# Patent

## Title
Application package to automatically identify some single stranded RNA viruses from characteristic residues of capsid protein or nucleotide sequences

## Abstract

A system implemented on a computer is disclosed for automatically identifying strains of partial or complete capsid sequences of picorna and caliciviruses, two of the most highly diverse ssRNA virus families.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/846,206, filed 21 Sep. 2006 (Sep. 21, 2006).
Sequence Listing
The specification includes a list of sequences including sequence ID numbers
Computer Code Listing
The specification includes a listing of a representative program for implementing the methods of this invention.


GOVERNMENTAL INTEREST
Governmental entities may have certain rights in and to the contents of this application due to funding from the Texas Learning and Computational Center (TLCC), grants from the National Aeronautics and Space Administration Office of Exploratory Research (NNJ04HF43G) and the Department of Homeland Security Advanced Research Projects Agency (W81XWH-05-2-0040).


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a software product that uses unique characterizing residues to automatically identify strains of partial or complete capsid sequences of picorna viruses and calici viruses, two of the most highly diverse single stranded RNA (ssRNA) virus families.
More particularly, the present invention relates to a computer program product stored on a computer readable medium for predicting strains of some ssRNA viruses from their limited sequence data, said computer product including a graphical user interface (GUI) code operable to carry out all data input-output (I/O) operations; storage codes operable to store virus sequence databases in the form of multiple data arrays containing information about phylogenetic trees, sequence groups and characteristic residues of these groups; sequence comparison codes operable to compare input virus sequences with the stored database sequences on a residue-by-residue basis; identification codes operable to identify the strains of input virus sequences based on comparisons (iii) and subsequent decision making algorithms.
2. Description of the Related Art
Currently, no equipments or kits can unambiguously distinguish among a particularly important class of viruses that cause alarming epidemic outbreaks all over the world and consequently, pose high bio-terrorism related threats. This class comprises of the single-stranded RNA (ssRNA) viruses which include diverse virus families ranging from those that cause flu (including the cruise-ship flu and the bird flu) to the AIDS causing viruses. The main difficulty in distinguishing among these viruses and detecting them lies in their molecular details. As the name suggests, each of these viruses consists of a single genomic RNA strand enclosed within a protein shell called the capsid. This encapsidated RNA strand undergoes rapid sequence mutations to generate a large number of virus strains that are often associated with different epidemic outbreaks. Sequence differences among these strains are so subtle and intricate that they appear to be almost random. There are no reliable methods to unambiguously distinguish among the strains by systematically tracking these variations. Most often, the capsid sequences show the maximum variations relative to the other genomic regions for a given ssRNA virus families. This is mainly because the capsid residues undergo most mutations in response to host immunity. Consequently, capsid sequences provide the best regions to identify strains as they truly represent the diversity of ssRNA virus families. Thus, any reliable method to uniquely identify the ssRNA virus strains would be based on capsid sequences even though the problem of strain identification on the basis of these sequences may appear intractable.
Two diagnostic methods are most widely used to detect ssRNA viruses. One of them relies on immuno-based techniques while the other is based on reverse transcriptase polymerase chain reaction (RT-PCR) assays. The immuno-based assays distinguish strains on the basis of epitope differences in the capsid protein while the RT-PCR based assays use nucleotide primers to amplify differences in the viral genome. However, experimental constraints limit the ability of both of these assays in distinguishing among the strains. Most often, these methods are useful in detecting only significant sequence differences but they fail to detect subtle sequence variations among the strains which occurs, for example, when the sequence identity falls to approximately 10% or below as in the case of noroviruses
Accurate strain recognition in uncharacterized target capsid sequences is essential in understanding the epidemiology and diagnostics of these viruses and for efficient vaccine development. Experimental techniques to detect ssRNA virus strains are inadequate when the number of strains are very large as is the case with picornaviruses and caliciviruses, or, the strains are non-cultivable like those of the human caliciviruses. Additionally, existing homology comparison based computational methods to recognize strains are of limited use as they most often rely on similarity scores between target sequences and sequences of homology matched reference strains. Methods based on such scores are often time consuming and ambiguous especially if only partial target sequences are available or, if different ssRNA virus families are jointly analyzed. In such cases, knowledge of residues that uniquely distinguish among known reference strains is critical for rapid and unambiguous strain recognition of target capsid sequences. Conventional sequence comparisons are unable to identify such capsid residues due to high sequence divergence among the ssRNA virus reference strains. Consequently, automated general methods to predict strains from sequence data of such viruses on the basis of strain distinguishing residues are not available.
One of the main challenges, therefore, in making efficient detection systems for ssRNA viruses is to devise methods to unambiguously distinguish subtle sequence variations among the different ssRNA virus strains using stain distinguishing residues. This challenge becomes significantly tough when only partial sequences of these viruses are available. The only feasible way to address this problem is through computational techniques. However, all such known techniques are based on criteria that allow them to distinguish only significantly different strains. In contrast, the intellectual property described here is a software product based on different computational criteria. The product successfully demonstrates a way to distinguish among very closely related virus strains of two important ssRNA virus families. The only requirements of the software are the availability of accurately known (complete or partial) genomic or protein capsid sequences of these viruses. Given the rapidly improving sequencing techniques, this should not be a major problem, and, it should therefore be possible to design and manufacture efficient ssRNA virus detection systems based on the described software. It is anticipated that the software will reduce both time and costs in identifying closely related ssRNA viruses from their sequences by substantially reducing the throughput time.
Most non-bacterial epidemic outbreaks are caused by single stranded RNA (ssRNA) viruses. Typically, these viruses undergo rapid genetic mutations that result in a large and dynamic population diversity seen as different virus strains utilizing multiple hosts [1]. Caliciviruses and picornaviruses are two of the most highly divergent ssRNA virus families each containing several hundred reference strains showing very low sequence identity even within families [2-4]. The software determines relationships among the strains using a unique algorithm in contrast to existing methods. Relationships among the strains are usually inferred through conventional homology based comparisons using complete capsid sequences or other genomic regions. These comparisons seek to identify clusters of similar sequences that comprise the major sequence groups (genogroups or genera) and their sub-groups leading to various diagnostics [5-16] and classification schemes for these viruses.
The four calicivirus genera (noroviruses, sapoviruses, lagoviruses and vesiviruses) [3, 4, 17] and the nine picornavirus genera (apthoviruses, cardioviruses, enteroviruses, erboviruses, hepatoviruses, kobuviruses, parechoviruses, rhinoviruses and teschoviruses) [2] are classified using such schemes [2, 4]. Further divisions of these genera reflect more detailed sequence relatedness among these viruses. For example, among the diverse caliciviruses [17], noroviruses are divided into two genogroups GI and GII [18-20] each of which contains seven sequence clusters (GI.1-GI.7 and GII.1-GII.7) [4], sapovirus sequences are grouped into 2-5 genogroups each of which contains several clusters [2]-23], vesivirus sequences are known to contain at least 40 immune response related antigenic serotypes and lagovirus sequences cluster into proposed sero-specific groups [24]. Similarly, classification of the 9 picornavirus genera into species, each of which consists of several serotypes [2, 25] reflects finer relations among these virus sequences (Table 1).







TABLE 1







Species and Serotypes of All Picornavirus Genera










Genera
Species (Abbreviation) [Number of serotypes]







Aphthoviruses
Foot-and-mouth disease virus (FMDV) [7]




Equine rhinitis A virus (ERAV) [1]



Cardioviruses
Encephalomyocarditis virus (EMCV) [1]




Theilovirus [2 or 3]



Enteroviruses
Human enterovirus A (HEV-A) [12]




Human enterovirus B (HEV-B) [36]




Human enterovirus C (HEV-C) [11]




Human enterovirus D (HEV-D) [2]




Bovine enterovirus (BEV) [2]




Porcine enterovirus A (PEV-A) [2]




Porcine enterovirus B (PEV-B) [2]



Hepatoviruses
Hepatitis A virus (HAV) [1]



Kobuviruses
Aichi kobuviruses (AKV) [1]




Bovine kobuviruses (BKV) [1]



Parechoviruses
Human parechoviruses (HPeV) [3]



Cardioviruses
Encephalomyocarditis virus (EMCV) [1]




Ljungan viruses [1]



Rhinoviruses
Human rhinovirus A (HRV-A) [75]




Human rhinovirus B (HRV-B) [25]



Teschoviruses
Porcine Teschoviruses (PTEV) [11]







Abbreviations for species are shown within parentheses and the number of serotypes for given species are shown within square brackets.






The available crystal structures of several calici and picornavirus capsids [26-32] further help understand such sequence relationships including those among the four subunits (VP1-VP4) of the picornavirus capsids [2].

Strain and genogroup predictions in uncharacterized target sequences of calici and picornaviruses depend critically on their sequence relationships. Most often, such predictions use conventional homology comparisons between the target and a large number of known reference sequences. However, there are difficulties in these approaches when applied to caliciviruses. Most prediction methods for these viruses are based on sequence similarity cut-off values that are arbitrarily derived from the homology based sequence comparisons [19]. Although recent reports indicate statistically significant estimation of such cut-off values in distinguishing the major norovirus genogroups [33], no uniform criteria exist to accurately estimate these values for the other caliciviruses. In addition, homology based sequence similarity cut-off values are even more difficult to estimate when different virus genera need to be analyzed together in situations for example, where the genus of the target sequences may not be known. These difficulties are compounded while determining the strains of partial sequences mainly because experimental considerations usually restrict these partial sequences to smaller and relatively more conserved regions [15, 19, 34-37] whose comparisons may often introduce ambiguities in strain identification.
Even if complete sequences of target virus capsids are compared [33, 38], strain determination using homology based similarity scores is still computationally challenging. This is because comparisons of a large number of complete capsid sequences demands significant computation time which increases exponentially with increasing sequence lengths and the number of sequences that are compared together. Such limitations may severely reduce the number of usable reference capsid sequences thereby creating major computational bottlenecks.
Recent methods to genotype sequences belonging to certain virus families [39] suggest ways to reduce such bottlenecks. These methods efficiently align sliding windows of target sequences with databases of reference sequences and genotype the target sequences essentially using highest overall alignment scores. However, such methods, primarily designed to detect recombination breakpoints within virus genomes, critically depend on parameters such as window sizes and choice of reference sequences. Smaller windows may significantly increase the computation time while larger windows may overlook fine sequence variations. Similarly, incorrect choices of reference sequences may introduce possible error inducing biases. Time consuming repetitive runs using different trial settings of these parameters may be necessary to correctly genotype virus strains in such cases [39].
Thus, strain recognition methods using sequence identity based scores have not been easily amenable to reliable and robust automation across ssRNA virus families. Based on earlier analysis of noroviruses [40], we describe here the generalized implementation of a residue-wise comparison based approach to automate strain predictions in complete and partial amino acid capsid sequences of calici and picornaviruses.
SUMMARY OF THE INVENTION
The present invention provides a software product implemented on a computer or a distributed computer environment such as an intranet (e.g., secure institution, corporate, etc. network) or an internet (e.g., the world wide web) that uses unique characterizing residues to automatically identify strains of partial or complete sequences of pathogens or nonpathogens, where the sequences can be nucleic acid sequence (DNA, RNA, DNA/RNA hybrids, RNA/protein hybrids, DNA/protein hybrids or any other biomolecules that include a sequencable nucleotide moiety), proteins, or any other biomolecule that includes a sequence of monomers that is unique to an organism or virus, e.g., a pathogen. These residues are possible adaptive mutation sites in the pathogens considered.
The present invention provides a software product implemented on a computer or a distributed computer environment such as an intranet (e.g., secure institution, corporate, etc. network) or an internet (e.g., the world wide web) that uses unique characterizing residues to automatically identify strains of partial or complete capsid sequences of picorna and caliciviruses, two of the most highly diverse ssRNA virus families.
The present invention also provides a computer program product stored on a computer readable medium for predicting strains of some single stranded RNA viruses from their limited sequence data, the computer product including a graphical user interface (GUI) code operable to carry out all data input-output (I/O) operations; a storage code operable to store pathogen sequences such as viral sequences, in one sequence database or a plurality of sequence database in the form of multiple data arrays containing information about phylogenetic trees, sequence groups and characteristic residues of these groups; a sequence comparison code operable to compare input sequences such as viral sequences with the stored database sequences on a residue-by-residue basis; an identification code operable to identify the strains of the input sequences such as viral sequences based on comparisons (iii) and subsequent decision making algorithms.
The present invention also provides a method of evaluating comparisons including the steps of identifying tree branches containing maximum number of characteristic residue matches obtained from comparisons; identifying tree branches to carry out further searches after identifying maximally matching tree branches; identifying tree branches to carry out further searches in case there are no unique maximally matching tree branches; and confirming that the chosen maximally matching tree branches do not lead to ambiguous comparisons.



