kidney cells (tMK; Cynomolgus) and R-HeLa cells. A CPE was detected exclusively on tMK cells and first noted at eight days post-inoculation. The CPE was diffuse with a refractive appearance in the affected cells followed by cell detachment after 7 days. More pronounced CPE was observed upon passage onto LLC-MK2 cells. Besides overall cell rounding, moderate cell enlargement was observed. Additional subculturing on human endothelial lung cells, HFL, Rhabdomyosarcoma cells and Vero cells remained negative for CPE. Immunofluorescent assays to detect influenzavirus A and B, RSV, adenoviruses or parainfluenza virus types 1, 2 or 3 in the culture remained negative
The culture supernatant of infected LLC-MK2 cells was subsequently analyzed by VIDISCA. As control we used the supernatant of uninfected LLC-MK2 cells. After the second PCR amplification step, several DNA fragments were present in the test sample but not in the control. These fragments were cloned and sequenced. A Blast search in GenBank revealed that 8 of 16 fragments had sequence similarity to the family of corona viruses with the highest homology the human corona virus 229E (Tables 4 and 5).
Phylogenetic analysis of a 270 nt fragment of the replicase 1B region indicated that we identified a distinct new member of the coronavirus group 1. With the VIDISCA technique, 8 HCOV-163-specific fragments, named 163-2, 163-4, 163-9, 163-10, 163-11, 163-14, 163-15 and 163-18 were isolated, cloned, sequenced and aligned with the relevant sequences from GenBank. The Genbank accession number of the used sequences are: MHV (mouse hepatitis virus): AF201929; HCoV-229E: AF304460; PEDV (porcine epidemic diarrhea virus): AF353511; TGEV (transmissible gastroenteritis virus): AJ271965; SARS-CoV: AY278554; IBV (avian infectious bronchitis virus): NC—001451; BCoV (bovine coronavirus): NC—003045; FCoV (feline coronavirus): Y13921 and X80799; CCoV (canine coronavirus): AB105373 and A22732; PRCoV (porcine respiratory coronavirus): M94097; FIPV (feline infectious peritonitis virus): D32044. Position of the HCoV-NL63 fragments compared to HCoV-229E (AF304460): Replicase 1AB gene: 15155-15361, 16049-16182, 16190-16315, 18444-18550, Spike gene: 22124-22266; Nucleocapsid gene: 25667-25882 and 25887-25957; 3′UTR: 27052-27123. Branch lengths indicate the number of substitutions per sequence. From the most closely related species sequence identity scores were calculated (Tables 5 and 6).
Also the deduced amino acid sequence were aligned to the corresponding domains in the open reading frames of related corona (-like) viruses (Table 6).
The human corona viruses account for 10 to 30% of the common colds in man7, and it is not unusual to find a coronavirus in a child with a respiratory illness. However, it is striking that the virus HCoV-NL63 was harvested from LLC-MK cells. Human Corona virus 229E and OC-43 are known for there inability to replicate on monkey kidney cells. Intriguingly, the newly identified human corona virus that is responsible for SARS is also able to replicate in monkey kidney cells30.
Propagation of HCoV-NL63 in Cell Culture
A nasopharyngeal aspirate was collected 4 days after the onset of symptoms. The specimen was tested for the presence of adenovirus, RSV, influenza A, influenza B, and parainfluenza type 1, 2 an 3 using the Virus Respiratory Kit (Bartels: Trinity Biotech plc, Wicklow Ireland). In addition, PCR diagnosis for rhinoviruses, meta-pneumovirus and HCoV-OC43 and HCoV-229E were performed2, 10. The original nasopharyngeal aspirate was subsequently inoculated onto a variety of cell cultures including HFL cells, tMK cells and R-HeLa cells. The tubes were kept in a roller drum at 34° C. and observed every 3 to 4 days. Maintenance medium was replenished every 3 to 4 days. Two different types of medium were implemented: Optimem 1 (Gibco) without bovine fetal serum was used for the tMK cells and MEM Hanks'/Earle's medium (Gibco) with 3% bovine fetal serum was used for the remaining cell types. On the virus culture direct staining was performed with pools of fluorescent-labeled mouse antibodies against influenzavirus A and B, RSV and adenoviruses (Imagen, DAKO). Indirect staining was performed for parainfluenza virus types 1, 2 or 3 with mouse antibodies (Chemicon, Brunschwig, Amsterdam Netherlands) and subsequent staining with labeled rabbit anti-mouse antibodies (Imagen, DAKO).
Method to Detect HCoV-NL63 in Nasopharyngeal Swabs.
For the diagnostic RT-PCR, nucleic acids were extracted by the Boom method4 4 from 50 μl virus supernatant or 50 μl suspended nasopharyngeal swab. The reverse transcription was performed as described above with the exception that 10 ng of reverse transcription primer repSZ-RT (Table 7) was used. The entire RT mixture was added to the first PCR mixture containing 100 ng of primer repSZ-1 and 100 ng of primer repSZ-3. The PCR reaction was performed according to the profile 5 min 95° C.; 20 cycles of: 1 min 95° C.-1 min 55° C.-2 min 72° C.; 10 min 72° C. A nested PCR was started using 5 μl of the first PCR with 100 ng of primer repSZ-2 and 100 ng of primer repSZ-4. Twenty-five PCR cycles were performed of the same profile as the first PCR.
Ten μl of the first and 10 μl of the nested PCR was analyzed by agarose gel electrophoresis (FIG. 2). Cloning and sequencing of the fragments was performed essentially as described above.
Method of Raising Polyclonal Antibodies
Appropriate domains within the HCoV-NL63 surface proteins (e.g. S-glycoprotein or HE-glycoprotein) 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 previously described38). 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, tranquillized 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 S-glycoprotein perhaps combined with the HE, M and N proteins, could be expressed in a suitable eukaryotic host (e.g. Y. lipolytica or LLC-MK2 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 HCoV-NL63 virus can be propagated in a suitable cell line as described above and subsequently treated as described by Wu11. 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 Blondel3.
