Patent Application
20100211327
2.1 Field of the Invention
The present invention provides compounds, software, systems, devices and methods for diagnosing viral infections. More particularly, the present invention provides compounds, software, systems, devices and methods for diagnosing viral infections by identifying the family of the virus infecting a mammalian, more specifically human, host. The present invention has applications in the areas of public health, virology, immunology, medicinal chemistry, medicine, and computer science.
2.2 The Related Art
Every day thousands of people suffer aches, fatigue, and lethargy associated with the onset of a viral disease. But these initial symptoms are usually so nondescript that the underlying disease cannot be diagnosed quickly. Instead, the afflicted must endure these discomforts as the viral infection progresses to cause more pronounced and specific symptoms that can be associated with a specific disease. During that interim, however, the virus is replicating and causing damage to the afflicted.
Making an early diagnosis of a viral infection is difficult, because many different viruses present the same, or substantially the same, nondescript symptoms in the early stages of infection. In addition, traditional medical practice has focused exclusively on rendering diagnoses of viral infections in terms of specific viruses or diseases, ignoring as irrelevant any attempt to diagnose an illness in terms of the more general family that encompasses the virus causing the illness. This approach would appear only reasonable in view of the fact that the few available anti-viral drugs are directed to act on specific viruses, not across viral families. The only treatments available during the phase before the virus can be positively identified are palliatives, like aspirin and decongestants, to help comfort the patient.
The traditional insistence on identifying and treating viral diseases carries a large price in addition to the above-mentioned suffering of the afflicted. Potentially catastrophic is that many of those infected are themselves viral vectors during the interim phase before diagnosis. During this time, the afflicted can spread the virus through common contact in public and private spaces. While this often is a nuisance for those in the vicinity of the sick, the growing concern over biological terrorism and warfare, and the expected return of such devastating “natural” viruses like avian influenza or the so-called “Spanish flu” pandemic of 1918, make the need for rapid diagnoses of viral infections all the more urgent.
Perhaps the greatest factor forcing health care givers to wait for a set of symptoms sufficient to support a diagnosis of a single virus (or one of a few closely related possibilities) is the lack of any “broad spectrum” anti-viral drugs, i.e., drugs that are clinically effective against a variety of viruses in a manner akin to certain antibiotics that have efficacy against a variety of bacterial infections. Recently, however, a number of such broad-spectrum anti-viral compounds were reported in PCT Application Serial No. WO 2008/124550, titled: Phenothiazin Derivatives for Antiviral Treatments.
In addition to the development of new anti-virals, methods and apparatus for detecting viruses in biological samples have been described that could aid in the rapid detection of viral infections and public outbreaks. For example, Briese, et al. (“Diagnostic System for Rapid and Sensitive Differential Detection of Pathogens”, Emerging Infectious Diseases 11(2) February 2005 310-314) describe an apparatus for using Mass Tag PCR techniques to identify viruses in samples. Similarly, Jabado, et al. (“Comprehensive viral oligonucleotide probe design using conserved protein re-Bions”, Nucleic Acids Research, 36(1) 2008 e3) describes the use of arrays of oligonucleotide probes to identify viruses in samples. Griffiths, et al. also describe using arrays of primers to identify viral infections in WO 02/099130. However, none of these publications describes the identification of viral families in biological samples.
Thus, although the compounds described in the above-referenced publication hold the promise of a new era in treating diseases, their use is hampered by the lack of diagnostic methods and systems that are capable of identifying viruses by familial or other generic qualities. The present invention meets these and other needs.
In a first aspect, the present invention provides methods, apparatus, and software for identifying sets of genomic oligonucleotides that identify a viral family and for determining sets of probes and primers that can be used to identify members of viral families in a sample, such as a biological sample.
In one embodiment, the method of the present invention includes normalizing viral genomic oligonucleotide sequence data from at least one viral family, and identifying at least one oligonucleotide sequence that is unique to each viral family. In more specific embodiment, at least three such unique oligonucleotide sequences are identified. An array of the identified oligonucleotide sequences is made. Primers provided by the present invention are used to amplify viral family genomic sequences in a biological sample of interest, and probes provided using the present invention, e.g., attached to an array or biochip, are then used to detect the presence of the corresponding virus(es) present in the sample by hybridization. More particularly, the above-described method is implemented using an electronic computing device that is configured to execute the method on an electronic database of viral oligonucleotide sequences that have been electronically encoded and stored in a electronic memory device in electronic communication with the electronic computing device.