BRIEF DESCRIPTION OF DRAWINGS
The invention can be better understood with reference to the following detailed description together with the appended illustrative drawings in which like elements are numbered the same.
Figures for the Capsid Embodiment
FIG. 1A depicts sequence groups and characteristic residues in partitioned phylogenetic tree: Representative phylogenetic tree with sequence groups s1-s12 forming the branches of the tree. Each group may consist of one or more sequence clusters. Vertical lines divide the tree into phylogenetic distance based partitions 1-10. Nodes closest to a given partition line and located to the left of the lines define the sequence groups belonging to that partition. Root node and other nodes (A-F) up to partition 5 are shown. Groups corresponding to the different nodes are denoted as “Group n” where n is the node name. For a given partition, characteristic residues (i.e. those residues which are conserved within individual groups but not across the groups) are designated as X and color matched with their node and group names. Subscripts of X denote residue locations which are numbered with respect to a user-defined reference sequence.
FIG. 1B depicts a software package GUI screenshot of this invention. Three options are shown in the orange encircled region at the top left hand corner of the GUI; File: Allows the output display to be saved in a file; Tree view: Displays genus tree in database; Help: Provides only limited help as most of the GUI options are self-explanatory through mouse attached tips. The user input and the program output parts of the GUI are indicated at the bottom. The program output part of the GUI displays the output results of the program. The user input area is divided into three parts shown using green arrows: “query sequence input”, “database input” and the “output control options”. These three parts correspond to the “select sequences”, “database” and “view results” options in the GUI. Details of each of these parts are shown using differently colored arrows. The white board/“browse files” button (grey arrow) is used to input query sequence(s). Drop-down menus and toolbars in GUI's database input part are shown using orange arrows along with accompanying numbers; 1: “Choose a database” drop-down menu allows users to select the input target sequence genus, if known. An “unknown” option may be chosen if the genus is not known; 2: “Select a reference” allows users to select a reference sequence from the drop-down menu; 3: “View individual database sequence” option allows users to display a sequence from the selected database; 4: “View all sequences in the database” option allows users to display all the sequences in the selected database; 5: “View group members in a partition” allows users to select a partition whose all sequences within the different groups are displayed; 6: “Run” button allows program activation. The yellow arrows and their accompanying numbers in GUI's “View results” part indicate output display options: 7: “Sequence order”: displays ID number of each input target sequence; 8/9: Display brief summary/details respectively of partition-wise matches of characteristic residues for an input target sequence identified by its ID in box shown using arrow ‘(a)’; 10: Displays details of characteristic residue matches for a chosen partition specified in box shown using arrow (b).
FIGS. 1C&D depict incongruous phylogenetic trees to explain recombination detection using the software: Representative phylogenetic tree with sequence groups s1-s12 forming the branches of the trees. Sequences s1 and s10 are interchanged in the two hypothetical trees to highlight a simple case of recombination-induced incongruities between the two trees.
FIG. 2A depicts partition-wise matching of norovirus characteristic residues using the software. The matches are shown as a wheel representation of the 10 partitions P1-P10 showing partition-wise matches for the aligned input target sequence (norovirus “Seacroft”; NCBI accession no. AJ277620). Single letter codes of input target sequence residues that match the characteristic residues in the norovirus database are shown within square brackets for partitions P1-P7. For each partition P1-P6, all sequence groups Gn, where n represents letter combinations, are color coded within parentheses outside the wheel. Of these, sequence groups that match the database characteristic residues are shown in black. Details about the remaining partitions are available from website prion.bchs.uh.edu/recovir. Symbols Pn+ indicates that all characteristic residues of the previous partition “n” are also included in the current partition.
FIG. 2B depict detailed partition-wise mapping of characteristic residue locations of aligned target sequence (norovirus “Seacroft”; NCBI accession no. AJ277620) on to reference Norwalk virus (PDB ID: 1IHM) sequence. The last residue in each line is numbered and every tenth residue is marked using the V symbol. The symbols and  map all of such residues from partitions 1, 2, 3, 4, 5 and 6 respectively of FIG. 2A where these symbols are shown near the edge of each partition.
FIG. 3 depicts a sample output from ‘overall classification’ button in GUI: Output summary of partition-wise matches of characteristic residues for a typical norovirus query sequence with output line numbers shown within parentheses in red. Details of the query sequence such as its number, input file, organism and sequence accession number are shown in lines 1-3. The partition-wise search results are shown in line 4. Each arrow indicates search progression to partition ‘Pn’ shown in blue, where n is the partition number. Line 5 shows the matched sequence groups of FIG. 2A within parentheses and in magenta color. The ‘.’ symbol indicates a partition which is identical with the previous one. The block of lines indicated as “6” shows the identified strain of the target sequence and details the sequences of the strain containing group.
Figure for the Adaptive Mutations Embodiment of this Invention
Strain Diversifying Locations in Human Rhinovirus (HRV) and Schematics to Explain Proposed Adaptations Through Strain Diversifying Mutations
FIG. 4A depicts a partitioned phylogenetic tree of 100 HRV-VP1 capsid sequences of serotypes denoted using numbers. Red lines indicate the 10 partitions P01-P10. The P02 classes A and B (nodes 1 and 2) are boxed. Green highlighted strains belong to the minor group while the remaining strains constitute the major group. The orange highlighted HRV-A strains are not consistent with the anti-viral grouping of Andries et al. (1990). 1AYM (PDB code) denotes HRV16 serotype.
FIG. 4B depicts HRV serotypes belonging to different classes (shown boxed and alphanumerically labeled: A, B A1-A29, B1-B5) in P02-P08 partitions of FIG. 1a. Boxes with these labels inside them indicate classes that remain unchanged in subsequent partitions. Emerging classes are colored green. Boxes highlighted in green indicate P09 classes.
FIG. 4C depicts green spheres indicate some of the HRV-VP1 class-specific (or strain diversifying mutation) locations (HRV16 numbering) that distinguish between the serotypes of A and B classes of partition P02 (FIGS. 1a, 1b). The N- and C-termini are shown in blue and red respectively. The red spheres marked as 88 and 89 within a red box indicate the HRV16 locations corresponding to the NIm-IA sites of HRV14. Strain diversifying residue 213 (magenta) interacts with HRV16 receptor. The magenta colored pentagon denotes the approximate icosahedral 5-fold axis location.
FIG. 4D depicts structural pockets (P1, P3) and cavities (C2) localize strain diversifying mutation residues that are also involved in receptor binding (RB) and antigenicity. This causes interplay between strain diversification and the capsid related functions in and around the pockets and cavities, thereby, facilitating adaptations. Capsid region (green) depicting strain diversifying, RB and antigenic sites bound to receptor and antibody. Pockets P1 & P3 are solvent-accessible through regions N1 and N4 respectively while cavity C2 is solvent-inaccessible. However, cavity C2 is connected with pockets P1 & P3 through residues in regions N2 & N3 thereby depicting a network of cavities and pockets. Blue dots on P1, C2 and P3 indicate strain diversifying residues.
FIG. 4E depicts arrow showing strain diversifying mutations induced by host immune responses. These mutations may create altered receptor binding and antigenic characteristics by changing the surface and possibly regions N1 and N2. The altered regions are indicated within the red rectangle.
Partitioned Tree of Poliovirus Serotypes
FIG. 5 depicts partitions are shown as P01-P10. The two P02 classes PV1 and PV2+3 are boxed. These two classes originate from nodes 1 and 2 respectively. The three poliovirus serotypes 1, 2 and 3 are denoted as PV 1, PV2 and PV3 respectively. Nodes 3 and 4 which separate the PV2 and PV3 respectively are shown in partition P03. Names of some of the well known serotypes are explicitly shown. Sequence accession numbers are appended at the end of each entry. Partitions are shown as P01-P10.
Rhinovirus Class-specific Capsid Residues Lining the Largest VP1 Pocket
FIG. 6A depicts a surface representation of five copies of the largest computed VP 1 pocket viewed down the icosahedral 5-fold axis denoted by the magenta pentagon. The VP 1 residues lining each of these pockets are (I77, W96, I98, N99, L100, Q101, R107, F110, E111, F118, D119, S120, I122, M124, Y142, M143, Y144, P147, A166, S167, V168, F179, L181, P182, L184, S185, I186, A187, A189, Y190, Y191, M192, F193, Y194, Y206, G207, T208, T211, N212, D213 (contacts HRV16 receptor), M214, L217, I236, H238, R252, Y256, H260, T261, T262 and N263) (HRV16-numbering). These residues are not shown. The pockets are enclosed within the magenta ellipses and their exposed parts are colored green.
FIG. 6B depicts details of FIG. 6A pockets: The P02 class-specific (strain diversifying) VP1 residues 102, 103 and 239 (FIG. 1c) are shown in each of the 5 icosahedral symmetry related copies of the largest computed pocket. These residues, along with residue 217, form parts of the roofs and the floors of the pockets. Labels are shown for only one of the pockets. Orange arrow points to representative antiviral compound (PDB ID: 1ND3) shown in the largest computed pocket.
Class-Specific (Strain Diversifying) Capsid Residues Line Most Norovirus Pockets
FIG. 7A depicts sphere representation of norovirus S, P1 and P2 domain class-specific (strain diversifying) residues in P02-P06 partitions superposed on the A-B dimer of the Norwalk virus capsid (PDB ID: 1IHM). Residues of partitions P02-P04, P05 and P06 are shown in green, red and cyan respectively. The vertical arrow shows the dimer axis. Letters N and C indicate corresponding termini in the two monomers. The purple colored residues (44 and 514) are the only exposed P02 class-specific residues (Chakravarty et al. (2005))
FIG. 7B depicts some of the exposed parts of the largest pockets in Norwalk virus dimer surface (wheat color). Exposed regions in S and the P1-domain pockets are shown in blue and green. Similar regions in the P2-domain pocket (#161 in Table 3) are indicated using a cyan arrow. Orange colors indicate the exposed regions near pockets around P1-P2 domain interface while the magenta colored region indicates the exposed parts of the hinge region pocket. Dimer orientation is that of FIG. 4a. 
FIG. 7C depicts P2-domain pocket of FIG. 4B. Green spheres represent inaccessible atoms of the pocket. Some class-specific (strain diversifying) residues that putatively bind norovirus receptor, line this pocket. These residues are indicated using arrows. The 4-letter codes denote subunit and residue numbers, e.g. W1375 and W2375 indicate residue 375 in subunits A and B respectively. The view is a 90° rotation of FIG. 4b about the horizontal direction.
FIG. 7D depicts the P2-domain pocket and class-specific residues (CSRs) of FIG. 4c shown against secondary structure of the domain. The view is that of FIG. 4c. Residue 331 forms part of the class-specific region formed by the CSRs shown and their neighbors.
FIG. 7E depicts close-up of the green colored P2-domain pocket region of FIG. 4C. The 4-letter codes of FIG. 4C are used to number the residues. Additional exposed residues 291-293 (purple) are located in the vicinity of the pocket.
Picornavirus Diversity Obtained by Partition-Wise Comparisons of Capsid Sequences
FIG. 8 depicts only 5 partitions P02-P06 are shown. Numbers in cyan within parentheses above each partition indicate the number of iterative sequence comparisons required to generate new P02 classes that are identical with that of the given partition. The starting P02 partition for such iterations is indicated in orange with an iteration number 0. Each rectangular box represents a class within the partition. Each class is separately numbered using color-coded pentagons. All P02 classes are shown within boxes that are outlined in magenta. Symbol used: ‡: Possible genera (P02 classes); Abbreviations used: HRV: Human rhinoviruses; Entero (HEV-A, B, C, D): enterovirus genus (A-D species); The HEV-D-68 & 70 represent serotypes 68 and 70 of the HEV-D species; Rhino-A and B: Rhinovirus genus and A & B species; Cardio (EMCV & TME): cardiovirus genus (encephalomyocarditis and the Theiler murine encephalitis species); Erbo (A & B): Erbovirus genus (A & B species); Aphtho (fmA, fm-Asia, fmC, fmO, fmSAT1, fmSAT2): aphthovirus (fm(Foot-and-mouth) species and A, Asia, C, O and South African (SAT) serotypes 1 & 2); SEV: Simian enteroviruses. Abbreviations of the remaining genera (hepatovirus, parechovirus, erbovirus, kobuvirus and the teschovirus) are self explanatory. Arrows indicate emergent classes in different partitions. Classes that show changes in subsequent partitions are connected using magenta arrows while classes that remain unchanged are connected using grey arrows.
Supplementary Figures
FIG. S1 depicts partition P02 insertions contain a human rhinovirus (HRV) neutralizing immunogenic (NIm) site of VP1 capsid protein: Conservation patterns among HRV classes for partitions P02-P09 are shown. Classes that are horizontally shaded in light blue and light green color contain the HRV14 and HRV16 (PDBID: 1AYM) sequences respectively. These classes are also indicated within parentheses next to each partition header. Square brackets show classes 1-35 that emerge in partitions P06-P09 from the two P02 classes A and B (shown within black square boxes). Dots (.) and dashes (-) indicate insertions and variable regions respectively. Highlighted green and red vertical bars indicate residues 91 and 95 respectively that belong to the NIm-Ia site of HRV14 serotype. Symbols “X” refer to class-specific locations.
FIG. S2 depicts a partitioned (P01-P10) tree of the human enterovirus (HEV) A and B species: Partition P02 classes are boxed as HEV-A and HEV-B. Nodes corresponding to these classes are shown using green circles. The HEV-C sequence 1Z7S has been included to serve as a marker as it does not belong to any one of these classes. Accession numbers are appended at the end of each sequence. Abbreviations used: cox: Coxsachievirus; echo: Echovirus; pHEV-nn: Proposed HEV-nn where nn is the serotype number; HK—1: Hongkong strain; PDBID: Protein Data Bank 4-letter code (the subunit name is also shown); SVDV: Swine vesicular disease virus.
FIG. S3 depicts partitions P01-P10 of the human enterovirus (HEV) A, B and C serotypes: The two boxes “HEV-A” and “HEV-(B&C)” denote the two P02 classes. The former consists of strains of the known A serotype while the latter consists of B and C serotypes together. A1-A4 indicates nodes from which HEV-A classes originate in subsequent partitions. The HEV-C strains are shown using a square bracket. B′ and C′ indicate nodes from which the HEV-B and C serotypes originate. Polioviruses are indicated within a red box. Accession numbers are appended at the end of each sequence entry. Abbreviations used: pv: Poliovirus; cv: Coxsachievirus; echo: Echovirus; pA, pB, pEcho: Sequences currently proposed as HEV-A, HEV-B and echovirus respectively; pEcho: Proposed echovirus; pB: Proposed HEV-B species; HK—1: Hongkong strain; PDB: Protein Data Bank 4-letter code. Serotypes are appended to these abbreviations e.g. cv_B3 implies coxsachievirus B3 serotype.
FIG. S4 depicts partitioned comparisons of the human enterovirus (HEV) A, B, C and D species: Species A, B, and C form two P02 classes that are shown within 2 boxes. One of these classes contains strains of HEV-A species while the other one contains HEV-B & HEV-C species together. The HEV-D class is indicated separately. Accession numbers are appended at the end of each sequence entry. Abbreviations used: cv: Coxsachievirus; echo: Echovirus; pA, pB, pEcho: Sequences currently proposed as HEV-A, HEV-B and echovirus respectively; pHEV-nn: Proposed HEV-nn where nn is the serotype number; PDB: Protein Data Bank 4-letter code. Serotypes are appended to these abbreviations e.g. cv_B3 implies coxsachievirus B3 serotype. The square bracket indicates HEV-C serotypes. The polioviruses have been omitted for clarity.
Database Structure Used in Adaptive Mutations Embodiment
FIG. D1 depicts the rhino cDNA database used in the adaptive embodiment.
FIG. D2 depicts the code used read the database used in the adaptive embodiment.
FIG. D3 depicts the group information used in the structured data of the database used in the adaptive embodiment.
FIG. D4 depicts the partition trees of the database used in the adaptive embodiment, consisting to ten partitions.
FIG. D5 depicts partition one (P1) of the database used in the adaptive embodiment.
FIG. D6 depicts Partition one (P2) of the database used in the adaptive embodiment.
FIG. D7 depicts Partition one (P3) of the database used in the adaptive embodiment.
FIG. D8 depicts Partition one (P4) of the database used in the adaptive embodiment.
FIG. D9 depicts Partition one (P5) of the database used in the adaptive embodiment.
FIG. D10 depicts Partition one (P6) of the database used in the adaptive embodiment.
FIG. D11 depicts Partition one (P7) of the database used in the adaptive embodiment.
FIG. D12 depicts Partition one (P8) of the database used in the adaptive embodiment.
FIG. D13 depicts Partition one (P9) of the database used in the adaptive embodiment.
FIG. D14 depicts Partition one (P10) of the database used in the adaptive embodiment.