Analysis of S Glycoprotein or any of the HCOV-NL63 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 S-glycoprotein 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 S-glycoprotein 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 (S-glycoprotein) were used, whereas for the measurement of koff, higher concentrations of ligate were employed (1 μM) to avoid any rebinding artefacts. 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.
Example 2
Methods
Virus Isolation
The child, who was living in Amsterdam, was admitted to the hospital with complaints of coryza and conjunctivitis since 3 days. At admission she had shortness of breath and refused to drink. The patient's temperature was 39° C., the respiratory rate was 50 breaths/min with oxygen saturation of 96% and her pulse was 177 beats/min. Upon auscultation bilateral prolonged expirium and end-expiratory wheezing was found. A chest radiograph showed the typical features of bronchiolitis. The child was treated with salbutamol and ipratropium at the first day, followed by the use of salbutamol only for 5 days. The child was seen daily at the out patient clinic and the symptoms gradually decreased. A nasopharyngeal aspirate was collected 5 days after the onset of symptoms. The specimen was tested for the presence of RSV, adenovirus, influenza A and B virus, and parainfluenza virus type 1, 2 and 3 using the Virus Respiratory Kit (Bartels: Trinity Biotech plc, Wicklow Ireland). In addition, PCR tests for rhinoviruses, enterovirus, meta-pneumovirus and HCoV-OC43 and HCoV-229E were performed (2, 10). The original nasopharyngeal aspirate was inoculated onto a variety of cells. The cultures were kept in a roller drum at 34° C. and observed every 3 to 4 days. Maintenance medium was replenished every 3 to 4 days. Two different types of medium were implemented: Optimem 1 (InVitrogen, Breda, The Netherlands) without bovine fetal serum was used for the tMK cells and MEM Hanks'/Earle's medium (InVitrogen, Breda, The Netherlands) with 3% bovine fetal serum was used for the remaining cell types. Cell cultures that were infected with the aspirate specimen were stained for the presence of respiratory viruses after one week of incubation. Direct staining was performed with pools of fluorescent-labeled mouse antibodies against RSV and influenza A and B virus (Imagen, DakoCytomation Ltd, Cambridge, UK). Indirect staining was performed for adenoviruses and parainfluenza virus type 1, 2 or 3 with mouse antibodies (Chemicon International, Temecula, Calif.) and subsequent staining with FITC-labeled rabbit anti-mouse antibodies (Imagen, DakoCytomation Ltd, Cambridge, UK).
VIDISCA Method
To remove residual cells and mitochondria, 110 ill of virus culture supernatant was spun down for 10 min at maximum speed in an eppendorf microcentrifuge (13500 rpm). To remove chromosomal DNA and mitochondrial DNA from the lysed cells, 100 μl was transferred to a fresh tube and treated with DNAse I for 45 min at 37° C. (Ambion, Huntingdon, UK). Nucleic acids were extracted as described by Boom et al. (4). A reverse transcription reaction was performed with random hexamer primers (Amersham Bioscience, Roosendaal, The Netherlands) and MMLV-RT (InVitrogen, Breda The Netherlands) while second strand DNA synthesis was carried out with Sequenase II (Amersham Bioscience, Roosendaal, The Netherlands). A phenol/chloroform extraction was followed by an ethanol precipitation. The cDNA-AFLP was performed essentially as described by Bachem et al (1) with some modifications. The dsDNA was digested with the HinP I and Mse I restriction enzymes (New England Biolabs, Beverly, Mass.). Mse I- and HinP I-anchors (see below) were subsequently added with 5U ligase enzyme (InVitrogen, Breda, The Netherlands) in the supplied ligase buffer for 2 hrs at 37° C. The Mse I- and HinP I-anchors were prepared by mixing a top strand oligo (5′-CTCGTAGACTGCGTACC-3′ (SEQ. ID. NO: 3) for the Mse I anchor and 5′-GACGATGAGTCCTGAC-3′ (SEQ. ID. NO: 4) for the HinP I anchor) with a bottom strand oligo (5′-TAGGTACGCAGTC-3′ (SEQ. ID. NO: 5) for the Mse I anchor and 5′-CGGTCAGGACTCAT-3′ (SEQ. ID. NO: 6) for the HinP I anchor) in a 1:40 dilution of ligase buffer. A 20 cycle PCR was performed with 10 I of the ligation mixture, 100 ng HinP I standard primer (5′-GACGATGAGTCCTGACCGC-3′ (SEQ. ID. NO: 7)) and 100 ng Mse I standard primer (5′-CTCGTAGACTGCGTACCTAA-3′ (SEQ. ID. NO: 1)). Five I of this PCR product was used as input in the second “selective” amplification step with 100 ng HinPI-N primer and 100 ng MseI-N (the “N” denotes that the standard primers are extended with one nucleotide: G, A, T or C). The selective rounds of amplification were done with a “touch down PCR”: 10 cycles of [60 sec 94° C.-30 sec 65° C.-1 min 72° C.] and the annealing temperature was reduced with 1° C. each cycle, followed by 23 cycles: [30 sec 94° C.-30 sec 56° C.-1 min 72° C.] and 1 cycle 10 min 72° C. The PCR products were analyzed on 4% Metaphor® agarose gels (Cambrex, Rockland, Me.) and the fragments of interest were cloned and sequenced using BigDye terminator reagents. Electrophoresis and data collection was performed on an ABI 377 instrument.
cDNA Library Construction and Full Genome Sequencing
The cDNA library was produced as described by Marra et al17, with minor modifications. During reverse transcription only random hexamer primers were used and no oligo-dT primer, and the amplified cDNA was cloned into PCR2.1-TOPO TA cloning vector. Colonies were picked and suspended in BHI media. The E. coli suspension was used as input in a PCR amplification using T7 and M13 RP for amplification. The PCR products were subsequently sequenced with the same primers that were used in the PCR-amplification and the BigDye terminator reagent. Electrophoresis and data collection was performed on an ABI 377 instrument. Sequences were assembled using the AutoAssembler DNA sequence Assembly software version 2.0.
Diagnostic RT-PCR