In a second aspect, the present invention provides arrays of oligonucleotides (including related probes and primers) configured to identify the viral family corresponding to a virus in a biological sample exposed to the array.
In a third aspect, the present invention provides methods for identifying the viral family of a virus in a biological sample, comprising exposing the sample to an array of oligonucleotides configured to identify the viral family corresponding to a virus in a biological sample exposed to the array.
In a fourth aspect, the present invention provides methods for treating a viral infection in a mammal suffering from a viral infection, comprising exposing a biological sample from the infected animal to an array of oligonucleotides configured to identify the viral family of the virus, identifying the viral family of the virus, and administering a medication effective to treat viruses in the family identified.
In a first aspect, the present invention provides methods for identifying sets of oligonucleotide sequences that uniquely identify a viral family. One embodiment of the invention is illustrated at 1000 in
Adenoviridae
Arenaviridae
Astroviridae
Bornaviridae
Bunyaviridae
Coronaviridae
Flaviviridae
Filoviridae
Hepadnaviridae
Herpesviridae
Orthomyxoviridae
Papillomaviridae
Paramyxoviridae
Parvoviridae
Picornaviridae
Poxviridae
Reoviridae
Retroviridae
Rhabdoviridae
Togaviridae
In some embodiments, the above-described database is computerized, such as the NCBI database. Generally, such databases provide the viral genomic sequence data as a formatted set of encoded electronic signals that are stored either as corresponding formatted electronic signals, or magnetic domains, in the electronic or magnetic memory (respectively) of a electronic computer device. The provision, manipulation, and transmission of such data is understood by those having ordinary skill in the art.
In some embodiments, that above-described viral genomic data is normalized (1008) to facilitate processing as described below. For example, in more specific embodiments normalization includes truncation of lines to 80 characters. In other more specific embodiments, any comments provided with the data are removed. In still other embodiments, the lines are truncated and the comments are removed; in still more particular embodiments, the comments are removed and stored in separate directory. Still other embodiments, which can be included with any of the foregoing embodiments, include the removal of newline characters. In yet other embodiments, the viral genomic data is organized into an array such that one genomic sequence is assigned to a unique array element. In still other embodiments, the entire genome for a single virus is provided as a single string; with the genomes for segmented viruses including spacer characters (e.g., a space) as appropriate. Any of the foregoing normalizing operations, alone or in combination, can be performed on the viral genomic data encoded as electronic signals using methods and implements, including programmable electronic computing devices, known to those having ordinary skill in the art.
Following normalization (if any), the set of genomic viral sequence is searched to identify at least one sequence that is unique to each viral family the set of viral genomic families (1012). In some embodiments, the set is searched for more than once such unique sequence in order to increase the robustness of the identification of the presence of a member of a viral family in a biological sample as described below. In more specific embodiments, at least three (3) unique sequences are determined and saved (1016). The choice of a suitable number of unique sequences can be made by those having ordinary skill in the art. In some embodiments, the search is performed using a programmable computer that is configured to process the viral genomic sequence data in electronic format using the methods described herein.
In some embodiments, the size of the search window for the unique sequence(s) (i.e., the number of characters searched in each oligonucleotide) is between about 20 characters and about 40 characters. Other window sizes will be apparent to those having ordinary skill in the art. In other embodiments, the array of viral sequences is arranged from the longest to the shortest. Other arrangements will be familiar to those having ordinary skill in the art.
In some embodiments, two sets of inputs are created: one for the sequences of the viral family of interest, and one for all sequences of the other viral families. In those embodiments in which the processing is performed using a programmed electronic computing device, each input is a directory created on a storage device, such as a magnetic disk. Still other methods for managing output will be apparent to those having ordinary skill in the art.
In some embodiments, a subsequence is created starting from the first position of the first sequence, and having a length equal to a first value between about 40 and about 20. The subsequence is then compared against each sequence of the other viral families, e.g., using the well known Basic Local Alignment Search Tool (“BLAST”) methodology. If the subsequence is common among the family of interest, then it is stored in a “results” array and a results counter is incremented. The length value is then decremented by one, and the comparison is repeated. Alternative methods for searching the desired window sizes will be apparent to those having ordinary skill in the art. In some embodiments, allowances are made for near matches of sequence elements, i.e., counting different sequence elements as the same if they are considered to have relative equivalence. Such methods are known to those having ordinary skill in the art. In still more specific embodiments, a limit for the number of such equivalences is also provided, e.g., about 25%. Again, such limits are familiar to those having ordinary skill in the art.