DETAILED DESCRIPTION OF THE INVENTION
The inventors have found that a software product can be constructed and implemented on or in computer or a distributed computer network, where the software product encodes an unique method for characterizing residues constructed to automatically identify strains of partial or complete sequences of pathogens. The product includes databases of pathogen residues that uniquely characterize strains of the pathogens and were created by constructing partitioned phylogenetic trees of available complete capsid sequences of these viruses. Partition-wise comparisons of the database residues with the corresponding residues of complete and partial sequences of unknown pathogens results in correct strain identification for all pathogens for which data are stored in the software database. A Java based user-friendly graphical console interfaced with Perl-coded computational parts ensures high portability of the software. The package has been successfully tested on MS-Windows XP and the Linux and UNIX platforms. The code used in this implementation is attached as Appendix 1.
The inventors have found that a software product that uses unique characterizing residues can be constructed to automatically identify strains of partial or complete capsid sequences of picoma and caliciviruses, two of the most highly diverse ssRNA virus families. The software is an efficient implementation of an algorithm outlined in earlier by on of the inventors in structure based phylogenic analysis of some human calicivirus sequences. Databases of capsid residues that uniquely characterize strains of picoma and caliciviruses were created by constructing partitioned phylogenetic trees of available complete capsid sequences of these viruses. Partition-wise comparisons of the database residues with the corresponding residues of more than 300 complete and partial sequences of these viruses resulted in correct strain identification for all of these sequences.
In one embodiment, the present invention relates to creating databases of capsid residues uniquely distinguishing among virus reference strains and using these databases to automatically predict strains of other closely resembling target sequences. The method is demonstrated for complete and partial capsid sequences of calici and picornaviruses, which are two of the most highly divergent ssRNA virus families and therefore especially difficult to unambiguously identify using a uniform method.
Capsid Embodiment of the Invention
METHOD
Basis
The method relies on creating databases of capsid residues that uniquely distinguish among the reference calici and picornavirus sequences. Residue-wise comparisons of the complete or partial input target sequences with the databases yield those reference sequences that are closest to the target sequences. Such reference sequences, in turn, yield the genogroup and other classification characteristics of the target sequences thereby identifying their strains.
Partitioned Phylogenetic Trees Yield Groups of Similar Sequences
Following the procedure described earlier [40], evolutionary trace [41] based partitioned phylogenetic trees were constructed for each of the 4 calicivirus genera and the 9 picornavirus genera using a large number of complete capsid sequences from public domain databases [25, 42]. These sequences will be called the “reference” sequences and the corresponding trees will be called the “reference” or the “genus” trees as each such reference tree represents a calici or a picornavirus genus.
By sieving the reference sequences of a genus tree through “similarity filters”, sequence groups, each of which contains several similar reference sequences, were created in every partition of the tree. Examples of such sequence groups are illustrated for a hypothetical representative tree (FIG. 1A). Starting from the root node, the partition 1 contains all of the aligned sequences. Sequence comparisons in this partition, therefore, are equivalent to conventional sequence comparisons that consider all of the sequences together. Partitions 2 and 3 are identical and contain the sequence groups s1-s11 and s12 belonging to nodes A and B respectively. Similarly, the two nodes C and D belong to partition 4 while nodes E and F belong to partition 5. Node C of partition 4 contains sequence groups s1 and s2 while node D of the same partition contains nine (s3-s11) of the remaining groups. Similarly, the eight groups (s3-s10) in partition 5 belong to node E while group s11 belongs to node F in the same partition (FIG. 1A). Henceforth, sequence clusters or sequence groups will be referred to simply as groups.
Sequence Groups Comparisons Reveal Characteristic Residues Within Genus Trees
For each genus tree, partition-wise comparisons among the different groups identified “characteristic residues” that are conserved within each group but not among the different groups of the partition. For example, characteristic residue X1 at location 1 of partition 2 of a given tree may be a conserved Ala for Group A of node A in contrast to a conserved Gly (X1′) for Group B of node B (FIG. 1A). Such residues were generated for each genus tree following the procedure described earlier [40].
Database Creation
Entire information about the genus trees including their partitions, all the sequence groups for each partition along with all characteristic residues taken group-wise, were stored in multiple 2-dimensional arrays that formed the calici and the picornavirus databases.
Strain Identification Through Partition-Wise Comparisons
To identify the strain of the input query (“target”) sequence whose genus is known, the program matches the target residues with the characteristic residues of each group of a given partition stored in the appropriate genus database. This is done by first aligning the target sequence with a reference sequence of the database. Then, starting with the second partition from the root (partition 2 in FIG. 1A), each characteristic residue of a given group in this partition is compared with the target residue at the corresponding location. Such comparisons are carried out for all the groups of this partition. The group showing the maximum number of matches is accepted by the program as the most probable sequence cluster resembling the input target sequence in that partition.
The program proceeds to the next partition, where, instead of similarly testing all the groups present in the partition, the program tests only those groups that are directly tree-linked with the most recently accepted group. This considerably reduces the number of groups to be searched in the next partition. The process continues until all partitions have been searched. Testing only a limited number of connected groups per partition guarantees an optimal tree search time thereby making the program quite efficient.
Within a given partition, input target sequence residues once matched are flagged as “marked” and are never used again for residue matching in subsequent partitions. Exceptions to the flagging procedure are carried out only in case of ambiguities. For example, if all of the groups in a partition show an identical number of characteristic residue matches, an ambiguity is declared and no match is flagged. This ensures that all such matched residues of the input sequence are available again for matching purposes in subsequent partitions thereby helping resolve the ambiguity. Ambiguities may also occur when all groups within a given partition show no matches with the input sequence, or, if two successive partitions show identical numbers of characteristic residue matches. In both these cases, the program ignores the ambiguous partition(s) and proceeds to the next one without marking any residue at all. This allows all of the unmarked residues in the current partition to be compared again in subsequent partitions.
To illustrate the method, let us assume that the input sequence corresponds to a known genus whose characteristic residues X1, X2 and X3 for partition 2 are Ala, Pro and Ser respectively for group A and the corresponding group B residues (X1′, X2′ and X3′) are Gly, Thr and Met, respectively (FIG. 1A). If the aligned target sequence shows more matches for X1, X2 and X3 of group A in comparison with that of X1′, X2′ and X3′ of group B, it implies that the input sequence belongs to group A in partition 2 and not to group B. Thus, residues X1, X2 and X3 are flagged and comparisons in subsequent partitions follow along those branches that are connected to group A (FIG. 1A). The program therefore proceeds to groups C and D in partition 4 ignoring partition 3 as it is identical to the previous partition 2 (FIG. 1A).
In partition 4, if both groups C and D show equal number of matches i.e. say, X4 and X4′ are both Trp and X5 and X5′ are both Leu (FIG. 1A) and the aligned target sequence also contains Trp and Leu at these locations, then the program will not flag these characteristic residues but will instead carry them over to partition 5 where these residues will again be matched in groups E and F (FIG. 1A) to determine which of these two groups maximally matches the target sequence. Similar comparisons in subsequent partitions 6-10 unambiguously identify the database strain that most closely resembles the input target sequence thereby yielding its strain characteristics.
In case the genus of the target sequence is not known, this sequence is first compared with groups of representative reference sequences (<3 sequences per group) from each of the genus trees in the database using ClustalW [43] and the alignment scores computed for each group. The highest alignment score indicates the genus of the input sequence which allows the appropriate genus tree to be selected from the databases for further strain identification using the earlier described procedure. A low number of sequences per group ensure rapid genus determination regardless of the number of reference sequences present in the genus tree.
Detecting Recombination and Spontaneous Mutations
Partition-wise comparisons allow RECOVIR to detect abrupt changes in phylogenetic sequence groupings among different trees. Assuming that absence of recombination creates similar phylogenetic relationships among sequences from different regions of multiple sequence alignments, these abrupt changes indicate nodes that may possibly contain recombination sites. Such abrupt changes are shown schematically using a pair of hypothetical trees (FIGS. 1C&D). These trees may represent, for example, two different genomic regions of the same virus strains. A simple example of incongruence has artificially been built in by interchanging sequence group s1 and s10 between the two trees. Consequently, sequence groups belonging to nodes A and B may be distinguished by the same set of residues (X1, X2, X3) and (X1′, X2′, X3′) in both the trees. However, subsequent nodes C, D, E, and F have different strain distinguishing residues up to partition 5 of the two trees that correspond to the two genomic parts being compared (FIGS. 1C&D). For example, nodes C and D are distinguished by residues (X4, X5, X6) and (X4′, X5′ X6′) in one part of the genome (FIG. 1C) and by (Y4, Y5, Y6) and (Y4′, Y5′, Y6′) in the other genome part (FIG. 1D). Similarly, nodes E and F are distinguished by residues (X7, X8, X9) and (X7′, X8′, X9′) in one part of the genome (FIG. 1C) and by (Y7, Y8, Y9) and (Y7′, Y8′, Y9′) in the other genome part (FIG. 1D). Systematic node-wise comparisons of such strain distinguishing residues in all of the partitions P2-P10 clearly show the genomic regions that result in the incongruence between the two trees and thus, indicate parent strains along with their possible recombination or spontaneous mutation sites. Detection of these sites and the corresponding parent strains is done manually at present as an automated version of this feature has not yet been built into the software.
Program Testing and Validation
The program was initially validated by identifying the closest strains for five noro and five enteroviruses of known genera from their respective complete and partial amino acid sequences of the capsids (Table 2). These viruses were chosen because of their wide sequence divergence and their large number of strains or serotypes many of which have multiple sequences available in public databases. The enteroviruses included the complete VP1 sequences of Poliovirus-2 (serotype), human enterovirus (HEV)-B including the Coxsackie viruses, HEV-D (serotype) and the simian enteroviruses.
The program was then further validated using more than 200 complete and partial sequences of different caliciviruses and more than 100 such picornavirus sequences. Among the calicivirus sequences, nearly 120 sequences were those of noroviruses with the remaining ones being those of other caliciviruses. Similarly, in the case of picornavirus sequences, nearly 50 were enterovirus partial sequences including those of echoviruses and of other HEV-B serotypes [25] while the remaining sequences were chosen from the other picornavirus genera and species (Table 1).
Most of the partial sequences were randomly chosen from different regions of the capsid with the sequence lengths being only  ̃20% of the sizes of the corresponding complete capsid sequences. All of the noro and enterovirus sequences were selected from the NCBI databases [42] taking care that possible errors due to biased choice of these sequences were minimized by ensuring that none of the selected sequences were included in the program databases.
Software Description
All functional modules of RECOVIR were written in the Perl programming language on Windows XP, Linux, and UNIX platforms. A Java based graphical user interface (GUI) has been designed to wrap all functionalities and allow user-friendly I/O options (FIG. 1B).
Program Input and Output
Input selections on the GUI have been divided into 3 categories: sequence, databases and options to control output (FIG. 1B). Any number of query sequences may be input by either pasting them on the white board area or by browsing one or more directories for single or multiple sequence files. A given sequence file may contain any number of complete and partial sequences. Only FASTA formatted sequence files are accepted and all white spaces and non-alphabet characters are ignored in input sequence files.
A dropdown menu in the database section of the GUI allows the user to specify the input sequence genus, if known (FIG. 1B). A default reference sequence, used for aligning the input sequences and for assigning the aligned location numbers to the input sequence, then pops up in the “Select a reference” box (FIG. 1B). This default reference may be changed if required. In case genus of the input sequence is not known, the “unknown” option in this box (FIG. 1B) allows the program to automatically determine the genus and an appropriate reference sequence from the built-in databases.
The “Run” button activates the program, displays a progress bar indicating the percentage of input sequences processed and outputs results on the GUI's output section (FIG. 1B). Depending on the “View results” options chosen, the output may include a summary or the complete details of partition-wise matches between the database characteristic residues and the residues of the input target sequences. Many other details about the partitioned databases and run results may be viewed using various user-friendly options in GUI's output control section (FIG. 1B).
Results and Discussion
Strain Identification of Complete and Partial Norovirus Capsid Sequences
Databases
Detailed strain identification results are described for only some norovirus strains among caliciviruses and some enterovirus strains among the picornaviruses. Complete details for the other calici and picornaviruses can be found at web site prion.bchs.uh.edu/recovir.
Sequences belonging to the norovirus genus tree have been described elsewhere [40]. Briefly, these sequences form a single group in partition P1 (FIG. 2A). Partition P2 splits this sequence group into the 2 known major genogroups GI and GII of noroviruses (See additional file 1: Supplementary Table 1) [4, 40]. Group GI further divides into GIa and GIb in partition P3 while group GII divides into GIIa and GIIb in partition P4 (or P5 that is identical with P4) (FIG. 2A & See additional file 1: Supplementary Table 1). Partition 6 further splits both GI and GII groups. Group GIa is divided into GIa1 and GIa2 while the group GIa splits into 4 groups (GIIa1-a4). In partition 7, Group GIa splits into 4 groups GIa1-a4 while GIIa3 splits further into the GIIa3—1 and GIIa3—2 groups (FIG. 2A & See additional file 1: Supplementary Table 1). Groups GIb, GIIa4 and GIIb do not split up any further in this partition. Details of the remaining partitions P8-P10 are available at website prion.bchs.uh.edu/recovir.