From 492 persons a total of 600 respiratory samples collected between December 2002 and August 2002. The kind of material ranged from oral/nasopharyngeal aspirate, throat swabs, bronchioalveolary lavages and sputum. The samples were collected for routine virus diagnostic screening of persons suffering from upper and lower respiratory tract disease. One hundred 1 of the sample was used in a Boom extraction (4). The reverse transcription was performed with MMLV-RT (InVitrogen) using 10 ng or reverse transcription primer (repSZ-RT: 5′-CCACTATAAC-3′ (SEQ. ID. NO: 9)). The entire RT mixture was added to the first PCR mixture containing 100 ng of primer repSZ-1 (5′-GTGATGCATATGCTAATTTG-3′ (SEQ. ID. NO: 10)) and 100 ng of primer repSZ-3 (5′-CTCTTGCAGGTATAATCCTA-3′ (SEQ. ID. NO: 11)). The PCR reaction was performed according to the profile 5 min 95 C; 20 cycles of: 1min 95° C.-1min 55° C.-2 min 72° C.; 10 min 72° C. A nested PCR was started using 5 S1 of the first PCR with 100 ng of primer repSZ-2 (5′-TTGGTAAACAAAAGATAACT-3′ (SEQ. ID. NO: 12)) and 100 ng of primer repSZ-4 (5′-TCAATGCTATAAACAGTCAT-3′ (SEQ. ID. NO: 13)). Twenty-five PCR cycles were performed of the same profile as the first PCR. Ten μI of the PCR products was analyzed by agarose gel electrophoresis. All positive samples were sequenced to confirm the presence of HCoV-NL63 in the sample.
Sequence Analysis
Sequences were compared to all sequences in the Genbank database using the BLAST tool of the NCBI. For phylogenetic analysis the sequences were aligned using the ClustalX software package with the following settings: Gap opening penalties: 10.00; Gap extension penalty 0.20; Delay divergent sequences switch at 30% and transition weight 0.5 (9). Phylogenetic analysis was carried out using the neighbor-joining method of the MEGA program (5) using the information of all fragments within one gene. The nucleotide distance matrix was generated either by Kimura's 2 parameter estimation or by the p-distance estimation (6). Bootstrap resampling (500 replicates) was employed to place approximate confidence limits on individual branches.
Results
Virus Isolation from a Child with Acute Respiratory Disease
In January 2003 a 7-month-old child appeared in the hospital with coryza, conjunctivitis and fever. Chest radiography showed typical features of bronchiolitis and a nasopharyngeal aspirate specimen was collected five days after the onset of disease (sample NL63). Diagnostic tests for respiratory syncytial virus (RSV), adenovirus, influenza A and B virus, parainfluenza virus type 1, 2 and 3, rhinovirus, enterovirus, HCoV-229E and HCoV-OC43 remained negative. The clinical sample was subsequently inoculated onto human fetal lung fibroblasts (HFL), tertiary monkey kidney cells (tMK; Cynomolgus monkey) and HeLa cells. CPE was detected exclusively on tMK cells and first noted at eight days post-inoculation. The CPE was diffuse with a refractive appearance in the affected cells followed by cell detachment after 7 days. More pronounced CPE was observed upon passage onto the monkey kidney cell line LLC-MK2 with overall cell rounding and moderate cell enlargement (FIG. 1). Additional subcultures on HFL, rhabdomyosarcoma cells and Vero cells remained negative for CPE. Immunofluorescent assays to detect RSV, adenovirus, influenza A and B virus, or parainfluenza virus type 1, 2 and 3 in the culture remained negative. Acid lability and chloroform sensitivity tests demonstrated that the virus is most likely enveloped and not a member of the picornavirus group24.
Virus Discovery by the VIDISCA Method
Identification of unknown pathogens by molecular biology tools encounters the problem that the target sequence is not known and that genome specific PCR-primers cannot be designed. To overcome this problem we developed the VIDISCA method that is based on the cDNA-AFLP technique4. The advantage of VIDISCA is that prior knowledge of the sequence is not required as the presence of restriction enzyme sites is sufficient to guarantee amplification. The input sample can be either blood plasma/serum or culture supernatant. Whereas cDNA-AFLP starts with isolated mRNA, the VIDISCA technique begins with a treatment to selectively enrich for viral nucleic acid, which includes a centrifugation step to remove residual cells and mitochondria. In addition, a DNAse treatment is used to remove interfering chromosomal DNA and mitochondrial DNA from degraded cells, whereas viral nucleic acid is protected within the viral particle. Finally, by choosing frequently cutting restriction enzymes, the method is fine-tuned such that most viruses will be amplified. Using VIDISCA we were able to amplify viral nucleic acids from EDTA-plasma of a person with hepatitis B virus infection and a person with an acute parvovirus B19 infection. The technique can also detect HIV-1 in cell culture, demonstrating its capacity to identify both RNA and DNA viruses.
The supernatant of the CPE-positive culture NL63 was analyzed by VIDISCA. We used the supernatant of uninfected cells as a control. After the second PCR amplification step, unique and prominent DNA fragments were present in the test sample but not in the control. These fragments were cloned and sequenced. Twelve out of 16 fragments showed sequence similarity to members of the family of coronaviruses, but significant sequence divergence was apparent in all fragments. These results indicate that we identified a novel coronavirus (HCoV-NL63).
Detection of HCoV-NL63 in Patient Specimens
To demonstrate that HCoV-NL63 originated from the nasopharyngeal aspirate of the child, we designed a diagnostic RT-PCR that specifically detects HCoV-NL63. This test, based on unique sequences within the 1b gene, confirmed the presence of HCoV-NL63 in the clinical sample. The sequence of this PCR product was identical to that of the virus identified upon in vitro passage in LLC-MK2 cells (results not shown).