In some embodiments, sequences each of the sequences stored in the results array is compared against each of the non-family sequences to determine if the sequence is unique to the family. This can be accomplished in substantially the same manner as just described for determining the common in-family sequences. Sequences that are not unique to the family are discarded. The remaining sequences thus are both common among all sequences in a family, but they are also unique to that family. In some embodiments, the foregoing search is continued until at least one, in more specific embodiments at least two, and in still more specific embodiments at lest three unique sequences are identified. In some embodiments, the number of sequences is chosen to provide substantially enough unique sequences to provide robust identification of the viral family in a biological sample.
In a second search embodiment, the same two files of family and non-family viral genomic sequences are created and manipulated as described above. However, the second embodiment includes two additional data elements. One holds the following data obtained when searching against the family: (a) a Boolean; whether or not it is found; (b) an integer; where it is found (if it is found); and (c) other information relating to the error of the string matches. A second holds information relating to segments that match multiple family members: (a) a string: the matching segment; (b) an integer: how many family members it identified; and (c) other information used for the search against the non-family list.
In this second search embodiment, the search for common sequences and subsequences that are unique to a viral family genome includes searching through the whole genome of the smallest organism looking only for matches of length 20. Information relating to this search, e.g., whether and where a match is found for a given genomic sequence, is stored in an array comprised of the first data structure described above. In a more specific embodiment, if the number of matches for a window size greater than or equal to 20 is two or more, then a check of the characters at the position immediately after the match (i.e., character 21) is made for family members which were matched by the sequence of length 20, to determine how many match a sequence or subsequence having a length of 21 characters. This process is continued by analogous extension until the number matched by 20+(n+1) is no longer equal to number matched by 20+n. The match of length 20+n is then added to an array of matches, and the search continues starting at the next character in the sequence; thereby defining a substantially minimal set of common genomic sequences to verify as unique to the viral family. These operations can be implemented by those having ordinary skill in the art.
Once the set of common viral genomic sequences is found, those sequences are searched against the non-family genomic sequences as follows. First, the best match is searched against the non-family list. If that sequence is found in the non-family list, then a record is made of where it is found, and how long the match was in the list of failed matches. The second best match is then searched against the non-family sequences. If the second best match comes from a different place in the shortest family members genome, then the search starts from the beginning. If it has already been matched against the non-family list, then the search starts at the point where the shorter match failed. If the search of the second-best sequence also fails, then the third-best is tried. This pattern is continued until a unique sequence or subsequence is identified. These operations can be implemented by those having ordinary skill in the art.
The methods described herein can be encoded on a computer-readable medium as computer-readable program code devices that configured to enable a general purpose electronic computing machine (
In another embodiment, the present invention provides methods for making a set of oligomers that uniquely identify a particular viral family, and a set of primers used to amplify the regions containing the sequences matching these oligomers. These methods can also be encoded on a computer-readable medium as computer-readable program code devices that are configured to enable a general purpose electronic computing machine (
In one embodiment, a computer is programmed to perform a string comparison of various viral family genomic sequences to determine if substrings of viral family member genomes match multiple viral family members. Substring matches at different locations in different viral family members are used to construct a list of oligomers which each match (within a defined tolerance) a certain number of viral family members. The list is then cleaned of excess error; and each oligomer is then expanded to it's longest length according to a defined tolerance. The list is then sorted, and the best oligomer is added to a “results set”. The intersection of the results set and the list of oligomers is then filtered out, and the next best oligo is selected. This process of filtering and selecting next best oligomers is repeated until every family member is matched (to within a defined tolerance) by at least one oligomer in the set of results. The above set of results defines the probes of interest used to identify a viral family. Three such sets are generated in total. Primers are then selected for each probe in an analogous manner. The performance of these operations can be accomplished by a person having ordinary skill in the art using the disclosure and drawings herein.
In one embodiment, the methods use a simple computer-generated string comparison technique to determine if subsequences of a viral family member genome matches multiple viral family genomic sequences. In a more particular embodiment, a “match” is determined when a count of differences between two sequences is less than, or equal to, a defined limit for any possible substring of a viral genomic sequence. In a more particular embodiment, the defined limit is defined as:
└Ta/100×Sl┘
where Ta is the acceptable tolerance (%) and Sl is the sequence length.
Substring matches at different locations in different viral family members are collected as a list of sequences, which sequences are formed from sets of substrings which, when combined, match multiple viral family members; wherein a “match” is declared when the count of base differences between two equal-length sequences is less than the defined limit. Two bases are said to match if the intersection of the set of characters encoding the base is not empty.