SUPPLEMENTARY TABLE 1










Sequence



Partitions
groups
Norovirus sequences in groups





P2
GI
Norwalk_1IHM, Aichi_Aic[AB010145),




Kyoto89_Ky89[L23828), Chiba_Chb[AB042808),




Koblenz_Kob[Q91I85), Valetta_Val[CAB89102),




Thistlehall_Thi[CAB89102), Musgrove_Mus [CAB89095),




Southampton_Sou[Q04542), Whiterose_Wh1[CAB89091),




bs5_bs5[AF093797), Sindlesham_Si [CAB89096), Norway




Stav_Nor[AF145709), Potsdam_Pot[Q8VA02),




VA115_Va1[AY038598), Birmingham_Bi[CAB89093),




DSV_Sa[U04538), Winchester_Wi[CAB89090),




Jena_Bovine_Jen[AJ011099), Bovine_BO [AF542083)



GII
Beeskow_Bee [Q915C5), Dijon171_Dij[Q8QY55),




Berlin_Ber[Q915C2), Altenkirchen_Al[Q916E8),




Frankfurt_Fr[Q915D1), Grimsby_Gr [AJ004864),




VA387_Va3[AY038600), Parkroyal_Pa[Q9IV44),




Ludwigs_Lu[Q915C9), Koenigs_Ko[Q915C4),




Symgreen_Sy[Q9IV38), Bristol_Br[S40111),




Lordsdale_Lo[P54635), MD145_Md[Q8V0P2),




Camberwell_Ca[Q68537), Idaho_Id[Q913B7),




VA207_Va2[Q91H09), Amsterdam_Am[AF195848),




Leeds_Le[Q9IV49), Gwynedd_GW[AAL12980),




Chesterfield_Ch[Q913B6), Mexico[Q68291), Snow




Mountain_Sn[U70059), Hillingdon_Hi[Q9IV50),




MOH_MO[AF397156), White River_Wh2[AF414423),




Erfurt_Er[Q915C7), Chitta_Cht[Q9QMK6),




Schwerin_Sc[Q91I15), Wortley_Wo[Q9IV39),




Pirna_Pi[Q915C6), Dillingen_Di[Q916E6),




Wiesbaden_Wi[Q916E4), Hawaii_Ha[Q68104),




Seacroft_Se[Q9IV37), Bham132_Bh[Q9IV46),




Rbh_Rb[Q9IV40), Minireo_Re[U02030), Toronto_To[Q66296),




Melksham_Mel[X81879), Auckland_Au[U46039),




Bitburg_Bi[Q915D2), Ober_Ob[Q916E5), Japan_Ja[23830),




Arg320_Ar_Mex[Q9PYA7), Swine_Sw1[Q8V713),




Swine_Sw2[BAB83516), Alphatron_Al[AF195847), Ft




Lauderdale_Ft[AAL13031)


P3
GIa
1IHM, Aic, Ky89, Chb, Kob, Val, Thi, Mus, Sou, Wh1, bs5, Si,




Nor, Pot, VA1, Bi, Sa, Wi



GIb
Jen, BO



GII
Bee, Dij, Ber, Al, Fr, Gr, Va3, Pa, Lu, Ko, Sy, Br, Lo, Md, Ca,




Id, Va2, Am, Le, GW, Ch, Mex, Sn, Hi, MO, Wh2, Er, Cht, Sc,




Wo, Pi, Di, Wi, Ha, Se, Bh, Rb, Re, To, Mel, Au, Bi, Ob, Ja, Ar,




Sw1, Sw2, Al, Ft


P4 & P5
GIa & Ib
Same as in P3



GIIa
Bee, Dij, Ber, Al, Fr, Gr, Va3, Pa, Lu, Ko, Sy, Br, Lo, Md, Ca,




Id, Va2, Am, Le, GW, Ch, Mex, Sn, Hi, MO, Wh2, Er, Cht, Sc,




Wo, Pi, Di, Wi, Ha, Se, Bh, Rb, Re, To, Mel, Au, Bi, Ob, Ja, Ar,




Sw1, Sw2



GIIb
Al, Ft


P6



GIa1
1IHM, Aic, Ky89, Chb, Kob, Val, Thi, Mus, Sou, Wh1, bs5, Si



GIa2
Nor, Pot, VA1, Bi, Sa, Wi



GIIa1
Bee, Dij, Ber, Al, Fr, Gr, Va3, Pa, Lu, Ko, Sy, Br, Lo, Md, Ca



GIIa2
Id, Va2, Am, Le, GW



GIIa3
Ch, Mex, Sn, Hi, MO, Wh2, Er, Cht, Sc, Wo, Pi, Di, Wi, Ha, Se,




Bh, Rb, Re, To, Mel, Au, Bi, Ob, Ja, Ar



GIIa4
Sw1, Sw2



GIIb
Al, Ft


P7
G ((Ia1_1))
((1IHM, Aic, Ky89)) (Chb, Kob, Val, Thi, Mus) {Sou, Wh1}



to [Ia1_4]
[bs5, Si]



GIIa1 & a2
Same as in P6



GIIa3_1
Ch, Mex, Sn, Hi, MO, Wh2, Er, Cht, Sc, Wo, Pi, Di, Wi, Ha



GIIa3_2
Bh, Rb, Re, To, Mel, Au, Bi, Ob, Ja, Ar



GIIa4 &
Same as in P6



IIb









Norovirus strains in different sequence groups in partitions P2-P7. This distribution is based on norovirus capsid residues. Partition P2 entries show complete strain names along with their NCBI accession numbers enclosed within square brackets and strain codes that are indicated as “_X” where X refers to the 2 or 3 letter codes e.g X id 1IHM for Norwalk strain and it is Aic for the Aichi strain. These strain codes are used in all subsequent partitions P3-P7. The different GIa1 sequence groups in P7 are enclosed in different types of parentheses: GIa1—1: (( . . . )); GIa1—2: ( . . . ); GIa1—3: { . . . }; GIa1—4: [ . . . ]. The red boldfaced code in P7 indicates the minireovirus strain (NCBI accession no. U02030).

Characteristic Residue Comparisons and Strain Identification for Complete Sequences
The characteristic residues of the norovirus genus tree stored in the program databases unambiguously identified the closest strains of target norovirus complete capsid sequences (Table 2).







TABLE 2







Strains Detected for Some Partial and Complete Amino Acid Residues of Noro and Enterovirus Capsids








Input amino acid
Output









Complete capsid sequence
Partial capsid sequence
Detected strains corresponding to input cols














Col (1)
Col (2)
Col (3)
Col (4)
Col (1)
Col (2)
Col (3)
Col (4)


Noro
Entero
Noro
Entero
Noro
Entero
Noro
Entero





Seacroft
Polio_2
1UK1
Cox-B5
Minireovirus
Polio_3
Beeskow
Cox-B5


[AJ277620]
[DQ841140]
[DQ665819]
[AF114383]
[U02030]
[AF448782]
[Q915C5]
[AF114383]


Appalachicola
Simian_A
2JP1
Echo-11
Chiba
Simian-7
Chiba
Echo-11


[AF414406]
[NP_714932]
[AB264170]
[AB239122]
[AB042808]
[AF326759]
[AB042808]
[AF081326]


Baltimore_a
HEV-D
3JP2
Echo-13
Minireovirus
HEV-D
Norwalk
Echo-13


[AF414408]
[NP_740741]
[AB264158]
[AB239091]
[U02030]
[D17595]
[1IHM]
[AF081327]


Baltimore_b
HEV-71
4JP3
HEV-B
Chiba
HEV-A71
Potsdam
Cox-B3


[AF414404]
[AAY59418]
[AB264152]
[DQ842180]
[AB042808]
[AF135944]
[AF439267]
[AF231763]


Boxer
Cox-B5
5TP1
HEV-75
Potsdam
Cox_B5
Bitburg
Proposed


[AF538679]
[AAW71476]
[DQ263739]
[DQ468142]
[AF439267]
[AF114383]
[AF427112]
HEV-75









[AF152298]









Input target complete capsid sequences are shown in cols 1 and 2 while the input partial sequences are shown in cols 3 and 4. The corresponding closest database strains detected by the program are shown in the last 4 cols whose headers are color matched with those of the input sequence cols. The NCBI accession codes are enclosed within square brackets for all capsid sequences except for Norwalk virus whose PDB code is correspondingly shown. The enterovirus capsid sequences refer to those of VP1 subunits. Abbreviations used: HEV: Human enterovirus; Cox; Coxsackievirus.

As an example, the results for norovirus “Seacroft” sequence (NCBI: AJ277620) are described here. Comparisons of the characteristic residue locations with the corresponding residues of this sequence showed that the maximum number of matches in partition P2 occurred in the aligned locations 70V and 203T of group GII and not for GI (FIG. 2A). The corresponding GI locations were 701 and 201V respectively when mapped to the unaligned reference Norwalk virus (PDB ID: 1IHM) sequence (FIG. 2B). Thus, the program searched only along the GII branch of the norovirus genus tree in subsequent partitions. In partition P3, the query sequence maximally matched the GII characteristic residues 4A, 5S, 36V, 44P, 104A, 106G, 205S, 446I, 455P, 508R, 511N and 519F while the corresponding GI residues in the reference Norwalk virus sequence are 4A, 5S, 36V, 44A, 104V, 106N, 203A, 405L, 414F, 460H, 463D and 471G respectively (FIGS. 2a & 2b). In partitions P4 and P5, the maximum number of matches (102G 206C 228S 482D 562A) occurred in GIIa, indicating that further database searches in subsequent partitions should be restricted only to those groups that originate from GIIa.
Of the 4 possible GIIa choices (GIIa1-a4) in the following partition P6, the maximum number of characteristic residue matches occurred in group GIIa3 (FIG. 2A) corresponding to residues 11S, 13D, 82D, 83L, 103W, 181R, 185M, 227K, 250I, 306G, 329H, 397S, 409V, 436L, 492P, 516V and 519A of the unaligned reference sequence (FIG. 2B). Comparisons in partition P7 showed that of the 2 groups GIIa3—1 and GIIa3—2 originating from the GIIa3 group of the previous partition P6, the GIIa3—2 group maximally matched the characteristic residues (FIG. 2A). These matches occurred at 63A, 84S, 110R, 119T, 125V, 126S, 133G, 139I, 145F, 154T, 163E, 184C, 192T, 193G, 207M, 218F, 229R, 236L, 257P, 259N, 347T, 402A, 419V, 420F, 447S, 448E and 504V of the unaligned reference sequence (FIG. 2B). Similar comparisons in partitions P8-P10 conclusively showed that the query sequence is most similar to the minireovirus-like norovirus strain (FIG. 3)
Similarly, the input Appalachicola Bay, the Baltimore ‘a’ and ‘b’ and the Boxer strain sequences most closely resembled the Chiba, minireovirus and the Potsdam strains of noroviruses (see Table 2). Strains were also correctly predicted after removing random stretches of 10-15 amino acids from these sequences indicating thereby that the strain prediction capability of the program is quite robust and is independent of the locations of the input target sequences.
Strain Identification of Input Partial Amino Acid Target Sequences of Noroviruses
Strains of five partial amino acid capsid sequences of noroviruses were consistently predicted using both the “noro” and “unknown” database options of the GUI (FIG. 1B). Only one of the 5 input sequences (1UK1) explicitly included the N-terminus residues while the remaining sequences 2JP1, 3JP2, 4JP3 and 5TP1 were from different capsid regions (Table 3).








TABLE 3





Input Target Partial Capsid Sequences of Noroviruses (A)and



Partition-wise Distribution of Maximally Matching Residues (B)







Target Partial Amino Acid Residues of Norovirus



Capsid Sequences -A-








>1UK1|[NCBI:DQ665819]



MKMASSDANPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSP


RNAPGEILWSAPLGPDLNPYLSHLAR


 


>2JP1|[NCBI:AB264170]


SADGATGAGQLVPEVNTADPIPIDPVAGSSTALATAGQVNLIDPWIINNFVQAPQGEFTISPNNTPGDV


 


>3JP2|[NCBI:AB264158]


GASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTISPNNTPGDV


 


>4JP3|[NCBI:AB264152]


GTSGAGQLVPEANTAEPISMDPVAGAATAVATAGQINMIDPWIMSNFVQAPQGEFTVSPNNTPGDV


 


>5TP1|[NCBI:DQ263739]


DGAAGLVPEINNEAMALDPVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRNSPGEVLLNLEL


GPEI










-B-










Partition-wise maximally matching




characteristic residues


Partial
(Corresponding sequence group)













Sequence
P2
P3
P4
P5
P6
P7

















1UK1
70V
4A, 5S, 36V,


**
11S, 46A, 63A, 65G,




(GII)
44P



82S, 83A, 84P, 89L




(GII)



(GIIa1)


 


2JP1
20Q, 70I
26N, 36V,
—
—
11S, 13D,
**



(GI)
44A


14G




(GIa)


(GIa1)


 


2JP2
20Q, 70I
26N, 36V,
—
—
**
##



(GI)
44A




(GIa)


 


4JP3
**
26N, 36V,
—
—
**
##




44A




(GIa)


 


5TP1
70V
36V, 44P


##
13D, 14G, 46T, 63A,



(GII)
(GII)



65G, 82N, 83L, 84E,








89I








(GIIa3_2)





Ambiguities detected during search process:


**: Equal number of characteristic residue matches found in all of the groups


Blank entry: No matches found for the characteristic residues


—: Identical to previous partition


##: Ambiguity detected: More than one relevant group had equal no. of highest matches






In Table 3, part B above, the maximally matching sequence groups for each partition are shown within parentheses. Residues and sequence groups in Table 3, part B above use notations of FIGS. 2A & 2B. Residues are color matched in Table 3, parts A & B above.
Despite the short sizes of the input partial sequences and the variations in their capsid locations, the program unambiguously recognized the strains of all of them from their matches with the characteristic location residues. Location 70 in partition P2 determined the major genogroup in all but one sequence. Sequences 1UK1 and 5TP1, both containing 70V, belonged to genogroup GII while 2JP1 and 3JP2 belonged to genogroup GI based on the corresponding 70I residue (Table 3; FIGS. 2A&B). All of the GI residues (2JP1 & 3JP2) contained 20Q in partition P2. However, the major genogroup of sequence 4JP3 appeared to be ambiguous as it showed features of both GI and GII groups in partition P2 (ambiguity shown as ** in Table 3). It contained not only 70V, typical of GII sequences, but also the GI-indicator 20Q. In addition, this sequence also presented the additional ambiguity of having an equal number of characteristic residue matches in both GI and GII groups for partition P2 (Table 3). To resolve this ambiguity, the program carried over the partition P2 matches of this sequence (4JP3) to partition P3.
In partition P3, the program examined characteristic residue locations 4, 5, 26, 36 and 44 for all of the sequences (FIGS. 2A&B). Residue 44 unambiguously confirmed the distinction between the GI and the GII group sequences in this partition. Sequences 2JP1, 3JP2 and 4JP3, by virtue of 44A, were all characterized as GI sequences similar to the reference Norwalk virus sequence (FIG. 2B; Table 3). In contrast, sequences 1UK1 and 5TP1, containing 44P, are genogroup GII sequences. In addition, because 1UK1 has 4A and 5S and sequences 2JP1, 3JP2 and 4JP3 have 26N in partition P3, choices of their genogroups were unambiguously confirmed in this partition (FIGS. 2A&B; Table 3). Residue 36V, being conserved in all of the sequences in P3, was not of much help in determining the sequence groups in this partition (Table 3).
The program ignored partitions P4 and P5 for the 1UK1 and 5TP1 sequences due to ambiguities in residue matches. The next partition P6 showed another ambiguity of having more than one group with the highest number of matches for both these sequences (Table 3). However, matches in partition P7 clearly indicated that sequence 1 UK1 belongs to GIIa1 while 5TP1 belongs to group GIIa3—2 (Table 3; FIGS. 2A&B). Further matches in partitions P8-P10 (Table 3) confirmed that 1UK1 and 5TP1 were most similar to the Beeskow and the Bitburg strains respectively (Tables 2, 3; See additional file 1: Supplementary Table 1).
Thus, the identified strain of the partial sequence 1UK1 is consistent with its NCBI classification as a member of the GII.4 cluster. However, 5TP1 appears to belong to the GII.3 sequence cluster according to the present analysis and not to GII.4 as shown in the NCBI database. Similarly, partitions P6-P10 allowed the program to confirm that the remaining sequences 2JP1, 3JP2 and 4JP3 indeed belong to genogroup GI (Tables 2 & 3) which is consistent with the NCBI classification of these sequences. In addition, the program determined from residue comparisons that these sequences 2JP1, 3JP2 and 4JP3 are most similar to the norovirus GI Chiba, Norwalk and the Potsdam strains respectively (Tables 2 & 3). Such detailed strain information is seldom available for partial sequences in public domain databases.
Strain Detection Complete/Partial AA Sequences of Capsids
The program first correctly identified the strains for 5 complete and 5 partial enterovirus capsid sequences. The complete capsid sequences were those of the VP 1 subunits of poliovirus, simian enterovirus, echovirus and the coxsackievirus strains (one sequence per strain) (Table 2). Different reference sequences were tested for aligning the target sequences. The target sequence strains were all correctly identified regardless of the choice of the reference sequences (Tables 2 & 3). As an example, strain identification for the poliovirus strain (NCBI: DQ841140) using the porcine enterovirus-8 sequence (PEV-8: NCBI accession number AF406813) as reference is briefly described. The second partition P2 of enteroviruses contains only 2 reference groups 1 and 2 in the program database (Table 4). Of these, group 2 contains only the porcine enteroviruses (PEV) serotype 9 (PEV-9; NCBI: AF363453) indicating that the PEV-9 strains are distinct from the other enterovirus strains all of which (including the PEV-8 strain) belong to group 1. This group is characterized by nine residues 40P, 42L, 44A, 46E, 48G, 72E, 124T, 125Y, 127R, 150Q, 155P, 157G, 169W, 176S, 191P, 199Y, 202F and 203Y (reference sequence PEV-8 numbering) in the database (Table 4). Because all of these residues match the corresponding locations in the target poliovirus sequence, the program assigns group 1 to the target sequence in partition P2 and skips the next partition P3 as it is identical to P2 (Table 4).