Having confirmed that the cultured coronavirus originated from the child, the question remains whether this is an isolated clinical case or whether HCoV-NL63 is circulating in humans. To address this question, we examined respiratory specimens of hospitalized persons and individuals visiting the outpatient clinic between December 2002 and August 2003 for the presence of HCoV-NL63. We identified 7 additional persons that carried HCoV-NL63. Sequence analysis of the PCR products indicated the presence of a few characteristic (and reproducible) point mutations in several samples, suggesting that several subgroups of NL63 may co-circulate. At least 5 of the HCoV-NL63-positive individuals suffered from a respiratory tract illness, the clinical data of 2 persons were not available. Including the index case, five patients were children less than 1 year old and 3 patients were adults. Two adults are likely to be immuno-suppressed, as one of them is a bone marrow transplant recipient, and the other is an HIV positive patient suffering from AIDS with very low CD4 cell counts. No clinical data of the third adult was available. Only 1 patient had a co-infection with RSV (nr 72), and the HIV-infected patient (nr 466) carried Pneumocystis carinii. No other respiratory agent was found in the other HCoV-NL63-positive patients, suggesting that the respiratory symptoms were caused by HCoV-NL63. All HCoV-NL63 positive samples were collected during the last winter season, with a detection frequency of 7% in January 2003. None of the 306 samples collected in the spring and summer of 2003 contained the virus (P<0.01, 2-tailed t-test).
Complete Genome Analysis of HCoV-NL63
The genomes of coronaviruses have a characteristic, genome organization. The 5′ half contains the large 1a and 1b genes, encoding the non-structural polyproteins, followed by the genes coding for four structural proteins: spike (S), membrane (M), envelope (E) and the nucleocapsid (N) protein. Additional non-structural proteins are encoded either between 1b and the S gene, between the S and E gene, between the M and N gene or downstream of the N gene.
To determine whether the HCoV-NL63 genome organization shares these characteristics, we constructed a cDNA library with a purified virus stock as input material. A total of 475 genome fragments were analyzed, with an average coverage of 7 sequences per nucleotide. Specific PCRs were designed to fill in gaps and to sequence regions with low quality sequence data. Combined with 5′RACE (Rapid Amplification of cDNA Ends) and 3′RACE experiments the complete HCoV-NL63 genome sequence was resolved.
The genome of HCoV-NL63 is a 27,553-nucleotide RNA with a poly A tail. With a G-C content of 34% it has the lowest G-C content among the coronaviridae, which range from 37%-42%25. ZCurve software was used to identify ORFs26 and the genome configuration is portrayed using the similarity with known coronaviruses (FIG. 6). The 1a and 1b genes encode the RNA polymerase and proteases that are essential for virus replication. A potential pseudoknot structure is present at position 12439, which may provide the −1 frameshift signal to translate the 1b polyprotein. Genes predicted to encode the S, E, M and N proteins are found in the 3′ part of the genome. Short untranslated regions (UTRs) of 286 and 287 nucleotides are present at the 5′ and 3′ termini, respectively. The hemagglutinin-esterase gene, which is present in some group 2 and group 3 coronaviruses, was not present. ORF 3 between the S and E gene probably encodes a single accessory non-structural protein.
The 1a and 1ab polyproteins are translated from the genomic RNA, but the remaining viral proteins are translated from subgenomic mRNAs (sg mRNA), each with a common 5′ end derived from the 5′ part of the genome (the 5′ leader sequence) and 3′ coterminal parts. The sg mRNA are made by discontinuous transcription during negative strand synthesis27. Discontinuous transcription requires base-pairing between cis-acting transcription regulatory sequences (TRSs), one located near the 5′ part of the genome (the leader TRS) and others located upstream of the respective ORFs (the body TRSs)28. The cDNA bank that we used for sequencing contained copies of sg mRNA of the N protein, thus providing the opportunity to exactly map the leader sequence that is fused to all sg mRNAs. A leader of 72 nucleotides was identified at the 5′ UTR. The leader TRS (5′-UCUCAACUAAAC-3′ (SEQ. ID. NO: 14)) showed 11/12-nucleotide similarity with the body TRS upstream of the N gene. A putative TRS was also identified upstream of the S, ORF 3, E and M gene.
The sequence of HCoV-NL63 was aligned with the complete genomes of other coronaviruses. The percentage nucleotide identity was determined for each gene. For all genes except the M gene, the percentage identity was the highest with HCoV-229E. To confirm that HCoV-NL63 is a new member of the group 1 coronaviruses, phylogenetic analysis was performed using the nucleotide sequence of the 1A, 1B, S, M and N gene. For each gene analyzed, HCoV-NL63 clustered with the group 1 coronaviruses. The bootstrap values of the subgroup HCoV-NL63/HCoV-229E were 100 for the 1a, 1b and S gene. However, for the M and N gene the bootstrap values of this subcluster decreased (to 78 and 41 respectively) and a subcluster containing HCoV-229E, HCoV-NL63 and PEDV becomes apparent. A phylogenetic analysis could not be performed for the ORF 3 and E gene because the region varied too much between the different coronavirus groups or because the region was too small for analysis, respectively. Bootscan analysis by the Simplot software version 2.529 found no signs of recombination (results not shown).
The presence of a single non-structural protein gene between the S and E gene is noteworthy since almost all coronaviruses have 2 or more ORFs in this region, with the exception of PEDV and OC4330, 31. Perhaps most remarkable is a large insert of 537 nucleotides in the 5′ part of the S gene when compared to HCoV-229E. A Blast search found no similarity of this additional 179-amino acid domain of the spike protein to any coronavirus sequence or any other sequences deposited in GenBank.
Tables