The list is then cleaned of excess error. An error is defined as any character which is not one of the set {A, C, G, T}. The amount of error is tolerable when it is less than, or equal to, the defined limit above.
Each sequence in the list is then expanded to its longest tolerable length, which is determined by considering the next character past the end of each subsequence as now part of the sequence. The acceptability of each extended sequence is then reconsidered using the criteria defined above, and if it passes, the expansion continues; if not, the length of the sequence is reduced by one. The process is stopped if the length of the sequence exceeds a defined upper limit.
The list is then sorted in ascending order according to the number of viral matches using the criteria for matching defined above, and the best sequence, defined as the first oligo in the sorted list which does not match any oligo in the results set, or any virus outside the viral family, using 0% tolerance for both, is added to the results set. The intersection of the results set and the list of oligos is then filtered out by moving to the back of the list those sequences in the list which match (with 0% tolerance) any sequences in the set of results, and the next best oligo is selected. This process, of filtering and selecting next best sequences, is repeated until every family member is matched by at least one oligo in the set of results. The above set of results defines the probes of interest used to identify a viral family. Three such sets are generated in total, in which the second and third sets of results reuse the list of sequences used to make the first set of results, but ignoring sequences oligos which match (0% tolerance) anything in the previous sets of results. More or fewer results sets can be generated in other embodiments.
Primers are then selected for each probe in an analogous manner as follows: For each sequence in every set of results, viral genomic sequences that match the sequence are used to generate two lists of substrings. The two lists are made from substrings found to the left, and to the right, of the location where the sequence is matched as defined above; and the sequences are chosen such that when the left substring, sequence, and right substring are concatenated together, they are of a length optimal for amplification using PCR at a given length of base pairs (bp). In some embodiments, the length is between about 100 by and about 1,000 bp, and in more particular embodiments about 500 bp. From the left list, the same process used to make the probes (described above) is used, but with the following changes: (a) using the left list in place of the viral family list, (b) using sequence lengths of between about 10 and about 17 bases, (c) using tolerance of about 25%, and (d) skipping the check against non-family members. The same is done for the right list, but using the compliment of each primer.
The foregoing can be implemented using skills and information known to those having ordinary skill in the art in light of the disclosure and drawings herein.
In another aspect, the present invention includes genes and corresponding polypeptides, probes, primers (e.g., PCR primers), and arrays of such polypeptides, genes, probes, and primers defined using the methods, software, and programmed electronic computing machines provided herein. Methods and materials for making such polypeptides and corresponding genes encoding such polypeptides, probes, primers (e.g., PCR primers), and arrays of such polypeptides, genes, probes, and primers are familiar to those having ordinary skill in the art.
In another aspect, the present invention provides systems for diagnosing diseases including the polypeptides, genes, probes, and primers described herein. In some embodiments, the systems provided by the present invention include devices configured to identify individual or sets of polypeptides or genes (or both) in biological samples. The preparation, configuration, and operation of such systems is familiar to those having ordinary skill in the art.
In yet another aspect, once a viral family (or families) have been identified in a biological sample from an infected mammal (e.g., a human or animal), the treatment as described in WO 08/124,550 can be administered to either cure or alleviate the infection, or prevent additional harm to the infected mammal or the public. Such administration is familiar to those having ordinary skill in the art.
Still other useful methods, systems, and apparatus provided by the present invention include the following.
In another aspect, the present invention provides a system for diagnosing a virus comprising a first array of oligonucleotides configured to identify at least one viral family in combination with a as described above in combination with a second, and optionally subsequent, array or arrays of oligonucleotides configured to identify a specific virus. As will be understood by those of ordinary skill in the art, exposure of a biological sample to the first array provides identification of the viral family (or families) present in the sample; exposure of the sample to the second array then can be used to identify the particular virus. As will be appreciated by those having ordinary skill in the art, in medical practice a care giver can prescribe treatment with a broad-spectrum anti-viral agent (such as described in WO 08/124,550) until identification of the virus is made using the second array, at which time the caregiver can assign a treatment more specific to the virus. Still other embodiments include using an array to identify the virus's sub-type. In still other embodiments, the second (or subsequent) array is configured to identify single nucleotide polymorphisms and other variants of host factors that prove to be relevant for viral infection (e.g. once drug targets are known). In yet another embodiment, the second (or subsequent) array is configure to reproduce the result of the first array, but for a subset of viral families; thereby corroborating the conclusion and raising the sensitivity or specificity (or both) of the result of the first array. Those having ordinary skill in the art will understand from the foregoing that the invention provides caregivers the opportunity to tailor drug therapies to viruses quickly and while giving treatment.