TABLE 4







Partition-Wise Strain Determination of a Poliovirus-2 (PV-2) Sequence











Total no. of





groups



(Maximally
Matching residues
Reference database strains



matching
(Numbering based on
present in maximally


Partition
group no.)
reference PEV-8¶ sequence)
matching group





P2, P3
2(1)
40P 42L 44A 46E 48G 72E 124T
All enteroviruses including various




125Y 127R 150Q 155P 157G
human (Coxsackie, echo and polio)




169W 176S 191P 199Y 202F 203Y
strains and some animal strains like





bovine and simian enteroviruses.





Includes the PEV-8 strain but does not





include the PEV-9 strain (More than





100 strains)


P4
3(2)
P3+ [61L 62Q 78L 106S 115S
All of the P2/P3 partition strains except




131D 152L 170N 174S]
PEV-8 strains


P5
7(6)
P4+[43T 55G 64T 84L 156H]
32 echoviruses, 21 coxsackieviruses,





all polioviruses and 7 other human and





animal enteroviruses (Total of ~60





strains)


P6
8(7)
P5+[158G]
All polioviruses, 12 coxsackieviruses





and 1 echovirus


P7, P8
14, 44£
**


P9
72(62)
P6+[4K 19L 35N 47T 49E 51D
Poliovirus-2 strains




52T 60E 63A 66C 68F 69S 70L




73T 77Y 79M 80S 81R 83S 85M




90L 109T 113S 117I 119K 120F




123F 126W 129D 137L 138E 140K




153F 154T 166S 167Q 171A 172P




173N 175T 178Y 180R 184C 185P




187S 189R 192F 195V 197N 198Y




206D 207G 209F 216Y 217G 218I




221G 222D 225G 228S 230R 233N




242G 249F 250L 252P 253V 254N




256E 258Y 262P 264V 266Y




268A]


P10
75(64)
P9+[11I 17N 33M 36Q 37G 45A
Poliovirus-2 strains




50S 57S 58T 71R 76E 103Y 121K




122A 130L 146N 147L 159A 179T




186A 200T 210D 227I 231M 232A




248I 251R 263L 265S 281P]





**Ambiguity: 2 or more groups have identical number of matches in both these partitions.


¶Porcine enterovirus (NCBI accession no.: AF406813)


£No matching reference group for partitions P07, P08 due to ambiguity; Strains not shown for these two partitions. All strains of poliovirus serotypes (1, 2 & 3) cluster as an independent group in partition P8.






The complete capsid sequence of input PV-2 target sequence (NCBI accession: DQ841140) was used as input. Symbol Pn+: Indicates “including all matching residues of the earlier partition Pn”.

The program detects the separation of group 1 into two groups 1a and 1b in partition P4. This creates a total of 3 groups (1a, 1b and 2) in this partition (Table 4). While group 1a contains only the PEV-8 sequences which have independently diverged from group 1 of partition P2/P3, group 1b contains the remaining group 1 sequences of these partitions. Group 2 of partition P2/P3 remains unaltered in partition P4. On comparing the characteristic residues of these three groups, the program determines that the target sequence most closely resemble group 2 in partition P4 because of the matches with residues 61L, 62Q, 78L, 106S, 115S, 131D, 152L, 170N and 174S (PEV-8 numbering) that characterize this group (Table 4). Similarly, matches with residues 43T 55G 64T 84L 156H in partition P5 allow the program to assign group 6 to the target sequence in this partition which contains nearly 60 other similar enterovirus sequences including those of polioviruses (Table 4). Similarly, residue 158G distinguishes group 7 as the maximally matching group out of the eight groups in partition P6. This group includes many similar strains in the program database including 12 strains of different coxsackievirus serotypes, one echovirus strain and strains of all 3 poliovirus serotypes (Table 4).
The three poliovirus serotypes which remain clustered with several other human enteroviruses in partition P7 finally separate as an independent group in partition P8 (group details not shown). However, partitions P7 and P8 showed an ambiguity with reference to the input target sequence. Both of these partitions have more than one group having identical highest number of residue matches with the target. This ambiguity did not allow the program to decide the best group in these partitions. Consequently, all group information of these partitions was carried over to the subsequent partition P9 (Table 4).
Partition P9 contains a total of 72 sequence groups including an independent group (#62) containing only the poliovirus-2 strains. The program detected this group to be the maximally matching group for the target sequence and this detection was confirmed in the next partition P10. Thus, the input target sequence was correctly detected as a poliovirus-2 strain (Table 4). Strains for the remaining picornaviruses (Tables 2 & 3) were also unambiguously detected regardless of the choice of the reference strains.
Recombination and Spontaneous Mutations
Only a few enterovirus strains could be analyzed for recombination and spontaneous mutations using RECOVIR due to the manual nature of such analysis in the present version of the software. Partition-wise comparisons of strain diversifying residues VP1-VP3 genes of the hepatitis-A (HAV) strains at the nucleotide and the amino acid levels indicated that the SLF88 (AY032861) and the MBB (M20273) strains may be putative parental strains that yield the recombinant product strain 9F94 (AJ519487) (data not shown). This is consistent with recent results obtained using conventional topology comparisons of phylogenetic trees by sliding windows across the corresponding multiple aligned sequences [44].
Similarly, partition-wise matches in the norovirus partial sequence 6ST1 indicated spontaneous mutations. While residue 20Q in partition P2 indicates that 6ST1 belongs to GI, residue 70V in the same partition indicates that this sequence may belong to GII as seen for sequence 5TP1 (Table 3). The program, therefore, checks both these possibilities for maximum number of matches in subsequent partitions. Each subsequent partition confirms that this sequence indeed belongs to GI and is closest to the Potsdam strain (NCBI accession AF439267). Therefore, location 70, which is an iso-leucine for other GI sequences (Table 3), may be a spontaneous mutation site.
Despite the limited results, it appears that the present software may provide a more efficient way to detect recombination sites than those provided by most of the current widely used methods. This is because such methods are inherently time consuming as their accuracy depends on multiple runs with different sequence window parameters In addition, the entire procedure must be repeated every time a new sequence needs to be analyzed. In contrast, the present method does not repeatedly compare phylogenetic trees using sliding sequence windows. Instead, it efficiently performs node-wise comparisons of the trees using a one-time created database of strain distinguishing residues.
Approximate Processing Times
The program rapidly identified the calici and the picornavirus strains. In typical runs on outbreak sequence data available from public databases, it took only 6 and 9 minutes of real time respectively to identify the strains of a number of norovirus sequences contained in 2 files. The first file had 71 complete norovirus capsid sequences while the second file had 117 norovirus capsid sequences of which 22 were complete sequences while the remaining 95 were partial sequences each of which was between 75 and 85 residues long. This indicates a real time (including I/O) of approximately 5 seconds per sequence. Comparable speeds were also observed in processing other calicivirus and picornavirus sequences processed separately in different files as well as in files containing sequences of these virus families together. The “unknown” database option (FIG. 1B) was selected in all of these runs. Selecting the known database type in the GUI reduced the processing times even further. Complete benchmark details of such strain identification runs are available at website prion.bchs.uh.edu/recovir.
CONCLUSIONS
A software package RECOVIR that can efficiently and accurately characterize strains of the highly divergent caliciviruses and picornaviruses is described here. In contrast to the currently existing techniques that mainly use sequence similarity scores to identify strains in these viruses, this package relies on capsid residues that unambiguously distinguish among a large number of calici and the picornavirus reference strains. Such strain distinguishing residues are stored in partitioned databases and compared with the residues of target capsid sequences using efficient tree based search techniques to rapidly identify strains of the target sequences. This method implements a generalized algorithm of a method outlined earlier for noroviruses [40]. Consequently, the program allows strain identification of not only norovirus sequences but of all other caliciviruses and of the picornavirus sequences as well. Strains were recognized for more than 300 complete and partial capsid sequences of calici and picornaviruses with the average strain identification time (real time including I/O) being approximately 5 seconds per sequence. The method is general enough to be applicable to the nucleotide sequences of calici and picornavirus capsids thereby providing powerful alternatives that complement current strain determination techniques.
Availability and Requirements


    
    
        Project home pages: http://prion.bchs.uh.edu/recovir
        Operating systems: Windows-XP, Linux and UNIX
        Programming language: Perl and Java
        Other requirements: X-Windows support (such as Cygwin) is needed for remotely running the program under Linux/UNIX environments.

Author Contributions

    
    


Dianhui Zhu coded the software and designed the GUI. Sugoto Chakravarty developed the concept, designed the algorithm and its implementation and created the initial databases. Sugoto Chakravarty and Dianhui Zhu performed extensive troubleshooting with both synthetic and real data. Sugoto Chakravarty and George E. Fox wrote the paper. All authors suggested improvements at different stages of manuscript preparation and read and approved the final version of the manuscript.
REFERENCES CITED IN CAPSID EMBODIMENT
The following references are cited within the text:

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    3. Buchen-Osmond C (ed.): Caliciviridae. New York, N.Y., USA; 2003.
    4. Green K Y, R. M. Chanock, Kapiakan A Z: Human caliciviruses. In: Fields virology. 4 edn. Baltimore, Md.: Lippincott, Williams & Wilkins; 2001: 841-874.
    5. Matson D O, Szucs G: Calicivirus infections in children. Curr Opin Infect Dis 2003, 16:241-246.
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    8. Green K Y, Vinje J, Gallimore C I, Koopmans M, Hale A, Brown D W G: Capsid protein diversity among Norwalk-like viruses. Virus Genes 2000,20:227-236.
    9. Ando T, Noel J S, Fankhauser R L: Genetic Classification of “Norwalk-like Viruses. The Journal of Infectious Diseases 2000, 181:S336-S348.
    10. Katayama K, Shirato-Horikoshi H, Kojima S, Kageyama T, Oka T, Hoshino F, Fukushi S, Shinohara M, Uchida K, Suzuki Y et al: Phylogenetic analysis of the complete genome of 18 Norwalk-like viruses. Virology 2002, 299(2):225-239.
    11. Okada M, Shinozaki K, Ogawa T, Kaiho I: Molecular epidemiology and phylogenetic analysis of Sapporo-like viruses. Arch Virol 2002, 147(7):1445-1451.
    12. Knowles N: IAH Virus pages. In: Institute for Animal Health, U.K., at www website iah.bbsrc.ac.uk/virus/; 2003.
    13. Prasad B V V, Hardy M E, Dokland T, Bella J, Rossmann M G, Estes M K: X-ray Crystallographic Structure of the Norwalk Virus Capsid. Science 1999, 286(5438):287-290.
    14. Chen R, Neill J D, Estes M K, Prasad B V V: X-ray structure of a native calicivirus: Structural insights into antigenic diversity and host specificity. PNAS 2006, 103(21):8048-8053.
    15. Hadfield A T, Lee W, Zhao R, Olivera M A, Minor I, Rueckert R R, Rossmann M G: The refined structure of human rhinovirus 16 at 2.15 A resolution: implications for the viral life cycle. Structure 1997, 5:427-441.
    16. Kim S S, Smith T J, Chapman M S, Rossmann M G, Pevear D C, Dutko F J, Felock P J, Diana G D, McKinlay M A: Crystal structure of human rhinovirus serotype 1A (HRV1A). J Mol Biol 1989, 210:91-111.
    17. Rossmann M G, Arnold E, Erickson J W, Frankenberger E A, Griffith J P, Hecht H J, Johnson J E, Kamer G, Luo M, al. AGMe: Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 1985, 317(145-153).
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    20. Zheng D-P, Ando T, Fankhauser R L, Beard R S, Glass R I, Monroe S S: Norovirus classification and proposed strain nomenclature. Virology 2006, 346(2):312-323.
    21. Vinje' J, Hamidjaja R A, Sobsey M D: Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II Noroviruses. J Virol Methods 2004, 116:109-117.
    22. Kageyama T, Kojima S, Shinohara M, Uchida K, Fukushi S, Hoshino F, Takeda N, Katayama K: Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription PCR. J Clin Microbiol 2003, 41(1548-1557).
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Adaptive Mutations Embodiment of this Invention
Adaptive Mutations Explain Strain Diversification in Rhinoviruses and Noroviruses
Most single stranded RNA viruses adapt to host immune responses by rapidly mutating their capsid proteins to generate a large number of strains. An effective way to understand this strain diversity would be to decipher those capsid residues whose mutations may be linked to important determinants of host immune responses such as receptor binding and antigenicity. In the present study, we show using evolutionary trace based approach that a large number of the strain diversifying residues belong to the known receptor binding sites and some of the neutralizing sites of human rhinoviruses (HRV) thereby validating the ET approach earlier applied to noroviruses NoV). In both NoV and HRV, these capsid residues mostly line structurally spacious and inter-connected pockets. These pockets, perhaps, define major adaptation sites that structurally facilitate strain diversification. The adaptive sites explain the antigenic diversity of HRV by uniquely distinguishing among phylogenetic groups of antigenically related serotypes. Therefore, similarly constructed groups of NoV may indicate antigenically related strains, none of which have been identified for these non-cultivatable viruses. When applied to sequences of other caliciviruses and picornaviruses, the present approach reveals significant relationships among the currently known genera and species of these viruses on the basis of unique strain diversifying residues. Such residues may therefore distinguish and classify these distinct virus families.
Introduction
Rapid mutations in coat protein residues allow most single stranded RNA (ssRNA) viruses to adapt to host immune responses. Tracking and deciphering these mutation patterns is important in understanding adaptations in these viruses. However, this is seemingly impossible because of the apparent randomness of sequence locations where these mutations occur, especially when different ssRNA virus families are considered.
One way to understand adaptations in these viruses is to identify those capsid residues whose mutations may generate new strains and modulate important determinants of host immune responses such as receptor binding and antigenicity. For some ssRNA viruses having only a few serotypes, experimental approaches that combine analysis of neutralization escape mutants and conventional sequence alignment may identify such strain diversifying capsid residues, as has been done for polioviruses (6, 19, 21, 24, 58). However, these approaches are impractical when a large or unknown number of serotypes exist as in human rhinoviruses (HRV) and the noroviruses (NoV) of the Picornaviridae and the Caliciviridae families respectively.
Computational techniques effectively identify strain diversifying residues in such cases. However, conventional sequence comparisons among NoV and HRV (1, 17, 20, 23, 29, 42, 48, 72, 75) have not been able to identify these residues. This is because such comparisons attempt to identify maximally conserved regions across phylogenetic trees thereby masking biologically significant capsid regions in HRV and NoV due to high sequence divergence.
Evolutionary trace (ET) techniques appear to address these issues while determining functionally important residues in simpler protein systems (60, 67). This is done by identifying node-wise sequence conservation in phylogenetic trees followed by structural mapping of the conserved residues (45). We earlier applied ET-based techniques to predict capsid residues putatively involved in receptor binding, antigenicity and strain diversification in NoV (12). Recent confirmation of some of these results (3, 10, 31, 46, 53) indicated that such predictive approaches may be used to understand ssRNA virus strain diversification in a more general way provided the NoV approach is successful for viruses belonging to a different ssRNA family.
The relatively well characterized HRV have been chosen in the present study to reliably validate the NoV approach and extend it to other picornaviruses. Each serotype of HRV belongs to either the HRV-A or the HRV-B species (36, 61, 68). Capsid structures of five of the serotypes show that each icosahedral capsid consists of 60 copies of each of the four monomers VP 1-4 (26, 35, 59, 71, 74). The receptors bind in or near structural ‘canyons’ surrounding the icosahedral 5-fold axes in VP1 subunit and drug binding pockets are located underneath the canyons (4, 11, 25, 28, 65). Of the four neutralizing immunogenic (NIm) sites (IA, IB, II and III) (54, 59), only the NIm-IA and IB sites are located in VP1 towards the exposed parts of the canyons (59). We analyze here the capsids of HRV and other picornaviruses and caliciviruses in order to elucidate adaptations in ssRNA viruses.
Method
Partitioned phylogenetic trees of VP1 capsid sequences of 100 HRV serotypes were created following the ET method (45) applied earlier to NoV (12). The VP1 sequences were obtained from public databases (38, 51) where only one sequence per serotype was available. The sequences were first aligned using ClustalW (70) and Gonnet-250 series identity matrices were used to create phylogenetic distances. Penalties for gap opening, closing, extension and separation were set to 10, −1, 0.2 and 4 respectively for amino acid sequences. Rooted phylogenetic trees of the aligned sequences were created using the Kitsch algorithm on Phylip distances as implemented on the Cambridge server (33). Assuming uniform evolutionary rates of the sequences, these trees were divided into ten equally spaced partitions (P01-P10) with the inter-partition distance in any given tree being computed from the maximum evolutionary distance within the tree. Ten partitions sufficed for all trees as no further changes in node distributions were observed in any tree with further increase in partition numbers.
Every partition created classes each of which contained groups of similar sequences. Sequences belonging to different classes of a given partition were separately aligned and the resulting aligned classes were compared to obtain the consensus (trace) residues for that partition. Those sequences that remained strictly conserved within a class but differed between various classes were denoted as ‘class-specific’. These residues were mapped onto the HRV-16 (PDB ID: 1AYM) structure where class-specific residue (CSR) clusters were analyzed following the procedure described earlier (12).
To identify structural location preferences of the CSRs, void volume cavities and pockets were separately computed for the HRV-16 and the Norwalk virus (PDB ID: 1IHM) capsids. For HRV16, pseudo-dimers composed of the VP1 and VP3 subunits and other VP1-VP4 subunit assemblies were used while for the Norwalk virus capsid, dimers composed of the A and the B subunits (55), trimers (A, B and the C subunits) and the A-subunit pentamers were used. These calculations were done using 1.41 radius probes in the VOIDOO package (37) and on the CASTp server (5). Cavities were defined as void contiguous regions formed by atoms not in touch with surface atoms while pockets were defined as the corresponding regions partly or wholly accessible to surface atoms. Therefore, a pocket, by definition, must have at least one opening that allows access to the interior of the pocket while cavities have no such openings (5). Consequently, exposed regions smaller than the probe size were considered parts of cavities. Accessible and inaccessible surface areas and volumes of the pockets and cavities were computed using definitions of Lee and Richards (43) and Connolly (13, 14).
Capsids of Other Picornaviruses and Caliciviruses
Amino acid sequences of capsids of known serotypes of the human enterovirus (HEV) species A-D were partition-wise analyzed. A total of 388 capsid sequences of all nine known picornavirus genera (68) were then similarly analyzed. The procedure was repeated for a large number of amino acid sequences of all known genera of caliciviruses.
Results and Discussion
Partition-Dependent Classes Show Antigenic Relationships Among Serotypes of HRV
Known Antigenic Relationships
Various antigenic relationships are known among serotypes of HRV. Some serotype pairs (1A and 1B, 2 and 49, 3 and 14, 9 and 32, 12 and 78, 13 and 41, 15 and 74, 29 and 44, and 36 and 58) share reciprocal cross-neutralization with heterotypic antisera while other pairs share one-way antigenic relationships between the pair members (Table 1 & FIG. 4B). Still others, like serotypes 9, 32 and 67 are antigenically related because serotypes 9 and 32 are cross-reciprocally related while serotypes 9 and 67 show one-way relationships (15) (Table 5). In addition, four groups of serotypes share cross-reactivity among the group members as seen in neutralizations with antibodies raised against serotypes obtained from natural infections (50) (Table 5) (15, 16, 62).







TABLE 5







Known Antigenically Related Serotypes of HRV Group


Together in Partition dependent Classes









Cross-Neutralization After









Reciprocal Cross-
One-way Cross-Neutralization
Natural Infection (62)










Neutralization (15)
(15)
Serotype













Serotype Pair
Class/Partition
Serotype Pair
Class/Partition
Group
Class/Partition





1A, 1B
A3/P07+
5, 42
B3/P07+
1A, 1B
A3/P07+


2, 49
A7/P07+




B5/P08*
2, 49
A7/P07+






B5/P08+




A15/P08+
23, 30
A7/P07+






A15/P08+




A1* → A12,
13, 41, 82
A8/P08+


12, 78
A10/P07+

A19/P06*




A9* → A21 &


13, 41
A17/P08+




A4* → A12,

A22/P07*


15, 74
A16/P08+

A16/P07*


 






A22/P08+
17, 42
B* → B2, B3


 






A23/P08+

 /P06*


 




17, 70
B2/P07+




22, 61
A17/P08*




32, 67
A15/P08*








A1* → A23,





A13/P06*








A23/P08+




39, 54
A8* → A20,





A19/P07*




60, 38
A16/P08*




66, 77
A18/P08+




76, 11
A12/P08+









These serotypes are known using rabbit hyperimmune sera raised against prototype strains for serotypes 1A to 89 of HRV. Pairs having antigenic relationships that lead to mutual antigenic relationships among three or four serotypes are shaded using the same color e.g., relationships between serotypes 3 and 14 (cross-reciprocal) and serotypes 6 and 14 (one-way) ensures that serotypes 3, 6 and 14 are antigenically related. Therefore, the pairs (3, 14) and (6, 14) are shaded using the same color. The + sign indicates that these serotype pairs continue to remain grouped together in subsequent partition classes while the * indicate that the corresponding serotype pairs do not group together in the same class in subsequent partitions. Arrows indicate the target partitions and classes into which some of the one-way related serotype groups get split up from their parent partitions and classes.

Classes Group Antigenically Related Serotypes
Antigenically related serotypes of HRV group together in distinct partition-dependent classes. For example, in P02, serotypes 3 & 14 group together in class B (HRV-B species) while all of the remaining antigenically related pairs belong to class A (HRV-A species) (Table 5, FIGS. 4A & 4B). Subsequent partitions continue to cluster most of these related serotypes and also cluster those antigenically related serotypes that are obtained from natural infections (Table 5; FIG. 4B). Even serotypes sharing antigenic relationships with multiple other serotypes, like serotypes 9, 32 and 67, are grouped together in the same class in at least one partition (Table 5; FIG. 4B).
Classes Distinguish Between Reciprocal Vs. One-Way Related Serotypes
The different partitions distinguish between the reciprocally related serotypes from the one-way related ones. The reciprocal serotypes remain grouped together in a larger number of partitions as compared to the one-way related ones. For example, the mutually reciprocal serotypes 1A and 1B remain together in classes A (P02-P04), A1 (P05, P06) and A3 (P07-P09) (Table 5; FIG. 4B). In contrast, the one-way related serotypes 11 and 74 split up after class A4 in partition P07. These serotypes get distributed into classes A12 and A16 in P08 (FIG. 4B). Similar splits are seen in other one-way related serotype pairs ((11 & 40), (17 & 42), (36 & 50) and (39 & 54)) (Table 5).
Strain Diversifying Locations Include Known Neutralization and Receptor Binding Sites Csrs are Strain Diversifying Locations
While partitioned classes show that the phylogenetic trees of HRV portray antigenic diversity, the CSRs define the antigenic diversification locations. This is because class-specific conservation in these residues may be viewed as strain diversifying mutations that generate the antigenically related classes (FIG. 4B). For example, location 1, which is conserved as either Asn or Gly in partition P02, may be equivalently viewed as two possible mutations in this location: Asn generates all class A sequences while Gly generates sequences of class B. Similarly, class-specific mutations in locations 48, 56, 102, 103, 155, 170, 174, 223 and 239 (HRV16 numbering is followed throughout unless stated otherwise) generate the HRV-A and HRV classes in partitions P02-P04 (FIGS. 4B, 4C) while class-specific mutations in the other partition-dependent CSRs generate classes in the remaining partitions (data available with authors from bchs.uh.edu/HRVET).
P02 Class-specific Insertions Define Neutralization Sites
The two major neutralizing immunogenic (NIm) sites 1a & 1b (59, 63) share a common feature in sequence comparisons: both these sites appear as insertion regions that are class-specific in P02 and remain so in most subsequent partitions. Site 1a, which is common to immunogenic site 1 in polioviruses and consists of the VP1 residues 91 and 95 (HRV14 numbering), occurs as insertions in a majority of the HRV-A and HRV-B classes (FIG. S1). Similarly, of the residues 83, 85, 138 and 139 (HRV14 numbering) belonging to the NIm-1b site in VP1(2), residues 138 and 139 are class-specific insertions for the HRV-A class but not for the HRV-B class (data available from the website bchs.uh.edu/HRVET).
Structurally, neutralization sites 1a and 1b belong to exposed VP1 loop regions in HRV (2, 59). This makes the corresponding P02 class-specific insertion locations amenable to strain diversifying mutations. Thus, exposed P02 class-specific insertions/deletions are good indicators of important neutralizing strain diversification sites. However, these conditions alone are not enough to predict the neutralization properties of sites located at subunit interface regions. This is clearly seen in the NIm II & III sites located near the VP1-VP2 and VP1-VP3 interfaces respectively (63). These neutralization sites do involve exposed VP1 residues (210 and 287 of HRV14) but they do not involve class-specific insertions (FIG. S1). Therefore, exposed P02 class-specific insertions/deletions are good indicators of neutralizing strain diversification sites provided these sites are not located in subunit interfaces.
CSRs Identify Receptor Binding Regions as Important Strain Diversifying Sites
The class-specific residue D213 (FIG. 4C), whose mutations generate classes in HRV beginning partition P09, contacts the major group receptor in HRV16 (4, 41). Similarly, the P09-class-specific residue 226Q of HRV 16 (or 225H in HRV2 serotype), that is located near but outside the canyon (28), contacts the minor group receptor in HRV2. Thus, CSRs correctly identify receptor contacting residues in both major and minor group HRV indicating that the receptor binding regions constitute important strain diversifying sites.
Strain Diversifying Residues are not Randomly Located But are Structurally Well Localized
Some of the strain diversifying locations include known and putative capsid related function sites in HRV (present analysis) and in NoV respectively (12). The remaining such locations appear to be randomly distributed in sequences as seen from the CSRs. However, closer examination of the capsid structures reveals that the strain diversifying mutations do not occur at random sequence locations. Instead, a majority of such mutations are localized in and around cavities and pockets enclosed by the capsids of HRV and NoV.
Cavities and Pockets in HRV
The VP1-VP3 pseudo-dimer of the HRV16 capsid (34) consists of nearly 27 cavities and 49 pockets. All cavities are small but their sizes are consistent with those found in smaller proteins (30, 44, 57). In contrast, all pockets are larger. Thirteen pockets have inaccessible and accessible volumes less than 2013 and 313 respectively while twenty two of the remaining pockets are larger having inaccessible volumes varying between 20A3 and 10013. Nine of the remaining 15 pockets are still larger with volumes between 100A3 and 50013.
Each of the five largest pockets has inaccessible volumes greater than 50013 with three of them (#71, #74 and #75) having volumes between 80013 and 1400A3 (Table 6). The largest pocket (#75) encompasses the known anti-viral binding region (25, 34, 49, 65, 73) as seen from the agreement between computed parameters such as the pocket openings and the roof and floor residues of the pocket (Table 6; FIGS. 6A & 6B) with the corresponding known results (26, 34, 59, 65).







TABLE 6





HRV-VP1 Capsid Class-specific Residues Lining the Largest Pockets
















Partitions
Class-specific capsid residues (HRV16 numbering)









1N, 48E(#71), 56V(#68), 102E, 103M, 155R, 170W, 174Q, 223T, 239K(#73, #74)


 






P02+ 238H(#75, #73))


 






P05+ 28N, 65M(#74), 90T, 181L (#75)


 






P06+ 108R(#69, #73), 125V


 






P07+ 13E, 29N, 35D, 75G(#73), 78H, 126P, 136G, 149A, 171Q, 192M (#75); 250P(#69), 264Y(#70)


 






P08+ 2P, 11L(#74), 13V, 17V, 19N, 26T(#71), 28S(#71), 74S(#73), 82L, 2M, 9A, 142Y(#75), 144Y(#75), 145V, 157D, 161Q, 162S, 163G, 166A(#75), 183F, 199G, 210V,


















Pockets in HRV16 VP1-VP3 dimer













No. of


Residues lining the cavities/pockets


#
mouths
[S1][S2]*
(V1)(V2)¶
(HRV16 numbering)





#75
6
 [538] [1289]
 (202) (1359)






 


#74
2
 [289]  [437]
 (336)  (826)






 


#73
2
 [181]  [502]
 (56)  (504)






 


#72
4
 [183]  [334]
 (97)  (448)






 


#71
3
 [268]  [398]
 (372)  (818)






 


#70
1
 [83]  [179]
 (36)  (219)






 


#69
1
 [139]  [278]
 (76)  (346)






 


#68
2
 [117]  [235]
 (61)  (300)









*S1, S2: Solvent-accessible & solvent-inaccessible surfaces respectively (Å2) of cavity/pocket


¶V1, V2: Solvent-accessible & solvent-inaccessible volumes respectively (Å3) of cavity/pocket






The upper part of the table shows the partition-wise (P02-P09) distribution of HRV16-VP1 class-specific residues (CSRs) and the lower part of the table shows eight of the largest computed structural pockets in the VP1-VP3 pseudo-dimer of HRV16 capsid. In the upper part of the table, any CSR that also lines one of the pockets is clearly indicated by the # sign and the corresponding pocket number within parentheses. Similarly, each pocket residue that is also a CSR, or, its sequence neighbor, is highlighted in the lower part of the table using matching colors of the corresponding partitions in the upper part of the table and one of the (^) or (*) signs along with the partition number. The (^) sign denotes CSRs while the (*) sign denotes sequence neighbors of CSRs in color-matched partitions. The bold-faced residue 213 directly contacts the major group receptor (ICAM) molecules. Blue highlighted residues indicate representatives lining more than one pocket.