TABLE 1







cDNA- AFLP oligonucleotides for virus discovery








Oligo
Sequence





Top strand MSE adaptor
CTCGTAGACTGCGTACC



(SEQ. ID. NO: 3)


 


Top strand for HinP1 adaptor
GACGATGAGTCCTGAC



(SEQ. ID. NO: 4)


 


Bottom strand oligo for
TAGGTACGCAGTC


MSE adaptor
(SEQ. ID. NO: 5)


 


Bottom strand oligo for
CGGTCAGGACTCAT


HinP1 adaptor
(SEQ. ID. NO: 6)


 


HinPI standard primer
GACGATGAGTCCTGACCGC



(SEQ. ID. NO: 7)


 


MseI standard primer
CTCGTAGACTGCGTACCTAA



(SEQ. ID. NO: 8)
















TABLE 2







Oligonucleotide for PALM


extension of the HCOV-NL63 Sequence









Oligonucleotide




name
Application
Sequence 5′-3′





JZH2R
1st PCR
GCTATCATCACAATGGACNN




NNNG




(SEQ. ID. NO: 15)
















TABLE 3







Nucleotide- and corresponding


deduced amino acid sequences








Fragment
Sequence





163-2
GTATTGTTTTTGTTGCTTGTGCCCATGCTGCTGTTGAT



TCTTATGTGCAAAAGCTATGACTGTTTATAGCATTGAT



AAGTGTACTAGGATTATACCTGCAAGAGCTCGGGTTGA



GTGTTATAGTGGCT



(SEQ. ID. NO: 16)


 


163-2
Replicase polyprotein 1a


Translation
IVFVACAHAAVDSLCAKAMTVYSIDKCTRIIPARARVE



CYSG



(SEQ. ID. NO: 17)


 


163-4
ATGGGTCTAGATATGGCTTGCAAAACTTACTACAGTTA



CCTACTTTTATTATGTTAGTAATGGTGGTAACAATTGC



ACTACGGCCGTTATGACCTATTCTAATTTTGGTATTTG



TGCTGATGGTTCTTTGATTCCTGTTCGTCC



(SEQ. ID. NO: 18)


 


163-4
Spike protein


Translation
GSRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGIC



ADGSLIPVR



(SEQ. ID. NO: 19)


 


163-9
ATGATAAGGGTTTAGTCTTACACACAATGGTAGGCCAG


(3′-UTR)
TGATAGTAAAGTGTAAGTAATTTGCTATCATAT



(SEQ. ID. NO: 20)


 


163-10
ATGTCAGTGATGCATATGCTAATTTGGTTCCATATTAC



CAACTTATTGGTAAACAAAAGATAACTACAATACAGGG



TCCTCCTGGTAGTGGTAAGTCACATTGTTCCATTGGAC



TTGGATTGTACTACCCAGGT



(SEQ. ID. NO: 21)


 


163-10
Replicase polyprotein 1ab


Translation
VSDAYANLVPYYQLIGKQKITTIQGPPGSGKSHCSIGL



GLYYPG



(SEQ. ID. NO: 22)


 


163-11
ATCTAAACTAAACAAAATGGCTAGTGTAAATTGGGCCG



ATGACAGAGCTGCTAGGAAGAAATTTCCTCCTCCTTCA



TTTTACATGCCTCTTTTGGTTAGTTCTGATAAGGCACC



ATATAGGGTCATTCCCAGGAATCTTGTCCCTATTGGTA



AGGGTAATAAAGATGAGCAGATTGGTTATTGGAATGTT



CAAGAGCGTTGGCGTAT



(SEQ. ID. NO: 23)


 


163-11
Nucleocapsid protein


Translation
SKLNKMASVNWADDRAARKKFPPPSFYMPLLVSSDKAP



YRVIPRNLVPIGKGNKDEQIGYWNVQERWR



(SEQ. ID. NO: 24)


 


163-14
ACAAAAATTTGAATGAGGGTGTTCTTGAATCTTTTTCT



GTTACACTTCTTGATAATCAAGAAGATAAGTTTTGGTG



TGAAGATTTTTATGCTAGTATGTATGAAAATTCTACAA



TATTGCAAGCTGCTGGTTTATGTGTTGTTTGTGGTTCA



CAAACTGTACTTCGTTGTGGTGATTGTCTGCGTAAGCC



TATGTTGTGCACTAAAT



(SEQ. ID. NO: 25)


 


163-14
Replicase polyprotein 1ab


Translation
KNLNEGVLESFSVTLLDNQEDKFWCEDFYASMYENSTI



LQAAGLCVVCGSQTVLRCGDCLRKPMLCTK



(SEQ. ID. NO: 26)


 


163-15
AGGGGGCAACGTGTTGATTTGCCTCCTAAAGTTCATTT



TTATTACCTAGGTACTGGACCTCATAAGGACCT



(SEQ. ID. NO: 27)


 


163-15
Nucleocapsid protein


Translation
RGQRVDLPPKVHFYYLGTGPHKD



(SEQ. ID. NO: 28)
















TABLE 4







Identification of cDNA-AFLP fragments










Fragment
Identification best Blast hit







163-2
replicase polyprotein 1ab [Human coronavirus 229E]



163-4
spike protein [Human coronavirus 229E]



163-9
3′UTR Human coronavirus 229E



163-10
replicase polyprotein 1ab [Human coronavirus 229E]



163-11
replicase polyprotein 1ab [Human coronavirus 229E]



163-14
replicase polyprotein 1ab [Human coronavirus 229E]



163-15
nucleocapsid protein [Human coronavirus 229E]



163-18
replicase polyprotein 1ab [Human coronavirus 229E]

















TABLE 5







Pairwise nucleotide sequence homologies between the virus 


of the present invention and different corona (like) 


viruses in percentages sequence identity (%)