In another aspect, the present invention provides arrays of oligonucleotides, probes, and primers, and methods for using such as described herein wherein the viral families and viruses are plant and non-human animal viruses. In addition, the methods, systems, and devices provided by the present invention can be used to identify and treat emerging viruses, and engineered viruses (e.g., weaponized viruses).
The following Examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in the art in practicing the invention. These Examples are in no way to be considered to limit the scope of the invention in any manner.
This Example demonstrates the amplification of the EBOV and MARV nucleocapsid genes from a mixture of nucleocapsid DNA sequences using the methods and materials provided by the present invention. This Example further demonstrate that the amplified EBOV PCR product so obtained could be identified by a specific biotinylated probes provided by the present invention using Southern blot analysis.
A plasmid DNA template consisting of a mixture of genes encoding nucleocapsid proteins, chosen from viruses of different families, was prepared. The viruses and their corresponding families are shown below:
EBOV and MARV (Filoviridae);
RPXV (Poxviridae);
LASV (Arenaviridae);
RVFV and HNTV (Bunyaviridae);
FLUV (Orthomyxoviridae);
HIV (Retroviridae);
SARS (Coronaviridae);
DENV and HCV (Flaviviridae); and
EGFP (Enhanced Green Fluorescent Protein) (irrelevant template);
The cDNA sequences encoding various nucleocapsid genes were cloned into the Kpn I and Not I sites of the pTnT plasmid (obtained commercially from Promega Corp., Madison, Wis.). Clones were authenticated by restriction mapping and sequence analysis as illustrated in
The following primers were synthesized commercially (Bioneer Inc., Alameda, Calif.), and employed in the PCR amplification of the cDNAs:
The oligonucleotide 5′-AAGCAACTCCAACAATATGC-3′ (for EBOV a member of the Filoviridae) maps to the nucleocapsid gene. A 5′- and 3′-biotinylated version of this probe (with a 15-atom triethylene glycol (TEG) spacer) was synthesized by Bioneer, Inc., Alameda, Calif., and employed in a Southern blot analysis.
The following reagents were added sequentially in a 100 μl PCR reaction:
7. 1 μA YieldAce DNA Ploymerase (provided by supplier—Stratagene, Inc., La Jolla, Calif.).
The following reagents were added sequentially in a 100 μl PCR reaction:
PCR was performed using a TGradient Thermocycler (BioMetra, Goettingen, Germany) the following sequence:
A 2 μL aliquot of each PCR reaction was electrophoresed through a 1% agarose gel in Tris acetate (TAE) buffer, as shown in
1 100 by DNA Ladder from New England BioLabs (Cat. No. N3231L)
2 1 Kbp DNA Ladder from New England BioLabs (Cat. No. N3232L)
3 PCR product from MARV—Reference
4 PCR product from EBOV—Reference
5 Mixed plasmid DNA templates PCR'd with MARV-For and -Rev primers
6 Mixed plasmid DNA templates PCR'd with EBOV-For and -Rev primers
7 Mixed plasmid DNA templates PCR'd with pTnT-For and -Rev primers
8 Mixed plasmid DNA templates
9 1 Kbp DNA Ladder from New England BioLabs (Cat. No. N3232L)
The PCR products in lanes 4 and 6 were slightly bigger than the corresponding PCR products in lanes 3 and 5 respectively. Without wishing to be bound to any particular theory of action, this was expected as PCR amplification with pTnT primers adds on an additional 200 bp to the PCR products when compared to PCR amplification using gene-specific primers.
The DNA from the agarose gel was transferred to a Biodyne Nylon membrane as described by the manufacturer (Pall Corp., Port Washington, N.Y.), and the membrane-bound materia was hybridized to a 5′- and 3′-TEG-biotinylated probe, as described above and as published in Leukemia (2000) 14, 767-772 (
As can be clearly seen in Lanes 5 and 6 of the agarose gel (
The above description of the embodiments, alternative embodiments, and specific examples, are given by way of illustration and should not be viewed as limiting. Further, many changes and modifications within the scope of the present embodiments may be made without departing from the spirit thereof, and the present invention includes such changes and modifications.
The present application claims priority under 35 U.S.C. §119(e) to provisional U.S. Patent Application Ser. No. 61/149,384, filed 3 Feb. 2009, which application is incorporated herein by reference in its entirety and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61149384 | Feb 2009 | US |