Cavities and Pockets in NoV
The A-B dimer of the Norwalk virus capsid (55) contains 86 cavities and 77 pockets. All cavities are smaller than most of the pockets, among which, 64 are relatively small pockets having inaccessible volumes less than 10013 and ten are somewhat larger with volumes varying between 10013 and 40013. All pockets, including two of the largest ones (#160 and #162 in Table 7), involve only the S and/or the P1 domain residues. Only one major pocket (#161 in Table 7; FIG. 7B) involves P2 domain residues.







TABLE 7







NOV Capsid Class-specific Residues Lining the Largest Pockets








Pockets in Norwalk virus capsid



(1IHM) A-B dimer












No. of


Residues lining the cavities/pockets


#
mouths
[S1][S2]*
(V1)(V2)¶
(Norwalk numbering)





#162
5
[2459] [3722]
(2954) (6839)






 


#161
2
 [989] [1673]
 (950) (2764)






 


#160
2
 [316]  [618]
 (216)  (853)









*S1, S2: Solvent accessible & solvent inaccessible surfaces respectively (Å2) of pocket


¶V1, V2: Solvent accessible & solvent inaccessible volumes respectively (Å3) of pocket






Three of the largest computed structural pockets in the A-B dimer of Norwalk capsid (PDB ID: 1IHM). Pockets #161 and #162 are lined by residues of both the A and the B subunits. Pocket #160 is lined by only the A subunit residues. Only the A-subunit residues are shown for each pocket. Each pocket residue that is also a class-specific residue (CSR), or, its sequence neighbor, is highlighted using the partition coloring scheme of Table 6. The (^) sign denotes the CSRs while the (*) sign denotes some of the sequence neighbors of CSRs in color-matched partitions. Blue highlighted residues indicate representative residues lining more than one pocket.

CSRs Line the Inter-connected Pockets in HRV and NoV
A majority of the CSRs and their sequence neighbors line the pockets in HRV and NoV indicating that these pockets localize most of the strain diversifying mutations. Most of the cavities and pockets are inter-connected through common CSRs. Additionally, some of the CSRs lining the largest pockets are also involved in, or are putatively involved in, capsid related functions in HRV and NoV respectively (Tables 6 & 7; FIGS. 7C-7E) (12, 31, 53). Thus, capsids of HRV and NoV enclose a network of inter-connected cavities and pockets, the largest of which define regions where three major determinants of adaptations, namely, strain diversifying mutations, receptor binding and antigenicity occur together.
Pockets and Cavities May Define Capsid Adaptation Sites in HRV and NoV
The occurrence of strain diversifying mutations and capsid related functions in the largest pockets has important implications in HRV and NoV. The inter-connected pockets and cavities may be schematically represented as trapped bubbles in regions whose surface contains the receptor binding and antigenic sites (FIG. 4D). Upon binding the receptor, strain diversifying mutations occur in these regions in order to escape host immune responses. These mutations may change the shapes of the bubbles by squeezing or expanding them. This, in turn, modifies the capsid surface to create altered receptor binding and antigenic characteristics (FIG. 4E). The pockets and cavities, therefore, define regions where capsid related functions and strain diversifying mutations mutually influence each other. Hence, these pockets and cavities may define important adaptation sites in HRV and NoV. Inter-connectivity among such sites indicates that adaptations in one site may affect other distantly located sites, perhaps facilitated by solvent molecules in a manner similar to the influence of protein cavities on ligand binding (9, 22, 66, 69).
Analysis of all known capsid structures of HRV indicates that the number of CSRs lining the pockets increases with increase in the pocket sizes (Tables 6 & 7 and data available from bchs.uh.edu/HRVET). Thus, the size of a given pocket approximately determines the number of different types of strain diversifying mutations that the pocket may tolerate, indicating thereby, the relative evolutionary importance of the different adaptation sites.
Adaptive Sites May Reveal Strain Diversification in Enteroviruses
Because both HRV and HEV mutate at uniform rates that are well within limits proposed for ssRNA viruses (18), the HEV may be compared using evolutionarily equidistant partitions in a similar way as the HRV. Such comparisons reveal the strain diversifying sites in HEV.
Partitioned Comparisons Reveal Antigenic Relationships Among Species of HEV
Comparisons of different HEV species reveal known and unidentified antigenic relationships. When serotypes of species HEV-A and B are compared, the two P02 classes (A & B) correctly group the serotypes in their respective species (FIG. S2) each of which contains antigenically related serotypes (32). Even strains with unconfirmed species (HEV-75, 76, 78, 89, 90 and 91) are correctly placed in classes that correspond to their respective proposed species. The CSRs, which are also the strain diversifying locations, are clearly revealed for the two species (data available with authors at http://bchs.uh.edu/HRVET).
HEV-B and C May be Antigenically Related Through CSRs
Inclusion of the HEV-C species (Coxsackie-A1, A 1, A13, A15, A17-A22, A24, echovirus-34) in partitioned comparisons along with the HEV-A and the HEV-B species indicates new antigenic relationships in partition P02. The HEV-B and the HEV-C species group together in the same class (FIG. S3) thereby indicating that the capsids of these two species antigenically resemble each other more closely than the HEV-A capsids. This is further confirmed when serotypes of the known four species, HEV-A, B, C and D, are compared together. Only three P02 classes are observed, of which, the B and C species group together in the HEV-(B+C) class (FIG. S4). The remaining two classes respectively contain the HEV-A and the HEV-D species. The P02 class-specific residues 34, 40, 52, 81 and 157 (PDBID: 1Z7S numbering) are the strain diversifying locations that explain the similarities and differences among the classes. It is only in P03 that the HEV-B and the HEV-C serotypes are distinguished as individual species through their respective CSRs (details available with authors: prion.bchs.uh.edu/HRVET).
Polioviruses are Similar to HEV-C
Partition P02 shows correct antigenicity for polioviruses with respect to the HEV species. Polioviruses are grouped together with the HEV-C class in P02 (FIG. S3) indicating that the these viruses belong to the HEV-C species, in agreement with the current consensus about the identity of polioviruses (56) as well as with recently approved recommendations (8, 68). Interestingly, when all three known poliovirus serotypes PV-1, 2 and 3 (7, 32) are compared, only two distinct P02 classes are created (FIG. 5). Of these, the PV2+3 class shows that the PV-2 and the PV-3 serotypes share closer antigenic similarities among each other than with the PV-1 serotypes due to the class-specific locations 6, 76, 213, 278 and 290 (PDBID: 1P01 numbering). Such similarities may indicate shared epitopes among the PV-2 and PV-3 classes that separate into distinct serotypes only in P03 (FIG. 5).
Evolutionary Importance of Partition P02
The present analysis places the greatest emphasis on sequence relationships in partition P02. This is because it is assumed throughout that among all the partitions, P02 has the highest evolutionary importance or rank in determining such relationships. By definition, the evolutionary rank of a residue is inversely related to the minimum number of classes required to denote the residue class-specific (47). Thus, lower the rank of a residue, higher is its evolutionary importance and vice-versa. This is because a low rank indicates conservation of a residue even among distantly related sequence classes, whereas, a high ranking residue indicates that the residue varies even among closely related sequences implying a relatively lower evolutionary significance for the residue due to a high probability that random chance events may create such residue variations.
Consequently, the first partition P01, which compares sequences in one group like in conventional sequence comparisons, has negligible evolutionary importance because class-specificity cannot be defined for this partition. More simply, P01 intrinsically masks evolutionarily important residues that may define phylogenetic strain diversification. In contrast, the second partition P02 is evolutionarily most significant because each P02 class contains the maximum number of distantly related sequences. The CSRs have become fixed to the distantly related classes so early in phylogeny that they acquire the greatest evolutionary importance in defining class-specific functions such as receptor binding and antigenicity that drive strain diversification. Therefore, partition P02 displays the most significant sequence relationships in its classes that, in turn, spawn other relatively less significant classes in subsequent partitions (FIGS. 4A, 5, S1-S3).
P02 Classes May Indicate Adaptive Basis of Strain Diversification in Other Picornaviruses
If partitioned comparisons reveal possible adaptive sites that are responsible for strain diversification in each of the two different genera HEV and HRV of the Picornaviridae family, it should also be possible to partition-wise compare two or more genera of this family together. This is because it is reasonable to assume that each of the nine genera (enteroviruses, rhinoviruses, cardioviruses, aphthoviruses, hepatoviruses, parechoviruses, erboviruses, kobuviruses and the teschoviruses (36)) of the Picornaviridae family has comparably uniform mutation rates. If so, then, the P02 classes arising out of the partitioned comparisons multiple genera together will yield evolutionarily significant strain diversification patterns. Additionally, such joint comparisons may be useful for checking the consistency of the results obtained earlier individually for HRV and the HEV in the present analysis.
HRV and HEV are Similar
When amino acid sequences of the VP1-capsids of all nine genera of picornaviruses are compared together, four P02 classes (1-4) are created (FIG. 8). The HRV and the HEV group together in class 3 of partition P02 and continue to remain grouped in class 3a of subsequent partitions P03 and P04. This indicates significant similarities among the HRV and the HEV, in agreement with recently accepted recommendations (39, 68).
It is only in P05 that the HRV separate from the HEV to create the HRV-A and the HRV-B species in classes 3a1 and 3a2 respectively which remain unchanged in P06 (FIG. 8). Similarly, the HEV split into five classes (3b1-3b5) in P05. Of these, classes 3b2 and 3b3 consist of the HEV-A and the HEV-D species respectively and class 3b5 consists of the simian enteroviruses (SEV). Class 3b1 groups the HEV-B and the HEV-C species together including the polioviruses (FIG. 8) confirming the earlier results of the present analysis using HEV alone (FIG. S2, S3).
HRV87Serotype is Similar to HEV-D Species
Among the HEV classes 3b1-3b5, class 3b4 shows that a current rhinovirus serotype (HRV87) is more similar to two HEV-D serotypes 68 and 70 (FIG. 8) than it is to other rhinoviruses, in agreement with recent recommendations (42, 61, 68). Such non-rhinovirus like characteristics of HRV87 is confirmed in comparisons among the HRV, where, this serotype independently branches off at node 3 in partition P02 (FIG. 4A).
More P02-Based Predicted Relationships Among Picornavirus Genera
The importance of partition P02 in correctly elucidating so many sequence relationships among the HRV and the HEV allows predictions of more P02-based sequence relationships and the consequent strain diversification in other picornaviruses. The P02 class 1 (FIG. 8) indicates that the aphthovirus genus (including the erbovirus-A species), the erbovirus-B species and the cardiovirus genus (including the encephalomyocarditis (EMC) viruses and the Theiler's murine encephalomyelitis (TME) viruses) are closely related, consistent with some known sequence comparisons and cleavage mechanisms of some of these viruses (27, 40).
While kobuviruses alone form class 2 of P02 showing their distinctiveness from the other genera, the teschoviruses (TEV) group together in class 3 along with HRV and the HEV (FIG. 8). This shows that the TEV genus may share important adaptive function similarities with the HRV and the HEV. Similarly, class 4 shows that the hepatoviruses and the parechoviruses are closer to each other than they are to the other genera.
Later Partitions Reveal Species of Picornaviruses
Unlike partition P02 which reveals inter-relationships among the currently known genera, the later partitions systematically show the emergence of other classification hierarchies such as the species. For example, the P02 class 1 splits up into the aphthovirus genus (class 1a), Erbo-A species (class 1b), Erbo-B species (class 1c) and the cardiovirus genus (class 1d) in P03 and P04 (FIG. 8). Of these, classes 1c and 1d remain unchanged in all subsequent partitions while the aphthovirus genus (class 1a) further splits up into the known A, C, O, Asian and the African species. Similarly, the cardiovirus class 1d splits up into the known EMCV and the TME species in partition P06 (FIG. 8). Of the other P02 classes, class 4 splits up into the respective hepatoviruses and the parechoviruses genera in P03 (classes 4a and 4b). These remain unchanged in P05 and P06 after which further species emerge (data not shown).
Thus, comparisons of the picornavirus VP1 amino acid sequences indicate partition-dependent relationships among the currently known genera and species. Such relationships among the known genera in P02 may reflect shared receptors, antigenicity or both, unlike comparisons within a given genus where the P02 classes primarily yield antigenic relationships as seen for HRV and the HEV. It is only in later partitions that all of the currently known species and serotypes emerge independently (FIG. 8).
P02 Classes in Caliciviruses
Comparisons of amino acid sequences of all caliciviruses shows that the lagoviruses and the noroviruses form independent classes while the sapoviruses and the vesiviruses together form the third class (data not shown). This indicates that the sapoviruses and the vesiviruses share similarities in their capsid adaptation sites
Need for Partition Based Classification to Indicate Adaptive Sites
Thus, partition-dependent classes and the CSRs explain strain diversification in picornaviruses and caliciviruses by distinguish these viruses on the basis of adaptation site residues. Therefore, classification schemes based on capsid sequences should explicitly indicate partitions in sequence classes in order to reflect these adaptive sites.
One way of doing so would be to represent the genus of a virus as Gn, where the uppercase G denotes the genus and n denotes the genus number. As each genus has its own phylogenetic tree, any sequence class (or cluster) on these genus trees may be uniquely represented using a partition number and a class number in the format P(xx).g(n), where the lowercase g is used to denote clusters belonging to a given genus, xx denotes the partition number and n denotes the class number within the partition.
As an illustration, the four genera of the Caliciviridae family are represented by G1 (noroviruses), G2 (lagoviruses), G3 (sapoviruses) and G4 (vesiviruses) with each genus having its own partitioned tree. In the norovirus (G1) tree, class #4 in partition P05 would be denoted as G1.P05.g4 that represents all sequences in class 4 (g4) of partition P05 of the norovirus tree. Similarly, G1.P02.g2 would represent those noroviruses that belong to class g2 of partition P02. Likewise, the symbol G3.P06.g2 would represent class #2 (g2) in partition P06 of the sapovirus (G3) tree. Such a representation would uniquely reflect all partition-dependent classes within a given virus family.
Norovirus Classes May Reflect Antigenic Relationships
The present analysis shows that the phylogenetic trees of the two genera (HRV and HEV) of picornaviruses portray antigenic diversity. The partitioned classes of a given genus correspond to groups of antigenically related serotypes. Therefore, the phylogenetic trees of capsid sequences of NoV may reflect antigenic diversity among noroviruses. The corresponding partitioned classes of NoV (12) may actually represent antigenic relationships among groups none of which are identified due to the non-cultivability of these viruses. For example, genogroup I strains Norwalk (PDB: 1IHM), Aichi (NCBI: AB010145) and Kyoto-89 (NCBI: L23828) which cluster together in partitions P02-P10 (12), may be antigenically related to each other. Similarly, the genogroup II strains Chesterfield (NCBI: Q91386), Melksham (NCBI: X81879) and Snow Mountain (NCBI: U700059) may also be antigenically related (12). Additionally, the exposed regions that correspond to P02 class-specific insertions, such as those occurring in the P-domains near residues 298, 339, 362, 426 of Norwalk virus (12), may play important roles in neutralizing NoV.
CONCLUSIONS
The present study uses evolutionary trace based approach to show that although noroviruses (NoV) and human rhinoviruses (HRV) belong to different families of single stranded RNA (ssRNA) viruses, their coat protein sequences do not mutate at random residue locations to create strain diversification. Instead, these mutations are mostly localized in and around inter-connected structural pockets in the capsids of both NoV and HRV. Many of the strain diversifying residues belonging to the largest pockets are involved in capsid related functions such as receptor binding and antigenicity. Thus, these pockets may structurally facilitate adaptations in response to host immune responses. The pockets are, therefore, called adaptation sites. Combinations of strain diversifying residues belonging to different adaptation sites explain the antigenic diversity in HRV by uniquely identifying each phylogenetic group of antigenically related serotypes. Therefore, known phylogenetic groups of NoV may reveal unidentified antigenic relationships. Extension to other picornaviruses and caliciviruses reveals significant sequence relationships among different genera and species on the basis of unique strain diversifying residues. Such residues may, therefore, be used to distinguish and classify these viruses and other ssRNA virus families as well.
Database Partitioning for Rhino cDNA
The rhino cDNA database is shown in FIG. D1. The code used read the database is shown in FIG. D2. The group information used in the structured data is shown in FIG. D3. The partition trees is shown in FIG. D4, which consists of ten partitions. Partition one (P1) is shown in FIG. D5. Partition one (P2) is shown in FIG. D6. Partition one (P3) is shown in FIG. D7. Partition one (P4) is shown in FIG. D8. Partition one (P5) is shown in FIG. D9. Partition one (P6) is shown in FIG. D10. Partition one (P7) is shown in FIG. D11. Partition one (P8) is shown in FIG. D12. Partition one (P9) is shown in FIG. D13. Partition one (P10) is shown in FIG. D14. This data and the partitions was used above in the analysis of adaptive mutations.
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All references cited herein are incorporated by reference. Although the invention has been disclosed with reference to its preferred embodiments, from reading this description those of skill in the art may appreciate changes and modification that may be made which do not depart from the scope and spirit of the invention as described above and claimed hereafter.