Fragment
BcoV 
MHV
HcoV 
PEDV
TGE
SARS 
IBV

















Replicase 
59.6
61.2
76.7
70.5
64.3
65.8
64.3


1AB 163-2









Spike gene 163-4
31.7
26.5 
64.6
48.9
45.4 
33.7
25.9


3′UTR 163-9
29.5
34
81.9 
53.6
50
31.5
38


Replicase 
55.2
57.4
82
73.8
69.4 
64.1
65.1


1AB 163-10









Nucleocapsid 163-11
25.5
23.8 
54.9
51.5
44.6
23.3
27.6


Replicase 
52.1
52.1
78.7
72.9
76.3 
52.6
58.4


1AB 163-14









Nucleocapsid 163-15
29.5
35.2
71.8 
63.3
60.5
25.3
45


Replicase 
67.2
65.4
72.8 
65.4
61.6 
68.2
57


1AB 163-18
















TABLE 6







Pairwise deduced amino acid sequence homologies between different


corona (like) viruses in percentages sequence identity (%)














Fragment
BCoV
MHV
HcoV
PEDV
TGE
SARS
IBV





Replicase 
55.8
53.4
88.3 
79  
60.4 
67.4 
55.8


1AB 163-2









Spike gene 163-4
ND
ND
56.2
ND
ND
ND
ND


Replicase 
51.1 
53.3 
93.3
86.6
80  
57.7 
55.5


1AB 163-10









Nucleocapsid 163-11
ND
ND
48.4
ND
ND
ND
ND


Replicase 
50.7
50.7
86.9 
78.2
78.2
46.3 
47.8


1AB 163-14









Nucleocapsid 163-15
ND
ND
82.6
ND
ND
ND
ND


Nucleocapsid 163-18
63.8 
63.8 
77.7
69.4
69.4
58.3
55.5





ND = Not Determined













TABLE 7







Oligos for specific detection of HcoV-163










Primer
Sequence







repSZ-RT
CCACTATAAC




(SEQ. ID. NO: 9)



 



repSZ-1
GTGATGCATATGCTAATTTG




(SEQ. ID. NO: 10)



 



repSZ-2
TTGGTAAACAAAAGATAACT




(SEQ. ID. NO: 12)



 



repSZ-3
CTCTTGCAGGTATAATCCTA




(SEQ. ID. NO: 11)



 



repSZ-4
TCAATGCTATAAACAGTCAT




(SEQ. ID. NO: 13)

















TABLE 8







Molecule Features










Start
End
Name
Description





  287
12439
1a
ORF-1a


 4081
 4459

Pfam 01661


 9104
10012

3Cl protease


12433
12439

Ribosome slippery site


12439
20475
1b
ORF-1b


14166
14490

Pfam 00680


16162
16965

COG1112, Super family DNA and 





RNA helicase


16237
16914

Pfam 01443 Viral helicase


20472
24542
2
ORF-2 S(pike)-gene


21099
22619

S1 Pfam 01601


22625
24539

S2 Pfam 01601


24542
25219
3
ORF-3


24551
25174

NS3b Pfam 03053


25200
25433
4
ORF-4 Pfam 05780, Coronavirus NS4 E 





(envelope) protein


25442
26122
5
ORF-5


25442
26119

Matrix glycoprotein Pfam 01635 M-gene


26133
27266
6
ORF-6


26184
27256

Nucleocapsid Pfam 00937 N-gene





Via a −1 frame shift at the ribosome slippery site the 1a ORF is extended to protein of 6729 amino acid residues referred to as 1ab. ORF 1a and 1ab encode two polyproteins that are proteolytically converted to 16 largely uncharacterized enzymes that are involved in RNA replication (for review see Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. Spaan, and A. E. Gorbalenya. 2003. Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage. J. Mol. Biol. 331: 991-1004).













TABLE 9







Proteins from HcoV-NL63 ORFs










ORF
Number of AA
Mw prediction






1a
4060
451364
Polyprotein


1ab
6729
752822
Polyprotein


2
1356
149841
Spike


3
 225
 25658



4
  77
  9177
Envelope


5
 226
 25927
Matrix


6
 377
 42252
Nucleocapsid





The Mw prediction does not take into account post-translational modification like glycosylation or cleavage of a signal sequence.













TABLE 10







Amplification oligonucleotides for


HCoV-NL65 S, M and N encoding regions








Primer
Sequence





S1
ACAAGTTTGTACAAAAAAGCAGGCTTCAAACTTTTCTTGAT



TTTGCTTGTTTTGCCCC



(SEQ. ID. NO: 31)


 


S2
ACCACTTTGTACAAGAAAGCTGGGTCTTGAACGTGGACCTT



TTCAAATTCG



(SEQ. ID. NO: 32)


 


M1
ACAAGTTTGTACAAAAAAGCAGGCTTCTCTAATAGTAGTGT



GCCTCTTTTAGAGG



(SEQ. ID. NO: 33)


 


M2
ACCACTTTGTACAAGAAAGCTGGGTCGATTAAATGAAGCAA



CTTCTC



(SEQ. ID. NO: 34)


 


N1
ACAAGTTTGTACAAAAAAGCAGGCTTCGCTAGTGTAAATTG



GGCCGATG



(SEQ. ID. NO: 35)


 


N2
ACCACTTTGTACAAGAAAGCTGGGTCATGCAAAACCTCGTT



GACAATTTCTATAATGGC



(SEQ. ID. NO: 36)





The S, M and N 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







Overall full length genome DNA sequence identity
















HC229



HCoV-
HCoV-



BCV
E
IBV
SARS
TGV
NL63
OC43

















BCV
100
46
43
54
40
43
95


HC229

100
50
48
53
65
46


E









IBV


100
43
46
48
43


SARS



100
40
43
53


TGV




100
55
40


HCoV-









NL63





100
43


OC43






100





Overall DNA sequence identity percentages of HCoV-NL63 compared to other coronaviruses. From the SimPlot graph (FIG. 7), comparing HCoV-NL63 (query) with SARS associated coronavirus and HCoV-229E, can be deduced that local sequence identity never exceeds 85%













TABLE 12







Overall DNA sequence identity Spike encoding region














OC43
NL63
229E
SARS







OC43
100
 46
 40
 44



NL63

100
 59
 38



229E


100
 41



SARS



100

















TABLE 13







Overall DNA sequence identity in 5′UTR












OC43
NL63
229E
SARS





OC43 
100
 36
 34
 48


NL63

100 
 74
 33


229E


100 
 34


SARS



100












BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1
cDNA-AFLP allows amplification of nucleic acids without any prior sequence information.
Culture supernatants from CPE-positive and uninfected cells are subjected to the cDNA-AFLP procedure. Amplification products derived from the CPE-positive culture which are not present in the uninfected control sample are cloned and sequenced.
FIG. 2
LLC-MK2 cells infected with HCoV-NL163.
Panel A and B are unstained cells while panel C and D are stained with haematoxilin eosin. The typical CPE of HCoV-NL163 is shown in panel A and C. The control uninfected LLC-MK cells are shown in panel B and D.
FIG. 3
VD-cDNA-AFLP PCR products visualized by Metaphor® agarose gel electrophoreses.
The PCR products of 1 (HinP I-G and Mse I-A) of 16 primer pair combinations used during the selective amplification step. Lanes 1 and 2: duplicate PCR product of virus culture NL163; lanes 5 and 6 control supernatant of LLC-MK2 cells and in lane 7 and 8 the negative PCR control. Lanes M: 25 bp molecular weight marker (InVitrogen). The arrow indicates a new coronavirus fragment that was excised out of gel and sequenced.
FIG. 4
Phylogenetic Analysis of the HCoV-163 Sequences.
G1, G2 and G3 denote the group 1, group 2 and group 3 coronavirus clusters. The Genbank accession number of the used sequences are: MHV (mouse hepatitis virus): AF201929; HCoV-229E: AF304460; PEDV (porcine epidemic diarrhea virus): AF353511; TGEV (transmissible gastroenteritis virus): AJ271965; SARS-CoV: AY278554; IBV (avian infectious bronchitis virus): NC—001451; BCoV (bovine coronavirus): NC—003045; FCoV (feline coronavirus): Y13921 and X80799; CCoV (canine coronavirus): AB105373 and A22732; PRCoV (porcine respiratory coronavirus): M94097; FIPV (feline infectious peritonitis virus): D32044. Position of the HCoV-163 fragments compared to HCoV-229E (AF304460): Replicase 1AB gene: 15155-15361, 16049-16182, 16190-16315, 18444-18550, Spike gene: 22124-22266; Nucleocapsid gene: 25667-25882 and 25887-25957; 3′UTR: 27052-27123. Branch lengths indicate the number of substitutions per sequence.
FIG. 5
Schematic representation of Coronavirus and the location of the 163-fragments listed in table 3.
FIG. 6
Restriction map of HCoV-NL63′
Complete 27553 nt cDNA derivative of the ssRNA genome. Open reading frames (ORF) are depicted as numbered black arrows and the identified (PFAM) domains within these ORFs are indicated as gray boxes.
FIG. 7
Simplot analysis HcoV NL63 and other human Coronaviruses
The gap in the comparison of HCoV NL63 to SARS, HCoV-OC43 and HCoV-229E is cause by a unique 537 in-frame insertion in the Spike protein encoding ORF (see elsewhere herein). Sigmaplot analysis is described in Lole, K. S., R. C. Bollinger, R. S. Paranjape, D. Gadkari, S. S. Kulkarni, N. G. Novak, R. Ingersoll, H. W. Sheppard, and S. C. Ray. 1999. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73:152-160.
FIG. 8
Expression constructs for HCoV-NL63 Spike and Matrix protein Expression of a H is and StrepII tagged Spike fusion protein can be induced by addition of IPTG to the bacterial growth medium. Through attB1/B2-mediated recombination, the S gene insert can be transferred to other commercially available expression vectors, facilitating protein production in other hosts. Through an identical cloning procedure as for pGP7S, a Gateway compatible expression vector for HCoV-NL63 M-gene can be constructed. The plasmid directs IPTG inducible production of N and C-terminally affinity tagged Matrix fusion protein, allowing selective recovery of full-length fusion protein.
FIG. 9
Recombination site NL63-229E (SEQ ID NO: 38)
NL63-derived sequences are in underlined bold black print and the 229E derived sequences are in gray bold print.
FIG. 10
Restriction map cDNA Clone NL63/229E hybrid
The NL63 derived part is indicated as gray boxes and the 229E-derived region is indicated as a line. The junction between the two genomes is indicated by the succession of the two black arrows marked 1b′ and ‘ORF-1b indicating the hybrid 1b ORF.
A second chimeric genome was generated by a reciprocal recombination fusing nucleotide 19653 of HCoV-NL63 to nucleotide 20682 of HCoV-OC43 again creating a hybrid ORF 1b giving rise to a hybrid 1ab replicase polyprotein. Recombination occurred within the conserved sequence AATTATGG (SEQ. ID. NO: 37).
FIG. 11
Recombination site NL63/OC43 hybrid (SEQ ID NO: 39)
Again, NL63-derived region is in bold black underlined print and the OC43 derived sequences are in gray bold print. The resulting cDNA restriction map is depicted in FIG. 12
FIG. 12
Restriction map recombinant NL63/OC43 genome.
The NL63-derived part is indicated as gray boxes and the recombination site is depicted as the between the black arrows 1b′ and ‘1b.
FIG. 13
Similarity plot deduced protein alignments of ORF1b from HCoV-NL63, HCoV-229E, HCoV-OC43 and the two hybrids NL63/229E and NL63/OC43.
FIG. 14
Green fluorescent protein expressing HcoV-NL63 derivative.
Functional equivalent NL63/4GFP carries an in-frame C-terminal fusion of the E protein (ORF4) with a human codon optimised Green Fluorescent Protein (EGFP, Stratagene). Infected cells appear fluorescent after excitation of the 4-EGFP fusion protein. HCoV-NL63 can be used to elucidate the process of viral; infection and the translation of the polycistronic sub-genomic messengers.
FIG. 15
Restriction map of functional derivative NL63D2052021011.
This deletion derivative of NL63 lacks most of the insertion at the N-terminal end of the Spike protein. By deleting nucleotides 20520-21011 the unique domain is removed while retaining the predicted secretory signal sequence (Nielsen, H., J. Engelbrecht, S. Brunak, and G. Von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1-6).
FIG. 16
Sequence variation in HCoV-NL63 from additional patient samples
Direct sequencing of both strands of RT-PCR products from 6 patient samples revealed the presence of polymorphisms in the ORF 1a region. REF (SEQ ID NO: 40); 223 B (SEQ ID NO: 41); 246_B (SEQ ID NO: 42); 248_B (SEQ ID NO: 43); 251_B (SEQ ID NO: 44); 466_B (SEQ ID NO: 45); 496_B (SEQ ID NO: 46)
FIG. 17
HCoV-NL63 specific and generic human Coronavirus detection probes. Coronavirus polymerases generate several sub-genomic RNAs. The frequency of S, E, M and N protein encoding cDNA clones in the sequencing library of HCoV-NL63 and SARS (Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. Spaan, and A. E. Gorbalenya. 2003). Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 331:991-1004). Northern blot data demonstrate a high abundance of these sub-genomic RNAs in infected cells. Consequently, these genes are attractive targets for diagnostic tests.
Since the genomic and sub-genomic RNAs possess identical 3′ ends, probes containing the N gene would hybridise to all of them (Table 8).
Through alignment of the full-length sequences of all human Coronaviruses a conserved region in ORF1b was identified, allowing their detection with a nested RT-PCR assay. Oligo NL63NF1 (SEQ ID NO: 47); Oligo NL63NR1 (SEQ ID NO: 48); Oligo NL63NF2 (SEQ ID NO: 49); Oligo NL63NR2 (SEQ ID NO: 50)
FIG. 18
Generic Coronavirus detection primers. Oligo COR1F (SEQ. ID. NO: 51); Oligo COR1R (Seq. ID. NO: 52), Oligo COR2F (SEQ. ID. NO: 53); Oligo COR2R (SEQ. ID. NO: 54)
FIG. 19
Nucleotide sequence an HcoV′_NL63 (SEQ. ID. NO: 55)
FIG. 20
ORF 1a, replicase enzyme complex of an HcoV_NL63 (SEQ. ID. NO: 56)
FIG. 21
ORF 1ab replicase polyprotein of an HcoV_NL63 (SEQ. ID. NO: 57). Adenosine diphosphate-ribose 1′-phosphate (SEQ ID NO: 58). 3CIPro Coronavirus polyprotein processing endoprotease (SEQ ID NO: 59); RNA dependent RNA polymerase (pfam00680) (SEQ ID NO: 60); Exon 3′ to 5′ Exonuclease and helicase (SEQ ID NO: 61); XendoU (homolog of) polyU-specific endoribonuclease (SEQ ID NO: 62); 2′-0-MT 2:S-adenosvlmethionine-dependent ribose 2′-orthomethyltransferase (SEQ ID NO: 63)
FIG. 22 (SEQ ID NO: 64)
The spike protein (ORF3) contains an N-terminal secretory signal sequence of 16 AA (indicated on the first line of the continuous sequence listed below). (Nielsen, H., J. Engelbrecht, S. Brunak, and G. Von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1-6)
FIG. 23
ORF-4 Coronavirus_NS4 (SEQ ID NO: 65), Coronavirus non-structural protein 4. This family consists of several non-structural protein 4 (NS4) sequences or small membrane protein.
ORF-5 (SEQ ID NO: 66). This family consists of various coronavirus matrix proteins that are transmembrane glycoproteins. The M protein or E1 glycoprotein is implicated in virus assembly. The E1 viral membrane protein is required for formation of the viral envelope and is transported via the Golgi complex. The matrix protein is predicted to contain an N-terminal secretory signal sequence (indicated in the first part of the continuous sequence) (Nielsen, H., J. Engelbrecht, S. Brunak, and G. Von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1-6.)
ORF-6 Pfam 00937 (SEQ ID NO: 67), Coronavirus nucleocapsid protein. Structural protein forming complexes with the genomic RNA.