## Claims

1. A computer program product implemented on a computer or stored on a computer readable storage medium for predicting strains of some pathogens from limited sequence data, the computer product comprising:
a graphical user interface (GUI) code implemented on the computer or encoded on the computer readable storage medium and operable to carry out all data input-output (I/O) operations, where the GUI code uses a sequence category region of a screen of a display of an implementing computer to allow users to input sequence data in a specified format using a paste option or by reading in files containing the data, where the GUI code uses a database category region of the screen to use default reference sequences or allow users to pre-select specific reference sequence to facilitate sequence comparisons, and where the GUI code uses an output options category region of the screen to allow users to selectively display identifiers of each input sequence and summary/details of matches of characteristic residues for an input query sequence specified by its identifier;
a storage code implemented on the computer or encoded on the computer readable storage medium and operable to identify characteristic residues of individual virus sequences or their groups from program supplied external files and to store virus sequence databases in the form of multiple data arrays including information about phylogenetic trees, sequence groups and characteristic residues of these groups, where arrays are in the form tree-like multiple 2-dimensional arrays for efficient referencing and retrieval;
a sequence comparison code implemented on the computer or encoded on the computer readable storage medium and operable to compare input pathogen sequences with the stored database sequences on a residue-by-residue basis to produce comparisons corresponding to all pathogens or pathogen strains stored in the database that include some or all of the input pathogen sequences; and
an identification code implemented on the computer or encoded on the computer readable storage medium and operable to identify the pathogens or pathogen strains of input pathogen sequences based on the comparisons and subsequent decision making algorithms, where the algorithms identifies the comparison pathogens or pathogen strains that include maximal sequence identify.

2. The product of claim 1, wherein the pathogens or strains comprise single stranded RNA viruses.

3. The product of claim 2, wherein the storage code numbers the characteristic residues and the storage code identifies characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval with respect to the GUI code, which uses the database category region of the screen to use default reference sequences or allow users to pre-select specific reference sequences to facilitate sequence comparisons for sequence comparison purposes.

4. The product of claim 2, where the sequence comparison code selects individual residues of input sequences where the GUI code uses a sequence category region of a screen of a display associated with an implementing computer to allow users to input sequence data in a specified format using a paste option or by reading in files containing the data and compares these residues with the characteristic residues in the storage code numbers the characteristic residues and a storage code identifies characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval with respect to the GUI code, which uses a database category region of the screen to use default reference sequences or to allow users to pre-select specific reference sequences to facilitate sequence comparisons for sequence comparison purposes using a tree-like search method.

5. The product of claim 1, wherein the GUI code is adapted to divide the screen into 3 distinct categories including the sequence category, the database category and the output options category adapted to simplify data input/output (I/O).

6. The product of claim 5, wherein the GUI code uses the database category region of the screen to allow users to pre-select specific sequence databases that might contain close matches to the input sequence strain.

7. The product of claim 5, wherein the GUI code uses the database category region of the screen to allow users to specify “Unknown” in case sequence databases that might contain close matches to the input sequence strain are not known.

8. The product of claim 5, wherein the GUI code uses the database category region of the screen to allow users to select and display a particular sequence or all sequences from a database of sequences using a drop down menu.

9. The product of claim 5, wherein the GUI code uses the database category region of the screen to allow users to view groups of similar sequences.

10. The product of claim 5, wherein the GUI code uses the database category region of the screen to allow users to activate execution of the program.

11. The product of claim 5, wherein the GUI code uses the database category region of the screen to allow users to activate execution of the program.

12. The product of claim 1, wherein the identification code identifies the best strain matching the input sequences.

13. A computer program product implemented on a computer or stored on a computer readable storage medium for predicting strains of some pathogens from limited sequence data, the computer product comprising:
a graphical user interface (GUI) code implemented on a computer or encoded on a computer readable storage medium and operable to carry out all data input-output (I/O) operations, where the GUI code uses a database category region of a screen of a display of an implementing computer to allow a user to pre-select specific sequence databases that might contain close matches to the input sequence strain and to use default reference sequences or allow the user to pre-select specific reference sequence to facilitate sequence comparisons;
a storage code implemented on the computer or encoded on the computer readable storage medium and operable to number and to identify characteristic residues of individual virus sequences or their groups from program supplied external files and to store the residues, groups and other related information in one or a plurality of pathogen sequence databases in the form of multiple data arrays including information about phylogenetic trees, sequence groups and characteristic residues of the groups for efficient referencing and retrieval, where the arrays comprise tree-like multiple 2-dimensional arrays;
a sequence comparison code implemented on the computer or encoded on the computer readable storage medium and operable to compare input pathogen sequences with the stored database sequences on a residue-by-residue basis to produce comparisons corresponding to all pathogens or pathogen strains stored in the database that include some or all of the input pathogen sequences; and
an identification code implemented on the computer or encoded on the computer readable storage medium and operable to identify the pathogens or pathogen strains of input pathogen sequences based on the comparisons and subsequent decision making algorithms, where the algorithms identifies the comparison pathogens or pathogen strains that include maximal sequence identify.

14. The product of claim 13, wherein the pathogens or strains comprises single stranded RNA viruses.

15. The product of claim 14, wherein the GUI code is adapted to divide a screen of a display device connected to a computer implementing the GUI code into 3 distinct categories including a sequence category, a database category and an output options category adapted to simplify data input/output (I/O).

16. The product of claim 15, wherein the GUI code uses the sequence category region of the screen to allow users to input sequence data in a specified format using a paste option or by reading in files containing the data.

17. The product of claim 15, wherein the GUI code uses the database category region of the screen to allow users to specify “Unknown” in case sequence databases that might contain close matches to the input sequence strain are not known.

18. The product of claim 15, wherein the GUI code uses the database category region of the screen to use default reference sequences or allow users to pre-select specific reference sequence to facilitate sequence comparisons.

19. The product of claim 15, wherein the GUI code uses the database category region of the screen to allow users to select and display a particular sequence or all sequences from a database of sequences using a drop down menu.

20. The product of claim 15, wherein the GUI code uses the database category region of the screen to allow users to view groups of similar sequences.

21. The product of claim 15, wherein the GUI code uses the output options category region of the screen to allow users to selectively display identifiers associated with each input sequence and a summary/details of matches of characteristic residues for an input query sequence identified by its identifier.

22. The product of claim 14, wherein the storage code identifies characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval.

23. The product of claim 14, where the sequence comparison code selects individual residues of input sequences where the GUI code uses a sequence category region of a screen of a display associated with an implementing computer to allow users to input sequence data in a specified format using a paste option or by reading in files containing the data and compares these residues with the characteristic residues in the storage code numbers the characteristic residues and a storage code identifies characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval with respect to the GUI code, which uses a database category region of the screen to use default reference sequences or to allow users to pre-select specific reference sequences to facilitate sequence comparisons for sequence comparison purposes using a tree-like search method.

24. A computer program product implemented on a computer or stored on a computer readable storage medium for predicting strains of some pathogens from limited sequence data, the computer product comprising:
a graphical user interface (GUI) code implemented on a computer or encoded on a computer readable storage medium and operable to carry out all data input-output (I/O) operations, where the GUI code uses a sequence category region of a screen of a display associated with an implementing computer to allow a user to input sequence data of one or a plurality of input sequences in a specified format using a paste option or by reading in files containing the data and where the GUI code uses a database category region of the screen to use default reference sequences or to allow users to pre-select specific reference sequences to form selected individual residues of the input sequences to facilitate sequence comparisons;
a storage code implemented on the computer or encoded on the computer readable storage medium and operable to store virus sequence databases in the form of multiple data arrays including information about phylogenetic trees, sequence groups and characteristic residues of these groups and to number and to identify characteristic residues of individual virus sequences or their groups;
a sequence comparison code implemented on the computer or encoded on the computer readable storage medium and operable to compare the input pathogen sequences with the stored database sequences of known pathogen and pathogen strains on a residue-by-residue basis using a tree-like search method to produce comparisons, where the sequence comparison code selected individual residues of the input sequences and compares the selected residues with the characteristic residues to form comparisons comprising characteristic residues including their identifiers assigned by the storage code and stores the comparisons tree-like multiple 2-dimensional arrays for efficient referencing and retrieval; and
an identification code implemented on the computer or encoded on the computer readable storage medium and operable to identify the pathogens or pathogen strains of input pathogen sequences based on the comparisons and subsequent decision making algorithms, where the algorithms identifies the comparison pathogens or pathogen strains that include maximal sequence identify.

25. The product of claim 24, wherein the pathogens or strains comprise single stranded RNA viruses.

26. The product of claim 25, wherein the GUI code is adapted to divide a screen of a display device connected to a computer implementing the GUI code into 3 distinct categories including a sequence category, a database category and an output options category adapted to simplify data input/output (I/O).

27. The product of claim 26, wherein the GUI code uses the sequence category region of the screen to allow users to input sequence data in a specified format using a paste option or by reading in files containing the data.

28. The product of claim 26, wherein the GUI code uses the database category region of the screen to allow users to pre-select specific sequence databases that might contain close matches to the input sequence strain.

29. The product of claim 26, wherein the GUI code uses the database category region of the screen to allow users to specify “Unknown” in case sequence databases that might contain close matches to the input sequence strain are not known.

30. The product of claim 26, wherein the GUI code uses the database category region of the screen to use default reference sequences or allow users to pre-select specific reference sequence to facilitate sequence comparisons.

31. The product of claim 26, wherein the GUI code uses the database category region of the screen to allow users to select and display a particular sequence or all sequences from a database of sequences using a drop down menu.

32. The product of claim 26, wherein the GUI code uses the database category region of the screen to allow users to view groups of similar sequences.

33. The product of claim 26, wherein the GUI code uses the database category region of the screen to allow users to activate execution of the program.

34. The product of claim 26, wherein the GUI code uses the output options category region of the screen to allow users to selectively display identifiers associated with each input sequence and a summary/details of matches of characteristic residues for an input query sequence identified by its identifier.

35. The product of claim 25, wherein the storage code identifies characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval.

36. The product of claim 25, wherein the storage code numbers the characteristic residues and the storage code identifies characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval with respect to the GUI code, which uses the database category region of the screen to use default reference sequences or allow users to pre-select specific reference sequences to facilitate sequence comparisons for sequence comparison purposes.

37. A method of evaluating comparisons implemented on a computer or encoded on a computer readable storage medium, the method comprising the steps of:
identifying tree branches including maximum number of characteristic residue matches between an input sequence or a plural using a viral pathogen identification computer product implemented on the computer or encoded on the computer readable storage medium, where the product includes:
a storage code adapted to identify characteristic residues of individual virus sequences or their groups from program supplied external files and stores these residues, groups and other related information in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval;
a sequence comparison code adapted to select individual residues of an input sequence or a plurality of input sequences and to compare the selected residues with the characteristic residues of the input sequence or sequences to generate comparisons including maximum number of characteristic residue matches using a tree-like search and to store the matches in tree-like multiple 2-dimensional arrays for efficient referencing and retrieval,
a graphics user interface (GUI) code adapted to allow a user to enter the input sequence or sequences in a sequence category region of a screen of a display associated with an implementing computer in a specified format using a paste option or by reading in files, to allow a user to select default reference sequences or to pre-select specific reference sequences to facilitate sequence comparisons in a database category region of the screen, and to allow the comparisons to be displayed in the database category region of the screen,
searching the matches to identify maximally matching tree branches,
carrying out further searches in the case where no unique maximally matching tree branches from the identifying step are found, and
confirming that the chosen maximally matching tree branches do not lead to ambiguous comparisons.