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## Claims

1. A method for determining whether an individual suffers from an HCoV-NL63 infection, said method comprising:
obtaining a biological sample from the individual, and
detecting the presence of HCoV-NL63 virus sequences in said biological sample with a primer and/or probe capable of specifically detecting a HCoV-NL63 virus sequences.

2. The method according to claim 1, wherein said primer and/or probe is capable of specifically hybridizing to a sequence selected from the group consisting of SEQ ID NOS: 16, 18, 20, 21, 23, 25, 27 and 29.

3. The method according to claim 1, wherein said primer and/or probe is capable of specifically hybridizing to a nucleic acid encoding a proteinaceous molecule comprising a sequence selected from the group consisting of SEQ ID NOS: 17, 19, 22, 24, 26, 28 and 30.

4. The method according to claim 3, wherein the proteinaceous molecule comprises SEQ ID NO: 67.

5. A method for detecting an isolated or recombinant HCoV-NL63 virus comprising a nucleic acid sequence that is at least 90% homologous to a HC0V-NL63 nucleic acid sequence in a sample, the method comprising:
hybridizing and/or amplifying a HCoV-NL63 nucleic acid of said sample with a primer and/or probe capable of specifically hybridizing and/or amplifying said HCoV-NL63 nucleic acid, and detecting hybridized and/or amplified product.

6. The method according to claim 5, wherein said primer and/or probe is capable of specifically hybridizing to a sequence selected from the group consisting of SEQ ID NOS: 16, 18, 20, 21, 23, 25, 27 and 29.

7. The method according to claim 5, wherein said primer and/or probe is capable of specifically hybridizing to a nucleic acid encoding a proteinaceous molecule comprising a sequence selected from the group consisting of SEQ ID NOS: 17, 19, 22, 24, 26, 28 and 30.

8. The method according to claim 7, wherein the proteinaceous molecule comprises SEQ ID NO: 67.

9. The method according to claim 5, for diagnosing a HCoV-NL63 coronaviral genus infection.